Volcanic plumes contain reactive halogen species, especially bromine monoxide (BrO), which catalyzes ozone (O3) destruction. Therefore, local O3 depletion is commonly assumed inside volcanic plumes and has also been measured to varying degrees at different volcanoes. However, a calculation comparing atmospheric mixing with the rate of O3 destruction inside the plume suggests no significant reactive halogen catalyzed O3 loss (1% or less) in the plume. So far, O3 and its distribution in volcanic plumes have only been insufficiently determined since commonly used ultraviolet (UV) absorption O3 monitors show interference with sulfur dioxide (SO2), an abundant volcanic gas. This issue can be overcome by using a chemiluminescence (CL) O3 monitor, which has no known interference from trace gases abundant in volcanic plumes. However, field measurements with former CL O3 monitors are challenging, as they are heavy and bulky. Here we report on a lightweight version of the instrument (1.5 kg, shoebox size), which can be mounted onto a drone. In particular, the design, construction, and characterization are presented in this thesis. Measurements in the field were performed, including ambient measurements in Heidelberg and drone measurements during which a vertical O3 profile was measured. The main focus of the measurements is the drone-based O3 measurements in the plume of Etna. With the simultaneous SO2 measurement, an anti-correlation in the data can be observed, suggesting an O3 depletion of up to ~60% in the volcanic plume of Etna. This raises the question of which process leads to this observed O3 depletion.
All cells in a multicellular organism contain the same genetic information, yet individual cell types express different genes in order to fulfil their specific func- tions. Expression patterns are regulated dynamically throughout the cell cycle and in response to internal and external stimuli. Improper regulation of gene expres- sion patterns is associated with diseases such as cancer, where developmental gene expression profiles are often hijacked. The serine - arginine rich splicing factor (SR) proteins are a conserved family, consisting of 12 members in humans. They were first described as splicing factors, promoting the inclusion of weak splice sites and shown to work as a network, regulat- ing their own and each others expression in a cell type dependent manner. Various SR proteins have also been shown to have additional RNA processing functions, including regulation of transcription, translation initiation and non-sense mediated decay. Other than splicing the precise mechanisms by which SR proteins modulate gene expression were unknown. My study focused on family member SRSF2 and a recurrent mutant form of SRSF2 (P95H) commonly found in cancer. I directly compared the effects of conditional deletion or mutation of SRSF2 in mouse skin. Both perturbations caused a loss of cellularity and cell cycle arrest accompanied by impaired differentiation and increased DNA damage. I showed that SRSF2 acts as an important regulator of transcription and that the P95H mutation results in a loss of protein function. To decipher the molecular mechanisms of SRSF2 action it was knocked down in primary human keratinocytes, an established in vitro model for normal skin, and two squamous cell carcinoma cell lines. In all cell lines analysed SRSF2 depletion caused cell cycle arrest and increased DNA damage. In an attempt to look at the relative contributions of splicing and transcription both processes were analysed. No conserved changes to splicing were observed across cell lines, while analysis of transcription rates via SLAM-sequencing and RNA polmerase II (Pol II) occupancy via Cut&Run experiments revealed that depletion of SRSF2 was associated with reduced global nascent transcription due to enhanced stalling of Pol II at transcrip- tion start sites, suggesting a direct effect on transcription is the main cause of the effects of SRSF2. DNA replication and repair associated genes were most affected by loss of SRSF2 due to their specific genomic organisation as bi-directional gene pairs. These results provide insights into why SRSF2 is an essential gene, but how mu- tant forms of SRSF2 contribute to carcinogenesis remained unexplained. Therefore I generated cancer cell clones expressing reduced levels of SRSF2 using CRISPR-cas9 mediated gene editing approaches. Cancer cells with reduced SRSF2 function can cycle but accumulate mutations faster and thereby enable faster tumour evolution. Introducing the Srsf2 P95H mutation into one allele of the mouse skin inhibited tumour formation following exposure to carcinogens. However, within SRSF2 P95H established clones cell growth was not affected and clone size tended to be larger than that of control cells. In summary, my studies showed for the first time that the P95H mutation causes a loss of function and that SRSF2’s role in transcription, and not splicing, is required for proper expression of DNA replication and repair genes due to their organisation as bi-directional gene pairs.
Oncolytic vaccine strains of measles virus (MeVac) are studied as novel cancer therapeutics. By preferentially lysing tumor cells, these attenuated viruses induce systemic antitumor immunity. However, MeVac virotherapy alone is insufficient to achieve high rates of complete tumor remissions. Thus, in this study I aimed at identifying immunological mechanisms that limit or restrict the therapeutic potential of oncolytic MeVac.
Following the cancer-immunity cycle, I first focused on antigen presentation and T cell priming. I hypothesized that the immune response elicited by MeVac virotherapy is limited by impaired antigen processing, common in tumor cells, and reasoned that delivering pre-processed antigens to the tumor via MeVac vectors could circumvent this limitation. Using a murine system and chicken ovalbumin as model antigen, I showed that dendritic cells and tumor cells exposed to MeVac vectors encoding antigen-derived epitope variants present the encoded epitopes, especially when exposed to MeVac encoding six epitope copies targeted to the proteasome. Increased epitope presentation enhanced priming of naïve OT-I T cells and activation of cognate cytotoxic T lymphocytes. Thus, I proved the concept of using MeVac encoding antigen-derived epitope variants for T cell priming and activation. This project is now continued in the human context.
Subsequently, I focused on T cell migration and effector function. Based on efficacy and tumor gene expression data from previous studies, I hypothesized that the efficacy of MeVac virotherapy is limited by insufficient intratumoral expression of specific chemokines and cytotoxic molecules. To assess whether intratumoral overexpression of these molecules improves therapeutic efficacy, I conducted gain of function (GOF) efficacy studies in immunocompetent models of murine melanoma and colon adenocarcinoma. GOF studies with MeVac vectors encoding murine CXCL9, CXCL10, CCL19, or CCL21a, which I generated and characterized, showed that these chemokines do not limit the therapeutic efficacy of oncolytic MeVac. Loss of function studies will reveal whether these molecules are essential for MeVac virotherapy despite not being limiting. The identified cytotoxic molecules will be investigated following the same experimental approach.
MeVac virotherapy often results in PD-L1 upregulation on tumor cells. To address whether the PD-1/PD-L1 pathway restricts the efficacy of MeVac virotherapy, I investigated the combination of MeVac with PD-1/PD-L1 blockade in two systems. In an immunocompetent model of murine colon adenocarcinoma, I found that MeVac vectors encoding antibody-like molecules against PD-1 or PD-L1 induce stronger antitumor immune memory compared to MeVac alone. However, this effect was insufficient to improve therapeutic efficacy. In an immunocompetent model of murine pancreatic ductal adenocarcinoma (PDAC), local MeVac plus systemic anti-PD-1 antibody treatment was more effective than either monotherapy. In this model, I showed that MeVac was the main driver of systemic antitumor immunity, but required combination with anti-PD-1 to transiently induce an immune activation gene signature in the tumor. This study provides the basis for a Phase I clinical trial of MeVac plus Pembrolizumab in PDAC patients, currently in preparation.
While this work was conducted in wild-type mice, I also established CD46tg mice as a novel animal model to study oncolytic MeVac therapy. My investigations are the first to show that these mice develop systemic tumor-specific and measles virus-specific immunity upon intratumoral MeVac treatment. In gene expression studies, I identified a MeVac-induced tumor immune gene signature that warrants further investigation. Finally, I worked towards the establishment of patient-derived ex vivo tumor slice cultures as a platform to study early effects of oncolytic MeVac in a setting that preserves the patient-specific tumor heterogeneity and microenvironment.
Overall, identifying limiting factors of MeVac virotherapy will lead to the rational development of combination approaches that tackle treatment resistance. Furthermore, the refined models that I have established will increase the robustness of preclinical findings, thus improving translation into clinical research.
The addendum describes a preclinical study that I conducted to test the cellular immune response of MeVac-susceptible mice to a MeVac-based vaccine candidate against COVID-19.
Tumors are complex tissues with substantial intra-tumor heterogeneity, intricately linked to tumor progression and therapeutic resistance. Emerging single-cell RNA sequencing (scRNA-Seq) technologies empower researchers to elucidate these diverse tumor subpopulations. This thesis presents the characterization of a distinct glioblastoma (GB) cell population and introduces a novel bioinformatics tool designed to quantify similarity among cell populations.
Cell-to-cell connectivity through tumor microtubes (TMs) has been discovered among glioma tumor cells, conferring self-repair capabilities, augmenting therapy resistance, and driving tumor progression. Yet, a comprehensive molecular understanding and precise quantification of this connectivity have remained elusive. This study delves into the transcriptomic landscape of the highly connected glioma cell population using scRNA-Seq and RNA-Seq. I found that these highly connected cells exhibited a notable predominance of astrocyte-like (AC) and mesenchymal-like (MES) cell states, while lowly connected cells were characterized by a prevalence of neuronal progenitor-like (NPC) cell states. I established a 71-gene connectivity signature by comparing highly and lowly connected cells. A connectivity signature score (CSS) was developed based on the relative average expression levels of the connectivity signature. This CSS was then applied to several GB patient tumor scRNA-Seq and RNA-Seq datasets, consistently revealing higher CSS values for AC and MES cell states compared to NPC cell states. Furthermore, correlations were observed between CSS values and mesenchymal expression subtypes as well as between CSS values and the mutation status of NF1, PTEN, and TP53. One key finding is that higher CSS values were linked to poorer patient survival. Additionally, CHI3L1 — one of the connectivity signature genes — was identified as a robust marker for cell connectivity and a potential prognostic marker for GB patients. Investigating CHI3L1 overexpression RNA-Seq and proteomics datasets revealed that CHI3L1 upregulated multiple cell state markers and elevated CSS values. Notably, CHI3L1 overexpression also led to increased phosphorylation of the TM-connectivity marker GAP43.
In this thesis, I present a new bioinformatic tool named Interactive Explorer of Single-Cell Cluster Similarity (ieCS). This tool serves to link similar cell populations that share the same biological cell types/states across various donors or experimental conditions. ieCS utilizes an innovative metric to quantify similarity between cell populations. ieCS offers three distinct methods for identifying superclusters comprising similar cell populations. Featuring a user-friendly graphical interface, ieCS enables interactive and intuitive visualization of these superclusters. In a demonstration dataset, ieCS accurately, robustly, and quickly identified superclusters across various experimental conditions.
In conclusion, this thesis characterizes the highly connected GB cell population and introduces a bioinformatics tool for mapping similar cell populations.
Biology at the molecular level is driven by macromolecules interacting with each other to form multi-component complexes. To understand the function of macromolecular complexes, quantitative information about their composition and dynamics are essential. Single-molecule fluorescence microscopy offers unique possibilities to provide such information, but relies on the labeling of target molecules with fluorescent markers. In addition, the fluorescence signal emitted by a sample does not readily inform about absolute fluorophore numbers. To measure complex stoichiometries with fluorescence microscopy, it is therefore required to know the fraction of successfully labeled target molecules and to apply a method which infers the number of fluorescent markers from the recorded fluorescence signal. To approach these challenges, I have established and optimized tools to allow for a reliable determination of labeling efficiencies and secondly to successfully determine fluorophore numbers in fluorescently labeled complexes inside cells. In the first part of this study, I show that the labeling efficiency achieved with self-labeling protein tags in living and fixed cells can be calibrated by single-molecule colocalization analysis. An improved and validated data processing pipeline enabled me to systematically study the performance of the self-labeling protein tags, SNAPt-tag and HaloTag across different labeling conditions and with different fluorescent ligands. I found that labeling efficiencies for both tags depend on the ligand used and are limited to sub-stoichiometric levels for all tested ligands and labeling conditions. The highest labeling efficiency for either tag was achieved by labeling of SNAPt-tag with BG-SiR in fixed cells, where a maximum labeling efficiency of 65% was reached. The developed calibration approach provides a generalizable platform for the development and benchmarking of new labeling schemes for quantitative fluorescence microscopy. To address the need for a method to translate fluorescence intensity into absolute fluorophore numbers, quickPBSA was established as a new framework for fluorophore counting by photobleaching step analysis. quickPBSA was validated with simulations and in vitro measurements on DNA origami demonstrating an accessible counting range of up to 35 fluorophores and a 100-fold improvement in computational cost compared to previous algorithms. I could show that by combining improved data acquisition conditions with the quickPBSA framework for photobleaching step detection enables measuring stoichiometries on complexes labeled with up to 32 fluorophores inside cells. In combination, the developments presented in this study provide a comprehensive approach for measuring stoichiometries of protein complexes in situ and put applications in cell biology into perspective.
Environmental conditions significantly influence the phenotype and functionality of immune cells. Specifically, hypoxic conditions (oxygen concentration below 1%) prevalent in tissues and organs impact both the adaptive and innate immune systems. The transcription factor hypoxia-inducible factor 1-alpha (HIF-1α) plays a pivotal role in regulating immune cell adaptation to hypoxia. Natural killer (NK) cells are essential in tumor surveillance, as they target tumor cells by producing cytokines, chemokines, and cytolytic granules. Nevertheless, NK cells infiltrating solid tumors with hypoxic environments often lose their functionality. In addition, the high sensitivity of human primary NK cells to in vitro culturing conditions poses a challenge for classical transduction and transfection methods, hindering the study and generation of improved NK cells for immunotherapies. In this study, I investigated the effects of hypoxia and HIF-1α on human NK cells. My findings indicate that NK cells exposed to hypoxia (1 % oxygen) exhibited reduced IFNγ production, diminished degranulation response to cytokine stimuli and tumor targets, and impaired proliferation compared to NK cells exposed to normoxia (21 % oxygen). I explored the potential rescue of human NK cell functionality under hypoxia by genetically targeting HIF1A using CRISPR/Cas9 ribonucleoprotein-based transfection. HIF-1α-targeted NK cells, under hypoxia, restored IFNγ production upon IL-12/18 stimulation compared to mock control cells. To assess the practical applicability of HIF-1α-targeted NK cells, I employed patient-derived organoid lines from colorectal adenocarcinoma patients. The results revealed an enhanced cytotoxicity in HIF-1α-targeted NK cells against specific patient-derived organoids, particularly when combining Chimeric Antigen Receptor (CAR) targeting HER-2 with HIF-1α targeting. Additionally, I investigated the impact of hypoxia on cytokine-induced memory-like (CML) NK cells. CML NK cells exposed to hypoxia for seven days displayed a reduced IFNγ production upon IL-12/18 stimulation compared to normoxic controls. However, degranulation and cytokine production during K562 co-culture remained unaffected. These results suggest an oxygen sensitivity in CML NK cells, potentially regulated by HIF-1α, which warrants further investigation. Furthermore, I developed an efficient transfection protocol for human primary NK cells using electroporation and CRISPR/Cas9 ribonucleoprotein with dye-labelled sgRNAs. This allowed successful targeting of surface molecules such as TRAIL, NKG2A, TIGIT, CD112R, DNAM-1, and CD96, enabling the sorting of double and triple knockout populations of NK cells. In summary, my study highlights the importance of oxygen concentrations for human primary NK cells and introduces a cytotoxic NK cell product: HER-2 CAR HIF-1α NK cells, which may hold promise for CML NK cells. Moreover, I established an effective transfection system for human primary NK cells, facilitating the generation of gene edited NK cells to enhance tumor target killing.
Am Heidelberger Aeolotron, einem ringförmigen Wind-Wellenkanal, wurden mit aktiver Thermographie für einen Windgeschwindigkeitsbereich zwischen 2,1 und 5,5 m/s systematische Messungen durchgeführt, um die Geschwindigkeiten des Wasserkörpers und der Wasseroberfläche sowie die Geschwindigkeitsscherung in der viskosen Grenzschicht zu untersuchen. Mit einem Erbium-Faserlaser wurden dazu dünne Linien senkrecht zur Strömungsrichtung auf der Wasseroberfläche erhitzt, deren Entwicklung mit einer Wärmebildkamera beobachtet wurde. Aus der Verschiebung der Linienpositionen konnte direkt die Oberflächengeschwindigkeit bestimmt werden, die bei windstillen Bedingung auch der mittleren Wasserkörpergeschwindigkeit entspricht. Aus der zeitlichen Entwicklung der Linienbreiten ergab sich bei windstillen Bedingungen die Wärmediffusionskonstante für Wasser, sowie bei windinduzierter Schubspannung die Geschwindigkeitsscherung in der viskosen Grenzschicht. Vergleichswerte für die Geschwindigkeitsscherung konnten durch einen gezielten Messablauf außerdem mit der Impulsbilanzmethode direkt aus den Oberflächengeschwindigkeiten gewonnen werden. Um die Auswertung einfacher zu gestalten, wurde ein unlöslicher Film aus Hexadekanol auf die Wasseroberfläche aufgebracht, der die Wellenbildung unterdrückt.
Cnidarians (corals, jellyfish and sea anemones) constitute ideal model systems to investigate the dynamics and interactions of extracellular matrix (ECM) components. Many cnidarians undergo a complex life cycle characterized by transitions through different life stages with distinct morphologies. These morphological changes recapitulate developmental processes observed in vertebrates and involve the synthesis, degradation and remodeling of the ECM. The simple body plan of cnidarians provides exceptional accessibility for ECM imaging and manipulation, and previous studies have shown that the cnidarian ECM shares striking compositional and structural similarities with the vertebrate ECM. However, a major hurdle in the investigation of the cnidarian ECM is the lack of fully annotated ECM datasets allowing for a deeper understanding of its interactions and functions. In this thesis, I used the starlet sea anemone Nematostella vectensis as a model system to fully characterize the components of the cnidarian ECM by a combination of electron microscopy, single cell transcriptomics, in silico matrisome prediction and mass spectroscopic analysis of isolated ECM from three life stages (larvae, primary polyp and adult). I present the fully annotated in silico matrisome comprising 843 proteins, categorized into 246 core matrisome, 309 matrisome-associated and 289 other ECM-like proteins. 182 of these components are specific to the cnidocysts. My research revealed that the primary source of ECM is the gastroderm while the contribution of the ectoderm to ECM production is minimal. The integration of single cell transcriptome analysis and stage-specific mass spectrometry unraveled dramatic changes of the ECM during development, showing that the basal lamina gets established well in advance of the fibrillar interstitial matrix. Among the various ECM components upregulated during the larva-to-polyp transition, I detected an expanded polydom/SVEP1 family in Nematostella. This family encompasses several novel proteins, one of which emerges as a promising candidate for genetic perturbation using shRNAs. The resulting knockdown phenotype displayed defects in epithelial organization, body elongation, and development of internal mesenteries. This underscores that individual ECM components can heavily affect crucial developmental processes. In summary, my work establishes a foundational framework for future studies of the ECM by providing the first annotated molecular atlas for a cnidarian life cycle. In addition, it provides evidence for an ancient specification of ECM-producing cells within the endomesodermal lineage, along with a hierarchical function of basement membrane and interstitial matrix components in the life history of Nematostella.
Hintergrund: Die Verfügbarkeit von E-Mental Health in Form von digitalen Interventionen wie Smartphone-Apps kann dabei helfen, Behandlungsbarrieren zu überwinden und Versorgungslücken in der psychischen Gesundheitsversorgung bei Patient:innen mit Borderline-Persönlichkeitsstörung (BPS) zu verringern. Wir haben BPS-Patient:innen nach 12-wöchiger stationärer Dialektisch-Behavioraler Therapie (DBT) rekrutiert, die nach Entlassung über einen Zeitraum von 12 Wochen im Alltag eine Skills-App nutzten, um gewonnene Fortschritte weiter zu festigen, und die erlernten Skills im Alltag anzuwenden. Ziel: Diese Studie zielte darauf ab, die Wirksamkeit einer Skills-App in Bezug auf die BPS-Symptomatik, dissoziative Symptomatik, sowie auf die Häufigkeit der Anwendung von Skills nach stationärer DBT im ambulanten Setting zu evaluieren. Die Skills-App sollte Patient:innen im ambulanten Rahmen unterstützen, ihre Anspannung mit Hilfe von bereits erlernten Skills zu regulieren und neue funktionale Verhaltensweisen zu stärken und gleichzeitig dysfunktionale Verhaltensweisen weiter zu reduzieren. Methoden: Diese nicht-randomisierte kontrollierte Studie rekrutierte Patient:innen mit BPS im Alter zwischen 18 und 65 Jahren nach stationärer DBT für die Nutzung der Skills-App über 12 Wochen im ambulanten Setting (App-Gruppe). Die Patient:innen der Kontrollgruppe nahmen an einer ambulanten Skills-Gruppe Teil (TAU). Die Teilnehmer:innen der APP-Gruppe bekamen eine Skills-App zur Verfügung gestellt, die mehrfach am Tag die individuelle Anspannung abfragte und abhängig von der Höhe der Anspannung geeignete Skills vorschlug. Gemäß dem Algorithmus der Skills-App wurde dieser Vorgang alle 10 Minuten wiederholt, bis die Anspannung in den niedrigen Anspannungsbereich gesunken war. Die Teilnehmer:innen beider Gruppen füllten über einen Zeitraum von 12 Wochen alle zwei Wochen Fragebögen zur Schwere der BPS Symptomatik, Dissoziativen Symptomatik sowie Skills-Anwendung aus. Ergebnisse: Insgesamt wurden 14 Patient:innen der App-Gruppe mit 7 Proband:innen der TAU-Gruppe verglichen. Es zeigte sich in der App-Gruppe keine signifikante Verbesserung der BPS-Symptomatik und kein Rückgang der dissoziativen Symptome. In der App-Gruppe wurde ein Rückgang des funktionalen Verhaltens nachgewiesen bei unverändertem dysfunktionalem Verhalten. In der TAU-Gruppe ließ sich eine Verringerung der BPS-Symptomatik nachweisen. In einer genaueren Analyse der App-Nutzungsdaten (insgesamt 4277 Zeitpunkte) zeigte sich, dass die Anspannung mit Hilfe der Skills-App signifikant reduziert werden konnte. Dies war insbesondere der Fall, wenn die App im Hochstressbereich genutzt wurde. Bei einer Nutzung im mittleren Anspannungsbereich zeigte sich kein Effekt, im niedrigen Anspannungsbereich wurde sogar ein kontraproduktiver Effekt gefunden. Schlussfolgerung: Die alleinige Nutzung einer Skills-App nach stationärer DBT-Therapie ohne begleitende störungsspezifische ambulante Gruppen- oder Einzeltherapie ist nicht ausreichend, um die in der stationären Behandlung gelernten funktionalen Verhaltensweisen aufrechtzuerhalten und die BPS-Symptomatik weiter zu verbessern. Eine Evidenz für den Nutzen der digitalen Intervention für eine Verringerung der BPS-Symptomatik wurde nicht gefunden. Daher ist weitere Forschung erforderlich, um digitale Interventionen wie die Skills-App zu untersuchen, um mögliche Ressourcen für die Versorgung von BPS Betroffene zu schaffen.
This thesis discusses the pure spinor superfield formalism and its applications, specifically in the context of twisted eleven-dimensional supergravity. We start by developing the pure spinor superfield formalism as a framework for the construction of supermultiplets from a graded equivariant module over the ring of functions on the nilpotence variety. This perspective establishes a connection between algebrogeometric properties of the nilpotence variety and the physics of multiplets. Furthermore, it allows for efficient computations by means of homological algebra. After exploring the formalism in various examples, we extend it to the setting of derived geometry, show that this generalization establishes an equivalence of categories, and relate it to Koszul duality. In particular, this result establishes a method to construct superspace descriptions for any multiplet. As an application, we provide an extensive case study of supermultiplets with six-dimensional $\cN=(1,0)$ supersymmetry and classify all multiplets whose derived invariants for the supertranslation algebra define a line bundle on the nilpotence variety. In the second part, we consider eleven-dimensional supergravity and its twists. We compute the maximal twist in the free perturbative limit starting from the $L_\infty$ action of the super Poincar\'e algebra on the BV complex of component fields. Then, we use the pure spinor superfield formalism to construct a generalization of Poisson--Chern--Simons theory, defined on any supermanifold equipped with an appropriate odd distribution. This theory recovers Cederwall's formulation of eleven-dimensional supergravity, Costello's description of the maximal twist, and gives a pure spinor lift of the interactions in the minimally twisted theory. Compatibility between the pure spinor formalism and twisting implies that all these theories are related by twists. Motivated by holographic duality, we use these methods to explore (twisted) six-dimensional (2,0) supersymmetry. We give a pure spinor construction of the decomposition of the minimally twisted eleven-dimensional supergravity fields into $E(3|6)$-modules and provide an interpretation in terms of supergeometry which hints towards a generalization in the untwisted case.
Eine vielversprechende Zellquelle für das Tissue Engineering von Knorpelgewebe stellen mesenchymale Stromazellen (MSC) dar. Im Gegensatz zu artikulären Chondrozyten (AC) können MSC aus regenerierbaren Geweben wie Knochenmark isoliert werden ohne irreversible Schäden hervorzurufen. Bislang ist die Verwendung von MSC für die In-vitro-Knorpelneogenese allerdings durch ihre Differenzierung in einen hypertrophen Chondrozytenphänotyp limitiert, der aus der Wachstumsfuge bekannt ist und zu unerwünschter Knochenbildung führt. Um MSC zu einem AC-ähnlichen Phänotyp differenzieren zu können, müssen Signalwege identifiziert werden, die für diese enchondrale statt chondrale Fehldifferenzierung verantwortlich sein könnten. In diesem Kontext bisher nicht untersucht war der Phosphoinositid-3-Kinase(PI3K)/AKT-Signalweg, der in der Wachstumsfuge als pro-hypertropher Effektor an der Chondrozytenhypertrophie und Knochenbildung beteiligt ist. Obwohl AKT während der MSC-Chondrogenese in vitro durch die Behandlung mit Insulin kontinuierlich aktiviert wird, war bisher überraschenderweise nicht geklärt, ob der PI3K/AKT-Signalweg die enchondrale Differenzierung von MSC in vitro fördert. Das Ziel dieser Studie war es daher, die Funktion des PI3K/AKT-Signalweges für die MSC-Chondrogenese in vitro aufzuklären. Anfängliche Untersuchungen, ob enchondral differenzierende Zellen sensitiver auf AKT-Stimulierung reagierten als chondral differenzierende Zellen, ergaben eine deutliche Abhängigkeit der AKT-Aktivierung vom Differenzierungsstadium. Trotz identischer und konstanter exogener Stimuli starteten MSC mit niedrigerer AKT-Aktivierung in die Knorpelneogenese als AC, steigerten die AKT-Aktivierung bis zum Erreichen des Stadiums proliferierender Chondroblasten, woraufhin die pAKT-Spiegel in beiden Zelltypen mit voranschreitender Chondrozytenreifung abnahmen. Im Einklang mit der beobachteten Klimax der pAKT-Spiegel in einer Phase intensiver Proliferation trug endogenes FGFR-Signaling zur AKT-Aktivierung bei, das für seine proliferative Funktion bekannt ist. Im Gegensatz dazu inhibierte das in supraphysiologischen Konzentrationen verwendete TGFβ1 eine potentiell übermäßige AKT-Aktivierung. Eine Erhöhung der initial niedrigen pAKT-Spiegel steigerte zwar geringfügig die Proteoglykanablagerung, reduzierte aber vor allem die Zellzahl pro Pellet, so dass die niedrige initiale AKT-Aktivierung in MSC für das anfängliche Zellüberleben beim Übergang zur serumfreien chondrogenen 3D-Kultur notwendig war. Zur Beantwortung der Frage, welche Funktion die erhöhte AKT-Aktivierung in der späten MSC-Chondrogenese erfüllt, zeigten Inhibitionsversuche, dass die PI3K/AKT-Signalwegaktivität für die Aufrechterhaltung des Chondrozytenphänotyps und für die Knorpelmatrixablagerung essentiell war. Zum ersten Mal beschrieb diese Studie für die In-vitro-Knorpelneogenese die Notwendigkeit von PI3K/AKT-Aktivierung für adäquates TGFβ/SMAD2-Signaling und die SOX9-Proteinakkumulation und untermauerte die pro-chondrogene Rolle von AKT, die im Kontrast zu In-vivo-Knockout-Studien steht, wo der Verlust einzelner Signalwegkomponenten offensichtlich besser kompensiert werden kann. Im Gegensatz zur Wachstumsfuge war für die MSC-Chondrogenese keine pro-hypertrophe AKT-Funktion evident, was wahrscheinlich auf die verwendete hohe Konzentration von anti-hypertroph wirkendem TGFβ zurückzuführen war. Dass AKT-Aktivierung während der enchondralen MSC-Differenzierung die IBSP-Proteinspiegel reduzierte, deutete auf eine multifaktorielle AKT-Funktion für die MSC-Chondrogenese hin, die neben einer pro-chondrogenen und in Abwesenheit von TGFβ pro-hypertrophen auch eine selektiv anti-osteogene Wirkung beinhalten könnte. In dieser Arbeit konnte erstmals gezeigt werden, dass MSC im Verlauf der Chondrogenese unter konstanter Stimulation je nach Differenzierungsstadium ein variables Maß an AKT-Aktivierung benötigen. Da eine frühe AKT-Überstimulation den Zellverlust verstärkt und späte PI3K/AKT-Inhibition die Knorpelneogenese beeinträchtigt, müssen Modulationen des PI3K/AKT-Signalweges sorgfältig auf den chondrogenen Differenzierungsgrad abgestimmt werden. Die hier erstmals belegte Relevanz der steigenden AKT-Aktivierung für die MSC-Chondrogenese ist wegweisend für das Knorpel-Tissue-Engineering, da zukünftige Versuche, die Chondrozytenhypertrophie in vitro zu reduzieren, darauf abgestimmt sein müssen, nach initial niedriger AKT-Aktivierung einen differenzierungsabhängigen Anstieg der AKT-Signal-wegaktivität zu ermöglichen. Die Erkenntnisse dieser Arbeit erweitern unser noch lückenhaftes Verständnis von PI3K/AKT-abhängigen Differenzierungsprozessen, die entscheidend für eine erfolgreiche Knorpelneogenese mit MSC sind. Somit leistet diese Studie einen wichtigen Beitrag, um MSC in einen AC-ähnlichen Chondrozytenphänotyp differenzieren zu können, der uneingeschränkt für die klinische Knorpelregeneration einsetzbar ist.
Vaccine development relies on identifying target proteins that can elicit protective immune responses. However, highly immunogenic regions within these proteins can divert B cell responses from generating protective antibodies specific for less immunogenic regions. This is exemplified by the major surface protein circumsporozoite protein (CSP) of Plasmodium falciparum (Pf), the primary causative agent of malaria. The only available malaria vaccine, RTS,S/AS01, induces strong antibody responses against repeating NANP amino acid motifs in the central repeat domain of PfCSP. Despite their correlation with protection, these antibodies are not long-lived, and the vaccine's efficacy is moderate. Recent studies identified highly potent antibodies that target alternating NANP and NANP-like-motifs in the N-terminal junction of PfCSP, which is not part of RTS,S/AS01, with high affinity. However, these antibodies are overall rare and seem to develop frequently from NANP-specific precursors through affinity maturation. I hypothesized that a large number of repeating NANP-motifs in PfCSP prevents the development of anti-PfCSP-junction responses, while fewer repeating motifs would allow the induction of higher-quality antibody responses. To determine the impact of differing numbers of NANP-motifs on the anti-PfCSP-junction response, I immunized C57BL/6J mice with Helicobacter pylori-ferritin-based immunogens presenting the PfCSP-junction either in combination with a NANP5- or a NANP18.5-repeat. The humoral data showed that the NANP5-immunogen effectively directed the IgG response towards epitopes in the PfCSP-junction without compromising the strength of the anti-PfCSP-repeat response. Furthermore, immunization with the NANP5-nanoparticle induced a higher proportion of cross-reactive antibodies binding both the PfCSP-repeat and -junction. Additionally, reducing the number of repetitive amino acid motifs led to antibodies with overall higher avidity for PfCSP and an increased parasite inhibitory capacity. These differences were also reflected in the immunoglobulin repertoire of the B cells. I found that heavy chain variable (VH) and light chain variable (VL) gene segment combinations associated with antibody cross-reactivity and high affinity were enriched in germinal center B cells and plasma cells of mice immunized with the NANP5-immunogen. Finally, I showed that reducing the number of repetitive amino acid motifs led to higher frequencies of PfCSP-reactive memory B cells after the booster immunization. By highlighting the impact of epitope repetition on B cell affinity maturation, antibody quality, and the development of robust B cell memory, these findings hold promising implications for optimizing malaria immunogens and for guiding the rational design of other vaccine candidates, unlocking the ideal immunization strategy against various pathogens.
Recent technological strides in cancer research have unveiled the landscape of somatic mutations in propelling uncontrolled growth. However the intricate organization of cellular hierarchies governing and fueling progression remains uncharted. Adult neural stem cells are specialized astrocytes in the neurogenic niches of the brain, continually generating de novo lineages under strictly governed conditions, and capable of responding to injury. Viewing astrocytic origin of glioblastoma as a misappropriated regeneration attempt in the aging brain, adult neural stem cell lineage represents the ideal model to draw direct comparisons and contextualize malignant organization of neoplastic cells. In this dissertation, I demonstrate that glioblastoma cells - much like their healthy counterparts - establish a cellular hierarchy spanning quiescence, activation and differentiation stages, respectively. Enabled by the single cell workflows I established to quantify canonical Wnt-activity in health and disease, I found out that; unlike during normal neurogenesis, key Wnt pathway modulators are recurrently dysregulated during glioblastoma progression. Directly comparing healthy and cancerous trajectories in the adult mammalian brain, I identified SFRP1 - a secreted Wnt antagonist functioning as the gatekeeper of neurogenic activation of astrocytes - to be lost in glioblastomas. Re-introducing the expression of SFRP1 in a patient derived xenograft model robustly blocked tumor progression and significantly increased overall survival in mice. Single cell RNA sequencing validated by dual immunofluorescence-spatial transcriptomics revealed that SFRP1 overexpression induces a quiescent astrocyte-like phenotype that is concomitantly reflected at tumor’s methylome. Together, I present an innovative framework to re-interpret cellular structures in tumors informed by the tissue of origin trajectories, introducing the use of DNA methylation as a means to stratify patients and monitor disease evolution as well as discover actionable targets for precision medicine.
Mucosal tissues act as a protective barrier in constant contact with microbiota. Epithelial cells on these surfaces must mount an effective immune response for protection from pathogens while minimizing adverse reactions to commensal microbiota. Each cell within these tissues has a specific population context, which is determined by the local cell density, cell-to-cell contacts, and relative location within the population. The aim of this thesis was to comprehensively elucidate how the cellular population context shapes viral infection and immune signaling pathways in epithelial tissues. To this end, I employed human intestinal epithelial cells (IECs) as a model system for mucosal tissues. I developed a density-based approach and a micropatterning system to place cells in controlled microenvironments, which I combined with bio-molecular methods and microscopy/ sequencing-based high-content data bioinformatics analyses. My findings show that IECs embedded in a dense monolayer polarize, leading to a basolateral localization of the interferon (IFN) receptors. Hence, cells located in the center of a dense population did not induce antiviral protection upon apical IFN treatment due the receptor inaccessibility. Additionally, I discovered that the cellular microenvironment, especially the local cell density, controls homeostatic immune pathways. Confluent IEC expressed significant basal levels of IFNλ3 and downstream interferon stimulated genes, while sparse cells elicited no basal IFN-signaling. The basal type III IFN expression was induced by the cGAS-STING pathway after recognition of mitochondrial DNA. Importantly, the Hippo pathway emerged as the master regulator controlling homeostatic immune signaling, which senses the population context (e.g. cellular density and cell-to-cell contacts) to adjust cell behavior to its microenvironment. Finally, I established that the population context governs virus particle binding and active pathogen replication within a cell colony, leading to increased edge cell infection while center cells remained protected. This thesis shows that the population context in vitro directly shapes homeostatic and antiviral processes in epithelial cells. Cells embedded in the intact monolayer elicit higher basal IFNλ3 expression and less virus infection, demonstrating that a physiological micro-environment supports the barrier function of mucosal surfaces. My work strongly suggests that the population context should be considered when planning experiments in vitro, and it also underlines the importance of studying the population context and its implications in vivo. In light of the COVID-19 pandemic, I additionally characterized the role of type I and III IFNs signaling in controlling SARS-CoV-2 infection in the gut. My results indicate that type III IFNs are more efficient than type I IFNs in clearing SARS-CoV-2 infection in human intestinal epithelial cells. These findings further our understanding of host-pathogen interactions and could contribute to a development of improved treatment options.
The controlled release of therapeutic agents offers certain benefits in pharmacology, patient compliance and tolerability compared to conventional dosage forms. Nowadays, long acting injectables for peptide and protein therapeutics gain in importance as increasing numbers of biotherapeutics receive approval and subcutaneous self-administration attracts attention due to cost reductions and improved patient acceptance. The innovative polymer platform SynBiosys® claims to overcome weaknesses of conventional polymers for microparticle-based controlled release. As objective of this work, the suitability of these polymers for controlled release of biotherapeutics was evaluated based on various integrity analyses of representative models after in vitro and in vivo release from SynBiosys®-based microparticles. Microparticle formulations of the anti-diabetic peptide drug exenatide were examined in vitro and in vivo in comparison with the marketed benchmark formulation Bydureon®. Exenatide was released in vitro with high linearity over two to four weeks and integrity of liberated peptide was preliminarily confirmed with a cell-based functional assay. Likewise, SynBiosys® formulations exhibited a more continuous release behavior in vivo compared to the benchmark Bydureon®. Single subcutaneous injection of SynBiosys®-based microparticles significantly reduced non-fasting blood glucose between 25-50% over approximately four weeks and decreased glycated hemoglobin significantly by 3.4 - 4.6% in a rat model of type II diabetes (high fat diet/streptozotocin induced). Formulation of the monoclonal antibody mAbB in differently composed SynBiosys® polymer matrices allowed the choice of a suitable polymer composition for the controlled release of proteins with high molecular weight. The structural integrity of in vitro released mAbB was confirmed by circular dichroism and fluorescence spectroscopy. Based on these results, mAbX as oncological model drug was encapsulated and its integrity after linear in vitro release over 10-14 days was confirmed by a powerful combination of SE-HPLC, circular dichroism and fluorescence spectroscopy, ELISA as well as a cell-based functional assay. The in vivo liberation of mAbX resulted in highly dose-dependent antibody plasma levels as well as reproducible values for the absolute bioavailability of 25.7-30.6% in xenograft mouse models. Pharmacodynamic efficacy of mAbX released from SynBiosys® microparticles was demonstrated by significant growth reduction of A-431 epidermoid carcinoma cell tumors between 50 and 100%. In conclusion, the compatibility of SynBiosys® polymers with sensitive peptides and large protein therapeutics was demonstrated herein by in vitro and in vivo release of model biotherapeutics in highly functional condition as confirmed by a comprehensive analytical method composition. In the case of exenatide, the accelerated onset of therapeutic effect, the lower number of treatments and the significantly reduced total dose required were remarkable. The SynBiosys® platform is a promising tool for the improved controlled release of biologicals.
Pediatric cancer is the third leading course of death among children and adolescents in the USA despite its low incident and high survival rate. Next-generation sequencing technologies allow the profiling of tumor genetics and the prediction of disease progression and response to therapies. However, tumor temporal and spatial heterogeneity could complicate the success of the selected therapy. Serial sampling of tumors at multiple time-points can accurately track the dynamics of clonal evolution during treatment. Multiple sampling of tumors at different locations can reveal all clonal genetic structures of the tumor. Nevertheless, both strategies might post discomfort or critical risk to the patient. Liquid biopsy has become an attractive strategy for obtaining tumor biomarkers non-invasively. Sequencing of cell-free DNA (cfDNA), DNA fragments in the liquid sample such as blood, has become a strategy to detect tumor-derived genetic markers known as circulating tumor DNA. Recently, cfDNA has been extensively evaluated its clinical value with different high-throughput sequencing technology in many adult cancers. Hence, cfDNA could also have a potential benefit to the management of pediatric cancer patients.
In this thesis, we developed bioinformatics workflows for analyzing cfDNA derived from an extensive group of pediatric cancer patients. The workflow aims to detect genetic alterations from three sequencing strategies, including low-coverage whole-genome sequencing (lcWGS), whole-exome sequencing (WES), and deep gene-panel sequencing (Panel-seq). The capabilities of detecting copy-number aberrations and point mutations have been compared between those strategies. We also compared the detectability of plasma cfDNA across tumor entities, including brain tumors, sarcomas, and other pediatric cancers. Sequencing strategy and tumor location have influences on the success of cfDNA in detecting tumor genetic alterations. An R package, cfdnakit, was developed to extract the length of cfDNA fragments and perform genome-wide fragment-length analysis using lcWGS dataset. The fragment-length analysis shows that the enrichment of short-fragment cfDNA is correlating with copy-number aberrations. In addition, this package calculates a comprehensive copy-number aberration (CPA) score that combines copy-number aberration and short-fragmented cfDNA ratio. This CPA-score is correlating with a higher level of ctDNA and could suggest the use of subsequent detection methods such as WES to detect actionable mutations with more sensitivity. Moreover, we applied TelomereHunter, a telomeric DNA analysis tool. It showed that telomeric DNA exists which opens an opportunity to detect telomeric aberration in plasma cfDNA. Analyzing plasma cfDNA of the pediatric cohort has shown the declining of telomere content. However, elongation and integration of telomeric variant repeats were found among brain tumor and sarcoma patients.
Finally, we demonstrated the utility of liquid biopsy cfDNA in the management of pediatric cancer. cfDNA reveals heterogeneous mutations possibly shred by tumor at metastasis site in a child with bilateral nephroblastoma. This finding supports the utility of cfDNA as a comprehensive source of genetic information derived from the tumor population in the body without invasive multiple tumor biopsies. In addition, we found that cfDNA can detect tumor temporal heterogeneity in several sarcoma patients through serial biopsy. This finding supports the idea of utilizing cfDNA to follow-up patients during the course of therapy.
Cancer persists as one of the prevailing causes of death in children and adolescents aged 0 to 19 years. There remains to be an unmet need for identification of therapeutic biomarkers and better treatment interventions for these patients. Advancements in state-of-the-art molecular profiling techniques have resulted in better understanding of pediatric cancers and their driver events. It has become apparent that pediatric malignancies are significantly more heterogeneous than previously thought as evidenced by the number of novel entities and subtypes that have been identified with distinct molecular and clinical characteristics. For most of these newly recognized entities there are currently extremely limited treatment options available. Unfortunately, there is also a lack of compiled and consistently analysed molecular data available, along with limited data of characterization and documentation of patient-derived models and/or genetic mouse models from high-risk pediatric tumors. Both my studies fall under the “Innovative Therapies for Children with Cancer Pediatric Preclinical Proof-of-concept Platform” (ITCC-P4) consortium which is an international collaboration between different European academic institutes, several partnering pharmaceutical companies and three contract research organizations. The two studies aim to shed light on identification of potential promising treatment options that specifically match the patient’s specific molecular tumour characteristics and the patient’s genetic data. Genetic information at the molecular level from pediatric tumors in relapsed patients has contributed to advancing our understanding of disease progression and treatment resistance. The first study overall aims to establish a sustainable platform of >400 molecularly well- characterized PDX models of high-risk pediatric cancers, including the analysis of their original tumors and matching controls. This will enable the selection of PDX models for in vivo testing of novel mechanism-of-action based treatments. Hence, facilitating the prioritization of pediatric drug development and clinical stratification of patients across entities. In a first batch, 251 models were fully characterized, including 180 brain and 71 non- brain PDX models, representing 112 primary models, 93 relapse, 42 metastasis and 4 progressions under treatment models. Using low-coverage whole-genome and deep whole exome sequencing, complemented with total RNA sequencing and methylation analysis, the aim was to define genetic features in the ITCC-P4 PDX cohort and assess the molecular fidelity of PDX models compared to the original tumor. Based on DNA methylation profiling 43 different tumor subgroups within 18 cancer entities were included. Mutational landscape analysis identified key somatic and germline oncogenic drivers where Ependymoma PDX models displayed the C11orf95-RELA fusion event, YAP1, C11orf95 and RELA structural variants. Medulloblastoma models were driven by MYCN, TP53, GLI2, SUFU and PTEN. High-grade glioma samples showed TP53, ATRX, MYCN and PIK3CA somatic SNVs, along with focal deletions in CDKN2A in chromosome 9. Neuroblastoma models were enriched for ALK SNVs and/or MYCN focal amplification, ATRX SNVs and CDKN2A/B deletions. Sarcoma models displayed characteristic alterations with PAX3-FOXO1 fusions detected in embryonal rhabdomyosarcoma, along with TP53, CDKN2A, NRAS SNVs, NCOA1 gains, NF1 and CDK4 SVs. Ewing sarcoma PDX models displayed the defining EWSR1-FLI1 gene fusion in most cases, along with two rarer cases of EWSR1-ERG and EWSR1-FEV observed in the cohort. Osteosarcomas were defined by highly unstable genomes with large chromosomal alterations, TP53 and RB1 tumor suppressor genes were frequently altered and ATRX loss and MYC gains were observed. Additional sarcomas such as clear cell sarcoma of the kidney showed CDKN2A loss, MYC gain, NF1 loss, TP53 mutations, while Synovial sarcoma models were driven by SSX gene fusions and alterations. Large chromosomal aberrations (deletions, duplications) detected in the PDX models were concurrent with molecular alterations frequently observed in each tumor type –isochromosome 17 was detected in five medulloblastoma models, while deletion of chromosome arm 1p or gain of parts of 17q in neuroblastomas which correlate with tumor progression. Tumor mutational burden across entities and copy number analysis was performed to identify allele-specific copy number events in tumor-normal pairs. Clonal evolution of somatic variants was not only found in certain PDX-tumor pairs but also between disease states. Across the 16 serial model cases, discordance in targetable SNV, SV and CNV, alterations were observed in later disease progressed states compared to the primary models. The multi-omics approach in this study provides insight into the mutational landscape and patterns of the PDX models thus providing an overview of molecular mechanisms facilitating the identification and prioritization of oncogenic drivers and potential biomarkers for optimal treatment. The second study was a Target Actionability Review on replication stress. Detrimental long-term side effects due to chemotherapy drastically affect the lives of patients under treatment, hence there is an urgent need to identify novel target driven therapies. Decades of published data provide evidence for targeting replication stress therapeutically. Hence, in this study, we evaluated specific targets within the replication stress response (RSR) pathway. A comprehensive, well-structured, and critically evaluated overview of literature related to replication stress across 16 pediatric solid malignancies was generated. The methodology focuses on the systemic extraction and structured evaluation of replication stress as a target. This aims to align targeted anti- cancer therapeutic interventions with specific cancer subtypes based on clinical studies. ATR, ATM, PARP, WEEI were observed to represent the most promising targets either using single agents or in combination with chemotherapy or radiotherapy. Evidence on CHK1 and DNA-PK although limited, showed potential to further investigate these promising targets over broader tumor types. The collective data and results from both studies, the “ITCC-P4: Molecular characterization and multi-omics analysis of Patient-Derived Xenograft (PDX) models from high-risk pediatric cancer” and the “Target actionability review on replication stress”, can be explored further on the interactively designed R2 platform, once users create an account to gain access to the cohort data. (https://r2-itcc-p4.amc.nl/).
Advances in light microscopy have allowed circumventing the diffraction barrier, once thought to be the ultimate resolution limit in optical microscopy, and given rise to various superresolution microscopy techniques. Among them, localization microscopy exploits the blinking of fluorescent molecules to precisely pinpoint the positions of many emitters individually, and subsequently reconstruct a superresolved image from these positions. While localization microscopy enables the study of cellular structures and protein complexes with unprecedented details, severe technical bottlenecks still reduce the scope of possible applications. In my PhD work, I developed several technical improvements at the level of the microscope to overcome limitations related to the photophysical behaviour of fluorescent molecules, slow acquisition rates and three-dimensional imaging. I built an illumination system that achieves uniform intensity across the field-of view using a multi-mode fiber and a commercial speckle-reducer. I showed that it provides uniform photophysics within the illuminated area and is far superior to the common illumination system. It is easy to build and to add to any microscope, and thus greatly facilitates quantitative approaches in localization microscopy. Furthermore, I developed a fully automated superresolution microscope using an open-source software framework. I developed advanced electronics and user friendly software solutions to enable the design and unsupervised acquisition of complex experimental series. Optimized for long-term stability, the automated microscope is able to image hundreds to thousands of regions over the course of days to weeks. First applied in a system-wide study of clathrin-mediated endocytosis in yeast, the automated microscope allowed the collection of a data set of a size and scope unprecedented in localization microscopy. Finally, I established a fundamentally new approach to obtain three-dimensional superresolution images. Supercritical angle localization microscopy (SALM) exploits the phenomenon of surface-generated fluorescence arising from fluorophores close to the coverslip. SALM has the theoretical prospect of an isotropic spatial resolution with simple instrumentation. Following a first proof-of-concept implementation, I re-engineered the microscope to include adaptive optics in order to reach the full potential of the method. Taken together, I established simple, yet powerful, solutions for three fundamental technical limitations in localization microscopy regarding illumination, throughput and resolution. All of them can be combined within the same instrument, and can dramatically improve every cutting-edge microscope. This will help to push the limit of the most challenging applications of localization microscopy, including system-wide imaging experiments and structural studies.
Characterisation, compression and shaping of mid-infrared (MIR) pulses are demonstrated in an acousto-optic modulator (AOM) shaper based setup. Characterisation of the pulses’ spectral phase is accomplished via an AOM shaper based variant of dispersion scan (d-scan). By a combination of d-scan and an evolutionary algorithm, broadband MIR pulses centred at 3.2 µm are compressed to below 50 fs FWHM autocorrelation. The shaping and characterisation capabilities of the setup are demonstrated by imprinting and retrieving a set of spectral phases of increasing complexity on compressed broadband MIR pulses. Moreover, the effect of excitation of the C=N=N stretching vibration on the photoreaction of 2-diazo-1-naphthol-5-sulfonate dissolved in methanol is investigated. The C=N=N stretch mode is excited up to at least v=3 by ultrafast MIR pulses. Molecular dissociation in the electronic ground state is not observed. Vibrational excitation decays within 20 ps. Experiments on vibrationally mediated photodissociation show that vibrational pre-excitation increases the photoreaction’s quantum yield by at least 10% compared to excitation of the molecule by a mere ultraviolet pulse. This is explained by an increased Franck-Condon overlap of the edge of the electronic ground state’s potential energy surface with the reactive part of the potential energy surface of the electronic excited state.
Diabetes is a major metabolic disorder and type 2 diabetes (T2D) is the most prevalent form of diabetes where insulin resistance serves as the reason for hyperglycemia. Insulin receptors are a crucial part of the insulin signaling pathway that mediates glucose uptake in skeletal muscles and adipose tissues as well as in other organs. Insulin signaling promotes glycogen and protein synthesis, cell growth, inhibits apoptosis through several downstream pathways. Different aspects of Insulin receptor depletion have been conducted in mice and zebrafish yet the implication with microvascular complications in zebrafish has not been carried out. Thus, this study was directed towards finding the abnormalities caused by insulin receptor knockout in zebrafish. My study was conducted in zebrafish where I have taken advantage of zebrafish as a model organism and the availability of Tg(fli1:EGFP) and Tg(wt1b:EGFP) transgenic zebrafish. Zebrafish depleted of Insulin receptors (insulin receptor a: insra-/- and insulin receptor b: insrb-/-) were generated using CRISPR/Cas9 gene-editing technology and then I characterized the morphology of the mutants during the early and adult stages. I have found that the larvae showed no hyperglycemia, however, overfeeding can lead to fasting high glucose in insra-/- fish. I also observed that while trunk vasculature is affected only in insra-/- larvae, retinal hyaloid vasculature is affected in both insra-/- and insrb-/- larvae. Furthermore, abnormalities in retinal vasculature were also found in over-fed insra-/- fish and in normalfed insrb-/- fish. I have observed metabolic shift in those mutants as saturated & unsaturated fatty acids and cholesterol increased in insra-/- and insrb-/- larvae. Also in adult fish, we found a higher amount of fatty acids in skeletal muscle. Altogether, the data acquired from this study show that despite maintaining a euglycemic state, microvascular complications can arise when insulin receptors are knocked out in zebrafish. Therefore it can be said that high glucose is not the only factor behind the development of microvascular complications in zebrafish.
The ventricular-subventricular zone (vSVZ) of adult mammalian brains harbors specialized astrocytes, called adult neural stem cells (NSCs), which are capable of generating both neurons and glial cells. In contrast, common parenchymal astrocytes perform a wide range of structural, metabolic, homeostatic and neurosupportive functions. Despite these distinct functions, studies employing immunostaining and single-cell RNA-sequencing have demonstrated that NSCs and astrocytes largely express the same set of genes, which raises the question how stem cell function is molecularly encoded. To address this question, I analyzed a single-cell triple-omic data set that contains information on gene expression, chromatin accessibility and DNA methylation for hundreds of cells of the adult NSC lineage, as well as common parenchymal astrocytes.
To enable this analysis, I developed "scbs", a Python software for the analysis of single-cell methylation data. I devised and implemented two major improvements over the current state of the art analysis workflow: First, instead of segmenting the genome into fixed intervals, I devised an approach to scan the entire genome for informative regions called variably methylated regions (VMRs). Second, instead of simply averaging methylation values within these tiles, I devised a more robust measure of DNA methylation.
By making use of these new methods, I demonstrated that adult NSCs possess a unique DNA methylation profile that is not found in common parenchymal astrocytes. This NSC methylome is characterized by hypomethylation of genes required for neurogenesis. I propose that this feature contributes to the neurogenic capabilities of NSCs by enabling the transcriptional activation of these genes. In contrast, common parenchymal astrocytes are locked in their current astrocyte fate by DNA methylation. To test this hypothesis, I analyzed single-cell multi-omic data from mice that were subjected to ischemia, as ischemia is known to induce a neurogenic response in common parenchymal astrocytes and NSCs. My analysis suggests that this gain of neurogenic capabilities is accompanied by gain of an NSC methylome, which supports the idea that a specific DNA methylome is required for stem cell function. Overall, my results demonstrate that DNA methylation is dynamic even in adult tissues and not just in embryonic development, and furthermore unveils DNA methylation as a crucial factor that constrains or enables alternative cellular fates.
Proteins are essential components of life. Their functions rely on intrinsic activities and specific interactions which together constitute the functional identity of a cell. Within this network of interactions, proteins (and other macromolecules) form dense and functional discernable modules that exert new, module-specific functions and are referred to here as cellular functional modules (CFMs). Examples of CFMs are protein complexes and regulatory or metabolic pathways. CFMs are evolutionary traceable, heritable and able to duplicate and diversify, and thus can be regarded independent units of selection. This opens up new avenues towards solving challenging questions in evolutionary biology, such as the origin of complex cellular traits. In particular, the nervous and muscular systems are essential for the evolution of coordinated behavior in animals, yet their origin remains obscure. This thesis summarizes my efforts investigating the evolution of CFMs, with a focus on actomyosin CFMs, using the freshwater sponge Spongilla lacustris as a model species. Sponges (Porifera) are one of the earliest-branching animals, lacking “conventional” neurons and muscle cell types, yet are shown to express neuronal and actomyosin CFMs. Strikingly, external stimuli evoke an intriguing coordinated whole-body movement which remains poorly understood. To advance on this, (1) I improved the annotation of the S. lacustris proteome by ~50%, designing a custom pipeline for protein structure-based functional transfer, termed MorF (Morpholog Finder), in a collaborative effort. This work confirmed that morphologs (= structural similar proteins) are homologs in most cases and share functions even when homology is no longer detectable with conventional sequence-based methods. Novel annotations revealed new cell-type specific CFMs. Leveraging this, (2) I specifically focused on shape changes and molecular mechanisms of the whole-body movement using live 3D imaging, pharmacological profiling, and a survey of sponge cell-type specific expression of actomyosin CFMs. I found that - in contrast to prevailing views - the movement is triggered by the relaxation of epithelial actomyosin stress fibers via an Akt/NOS/PKG pathway, conserved in vertebrate smooth musculature. These stress fibers likely present an early form of an actomyosin CFM that was ultimately incorporated into specialized myocytes. Functional proteomic profiling of the movement (3), in particular Thermal Proteome Profiling, quantitative phosphoproteomics and secretomics, reaffirmed the “smooth-muscle”-like regulation and further suggested a mechanosensory function of the stress fibers, serving both as “sensor” and “actor”. The secretion of paracrine and diffusible signals sheds new light on the whole-body coordination in an otherwise nerve-less animal. During the movement induced by mechanical stimulation, the sponge activated an inflammation-like state. This “relaxant-inflammatory” CFM represents a conserved reaction of fluid-carrying metazoan systems - such as the vertebrate vascular system - to oscillatory shear stress. Last, (4) I outline my efforts towards a sponge-specific protein-protein interactome, using Co-fractionation mass spectrometry and single cell RNAseq co-regulation, presenting preliminary sponge complex CFMs. Together, the evolutionary origin and diversification of the relaxant-inflammatory CFM in animal movement is a prime example how complex cellular functions can emerge through the step-wise integration of existing CFMs.
Tissues are a major focus of clinical research and histopathological diagnosis for a wide range of diseases. Understanding the complex biomolecular manifestations of disease within tissues by characterizing its morphology and biomolecular information content paves the way for exploring the fundamental mechanisms of pathogenesis and for identifying diagnostic and prognostic biomarkers and potential therapeutic targets. Among the many tissue-investigation techniques, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has evolved into a label-free core technology for visualization and spatially-resolved ex vivo analysis of biomolecules directly from tissue samples.
Ion images, i.e. false color renderings of mass-to-charge ratio (m/z) intervals of interest, are used as the fundamental investigation tool in MALDI-MSI for conveying the spatial distribution of molecules-of-interest (MOIs, e.g., metabolites, drugs, lipids or proteins) within biological tissues that are often compared to external histopathology annotations. They are considered as the gold-standard by MSI researchers against which the biomarker discovery methods are validated. However, the conversion of raw MSI data into ion images for visualization, spatial interpretation and molecular analysis, has not changed since the inception of the technology. Moreover, the generated ion images can be prone to technical artifacts, user input- and user perception-bias.
This work introduces a computational framework, moleculaR, which proposes a coherent spatial probabilistic approach for mapping tissue MOIs and allowing for a user-independent spatial visualization and interpretation of MOIs' distribution in tissue samples via MSI. moleculaR uses user-independent assignment of m/z intervals for capturing MOIs based on the device- and measurement-dependent mass resolving power along with Gaussian-weighting of observed peak intensities for improved reliability of metabolite/lipid/drug signals in MSI. Instead of relying on a subjective qualitative judgment of the end-user concerning the observed spatial distribution of an MOI within a tissue sample, moleculaR proposes molecular probabilistic maps (MPMs), which apply pixel-wise spatial significance testing of MOI intensities against a complete spatial randomness (CSR) model inferred from the signal intensities of that same MOI. The framework also allows for spatial statistical comparisons of different tissues (cross-tissue MPMs, or CT-MPMs) and for collective projections of metabolite ensembles onto a single tissue plane, followed by computation of collective projection probabilistic maps (CPPMs). Ultimately, computed "hotspot" and "coldspot" spatial contours provide user-independent and probabilistic localization of tissue areas where an MOI has a statistically significant non-random relative spatial abundance or deficiency, respectively. Furthermore, this work extends the above concept to spatial quantitative mapping in tissues based on drug dilution series proposing a generalized nonlinear calibration model as a replacement for the traditional linear model that could better model drug-intensity response in the presence of noise and technical variability.
The framework has been tested and validated on data acquired from various MALDI-MSI instrument platforms featuring different tissue samples including isocitrate dehydrogenase-wild type (IDH-WT) glioblastoma (GB), IDH-WT and -mutant glioma, gastrointestinal stromal tumor (GIST), wild-type mouse brain, porcine liver and APP NL-G-F Alzheimer’s disease mouse model tissue samples in addition to simulation-based experiments mimicking a MALDI-MSI ground-truth which have been developed and utilized to test the proposed workflows. The results highlight moleculaR’s capabilities of i) improving MOI signal reliability, ii) providing objective and data-driven designations of areas which exhibit statistically significant non-random spatial patterns of MOI intensities independent of how an end-user may perceive its (i.e. MOI's) spatial relative abundance or deficiency and iii) enabling spatially-resolved investigation of ion milieus, lipid remodeling pathways or complex scores like the adenylate energy charge within the same image. On the other hand, spatial quantitative mapping based on generalized nonlinear calibration and cross-tissue probabilistic mapping can be used to provide valuable insight into drug-tissue penetration. In particular, spatial quantitative mapping of the drug imatinib in a cohort of GIST tissue samples revealed striking inefficiency in imatinib penetration into GIST liver metastases, despite the abundant imatinib levels beyond the limit of quantification (LOQ) observed within the corresponding healthy liver tissues surrounding the metastatic GIST.
In conclusion, the results suggest that moleculaR, with its core concept of spatial probabilistic mapping of biomolecules in tissues, shall replace or complement ion images for the spatial analysis of MOIs because of its valuable benefit of enabling probabilistic localization of non-random patterns of MOI signal intensities and shall further foster the role of MALDI-MSI as a valuable technique for investigating the spatial distribution of biomolecules and drugs in tissue samples. moleculaR has been made available for the scientific community as an open-source R package.
For the majority of cancer patients, tumor-derived factors systemically induce metabolic and inflammatory changes which cause involuntary body weight loss, known as cancer cachexia (CC). The extensive changes in the body’s metabolism induce an imbalance in energy expenditure, reduced quality of life and heightened treatment toxicity resulting in increased morbidity and mortality. Endothelial cells (ECs) play a central role during chronic inflammation and cancer progression. In response to primary tumor-derived signals, quiescent ECs become activated and secrete paracrine, so called angiocrine, factors that serve as instructive signals to modulate the microenvironment. In a previous study, we could show that ECs act as amplifier of tumor-derived signals supporting the colonization of disseminated tumor cells in the lung. Yet, the role of angiocrine signals during CC progression remains largely elusive. This project focuses on the endothelial signaling during CC and the relevance of angiocrine signaling during CC progression. I propose that tumor-derived factors drive CC progression by systemically reprogramming angiocrine signaling mechanisms.
To study the role of angiocrine signaling in CC in a homogenous and clinically relevant setting, a resection model was established which enabled the establishment of lung metastasis independent of primary tumor growth. The resection of the primary tumor recapitulates the process performed in the clinics and creates the opportunity to study (1) primary tumor-induced cachexia, (2) recovery after tumor removal and (3) secondary cachexia during metastasis in one mouse model. Following the tumor resection, body composition normalized, and cachectic symptoms were re-established upon subsequent outgrowth of overt lung metastases. Longitudinal transcriptomic and plasma proteomic changes were determined at the distinct biological timepoints. By comparing the gene expression of ECs in wasted organs (heart, adipose tissue) with a metastatic organ (lung), I observed that CC initiated systemic EC reprogramming which was largely mitigated after surgical tumor removal. Interestingly, the transcription factor STAT3 was similarly regulated in ECs indicating endothelial involvement in inflammatory responses during cachexia. The endothelial-specific deletion of Stat3 diminished adipose tissue wasting and prolonged survival verifying that ECs are actively involved in CC progression. Additionally, STAT3-regulated acute phase proteins (APP) were increased in the plasma during cachexia. Notably, the most upregulated EC-derived APP group in cachectic mice was the serine protease inhibitor family, SERPINA3. Mechanistic in vitro studies confirmed that the reduction of EC-derived SERPINA3 diminished muscle wasting. The analysis of the recently published lung cancer dataset of the TRACERx study confirmed the correlation of SERPINA3 plasma concentration and the cachectic status of the patients.
The first results of the German pediatric precision oncology program INdividualized Therapy FOr Relapsed Malignancies in Childhood (INFORM) showed the significance of high evidence levels for successfully matched targeted therapy based solely on molecular diagnostics. Yet, only a small number of patients (8%, 42/519) (1) actually present with a high evidence target, highlighting an unmet need to improve drug response predictions and clinical treatment recommendations. Therefore, the aim of this thesis is to integrate pharmacodynamic (PD) parameters from Drug Sensitivity Profiling (DSP) with pharmacokinetic (PK) parameters, and improve drug response prediction in high risk pediatric patients. To achieve this aim, a literature review was conducted, and nine PK parameters focused on the pediatric population were collected for the drugs from the DSP drug library in the INFORM study. In addition, a database of primary patient tumor (PPT) samples (n=68) and a database of positive control cell (PCC) line models (n=7) were generated. The PCC models habor a specific molecular alteration (e.g., BRAF V600E, NTRK fusion) with a clinically proven drug- target relationship. Among the 68 PPT samples, five samples (PPT subgroup I) harbored a very high priorty (INFORM priorty score 1) alteration with a clinically proven drug-target relationship. Both the PPT samples and PCC models underwent DSP using a library of 79 clinically relevant oncology drugs. Hit selection was based on dose-response curves-derived PD parameters and PD-PK integrated parameters. These parameters were evaluated for their predictive value in the PCC models and the PPT subgroup I samples. Subsequently, the parameter with the best predictive value was investigated in the PPT samples without a defined drug-target relationship. A PK database of 74 drugs and nine PK parameters for each drug focusing on the pediatric population was successfully created and published for the scientific community. When investigating the predictive power of PD parameters, the drug sensitivity score (DSS) z-score showed the best predictive power in identifying the matching drug in the PPT subgroup I samples based on the molecular background. However, the DSS z-score could not capture the patient's clinical history. Conversely, the integrated PD-PK parameter, the DSS Cmax z- score, could effectively capture the patient's clinical history in the PPT subgroup I samples. In the PPT samples without a defined drug target match and no clinical treatment history, the DSS Cmax z-score provided additional insights for 77% (n=53/68) of the patient samples that were not detected by NGS molecular analysis. In summary, a previously unavailable and comprehensive pediatric PD database was generated and published to serve the scientific community. The PK parameter Cmax was identified and successfully integrated with the DSS, introducing a novel DSP metric for drug response prediction. The groundwork established by testing and describing the DSS Cmax z- score in this thesis serves as a foundation for further investigation in larger datasets with clinical outcomes. This could refine the prediction of drug response for pediatric high-risk patients and improve their treatment selection without relying on time-consuming and costly techniques.
Radiotherapy with protons or light ions is a highly precise form of cancer treatment. In treatment planning for particle therapy, ion stopping power ratio (SPR) maps of patient tissues are used to predict particle ranges and calculate dose distributions. To more accurately calculate dose distributions and minimize irradiating healthy tissue, it is crucial to improve SPR prediction. To this end, this thesis investigated dual-layer spectral computed tomography, a dual-energy CT (DECT) technique, as an alternative to conventional single-energy CT (SECT). The SECT-based method relies on converting CT numbers to SPR, yet CT numbers acquired from photon attenuation cannot be used to accurately predict energy loss by ions, which makes the approach indirect and heuristic. The DECT-based method, however, uses measurements of relative electron density and effective atomic number to directly and patient-specifically predict SPR. SPR prediction using DECT was evaluated in tissue-equivalent materials, anthropomorphic phantoms, and non-tissue materials; clinically analyzed in a retrospective patient study; and experimentally investigated for patients with dental materials. DECT-based SPR prediction improved dose calculation accuracy in particle therapy compared to SECT with a remaining range uncertainty of about 1% in controlled experimental scenarios. DECT may thus substantially improve range prediction for highly accurate particle therapy.
In quantum chemistry, a full quantum dynamical description of large many-body systems is not currently feasible. One can consider both classical and semi-classical treatments of approximating the quantum dynamics of molecular systems to simulate simpler dynamics. Motivated by their cost-effectiveness and the fact that chemical dynamics take place often in an energy and density-of-states regime where a classical description can be meaningful, a classical description of the quantum dynamics of systems is explored in this dissertation. We first illustrate how the reaction rate is affected by the cavity effect. cis-trans iso�merization of HONO is used as an example to demonstrate the cavity-controlled reactivity. Due to the high dimensionality of the potential energy surface, we describe the reaction rate through a classical reactive flux method. The quantum Hamiltonian for simulating cavity-modified molecular dynamics is transformed into a classical mapping Hamiltonian. We consider a single molecule inside the cavity. For simplicity, we assume the cavity is coupled to an aligned molecule. The x− aligned case is studied in both low-friction and strong-friction regimes of the reaction coordinate. The low(strong)-friction regime is also known as the underdamped(overdamped) regime, which is mentioned in Grote-Hynes theory. In the underdamped regime, we illustrate the key difference between a single molecule and a collective of molecules with fixed Rabi splitting. We also show a modification of the reaction rate with different cavity frequencies for different aligned cases. Our results show that the modification of the reaction rate is related to the solvent environment. This will be described in chapter 3. We then consider free-orientated molecules inside the cavity within the underdamped regime. Compared with aligned cases, the free orientation of molecules leads to a disorder of light-matter coupling, which should be observed in experimental results. Since a thermally excited molecule passing through the barrier is a rare event, we consider N molecules inside the cavity with 1 activated molecule and N −1 non-activated molecule. We aim to see how the reaction rate is affected by the number of molecules with fixed coupling strength. We connect the enhancing rate by increasing the number of molecules with the energy transfer efficiency from the activated molecule to the cavity. And the efficiency is sensitive to the resonant frequency. Based on this observation, we also show the modification of the reaction rate by tuning the lifetime of the cavity. Our findings shed important new light on the question of collective effects in chemical reactivity under vibrational strong coupling. This will be described in chapter 4. On the other hand, we turn to describe the fermionic dynamics through Meyer-Miller mapping. In chapter 5, We proceed by describing the relation between the initial phase space density of the classically mapped system and the initial configuration of the electrons, and propose strategies to sample this phase space density. We compare the MM mapping with exact quantum results and with different mappings explicitly designed for fermions, namely the SM with and without the inclusion of antisymmetry (the latter corresponds to the original MW mapping), and to the LMM. We then compare Hubbard and impurity Hamiltonians, with and without interactions, and consider as well a model for excitonic energy transfer between chromophores. In this model with interactions we show that the classical MM mapping is able to capture interference effects caused by the presence of different energy transfer pathways leading to the same final state, both when the interferences are constructive and destructive. Our results show that the construction of the maximal fermionic occupation does not seem to be necessary. Also, the performance of the mappings is sensitive to sampling strategies of the initial phase-space distribution for fermions
Here I developed a novel method to investigate the growth of cell colonies in vitro. The method is inspired by and augments the standard in vitro clonogenic assay (IVCA). While the field of application is radiobiological research, the approach can be applied to any domain where colony growth of adherent cells is of interest. The method utilizes high numbers of time-resolved microscopy image series and hence requires largely automated image data acquisition, image processing, quantitative data extraction and single-colony growth characterization. I designed a multi-step analysis framework to implement these steps. This contrasts with traditional approaches relying on visual examination of cell culture containers and manual classification of cell colonies. This new approach allows yet unattained insights into growth behaviors and growth rates of large numbers of individual cell colonies. In applying the new method to five different cell lines (H3122, H460, RENCA, SAT, UTSCC-5) in different experimental settings, the following main results were found: a) For some of the cell lines, the initial seeding density influences the growth dynamics of the resulting colonies in densities commonly used in standard experiments. b) Pre-experimental cell culture conditions influence the growth dynamics in two tested cell lines (SAT, UTSCC-5) without irradiation. c) Exponential growth rates of two tested cell lines (H3122, RENCA) are normally distributed independent of irradiation dose, but the average growth rate decreases linearly across commonly used doses. d) Some colonies growing from photon-irradiated cells exhibit a distinct delayed abortive growth behavior, as observed for the two analysed cell lines (H3122, RENCA). The frequency of this behavior increases with increasing dose. e) Survival rates, as traditionally determined via the standard IVCA, clearly depend on experimental readout choices, namely the time of readout and the size threshold used to score survival of colonies. My analysis indicates that this dependence emerges from observations c) and d). f) The observed influence of readout choices propagates into relative biological effectiveness quantification for carbon irradiation for three examined cell lines (H460, RENCA, UTSCC-5). Hence, I demonstrate that the presented method can be used to inform experimental design decisions in standard IVCA experiments, to perform robustness analyses on these assays, and to find distinct types of growth behavior. Still, the application in its current form is limited to adherently growing cell lines forming contiguous colonies. In addition, due to the multi-step procedure,some underlying assumptions and methodological decisions need to be made which potentially influence the resulting findings. I discuss these aspects in a dedicated chapter. In future work, potential extensions and combinations with quantitative single-cell analysis methods such as FACS, fluorescent live-cell imaging or single cell omics methods can make this method a cornerstone application to build on in order to understand not only how, but also why colonies grow the way they do. In conclusion, the presented method elucidates colony growth in unprecedented detail. The presented results showcase the potential relevance of these details. However, to establish this method as a standard tool for applied research, a unified analysis framework is necessary to standardize the methodological aspects, from image acquisition to colony growth type classification.
DNA methylome remodeling is an essential molecular mechanism underlying all stages of hematopoietic differentiation. However, current datasets only cover a fraction of the genome and are often limited to specific hematopoietic cell types. A comprehensive, genome-wide atlas of the DNA methylation dynamics during hematopoietic differentiation is still missing. Preliminary evidence suggests that the single-cell landscape of the hematopoietic stem and progenitor cell (HSPC) compartment is characterized by a structured continuum of epigenetically-defined cell states. Significant advances in charting this epigenetic state manifold have recently been achieved for the chromatin accessibility and histone modification layers. However, despite its potential importance, the landscape of single-cell DNA methylome states in the HSPC compartment remains largely unexplored. This project aimed to comprehensively map the genome-wide DNA methylation dynamics during hematopoietic differentiation and leverage this atlas as a reference to analyze the single-cell DNA methylome landscape in the HSPC compartment and among mature hematopoietic cells. The functional importance and rich information content of differentially methylated regions (DMRs) are well-established. However, the DNA methylation layer inherently possesses the capability to encode information at CpG resolution. The role and extent of differentially methylated CpG (DMCpG) programming within DMR regions is largely unexplored. This project therefore aimed to evaluate the role and mechanisms of DMCpG programming during hematopoietic differentiation. Using high-coverage tagmentation-based whole-genome bisulfite sequencing data for 25 hematopoietic populations, I have compiled a genome-wide, dual-layer DMR/DMCpG atlas, which maps, annotates, and integrates DMR and DMCpG programming during hematopoietic differentiation. Loss of stemness was associated with lineage-independent gain of DNA methylation, while lineage specification was accompanied by hierarchical DNA methylation dynamics, characterized by unidirectional loss of DNA methylation. Different DMCpGs within focal DMR intervals were often distinctly programmed and thus contained heterogeneous information content. In particular, most of the DMRs were seeded and progressively expanded through subsequent programming of specific DMCpGs at different stages of differentiation. Mature hematopoietic cells exhibited systematic seed DMCpG hypomethylation in DMRs associated with alternative cell fates. This seed hypomethylation likely represents epigenetic memory of alternative fate explorations in progenitor cells. Collectively, these findings suggest a hierarchical model of DNA methylation programming, in which information is encoded through DMR programming and through DMCpG programming within DMR regions. This model represents a significant extension of the commonly accepted paradigm of regional DNA methylation programming. Using the dual-layer DMR/DMCpG atlas as a reference, single-cell methylome states for 312 HSPCs, as well as for a total of 136 mature B cells, T cells, CFU-Es, and monocytes, could be dissected with high resolution. The HSPC compartment was characterized by a structured continuum of single-cell DNA methylome states. Multiple lines of evidence suggested that differentiation starts from apex HSCs possessing a lineage-naive DNA methylome state. Exit from the apex HSC state was initiated by balanced, multi-lineage DMR seeding. This early DMR programming was strictly restricted to specific DMR seeding regions, which often comprised only one or two DMCpGs. This contrasts with the conventional paradigm that functionally relevant DMRs always contain at least several DMCpGs. Further differentiation within the HSPC compartment was accompanied by continuous, gradually more lineage-specific accumulation of hypomethylation, leading to progressive DMR expansion. The dual-layer DMR/DMCpG atlas provides an essential resource for studying the epigenetic regulation of the hematopoietic differentiation process and serves as a valuable reference for the analysis of single-cell bisulfite sequencing data. This work highlights the highly-resolved, progressive, and stable nature of DNA methylome remodeling during hematopoietic differentiation and reveals several aspects of the structure and information content of the DNA methylome layer which go beyond the currently accepted paradigms. It appears likely that the DNA methylome remodeling mechanisms active in other differentiation systems and related processes, such as tumor evolution, share the same principles of hierarchical DNA methylation programming with CpG resolution. However, in many systems, the information content of the DNA methylome may be convoluted by a combination of this programming mechanism and other programming mechanisms characterized by stochastic regional accumulation of DNA methylation alterations. The analysis strategies presented in this work provide a basis for the further development of computational methods capable of dissecting the rich but complex information content of the DNA methylome with high resolution.
To survive, animals have to finely regulate their threat assessment and escape behavior. In mice, the brainstem dorsal periaqueductal grey (dPAG) generates innate defensive behaviors towards a multitude of threats, like predators, prey and aggressive conspecifics. dPAG acts as a switchboard between forebrain areas like the anterior cingulate cortex (ACC) and the brainstem. Glutamatergic neurons in the ACC project to dPAG and have an inhibitory effect on the structure, promoting approach. In dPAG, separate neuron ensembles correlate with risk assessment or with escape. However, the differential connectivity and gene expression of these two classes remains unknown. In this thesis, I aimed at better understand the anatomy and function of the dPAG microcircuits controlling assessment and escape behavior. To do so, I performed single neuron calcium imaging recordings with a miniaturized fluorescent microscope in freely moving mice in glutamatergic and GABAergic neurons in dPAG, showing that both cell types correlated with assessment and escape in a social, predatorial and prey threat exposure test. Furthermore, I used volume electron microscopy combined with multiplexed labeling of pre- and postsynapses, showing that ACC axons establish synapses onto Vglut2+ and non-glutamatergic, non-GABAergic neurons in dPAG, pointing to the role of a neuromodulator neuron class mediating dPAG inhibition. Further experiments will analyze the expression profile of dPAG neurons, with a focus on ACC synaptic partners, using spatially resolved transcriptomics, and will explore the functional role of dPAG candidate neuromodulatory neurons in defensive behaviors, such as enkephalin expressing neurons. In conclusion, my PhD work characterized the connectivity and function of a brainstem neural circuit that modulates innate defensive behaviors towards aggressive conspecifics, predators and prey in mice.
O-GlcNAcylation is a reversible posttranslational modification (PTM) found on thousands of nuclear and cytoplasmic proteins catalyzed by OGT and OGA enzymes. Because of the embryonic and cellular lethality resulting from Ogt mutation, the mammalian function of OGlcNAc modification is still unknown. Despite the extensive studies since 1987, when OGlcNAcylation was identified for the first time, the role of O-GlcNAc and OGT in regulating gene expression remains still enigmatic. With my PhD project I aim at studying the role of O-GlcNAc modification in mouse chromatin by combining genomic and proteomic approaches. Pan-GlcNAc chromatin profiling revealed that O-GlcNAc proteins densely occupy promoter regions of pluripotent and neuronal differentiated cells. Unbiased bioinformatic screening for co-occupancy with public ChIP-seq data sets revealed that O-GlcNAc strongly co-localizes with RNA Polymerase II (RNA Pol II) at promoters. RNA Pol II was previously shown to be modified by O-GlcNAc modification; while it is well described how RNA Pol II catalyzes the DNA-directed mRNA synthesis of proteincoding genes, the mechanism by which O-GlcNAc regulates its activity is still unknown. OGlcNAc nuclear perturbation followed by super-resolution imaging revealed a novel role for OGlcNAc modification in regulating RNA Pol II localization at nuclear transcription factories. To gain deeper insight into O-GlcNAc mechanism in modulating RNA Pol II activity, I have established new tools to perturb and detect O-GlcNAc modification selectively on the Cterminal domain (CTD) of RNA Pol II. To investigate how O-GlcNAc regulates transcription at promoter regions, we have engineered and validated a novel inducible perturbation system to deplete O-GlcNAc at CpG dense promoters in pluripotent and differentiated cells. Our transcriptomics analysis revealed that targeted O-GlcNAc depletion at CpG-dense promoters causes the perturbation of several metabolic and mitochondrial genes, as well as a significant upregulation in ribosomal subunits expression. qPCR analysis of rRNA demonstrated that O-GlcNAc perturbation at CpG-rich sites leads to a strong deregulation of rRNA expression. Overall, I identified a novel function for O-GlcNAc in the regulation of ribosome biogenesis. This finding is consistent with my proteomic data obtained from the characterization of the OGT interactome, which revealed that nuclear OGT strongly interacts with a large number of nucleolar and ribosomal subunits. Preliminary mass spectrometry analysis of the nuclear O-GlcNAc proteome showed that the majority of nuclear O-GlcNAc proteins are factors involved in RNA Pol I and III transcriptional regulation, the two RNA Polymerases that control rRNA transcription. Taken together, my results demonstrate how O-GlcNAc chromatin proteins are not uniformly distributed all over the genome, but instead occupy predominantly gene promoters: part of these promoters are transcriptionally silenced, while the majority of them are active genes. Chromatin enrichment analysis revealed that O-GlcNAc highly correlates with RNA Pol II occupancy, suggesting that O-GlcNAc might regulate the expression of RNA Pol II-rich genes. Finally, by transcriptomics and proteomics approaches I have described a novel mechanism of nuclear O-GlcNAcylation: my analysis demonstrated how nuclear OGT regulates, via OGlcNAc modification, ribosomal biogenesis by controlling the expression of ribosomal proteins and of rRNA. These results bring new avenue into the possible mechanism of OGT in adapting the cellular metabolic state in response to the intracellular O-GlcNAc levels.
This guide provides a detailed step-by-step procedure for the dispersion of (6,5) single-walled carbon nanotubes by shear force mixing with the conjugated polymer PFO-BPy in organic solvents. All processes presented here were developed in the Zaumseil group at Heidelberg University since 2015 and represent best practices to the best of our knowledge. In addition to the detailed instructions, we discuss potential pitfalls and problems, that we have encountered over eight years of operation and show how to solve them. This also includes a detailed description of how to maintain and service a shear force mixer to ensure long operation lifetime. Finally, we show how to expand our process to the dispersion other nanotube chiralities in electronic-grade quality and how to treat dispersions for subsequent processing (e.g., thin film deposition or functionalization).
Stem cells have fascinated humans for a long time because of their fundamental importance in building and sustaining many forms of life. Stem cells are able to divide and maintain themselves while producing new cell types. Like animal stem cells, plant stem cells are a fascinating research object. They provide the cells for plant growth and allow many plants to continue growing throughout their lives, thereby producing enormous amounts of plant biomass on this planet. In this study, I specifically focussed on the plant stem cells that produce wood and bast. These stem cells form a cylindrical stem cell niche, the vascular cambium, which is found in the root, the stem and the tissue that connects the root and stem, the hypocotyl. In my analyses, I focused on the hypocotyl of the model plant Arabidopsis thaliana, since wood and bast production is very active there and the tissue patterning resembles the patterning of the vascular cambium of trees.
A vascular cambium stem cell produces both, wood progenitor cells inward and bast progenitor cells outward. Thus, it needs to “choose” between the following cell fates: becoming a wood progenitor cell, becoming a bast progenitor cell, or remaining a stem cell. The basis on which this first cell fate decision is made has not yet been clarified. To better understand this decision-making process, in a first step I mathematically modelled gene regulatory networks in collaboration with the Mironova laboratory. This has shown that a network of just three genes that regulate each other in a specific way is sufficient to bring about the three cell fate possibilities mentioned above. I then hypothesized that this network could consist of two mutually inhibitory auxin and cytokinin signalling pathway components. Furthermore, these components could be additionally regulated by WUSCHEL-RELATED HOMEOBOX4 (WOX4), an important transcription factor for stem cell regulation in the vascular cambium. Integrated into a 1D model, this network generated bidirectional growth. Using in planta analysis, I was able to show that both auxin and cytokinin signalling levels are low specifically in the vascular cambium. To test my hypothesis, I investigated which genes of the auxin and cytokinin pathways are regulated by WOX4, also in comparison to WUSCHEL, a stem cell regulator of the shoot apical meristem. In the course of my investigations, I identified specific type-A ARABIDOPSIS RESPONSE REGULATORs (ARRs), which negatively regulate cytokinin signalling, as putative WOX4 targets. I found that the expression of the type-A ARR genes ARR5, ARR6, ARR7 and ARR15 is reduced in the hypocotyl vascular cambium. Furthermore, I observed that the expression of ARR6 and ARR15 is downregulated by WOX4. WUSCHEL also exerts this type of regulation on type-A ARRs in the shoot apical meristem and therefore this could represent a general concept for the regulation of plant stem cells. In addition, findings in this study propose that WOX4 itself is regulated by cytokinin via the DNA BINDING WITH ONE FINGER 2.1 (DOF2.1) protein. Importantly, when ARR7 and ARR15 are mutated, the cell fate decisions of vascular cambium stem cells are altered and more wood cells are produced than in wild type plants. In conclusion, this study suggests that a mechanism of cell fate decision making in vascular cambium stem cells is based on the regulation of cytokinin signalling by WOX4 and by type-A ARRs.
Mild replication stress in neural stem and progenitor cells leads to the formation of recurrent DNA break clusters (RDC). Genes containing these RDCs (RDC-genes) play important roles in brain functions such as synaptogenesis and cell-cell-adhesion. Most RDC-genes do not harbor break clusters in cell types in which the RDC-genes are not being actively transcribed, but the link between transcription activity and the formation of DNA breaks in RDC-genes has not been investigated before.
To examine whether transcription is the licensing factor for RDC formation, the promoters of two robust RDC-genes, Catenin Alpha 2 (Ctnna2) and Neurexin 1 (Nrxn1), have been independently successfully deleted in multiple ESC-NPC cell lines in vitro. In these cell lines, the transcription of Ctnna2 and Nrxn1 genes was abolished. Moreover, the number of DNA double-strand breaks was reduced in the gene-of-interest while the amount of DNA breaks in other RDC-genes remained unaffected. Additionally, also the replication timing did not change significantly when comparing the cells. Using the same unbiased genome-wide nucleotide resolution assay to detect the recurrent DNA break clusters, I was able to detect the movement of stalled/collapsed replication forks across increasing levels of replication stress and identify the observed breaks as single-ended double-strand breaks. Taking these findings together with transcription and replication directionality, it is evident that there is a bias towards head-on collision (40% more DNA double-strand breaks) versus co-directional collision of the replication fork and transcription machinery. These data fit to the hypothesized transcription/replication conflict, which is believed to play an essential role in the formation of the recurrent DNA break clusters. All things considered, my project illuminates the relationship between transcription, replication, and DNA damage in the form of double strand breaks in replication stress conditions, which can be translated to influences on the developing brain and its genome.
Das Synovialsarkom ist ein aggressives Weichteilsarkom, welches durch die SS18-SSX1/2/4 (SS18-SSX) Genfusion ausgelöst wird. Der SS18-Teil des Fusionsgens ist Teil des BRG1/BRM-assoziierten Faktors (BAF)-Komplexes, welcher in der Chromatinreorganisation und der Transkriptionsaktivierung von Bedeutung ist. Der SSX-Schwanz lenkt den Komplex auf Ziele des Polycomb repressiven Komplexes 1 (PRC1), welcher als Transkriptionsrepressor wirkt. Die resultierende abnorme Hochregulierung von Genen durch SS18-SSX führt zur Tumorigenese und Aufrechterhaltung von Synovialsarkomen. Bedauerlicherweise fehlen bis heute Strategien, die herausgehobene Rolle von SS18-SSX therapeutisch nutzbar zu machen. In diesem Projekt konzentrierte ich mich daher auf die Identifizierung der kritischen Domänen der SS18-SSX-Genfusion sowie ihrer nachgeschalteten Zielgene. Zunächst konnte ich SSXRD als wichtigste Domäne von SS18-SSX identifizieren. Diese Domäne ist für die Bindung von SS18-SSX an die Loci seiner Zielgene durch Erkennung von ubiquitiniertem Histon H2A verantwortlich. Um funktionell besonders relevante Zielgene zu identifizieren, führte ich einen CRISPR-Dropout-Screen durch, welcher auf Gene abzielte, deren genomische Loci entweder direkt von SS18-SSX gebunden werden oder anderweitig in Synovialsarkomen überexprimiert sind. Ich identifizierte PCGF3, eine Komponente von PRC1.3, als starke und selektive Abhängigkeit in Synovialsarkomen. Bei weiteren Untersuchungen zeigte sich von allen kanonischen und nicht-kanonischen PRC1-Komplexen lediglich für PRC1.1 (PCGF1) und PRC1.3 (PCGF3) eine funktionelle Abhängigkeit in Synovialsarkomen. Ich führte Transkriptionsanalysen und Onkofusions-Chromatin-Belegungsprofile durch, um festzustellen, dass, obwohl PCGF1 und PCGF3 beide Teil von PRC1-Komplexen sind, sie im Synovialsarkom unterschiedliche Wirkungsweisen haben. Der Knockout von PCGF3 führt hauptsächlich zu einer Hochregulierung von Genen, die als Transkriptionsrepressoren in Synovialsarkomen wirken. Bei der weiteren Analyse der Auswirkungen des PCGF3-Knockouts auf die Bindung von SS18-SSX stellte ich fest, dass SS18-SSX in Abwesenheit von PCGF3 umverteilt wird und an bestimmten Stellen verstärkt bindet. Diese Stellen entsprechen Loci von Genen, die durch das Ausschalten von PCGF3 stark hochreguliert werden. PCGF3 schränkt also die SS18-SSX-vermittelte transkriptionelle iv | P a g e Aktivierung einer Untergruppe von Genen ein, die von SS18-SSX und PCGF1 gemeinsam gebunden werden. Während PCGF1, wie zuvor beschrieben, für die Chromatin-Rekrutierung von SS18-SSX verantwortlich ist, zeige ich hier, dass PCGF3 für die Aufrechterhaltung optimaler Konzentrationen von SS18-SSX an Schlüsselstellen verantwortlich ist und so für die Feinabstimmung des Expressionsniveaus seiner Zielgene sorgt. Diese Arbeit identifizierte eine Schlüsselanfälligkeit beim Synovialsarkom und kann Aufschluss über die Rolle von nicht-kanonischen PRC1-Komplexen in anderen Zusammenhängen geben.
Immunotherapy is a promising tool for cancer treatment and there is great interest to implement T cell-based therapies in patient care. Development of T cell-based therapies has focused on highly immunogenic tumors. Due to their low-mutational load and limited immune infiltration, gliomas are considered difficult targets for immune intervention and thus remained understudied, despite the urgent need to define new therapeutic options for these aggressive brain tumors. This thesis aimed at (I) identification and characterization of T cell responses elicited upon vaccination of glioma patients against the recurrent driver mutation R132H in isocitrate dehydrogenase 1 (IDH1), (II) identifying tumor-reactive T cell receptors (TCRs) targeting tumor-private antigens and (III) antigen-agnostic TCR identification in non-vaccinated patients with the overall aim to define a gene expression signature characteristic for tumor-reactive T cells. The IDH1R132H oncogenic driver mutation is found in about 70% of WHO grade 2 and 3 gliomas. Its homogenous exclusive expression in tumor tissue makes it an attractive target for immunotherapy, and vaccinations with an IDH1R132H encoding long peptide were shown to efficiently mediate tumor shrinkage in pre-clinical mouse models. The vaccine was proven to be immunogenic in primary WHO grade 3 and 4 IDH1-mutant astrocytomas in the completed NOA16 phase I clinical trial, and also shows immunogenicity in the ongoing NOA21 phase I clinical trial, where combination of the vaccine with the PD-L1 inhibitor avelumab is tested in patients with IDH1 mutant recurrent gliomas. To gain a deeper understanding of the T cell response following vaccination, I established “epitope specific expansion cultures” (ESPEC) with “subsequent identification of TCRs” (SUIT) to pre-select TCRs for testing their reactivity. Based on ESPEC, a total of 120 CD4-derived TCRs from seven patients were selected for cloning and functional validation in in vitro co-culture assays, of which 106 were shown to be reactive. IDH1R132H-reactive clones were found to be enriched intratumorally as compared to concurrently sampled blood of vaccinated patients. 49 identified IDH1R132H-reactive TCRs showed in vitro reactivity against wildtype IDH1 if the peptide was used at supraphysiological concentrations, but not at lower peptide concentrations as shown for a selection of TCRs. Analysis of HLA restrictions indicates promiscuous binding of the IDH1R132H peptide to HLA-DR alleles, which is in accordance with the high immunogenicity of the vaccine in an HLA-diverse patient population. Contrary to what was reported so far, I also observed CD8+ T cell responses against IDH1R132H in two of three tested patients. Mass spectrometry confirmed presentation of short IDH1R132H peptides on HLA-B*07:05 and HLA-B*35:01, with these HLA alleles being representative for 12.3% of the German population taking HLA supertype families into account. Using ESPEC-SUIT, one CD8-derived TCR reactive against IDH1R132H was identified. The TCR was found to infiltrate the tumor, making up 0.24% of the T cell repertoire and being positive for granulysin, which could be an indication of cytotoxic function. Extending the screening for reactive CD8-derived TCRs to further patients is planned to gain deeper insights into the vaccine-induced immune response. ESPEC-SUIT was also used to identify TCRs reactive to patient-individual antigens. Private neoepitopes, both from SNVs and fusion events, were predicted based on whole exome and RNA sequencing data of the tumor, and used to stimulate autologous PBMCs. ESPEC cultures were performed with individual peptides or in peptide pools to screen for reactive TCRs against 18-189 peptides per patient. For a colon carcinoma patient (POC-001), 25 of 28 screened TCRs were reactive, covering reactivities against 14 of 18 predicted neoantigens. For patient POC-004 with liposarcoma (12 mutations included for testing) and patient POC-005 with metastatic melanoma (50 mutations included for testing), several hundred expanded TCRs have been identified and await functional validation. While POC-001 and POC-004 were vaccinated with long peptides representative for a selection of mutanome encoded antigens prior to ESPEC, patient POC-005 did not receive such vaccination, but T cells were found to be expanded in post-ESPEC cultures for three short peptides and 32 long peptides, underlining the sensitivity of the ESPEC approach. To further understand the role of anti-tumor T cell immunity in brain tumors, tumor infiltrating lymphocytes (TILs) of non-vaccinated patients were used for single cell sequencing with the overall aim to identify a gene signature that can distinguish between tumor-reactive clones and non-reactive bystander clones for future antigen-agnostic identification of reactive TCRs. While gene signatures based on published markers identified no TCRs in primary brain tumors, a total of 46 reactive TCRs were identified when screening the top 83 TIL clonotypes from a melanoma brain metastasis tumor sample. On basis of this data, Chin Leng Tan (DKFZ Heidelberg, Germany) is working on establishing a gene signature, which could be used for antigen-agnostic discovery of reactive TCRs. Collectively, this thesis presents two strategies on how putatively tumor-reactive TCRs can be selected and validated on a larger scale. Screening for IDH1R132H-reactive TCRs allowed to gain deeper insights into the peripheral and intratumoral immune response elicited upon vaccination with the IDH1R132H peptide vaccine in brain tumor patients. The antigen-targeted approach was shown to be highly efficient for selecting reactive TCRs, which could potentially help to implement TCR-transgenic T cell therapies in patient care.
Evolutionary theory has been the foundation of biological research for about a century now, yet over the past few decades, new discoveries and theoretical advances have rapidly transformed our understanding of the evolutionary process. Foremost among them are evolutionary developmental biology, epigenetic inheritance, and various forms of evolu- tionarily relevant phenotypic plasticity, as well as cultural evolution, which ultimately led to the conceptualization of an extended evolutionary synthesis. Starting from abstract principles rooted in complexity theory, this thesis aims to provide a unified conceptual understanding of any kind of evolution, biological or otherwise. This is used in the second part to develop Amee, an agent-based model that unifies development, niche construction, and phenotypic plasticity with natural selection based on a simulated ecology. Amee is implemented in Utopia, which allows performant, integrated implementation and simulation of arbitrary agent-based models. A phenomenological overview over Amee’s capabilities is provided, ranging from the evolution of ecospecies down to the evolution of metabolic networks and up to beyond-species-level biological organization, all of which emerges autonomously from the basic dynamics. The interaction of development, plasticity, and niche construction has been investigated, and it has been shown that while expected natural phenomena can, in principle, arise, the accessible simulation time and system size are too small to produce natural evo-devo phenomena and –structures. Amee thus can be used to simulate the evolution of a wide variety of processes.
Cell identity plasticity is a normal and essential feature of cells during development, but is also a hallmark of diseases such as cancer. The mechanisms involved in suppressing inappropriate cell fates are poorly understood. Gene repression in terminally-differentiated cells is typically thought to be mediated by passive epigenetic silencing, such as through DNA methylation and repressive histone modifications.
However, recent studies suggest that active repression by cell type-specific transcription factors may play an essential role in maintaining cell identity. MYT1L, a neuron-specific transcriptional repressor, has been shown to maintain neuronal identity by the inhibition of non-neuronal lineages. The observation that deficiencies in MYT1L may lead to diseases such as neurodevelopmental disorders and cancer demonstrates its important role in cell identity maintenance by inhibition of cellular plasticity.
In this thesis, I investigate whether active, lifelong transcriptional repression of alternate lineages – or safeguard repression – is a general mechanism for cell identity stabilisation. Using computational prediction and direct cell reprogramming, I demonstrate that PROX1 safeguards hepatocyte identity by directly suppressing alternate lineage programs, hence demonstrating safeguard repression in a second cell type and germ layer. With single-cell transcriptomics analysis, I find that PROX1 robustly silences donor and alternative cell identities.
I also show that PROX1 directly binds to promoters of key mesodermal transcription factors, including Prrx1 and Pparg, hence decreasing their chromatin accessibility and expression. In addition, repression of PROX1 target genes during hepatocyte reprogramming mimicked effects of Prox1 overexpression. On the other hand, Prox1 deletion or target gene activation permitted inappropriate gene expression.
Finally, in a hepatocellular carcinoma mouse model, Prox1 overexpression prevented tumour initiation, reducing overall tumour load and extending survival substantially. Prox1 depletion in the same model induced cholangiocarcinoma-like morphology and gene expression, demonstrating the role of PROX1 in cell identity maintenance.
These results show that PROX1 is a repressive safeguard of hepatocyte identity. They also support a model whereby continuous silencing of alternate lineage gene expression programs by safeguard repressors prevents cell fate plasticity, thereby maintaining cell identity.
Wnt pathway is one of the main signaling pathways required for development and homeostasis. Wnt proteins must be secreted into the extracellular space in order to trigger the signaling cascade. Regulation of Wnt protein secretion is therefore crucial for Wnt pathway activity. All intracellular membrane trafficking is regulated by Rab GTPases. Very few Rab GTPases, however, have been directly associated with Wnt secretion. I performed a systematic screen by perturbing the 26 Drosophila melanogaster RabGTPases and observing the effects on Wnt secretion in the wing imaginal disc through immunohistochemical analysis. I identified Rab7 as a regulator of Wnt secretion and characterized its role in this process. I found that overexpression of Rab7 induces the accumulation of Drosophila Wnt1 homolog Wingless (Wg) in both secreting and receiving cells, as well as moderate relocalization of Wg from predominantly apical side to the basal side of these cells. The changes in Wg protein localization did not, however, affect the level of secreted Wg in the wing discs or the downstream Wg signaling. By performing colocalization staining, I determined that upon Rab7 overexpression, Wg was localizing to LAMP1- and Arl8-positive lysosomes at the basal side of the wing disc cells. Knockdown of Arl8 in cells with Rab7 overexpression reduced the translocalization of Wg to the basal side of the disc.
In order to study the interaction between Rab GTPases and Wnt secretion in living tissue, I developed novel Drosophila tools, which will be of use to other studies. Firstly, I endogenously tagged the Wg carrier protein Evi/Wls with the fluorescent proteins mScarlet and EGFP. Additionally, I generated a Wg:mScarlet fly line based on the existing Wg:EGFP fly line. Using these tools, I characterized the different fluorescence localization of proteins tagged with EGFP and mScarlet. I confirmed that the fluorescent tags do not affect Wg secretion, but that the difference in signal localization is due to the intrinsic properties of the fluorescent proteins. Secondly, I adapted the LAMA (ligand-modulated antibody fragments) system, an acute protein trap-and-release system previously published in human cell culture, for use in Drosophila. I could show that the system was functional in wing discs and salivary gland cells and that it allowed the trapping of both overexpressed and endogenous GFP-tagged proteins on the mitochondrial membrane and the endoplasmic reticulum (ER). The addition of the release molecule triggered the relocalization of the tagged proteins from the mitochondrial membrane into the cytoplasm. Using the LAMA system, I was able to show that the Wg accumulation induced by Rab7 overexpression appeared within sixty minutes of the Rab7 release. In summary, my data indicate a role of Rab7 in the translocalization of Wg to the basal side through the late endosomal pathway. Furthermore, the development of experimental tools, especially the adaptation of a protein trap-and-release system for use in Drosophila melanogaster, provide a material contribution to the field.
Meningiomas are thought to arise from the arachnoid cells of the leptomeninx and make up the most common primary intracranial tumor in adults. They are usually benign, however in about 20 % of cases, tumors present with an aggressive phenotype and higher risk of recurrence. Risk stratification thereby remains challenging especially for NF2-mutated meningiomas, which make up about two thirds of all cases, as they can occur at the full spectrum of WHO grades in meningioma from 1 to 3. Recently, molecular profiling has gained importance for prognosis in meningioma with several classification systems that have been established mostly based on the DNA methylation of the tumors. However, the DNA methylation-based classification has not been extensively linked to phenotypic traits of the tumor. Nor have meningiomas been investigated regarding intratumoral subpopulations that may exist in parallel and may have different characteristics, especially regarding the stage of progression and ability to recur. In addition, the role of the immune microenvironment in meningiomas is poorly understood, despite the identification of an immune-enriched meningioma subgroup with beneficial outcome in two independent DNA methylation classification systems. In this dissertation, I investigated the consistency of subgroups initially defined on epigenomic level across molecular levels by comparison to transcriptomic and proteomic data. Further, I leveraged single nuclei transcriptomic profiling to dissect intertumoral differences in the expression profile specific to the tumor cell population depending on the tumor subgroup, and to investigate the abundance and phenotype of intratumoral tumor cell subpopulations across samples. Similarly, I analyzed the single nuclei transcriptomic data to characterize tumor-infiltrating immune cells with respect to their abundance and activation status. I furthermore correlated the differences in immune infiltration with the progression-free survival of patients by deconvoluting DNA methylation array data according to their cellular composition. These analyses underlined the coherence of epigenomic meningioma subgroups across transcriptome and proteome. Moreover, I identified six tumor cell subpopulations that were defined by distinct expression profiles und could be identified across samples at varying abundancies depending on the stage of progression. Similarly, I observed profound differences in infiltrating immune cells between tumor subgroups, with a significant enrichment of tumor-associated macrophages in a benign subgroup of NF2-mutated meningiomas as compared to more progressed tumors. In parallel to their abundancy, macrophages changed in activation between benign and malignant cases from an anti- to a pro-tumorigenic phenotype. The evaluation of progression-free survival data revealed a positive correlation to the proportion of infiltrating immune cells as estimated from epigenomic profiles. Altogether, these results highlight the role of multi-level molecular profiling for tumor grading in a paradigmatic, epidemiologically relevant tumor type. They further indicate an important role of tumor-infiltrating macrophages during meningioma progression with possible consequences for risk prediction as well as therapeutic targets in meningioma.
The innate immune system is the first wall of defense against many infectious pathogens, such as viruses or bacteria. The antiviral interferon-induced transmembrane protein 3 (IFITM3) is one of the key players against enveloped viruses like the influenza A virus. IFITM3 is localized in the endosomal-lysosomal system and is known to prevent viral cytoplasmic entry. Different hypotheses on the mode of action of IFITM3 were proposed, but the underlying molecular mechanism still needs to be fully understood. Here, I am using a combination of cryo-light microscopy and in situ cryo-electron tomography to study the antiviral function of IFITM3 within the natural cellular environment in the context of an influenza A virus infection. To visualize the antiviral actions of IFITM3, I established a novel cryo-correlative light and electron microscopy method. This novel approach allowed me to localize trapped influenza A virus particles in the endosomal-lysosomal system of an IFITM3-overexpressing human epithelial lung cell line A549, which allowed me to study them by cryo-electron tomography. Structural analysis of IFITM3-positive multivesicular bodies revealed that IFITM3 does not alter the ultrastructural morphology of the endosomal-lysosomal system and does not modulate the number of intraluminal vesicles (ILVs). These results contradict the ’fusion decoy hypothesis,’ which suggests that an increased number of ILVs in the late endosomal lumen could redirect viral membrane fusion from the limiting late endosomal membrane to fusion with ILVs. High-resolution in situ cryo-electron tomography of influenza A virus particles within late endosomes revealed that IFITM3 traps influenza A virus particles in a hemifusion state at the limiting late endosomal membrane and ILVs. These findings support the previously formulated ’hemifusion stabilization’ hypothesis as they are the first direct proof of IFITM3-mediated hemifusion stabilization within the natural cellular environment. Furthermore, ultrastructural characterization of the hemifusion sites revealed the post-fusion form of the viral fusion protein hemagglutinin (HA). Thus, IFITM3 does not inhibit low-pH triggered HA conformational changes, indicating that IFITM3 inhibits membrane fusion indirectly by modulating the membrane properties of the late endosomal-lysosomal system and thus stabilizing hemifusion.
Over the past decade, cancer therapy has witnessed significant advancements, particularly in the field of oncolytic virotherapy. Oncolytic viruses specifically target cancer cells and trigger an immune response through immunogenic cell death. H-1 Parvovirus (H-1PV) is an oncolytic virus which has shown extensive potential in various cancer models. Phase I and IIa clinical evaluation of H-1PV in pancreatic cancer and glioblastoma has demonstrated a safe and non-toxic pro- file of the virus. The virus treatment showed initial signs of effectiveness, such as a shift in the tumour microenvironment towards improved immune response, ef- fective distribution of the virus within the tumour bed, and overall better patient survival. However, it was also observed that monotherapy alone is insufficient for complete tumour eradication. Thus, there is a need for improving H-1PV virotherapy. A promising approach involves the utilization of H-1PV in combination with other drugs. H-1PV has already shown promising results when combined with different drugs, espe- cially HDAC inhibitor VPA and pro-apoptotic drug ABT-737. In this regard, this study aims to investigate the effects of combining clinically tested oncolytic H-1PV with two different classes of therapeutics- HDAC inhibitors and BH3 mimetics- in prostate cancer. In order to identify an effective combination treatment, I adapted an assay to test the efficacy of the combination using a cell viability assay. My findings reveal that the combination of H-1PV with BH3 mimetic, pro-apoptotic drug ABT-737 is synergistic in killing PC3 prostate cancer cells. The combination of H-1PV and ABT-737 was able to induce upregulation of ac- tivated caspase 3/7 and mitochondrial outer membrane permeabilization (MoMP) in PC3 cells, which are both markers of apoptosis. After rescuing the cells by us- ing an apoptosis inhibitor, Z-VAD-FMK, I was able to confirm that H-1PV/ABT-737 combination was inducing an apoptotic cell death in PC3 cells. The dying cells were also expressing cell surface calreticulin, Hsp70 and Hsp90, which are all Danger associated molecular patterns (DAMPs) associated with an immunogenic cell death. Moreover, I was able to show that the immunogenic cell death of PC3 cells trig- gered by the H-1PV/ABT-737 co-treatment was able to induce the maturation and activation of dendritic cells (DCs). These DCs were further capable of phagocyto- sis and T-cell priming, thus indicating that H-1PV/ABT-737 combination treatment triggers the DC/T-cell axis in engaging the adaptive immune system for tumour clearance. I could show that H-1PV and ABT-737 co-treated PC3 cells displayed an up- regulated cell surface expression of pro-cytotoxicity natural killer cell associated ligands, MICA/MICB, CD155 and ULBP 2/5/6. Furthermore, this was capable of inducing NK cell activation, engaging the respective cell cytotoxicity receptors, NKG2D and DNAM-1 on NK cells. Thus, accelerating NK cell mediated clearance of PC3 cells. This impact was notably higher in the combination treated cells as compared to the individual therapies alone. This emphasizes the potential of the combina- tion in enhancing immune cell-mediated clearance of the tumour. Furthermore, the combination of H-1PV and ABT-737 was able to synergize in killing patient derived LuCaP 136 and LuCaP 147 cells, further cementing the potential of this combination across multiple cell types. Overall, this study highlights the potential benefits of combining H-1PV with BH3 mimetic ABT-737 in improving the immune response against prostate cancer, thereby suggesting its potential as a valuable treatment strategy.
The scarcity of labels combined with an abundance of data makes unsupervised learning more attractive than ever. Without annotations, inductive biases must guide the identification of the most salient structure in the data. This thesis contributes to two aspects of unsupervised learning: clustering and dimensionality reduction.
The thesis falls into two parts. In the first part, we introduce Mod Shift, a clustering method for point data that uses a distance-based notion of attraction and repulsion to determine the number of clusters and the assignment of points to clusters. It iteratively moves points towards crisp clusters like Mean Shift but also has close ties to the Multicut problem via its loss function. As a result, it connects signed graph partitioning to clustering in Euclidean space.
The second part treats dimensionality reduction and, in particular, the prominent neighbor embedding methods UMAP and t-SNE. We analyze the details of UMAP's implementation and find its actual loss function. It differs drastically from the one usually stated. This discrepancy allows us to explain some typical artifacts in UMAP plots, such as the dataset size-dependent tendency to produce overly crisp substructures. Contrary to existing belief, we find that UMAP's high-dimensional similarities are not critical to its success.
Based on UMAP's actual loss, we describe its precise connection to the other state-of-the-art visualization method, t-SNE. The key insight is a new, exact relation between the contrastive loss functions negative sampling, employed by UMAP, and noise-contrastive estimation, which has been used to approximate t-SNE. As a result, we explain that UMAP embeddings appear more compact than t-SNE plots due to increased attraction between neighbors. Varying the attraction strength further, we obtain a spectrum of neighbor embedding methods, encompassing both UMAP- and t-SNE-like versions as special cases. Moving from more attraction to more repulsion shifts the focus of the embedding from continuous, global to more discrete and local structure of the data. Finally, we emphasize the link between contrastive neighbor embeddings and self-supervised contrastive learning. We show that different flavors of contrastive losses can work for both of them with few noise samples.
Cutaneous squamous cell carcinoma (cSCC) is a keratinocyte cancer with a rapidly increasing incidence and one of the most common cancer types in the fair-skinned population. Chronic exposure to ultraviolet radiation (UVR) is its main risk factor and can lead to the development of a premalignant skin lesion, actinic keratosis (AK), which might further progress into cSCC. In addition, invasive cSCC can also develop from the in situ carcinoma Bowen’s disease (BD). During this progression, resident dermal fibroblasts are transformed into cancer-associated fibroblasts (CAFs) that are known to promote tumorigenesis. However, a detailed characterization of cSCC-related CAFs with respect to their fibroblast subpopulation-specific origin, heterogeneity, and tumor-promoting functions was still missing. Therefore, in this thesis, more than 115,000 single-cell transcriptomes from healthy human skin, BD and cSCC samples were analyzed. The results revealed two main CAF subpopulations with distinct functions and origins. Inflammatory CAFs (iCAFs) seemed to develop mainly from pro-inflammatory fibroblasts and presented immunoregulatory functions, including cellular interactions with immune cells in the tumor microenvironment (TME). On the other hand, myofibroblastic CAFs (myCAFs) were observed to originate mainly from healthy mesenchymal fibroblasts and were involved in extracellular matrix (ECM) remodeling processes. Furthermore, multiplexed RNA fluorescence in situ hybridization (FISH) assays not only confirmed both CAF subpopulations in human BD and cSCC, but also provided valuable information about the time window of CAF activation, as no CAFs could be observed in AK tissue sections. Interestingly, these findings could not be transferred to basal cell carcinoma (BCC), the second major keratinocyte cancer. Taken together, this thesis provides novel insights into CAF development, stratification, and functions during cSCC initiation and progression.
Lysosomes are membrane-bound organelles that act as a central hub for the recycling of biomolecules derived from cellular processes such as autophagy, endocytosis, among others. Lysosomal dysfunction is often linked to severe pathologies such as lysosomal storage disorders (LSDs), which are characterized by the aberrant accumulation of substrates, such as lipids. While cholesterol efflux from lysosomes is well-understood, the transport of other biologically active lipids such as sphingosine remain unknown. This knowledge gap is attributed to a lack of functional tools to manipulate and investigate lipids within living cells and on a single organelle level. The recent development of organelle-targeted caging groups, photoaffinity labeling and in combination with biorthogonal reactions represents a valuable and non-invasive way to identify new protein interactors of single lipid species while acquiring an exquisite spatial-temporal control. This work presents the development, characterization and application of a method to investigate the previously enigmatic export of sphingosine from lysosomes. To this end, We have synthesized lysosome-targeted photoactivatable sphingosine (Lyso- pacSph) and lysosome-targeted photoactivatable cholesterol (Lyso-pacChol) that combine existing technologies such as photoaffinity labeling and a lysosome-targeted photoremovable caging group. In this way, the lyso-probes allow their controlled release within the lysosome using a flash of light. Their remaining modifications enable the study of their trafficking and metabolism as well as the capture of their unique lysosomal interactome. Excitingly, known cholesterol transporters, such as the abundant lysosomal protein SCARB2/LIMP-2 and Niemann-Pick type C1 (NPC1) were also identified as sphingosine interactors. Additionally, I show that both proteins play similar roles in sphingosine transport from lysosomes. Absence of either protein resulted in delayed sphingosine metabolism as observed by thin-layer chromatography as well as prolonged lysosomal localization of the sphingosine probe as shown in fluorescence microscopy experiments. The latter method also allowed me to analyze the impact of an approved drug for NPC, miglustat, on subcellular sphingosine and cholesterol trafficking. Additionally, artificial elevation of sphingosine levels in WT cells created a cholesterol export defect reminiscent of NPC disease, pointing towards a direct and causative role of sphingosine in the pathobiochemistry of this disease. Overall, the developed method presents a powerful tool to investigate the actions of biologically active lipid species with subcellular precision. This will likely inspire the generation of similar tools targeting different lipids and other organelles, thus contributing to a more detailed understanding of the intricacies of lipid-mediated signaling events.
Primary liver cancer (PLC) is among the five deadliest cancers worldwide, which represents a clinical challenge to the global health system. Anatomically arising from a regenerative organ with high capacity for plasticity as the liver, hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA) are the most frequent types of PLC. Although iCCA represents just 10-20% of liver tumors, the rise in its incidence in the past decades urges to develop new strategies that facilitate patient stratification and offer the best particular therapies and outcomes. Due to the limited treatment options and the considerable heterogeneity that this malignancy displays on distinct levels, integrative analyses have been key to identify targetable alterations that may improve the clinical management of this disease. Notably, gain-of-function mutations in the IDH1 gene, which lead to the accumulation of the oncometabolite 2-HG, have been commonly reported in 15-20% of iCCA cases. Mutant-IDH1 plays a pivotal role in the deregulation of homeostatic processes, strongly affecting the tumor immune microenvironment (TIME) and promoting cancer development. Nevertheless, further research is needed to decipher the intra- tumor heterogeneity and the molecular underpinnings by which mutant-IDH1 enhances cholangiocarcinogenesis.
Glioblastomas are primary brain tumors that aggressively invade the brain and resist current therapies as multicellular networks. Tumor Microtubes (TMs) play a crucial role in both hallmarks of malignancy. Targeting these malignant networks should therefore increase brain tumor response to therapy and improve patient outcomes. However, the development of drugs that interfere with TM and network formation suffers from the lack of established drug screening pipelines that study their specific anti-TM/network activity. Therefore, to identify anti- TM drugs, I developed and validated a combined, comprehensive in vitro/in vivo drug screening approach, including novel AI-based analysis tools of TM parameters. I found that of 87 tested compounds, two Protein Kinase C (PKC) activators, PMA and TPPB, exhibited robust inhibition of TM formation and TM-mediated, Connexin 43-dependent glioblastoma cell network communication in cell-based assays. Since unconnected tumor cells display increased sensitivity to cytotoxic therapy, brain tumor-bearing mice received radiotherapy, and long-term intravital 2-photon microscopy confirmed the first anti-TM and anti-tumor effects of TPPB administration. Mechanistically, RNA sequencing revealed that TPPB treatment caused a robust decrease of Tweety-homolog 1 (TTYH1) expression, a key TM driver. Additionally, immuno- histochemistry of patient-derived tumor organoids showed tumor cell network disruption and downregulation of TTYH1 after TPPB. This study establishes a novel screening pipeline for anti-TM drug development in glioblastoma and underlines the therapeutic potential of disrupting TMs and their networks.
CD40 signaling is a key modulator of immunological responses and serves several biological functions across multiple organs. While its primary purpose is restoring homeostasis, its unregulated activation drives various immune-modulated pathological conditions. Its role in the development of insulin resistance and the progression of diabetes is well documented. A further contribution of CD40 signaling in escalating systemic inflammation in diabetes leads to the initiation of vascular complications. The functional single nucleotide polymorphism (SNP) in the Kozak region of the CD40 gene (T-1C, rs1883832) impacts CD40 translation efficiency and is associated with several immune and inflammation-modulated diseases. Given the role of CD40 signaling in the progression of diabetes and systemic inflammation, this SNP may also represent a genetic risk factor for diabetes. This hypothesis was investigated by examining the distribution of the T-1C SNP in patients with type 1 (T1D) and type 2 diabetes (T2D). There was, in fact, a strong association of the C-allele with T2D (odds ratio = 1.43; 95% confidence interval: 1.09-1.86). T2D patients also revealed higher plasma levels of the presumably protective soluble CD40 receptor (sCD40), which is known to neutralize the CD40 ligand (CD40L) and its pro-inflammatory capacity. Hence, in T2D patients, CD40 signaling seems to be switched on, leading to an upregulation of the compensatory sCD40 in the blood. The possibility that the T-1C SNP differentially affects the genome of sCD40L-stimulated CC and TT-genotype human cultured endothelial cells (HUVECs) was examined through RNA sequencing. The CC-genotype HUVECs exhibited a higher degree of gene expression in general and in particular of genes involved in escalating inflammation, insulin receptor recycling, and driving endothelial to mesenchymal transition (EndMT). The quiescent TT-genotype HUVECs seemed to be protected against such changes. The impact of a pro-diabetic microenvironment on the pro-inflammatory CC-genotype HUVECs was further studied. Under these conditions, they revealed an increased expression of mediators promoting inflammation, atherosclerosis, and EndMT. In addition, they showed morphological changes characteristic of a partial EndMT-like phenotype. Expression of the osmoadaptive transcription factor nuclear factor of activated T-cells (NFAT) 5 and its nuclear abundance was also increased under these conditions. Blocking NFAT5 DNA-binding in the CC-genotype HUVECs by using a neutralizing consensus NFAT5 decoy oligonucleotide significantly reduced the expression of the mesenchymal marker SM22α. Several nuclear factor κB (NF-κB)/NFAT5 dual binding sites are located in promotor regions of genes involved in the progression of atherosclerosis and EndMT. While NF-κB is known to drive inflammation and EndMT in HUVECs, less is known about the role of NFAT5 in modulating these processes. This work provides novel insights into the role of NFAT5 in enhancing NF-κB mediated vascular inflammation and EndMT under pro-diabetic conditions.
Einführung: Consumer Health Informatics (ConsHI) ist eine Fachdisziplin, die basierend auf Methoden, Diensten und Informations- und Kommunikationstechnologie-Ausstattung Laien in die Lage versetzt, eine aktive Rolle hinsichtich ihrer Gesundheit sicher zu spielen. ConsHI verspricht, ein Heilmittel für die Myriaden an Gesundheitsherausforderungen zu sein, welche die Welt bedrängen, hauptsächlich nach der Covid – 19 Pandemie. Während Consumer Health Informatics (ConsHI) eine Menge verspricht, besteht ein Mangel an Modellen zur Bewertung der Annahme dieser Konzepte in verschiedenen Ländern, insbesondere Entwicklungsländern. Auch sind die zahlreichen Modelle der Annahme von Technologie und von Gesundheitsfürsorge uneinheitlich und der kritische Bedarf an einem zusammengesetzten Modell ist mehr ausgeprägt denn je. Diese Studie zielt darauf ab, Faktoren zu bewerten, die die Annahme von ConsHI in low-middle income countries (LMICs) zu erleichtern und die vorherrschenden Faktoren zu modellieren, welche Vorhersagen über die Reife ihrer Bürger für die Annahme von ConsHI treffen. Methoden: Wir haben eine umfassende Suche danach durchgeführt, wie Laien in Entwicklungs- wie in entwickelten Ländern IKT für ihre Gesundheit einsetzten und haben aus vielen Optionen drei wesentliche Modelle identifiziert. Die Modelle waren Unified Theory of Acceptance and Utilisation of Technology (UTAUT), Patient Activation Measure (PAM) und Consumer Health Informatics (ConsHI) Modelle, welche die individuelle Annahme und Teilnahme an Technologie und Gesundheitsfürsorge parallel überprüften. Wir haben unter Nutzung dieser drei Modelle ein zusammengesetztes Modell entwickelt. Wir haben es anschließend mittels Wilcoxon Signed Rank Test und Item Response Theory validiert. Wir haben eine mehrstufige ad-hoc-Datensammlung (convenience sampling) durchgeführt, um den Fragebogen 1800 Befragten aus sechs LMICs in einer Querschnittserhebung vorzulegen. Die Befragten waren z.T. gesund, z.T. bei eingeschränkter Gesundheit, 18 Jahre oder älter – der Rücklauf war fast 100%, von einigen fehlenden Daten abgesehen, da die Untersucher die Erhebungsblätter persönlich vorlegten. Die Daten wurden sowohl mit explorativen wie auch konformativen Faktoranalyse-Techniken analysiert, so zum Beispiel partial least square structural equation models in Rstudio und SmartPLS 4.0, je nach Gegebenheit. Unser Datensatz erfüllte alle Grundannahmen der explorativen und konfirmativen Faktorenanalyse ohne signifikante Probleme. Aus der explorativen Faktorenanalyse haben wir Faktoren (e – factors) extrahiert und Fakten aus empirischen Modellen (t – factors) gegenübergestellt. Auch haben wir mittels Strukturgleichungsmodellen Reifefaktoren (m – factors) aus den t – factors zusammengestellt. Ergebnisse: Wir haben zwei hauptsächliche Ergebnisse erzielt. Zunächst, in einer Voruntersuchung, haben wir einen Fragebogen mit 43 Items, gestaltet als 5-Punkt-Likert-Items zusammengestellt und validiert und acht demographische Items hnzugefügt, als Moderatoren der m – factors zur Vorhersage der ConsHI-Reife. Zum zweiten haben wir aus dem Datensatz sechs explorative Faktoren als erleichternde Elemente für ConsHI extrahiert. Die e – factors waren wesentlich durch ein Gestaltexperiment gekennzeichnet und spiegelten die drei theoretischen Faktoren (t – factors) unseres Modells wider. Wir haben auch konfirmative Modellierung höherer Ordnung angewandt, um vier m – factors (Aptitute, Attitude, Confidence und Motivation) als Prädiktoren der Reife von Bürgern in LMICs zusammenzusetzen. Attitude (Haltung) trug am meisten zur Vorhersage der Reife von Bürgern in LMICs bei, aptitude (Eignung) am wenigsten. Die prädiktive Relevanz und Stärke des Modells wurden validiert und waren auf dem 95%-Konfidenzniveau signifikant. Zu beachten ist, dass eine Mehrgruppenanalyse die statistische Signifikanz der beobachteten Heterogenität und den moderierenden Effekt mehrerer demogrpahischer Variablen, wie z.B. Alter, die Vorhersehbarkeit für ConsHI in LMICs bestätigten. Schlüsse: Soweit es uns bekannt ist, ist unsere Studie ein Vorreiter, indem sie ein zusammengesetztes Modell aus UTAUT, PAM und ConsHI erstellt; diese Forschung macht die Notwendigkeit von Politikformulierung zur Maximierung der Technologie in der Gesundheitsversorgung und zur Optimierung eines ausgeweiteten Zugangs zu mobiler Telefonie zum zentralen Thema. Die Studie hat ein prädiktives lineares Modell zur Bestimmung der Reife von Bürgern in LMICs für ConsHI formuliert. Die Ad-Hoc-Datenerhebung war eine wichtige Einschränkung der Studie; als Querschnittstudie, da sich Faktoren mit der Zeit ändern, könnten ihr auch dynamische Umgebungsfaktoren entgangen sein. Wir empfehlen, dass künftige Studien Zufallsstichprobenvefahren anwenden, und Anstrengungen, aktive Techniken in einer Verhaltensstudie zu verwenden, würden auch helfen. Schlüsselwörter: Verbrauchergesundheitsinformatik, Moderatoren, Reife der Bürger von LMICs, Ländern mit niedrigem mittlerem Einkommen und Vorhersagemodelle
We formulate extended mirror symmetry of Calabi-Yau threefolds with D- branes as an equivalence between variations of mixed Hodge structure under the mirror map. After an introduction to Hodge theoretic closed string mir- ror symmetry, we review the relation between D-branes, normal functions and extensions by algebraic cycles on the side of the B-model. We define an exten- sion of the A-model variation of mixed Hodge structure whose flat connection is derived from an enhancement of the quantum product by holomorphic disks ending on Lagrangian submanifolds. Our construction is based on the Solomon- Tukachinsky axioms for open Gromov-Witten invariants together with the open WDVV equations and matches the predictions from extended mirror symmetry. For the particular case of homology spheres, we define an extension of Iritani’s Gamma-integral local system and propose an extended version of the Gamma conjecture. We demonstrate the validity of the conjecture for the standard pair of branes in case of the quintic and prove a corresponding extended Mir- ror Theorem. Using the extended holomorphic anomaly equations, we explore novel invariants from one-loop amplitudes for cycles of van Geemen-type, whose A-model geometry is at present unknown.
To enable a quick response to a variety of different stresses, cells modulate mRNA translation by repressing global translation and permitting translation of certain transcripts, thereby reprogramming the cellular proteome and adapting to the stress. In my thesis, I study two different mechanisms of how mRNA translation can be selectively regulated on subsets of mRNAs to shape the proteome.
Usually, cells recruit mRNA to a small ribosomal subunit with the use of translation initiation factor eIF4E. Cellular stresses result in the inactivation of mTORC1, which is a central controller of cellular growth and metabolism, and consequently downstream activation of a group of proteins called 4E-BPs. 4E-BPs sequester eIF4E, thus hampering eIF4E-dependent translation initiation. Although general protein production is suppressed, the translational shut-down is not complete and cells still can translate in an eIF4E-independent manner. In the first part of this thesis, I study how this eIF4E-independent translation is realised. By applying the ribosome profiling technique in cells overexpressing a constitutively-active 4E-BP1, I determined the list of transcripts resistant to eIF4E-sequestration. These transcripts have longer-than-average 5’-UTRs and rely on cap- and 5’-end dependent initiation in a condition of inactive mTORC1. I show that when 4E-BPs are active the resistant transcripts are released from eIF4E-4E-BP complex and bind via cap to eIF3D to recruit ribosomes in an eIF4E-independent manner.
In the second part of this thesis, I focus on PRRC2A/B/C proteins, which were recently linked to mRNA translation in our lab. Although PRRC2 proteins are often dysregulated in many cancers and were linked to RNA metabolism, little is known about their biological function. In our lab, we identified them as translation initiation factors. I show that the PRRC2A/B/C knock-down affects only certain transcripts, which possess upstream ORFs (uORF). The presence of uORF is sufficient to make mRNAs dependent on PRRC2A/B/C. Repression of these mRNAs suggests that lack of PRRC2s increases translation of uORFs, thereby repressing main ORF translation. Two independent mechanisms to overcome uORF repression exist – translation reinitiation and leaky scanning. I present that PRRC2A/B/C proteins promote leaky scanning on uORFs, thus permitting translation of the main ORF of uORF-containing mRNAs. This is highly relevant during cellular stress since many uORFs play regulatory roles in the stress-response.
Many non-perturbative phenomena in quantum field theories are driven or accompanied by non-local excitations, whose dynamical effects can be intricate but difficult to study. Amongst others, this includes diverse phases of matter, anomalous chiral behavior, and non-equilibrium phenomena such as non-thermal fixed points and thermalization. Topological data analysis can provide non-local order parameters sensitive to numerous such collective effects, giving access to the topology of a hierarchy of complexes constructed from given data. This dissertation contributes to the study of topological data analysis and geometry in quantum field dynamics. A first part is devoted to far-from-equilibrium time evolutions and the thermalization of quantum many-body systems. We discuss the observation of dynamical condensation and thermalization of an easy-plane ferromagnet in a spinor Bose gas, which goes along with the build-up of long-range order and superfluidity. In real-time simulations of an over-occupied gluonic plasma we show that observables based on persistent homology provide versatile probes for universal dynamics off equilibrium. Related mathematical effects such as a packing relation between the occurring persistent homology scaling exponents are proven in a probabilistic setting. In a second part, non-Abelian features of gauge theories are studied via topological data analysis and geometry. The structure of confining and deconfining phases in non-Abelian lattice gauge theory is investigated using persistent homology, which allows for a comprehensive picture of confinement. More fundamentally, four-dimensional space-time geometries are considered within real projective geometry, to which canonical quantum field theory constructions can be extended. This leads to a derivation of much of the particle content of the Standard Model. The works discussed in this dissertation provide a step towards a geometric understanding of non-perturbative phenomena in quantum field theories, and showcase the promising versatility of topological data analysis for statistical and quantum physics studies.
The cell nucleus is organized into functional domains that form around chromatin, which serves as a scaffold composed of DNA, proteins, and associated RNAs. On the 0.1-1 µm mesoscale these domains can form spatially defined compartments with distinct composition and properties that enrich specific genomic activities like transcription, chromatin modification or DNA repair. In addition, extrachromosomal DNA elements and RNAs can separate from the chromatin template and assemble with proteins into nuclear bodies. The resulting accumulations of proteins and nucleic acids in the nucleus modulate chromatin-templated processes and their organization. The assembly of these compartments occurs in a self-organizing manner via direct and indirect binding of proteins to DNA and/or RNA. Recently, it has been proposed that multivalent interactions drive compartmentalization by inducing phase separation with a non-stoichiometric accumulation of factors into biomolecular condensates. Despite the importance of compartments for genome regulation, insights into their structure and material properties and how these affect their function is limited. To address this issue, it is important to devise approaches that can perturb nuclear compartments in a targeted manner, while also measuring changes in genome activities within the same cell. In this thesis, the methodology to reveal the underlying structure-function relationships of nuclear compartments has been advanced and applied to compartments involved in activation and silencing of chromatin, and telomere maintenance in cancer cells. I first established a toolbox of chromatin effector constructs to probe and perturb properties of nuclear compartments in living cells that comprised different combinations of DNA binding, transcription activation and light-dependent interaction domains. In addition, I developed workflows to quantitatively assess relevant compartment features by fluorescence microscopy. These methods were employed to study the compaction mechanism of mouse pericentric heterochromatin (PCH) foci and to investigate the interplay between transcriptional co-activators, phase separation and transcription at an inducible reporter gene cluster. It revealed determinants of PCH compaction and identified differential co-activator usage and multivalent interactions as contributors to transcription factor (TF) strength. The results furthermore challenged the model of TF phase separation as a general positive driver of gene transcription. In the second part, I focused on exploiting the detection of compartments for measuring activity of the alternative lengthening of telomeres (ALT) pathway used by cancer cells to extend their telomeres in absence of telomerase. I developed ALT-FISH, a scalable and quantitative imaging assay that detects ALT pathway-specific compartments containing large amounts of single-stranded telomeric nucleic acids. I applied the method to cell line models from different cancer entities and to tumor tissue from leiomyosarcoma and neuroblastoma patients. By devising automated ALT-FISH data acquisition and analysis IV workflows, I implemented an approach, which enabled ALT activity measurements in hundreds of thousands of single cells. These technological advancements provided a quantitative description of ALT activity at single cell resolution and were used to characterize the spatial distribution of ALT activity in relation to other biological features and in response to perturbations. Finally, a novel approach for studying the regulation of ALT in tumors could be established by integrating the method with the spatially resolved detection of single cell transcriptomes. In summary, this thesis introduced and utilized several methods to establish connections between nuclear compartment organization, chromatin features, transcription regulation, and telomere maintenance. These perturbation and imaging techniques are versatile and may be applied to dissect nuclear activities related to other compartments and biological model systems. Furthermore, the detection of ALT activity has demonstrated that compartments can offer valuable biological insights into how phenotypic cellular heterogeneity is encoded and linked to diseases such as cancer.
Methylglyoxal (MG) is a byproduct of glucose metabolism that displays high reactivity with many biological macromolecules, in particular proteins, giving rise to adducts known as advanced glycation end products (AGEs). Increased MG and AGEs are commonly observed in diabetes and, among diabetic patients, those affected by diabetic complications show the highest levels of MG and MG-adducts, raising the possibility that, besides hyperglycemia, a buildup of MG could play a direct causative role in the development of diabetes and its complications. Indeed, findings in Drosophila melanogaster and Danio rerio showed that disruption of glyoxalase I (Glo1), the rate limiting enzyme for MG detoxification, causes features of type 2 diabetes such as insulin resistance, hyperglycemia and obesity . Understanding how the activity of Glo1 is regulated and how exactly MG affects cellular metabolism is thus of the utmost importance to determine how MG detoxification goes awry in diabetes and how the ensuing MG accumulation contributes to the metabolic alterations typical of this disease. To this end, I worked on two complementary lines of investigation aimed at (1) assessing the role of post-translational modifications in the regulation of Glo1 activity and (2) studying which metabolic pathways are affected by high levels of MG in vitro and in mouse models of diabetes. My results show that phosphorylation of Glo1 at Y136 by multiple kinases, including those belonging to the Src family, promotes Glo1 activity. Consistent with impaired detoxification of MG in the pathogenesis of diabetes, I observed that phosphorylation at this residue and overall Glo1 activity are decreased when cells are cultured in high glucose (25 mM), as well as in diabetic mouse models. To study the metabolic alterations caused by MG, I generated cell lines knockout for Glo1 or acutely treated control cells with MG. Interestingly, chronic or acute exposure to MG was sufficient to increase glucose uptake, lactate production and impair fatty acid -oxidation. I found that MG inhibits the activity of pyruvate dehydrogenase (PDH), probably accounting for the increased glucose uptake and lactate production. The effect of MG on PDH activity is not mediated by altered phosphorylation of PDH, a well-established mode of regulating PDH activity, but rather by direct interaction of MG with the pyruvate dehydrogenase α (PDHA) subunit of PDH, together with formation of a DTT-sensitive modification on the PDH subunit dihydrolipoamide acetyltransferase (DLAT). I also observed decreased PDH activity in mouse models of diabetes, further strengthening the link between accumulation of MG and impaired PDH activity. Overall, my data point to a deleterious positive feedback loop whereby hyperglycemia leads to reduced Y136 Glo1 phosphorylation and activity, contributing to elevated MG levels,inhibition of PDH and changes of cellular metabolism to promote hyperglycemia and thus further production of MG.
In this thesis, efficient overlapping multilevel Schwarz preconditioners are used to iteratively solve Hdiv-conforming finite element discretizations of models in poroelasticity, and an innovative two-scale multilevel Schwarz method is developed for the solution of pore-scale porous media models. The convergence of two-level Schwarz methods is rigorously proven for Biot’s consolidation model, as well as a Biot-Brinkman model by utilizing the conservation property of the discretization. The numerical performance of the proposed multiplicative and hybrid two-level Schwarz methods is tested in different problem settings by covering broad ranges of the parameter regimes, showing robust results in variations of the parameters in the system that are uniform in the mesh size. For extreme parameters a scaling of the system yields robustness of the iteration counts. Optimality of the relaxation factor of the hybrid method is investigated and the performance of the multilevel methods is shown to be nearly identical to the two-level case. The additional diffusion term in the Biot-Brinkman model yields a stabilization for high permeabilities. Additionally, a homogenizing two-scale multilevel Schwarz preconditioner is developed for the iterative solution of high-resolution computations of flow in porous media at the pore scale, i.e., a Stokes problem in a periodically perforated domain. Different homogenized operators known from the literature are used as coarse-scale operators within a multilevel Schwarz preconditioner applied to Hdiv-conforming discretizations of an extended model problem. A comparison in the numerical performance tests shows that an operator of Brinkman type with optimized effective tensor yields the best performance results in an axisymmetric configuration and a moderately anisotropic geometry of the obstacles, outperforming Darcy and Stokes as coarse-scale operators, as well as a standard multigrid method, that serves as a benchmark test.
Many clinical applications currently rely on several imaging modalities such as Positron Emission Tomography (PET), Magnetic Resonance Imaging (MRI), Computed Tomography (CT), etc. All such modalities provide valuable patient data to the clinical staff to aid clinical decision-making and patient care. Despite the undeniable success of such modalities, most of them are limited to preoperative scans and focus on morphology analysis, e.g. tumor segmentation, radiation treatment planning, anomaly detection, etc. Even though the assessment of different functional properties such as perfusion is crucial in many surgical procedures, it remains highly challenging via simple visual inspection. Functional imaging techniques such as Spectral Imaging (SI) link the unique optical properties of different tissue types with metabolism changes, blood flow, chemical composition, etc. As such, SI is capable of providing much richer information that can improve patient treatment and care. In particular, perfusion assessment with functional imaging has become more relevant due to its involvement in the treatment and development of several diseases such as cardiovascular diseases. Current clinical practice relies on Indocyanine Green (ICG) injection to assess perfusion. Unfortunately, this method can only be used once per surgery and has been shown to trigger deadly complications in some patients (e.g. anaphylactic shock).
This thesis addressed common roadblocks in the path to translating optical functional imaging modalities to clinical practice. The main challenges that were tackled are related to a) the slow recording and processing speed that SI devices suffer from, b) the errors introduced in functional parameter estimations under changing illumination conditions, c) the lack of medical data, and d) the high tissue inter-patient heterogeneity that is commonly overlooked. This framework follows a natural path to translation that starts with hardware optimization. To overcome the limitation that the lack of labeled clinical data and current slow SI devices impose, a domain- and task-specific band selection component was introduced. The implementation of such component resulted in a reduction of the amount of data needed to monitor perfusion. Moreover, this method leverages large amounts of synthetic data, which paired with unlabeled in vivo data is capable of generating highly accurate simulations of a wide range of domains. This approach was validated in vivo in a head and neck rat model, and showed higher oxygenation contrast between normal and cancerous tissue, in comparison to a baseline using all available bands. The need for translation to open surgical procedures was met by the implementation of an automatic light source estimation component. This method extracts specular reflections from low exposure spectral images, and processes them to obtain an estimate of the light source spectrum that generated such reflections. The benefits of light source estimation were demonstrated in silico, in ex vivo pig liver, and in vivo human lips, where the oxygenation estimation error was reduced when utilizing the correct light source estimated with this method. These experiments also showed that the performance of the approach proposed in this thesis surpass the performance of other baseline approaches.
Video-rate functional property estimation was achieved by two main components: a regression and an Out-of-Distribution (OoD) component. At the core of both components is a compact SI camera that is paired with state-of-the-art deep learning models to achieve real time functional estimations. The first of such components features a deep learning model based on a Convolutional Neural Network (CNN) architecture that was trained on highly accurate physics-based simulations of light-tissue interactions. By doing this, the challenge of lack of in vivo labeled data was overcome. This approach was validated in the task of perfusion monitoring in pig brain and in a clinical study involving human skin. It was shown that this approach is capable of monitoring subtle perfusion changes in human skin in an arm clamping experiment. Even more, this approach was capable of monitoring Spreading Depolarizations (SDs) (deoxygenation waves) in the surface of a pig brain. Even though this method is well suited for perfusion monitoring in domains that are well represented with the physics-based simulations on which it was trained, its performance cannot be guaranteed for outlier domains. To handle outlier domains, the task of ischemia monitoring was rephrased as an OoD detection task. This new functional estimation component comprises an ensemble of Invertible Neural Networks (INNs) that only requires perfused tissue data from individual patients to detect ischemic tissue as outliers. The first ever clinical study involving a video-rate capable SI camera in laparoscopic partial nephrectomy was designed to validate this approach. Such study revealed particularly high inter-patient tissue heterogeneity under the presence of pathologies (cancer). Moreover, it demonstrated that this personalized approach is now capable of monitoring ischemia at video-rate with SI during laparoscopic surgery.
In conclusion, this thesis addressed challenges related to slow image recording and processing during surgery. It also proposed a method for light source estimation to facilitate translation to open surgical procedures. Moreover, the methodology proposed in this thesis was validated in a wide range of domains: in silico, rat head and neck, pig liver and brain, and human skin and kidney. In particular, the first clinical trial with spectral imaging in minimally invasive surgery demonstrated that video-rate ischemia monitoring is now possible with deep learning.
Hepatitis B führt zu über achthunderttausend Toten pro Jahr und stellt somit eine der bedeutendsten Erkrankungen der Menschheit dar. Mit Hepatitis D co-infizierte Patienten leiden unter der schwersten Form der Hepatitis. Hepatitis D kann jedoch seit kurzem mit dem Eintrittsinhibitor Myrcludex B (Handelsname: Hepcludex®) effizient behandelt werden. Diese nahezu nebenwirkungsfreie Therapie könnte eventuell noch weiter verbessert werden, da Myrcludex B zusätzlich zur Virusaufnahme auch die Wiederaufnahme von Gallensäuren, die eigentliche Funktion des NTCP, inhibiert. Die Aufnahme von Hepatitis B in Hepatozyten wird über NTCP vermittelt. Da dieser Rezeptor auch Gallensäuren bindet, wurden im Rahmen dieser Arbeit verschiedene Gallensäure-Peptid-Konjugate synthetisiert. Hierbei wurden monomere und dimere Gallensäuren verwendet und der Einfluss verschiedener Linker und Konformationen an der Bindestelle genauer untersucht. Die Peptidsequenz wurde vom Goldstandard Myrcludex B abgeleitet, zudem wurden weitere HBV-Genotypen charakterisiert. Um aussagekräftige Struktur-Wirkungsbeziehungen zu erhalten, wurden über 165 verschiedene Peptide und Gallensäure-Peptid-Konjugate synthetisiert und im ersten Schritt auf ihre HDV-Infektionsinhibition untersucht. Die chimären Substanzen erzielen hervorragende Effizienzen. Mit HBVpreS/2-21-yK-LCA und HBVpreS/2-21-yK-DCA, den beiden besten Substanzen, werden die inhibitorischen Eigenschaften von Myrcludex B erreicht. Für das Erreichen hoher inhibitorischer Aktivitäten müssen jedoch enge strukturelle Voraussetzungen eingehalten werden, beispielsweise führt die Verwendung von D-Lysin in der Verknüpfungsstelle zu einer zehnfach verringerten Effizienz. Da die beiden Substanzen diese Aktivität mit einer deutlich verkürzten Peptidsequenz erreichen, bieten sie, bei vereinfachtem synthetischem Zugang, großes Potential bezüglich einer Verbesserung der Pharmakokinetik. Zudem belegen physikochemischen Charakterisierungen ihre generelle Eignung als Medikamente. In weiteren in vitro-Untersuchungen konnte eine hohe Stabilität der beiden Gallensäure-Peptid-Konjugate gezeigt werden. Zudem waren weder Cytotoxizität noch hämolytische Eigenschaften beobachtbar. Die hervorragenden Daten in vitro konnten mit dem Erhalt der nahezu exklusiven Anreicherung in der Leber in vivo bestätigt werden. Somit stellen diese Substanzen aussichtsreiche Kandidaten für die zukünftige Weiterentwicklung als Medikamente dar.
Background: Machine learning approaches are becoming increasingly common in biological research, as these allow for a better understanding of the complex cell dynamics. Epigenetics encompasses processes able to modulate gene expression that do not depend on genomic sequence. Oftentimes, epigenetic alterations have been linked to disease. In this thesis, we applied several computational approaches to characterise the epigenetic landscape of diseased states caused by Human Immunodeficiency Virus infection and cancer in the brain.
Results: On the first part of this thesis, we applied non-negative matrix factorisation to build an epigenetic state map for the C20 microglial cell line and assessed the connection between integration and epigenetics in the context of HIV-1 infection. Through random forest models, we observed that genomic targets of HIV-1 integration are influenced by the initial epigenetic landscape and that infection leads to changes in the chromatin accessibility and TF binding. Furthermore, we found that regions often targeted by viral integration are associated to higher order chromatin structures, in particular topologically associated domains. On the second part of this thesis, we characterised CpG islands (CGI) of four glioblastoma subtypes and identified a new phenotype of CGI hypermethylation associated to RTK-II subtype, different from the one observed on the IDH subtype. We compared the CGI hypermethylation phenotypes associated to the IDH and RTK-II subtypes using random forests and use progenitor states to assess the tendency within each CGI to become hypermethylated. We observed that CGI most likely to become hypermethylated in cancer are marked already on undifferentiated cell states. Moreover, we observed that RTK-II CGI hypermethylation disturbs the astrogenic/neurogenic fate balance.
Conclusions: This thesis provides novel insights into the epigenetics of HIV-1 integration and CGI hypermethylation in glioblastoma. Through a genomic and epigenomic data-driven approach, we emphasise the importance of computational approaches like non-negative matrix factorisation, random forest, and bayesian networks into epigenetic research, as these provided an hollistic view of the global effects of viral integration and CGI hypermethylation in human cells.
Biological cells sense the mechanical properties of their surrounding environment and adapt their shape and function. Moreover, the mechanical properties of cells and tissues tightly correlate with their functions. The main thrust of this thesis is to quantitatively determine the mechanical proper- ties of cells and cell-repellent coating materials by the combination of unique experimental techniques by covering different spatio-temporal domains. In chapter 7 the viscoelastic shape relaxation of malaria-infected human red blood cells with a di- ameter of about 10 μm was monitored by the combination of a custom-designed microfluidic device and a high-speed imaging platform under collaboration with Prof. Dr. M. Lanzer (Center for Inte- grative Infectious Diseases, Heidelberg University). Using the binarised cell rims extracted from the live-cell images, the shape recovery of red blood cells upon the ejection from the narrow constriction was monitored with a time resolution of 30 μs per frame. The mechanical responses of the malaria- infected red blood cells were monitored through the entire life cycle of parasites. The systematic comparison of the red blood cells with genetically mutated hemoglobin (hemoglobinopathie) with normal red blood cells indicated a less pronounced change in the relaxation time in hemoglobinopa- thetic red blood cells, which might correlate with delayed protein synthesis in hemoglobinopathetic red blood cells. In chapter 8 the film elastic properties and internal structures of the monolayers of oligoethylene glycol-based dendrons for the coating of iron-oxide nanoparticles were studied by the combination of high energy X-ray reflectivity and high-speed atomic force microscopy. To achieve higher film sta- bility in blood stream, the dendrons, synthesized by the group of Prof. Dr. Felder-Flesch (Institut de Physique et Chimie des Materiaux , Univ. Strasbourg) were coupled to the oxide surface via two phosphonate groups. The interfacial force measurements were performed on planar silicon dioxide surfaces instead of iron oxide nanoparticle surfaces due to the technical limitations. The internal structures of dendron monolayers in water were probed by high energy specular X-ray reflectivity. An analytical model considering the transition from a soft layer to a hard layer was introduced to cal- culate the Young’s modulus from nm-thick monolayers. To gain deeper insights into the interfacial force interactions, the coarse-scale surface force-distance curves were measured by a cell-sized particle attached to an atomic force cantilever cantilever, while the size and distribution of nanoscopic pin- ning centers were monitored by fast force mapping with a pixel rate of 200 Hz. The capability of the dendron coating to prevent the platelet aggregation was assessed by observing the non-specific adhesion of human platelets on dendron-coated substrates. The dynamic uptake and localisation of fluorescent dendron-coated iron oxide nanoparticles into hypoxic mouse breast cancer cells was tracked using fluorescence imaging and cryo-transmission electron microscopy. Together, these meth- ods revealed a continuous uptake of iron oxide nanoparticles into in intracellular compartments such as endosomes via endocytosis. The iron oxide particles were found either agglomerated or as single nanoparticles.
During membrane protein biogenesis, cells need to detect and degrade faulty proteins. Despite a key role in cellular homeostasis and human diseases, little is known about the underlying mechanisms. In recent years, few endoplasmic reticulum (ER)-resident proteases have been linked to quality control by cleaving their clients and thereby facilitating membrane extraction and degradation via the ER-associated degradation (ERAD) pathway. The major ER-resident protease in mammalian cells is the signal peptidase complex (SPC), a tetra subunit complex discovered in the 1970s to be responsible for the removal of signal sequences from ER-targeted and secretory proteins. Until now, this was thought to be the only function of the SPC besides a few studies reporting a role in the maturation of viral polyproteins. In this work, I show that the SPC also acts as a membrane protein quality control factor. First, through proteome-wide computational analyses, I identified approximately 1500 membrane proteins containing SPC cryptic cleavage sites after N-terminal and internal type-II oriented transmembrane domains (TMDs). I then validated SPC cleavage for several candidate substrates (Cx32, Cx26, Cx30.3, PMP22, iRhom2 and Hrd1) and revealed that SPC cleavage relies on the accessory subunit SPCS1 as recognition factor to discern between signal sequences and TMDs. Moreover, I show that the SPC cleaves membrane proteins when they fail to fold properly or assemble correctly into their native complexes, thus exposing cryptic cleavage sites. I also show that this SPC cleavage mechanism cooperates with the ERAD pathway to help maintain a functional membrane proteome and confers a fitness advantage to cells exposed to ER stress. Finally, I report first data on the possible role of the SPC in controlling protein abundance beyond its quality control function. Overall, this thesis characterises a novel function of the SPC, expanding its substrate spectrum, extending the knowledge on the essential cellular functions performed by this protease and laying the foundations for future work at the organismal level in quality control-related diseases and beyond.
In this thesis, we focus on the image labeling problem which is the task of performing unique pixel-wise label decisions to simplify the image while reducing its redundant information. We build upon a recently introduced geometric approach for data labeling by assignment flows [ APSS17 ] that comprises a smooth dynamical system for data processing on weighted graphs. Hereby we pursue two lines of research that give new application and theoretically-oriented insights on the underlying segmentation task. We demonstrate using the example of Optical Coherence Tomography (OCT), which is the mostly used non-invasive acquisition method of large volumetric scans of human retinal tis- sues, how incorporation of constraints on the geometry of statistical manifold results in a novel purely data driven geometric approach for order-constrained segmentation of volumetric data in any metric space. In particular, making diagnostic analysis for human eye diseases requires decisive information in form of exact measurement of retinal layer thicknesses that has be done for each patient separately resulting in an demanding and time consuming task. To ease the clinical diagnosis we will introduce a fully automated segmentation algorithm that comes up with a high segmentation accuracy and a high level of built-in-parallelism. As opposed to many established retinal layer segmentation methods, we use only local information as input without incorporation of additional global shape priors. Instead, we achieve physiological order of reti- nal cell layers and membranes including a new formulation of ordered pair of distributions in an smoothed energy term. This systematically avoids bias pertaining to global shape and is hence suited for the detection of anatomical changes of retinal tissue structure. To access the perfor- mance of our approach we compare two different choices of features on a data set of manually annotated 3 D OCT volumes of healthy human retina and evaluate our method against state of the art in automatic retinal layer segmentation as well as to manually annotated ground truth data using different metrics. We generalize the recent work [ SS21 ] on a variational perspective on assignment flows and introduce a novel nonlocal partial difference equation (G-PDE) for labeling metric data on graphs. The G-PDE is derived as nonlocal reparametrization of the assignment flow approach that was introduced in J. Math. Imaging & Vision 58(2), 2017. Due to this parameterization, solving the G-PDE numerically is shown to be equivalent to computing the Riemannian gradient flow with re- spect to a nonconvex potential. We devise an entropy-regularized difference-of-convex-functions (DC) decomposition of this potential and show that the basic geometric Euler scheme for inte- grating the assignment flow is equivalent to solving the G-PDE by an established DC program- ming scheme. Moreover, the viewpoint of geometric integration reveals a basic way to exploit higher-order information of the vector field that drives the assignment flow, in order to devise a novel accelerated DC programming scheme. A detailed convergence analysis of both numerical schemes is provided and illustrated by numerical experiments.
Mammalian embryo development is highly regulative in nature, with cell fate being reversible to compensate for changes in the nascent environment during early stages. In such regulative paradigms, the complex interactions between cellular processes to accomplish precision in patterning of tissues present exciting open questions in biology. In this thesis, I explore the patterning mechanisms underlying blastocyst maturation during pre-implantation development of the mouse embryo. Development of the mammalian blastocyst involves the progressive segregation of the inner cell mass comprising the embryonic epiblast and the extra-embryonic primitive endoderm, and the expansion of a fluid-filled cavity. A gene regulatory network between fibroblast growth factor signalling and cell fate-specific transcription factors establishes the characteristic salt-and-pepper distribution of epiblast and primitive endoderm cells in the early blastocyst. Following the establishment of the two precursor populations, cells spatially segregate into distinct domains within the embryo. The primitive endoderm forms an epithelial layer at the surface of the fluid cavity, with the pluripotent epiblast enclosed between the trophectoderm and the primitive endoderm. Symmetry-breaking in the blastocyst has been the focus of much research in the past decades. However, a coherent mechanism of blastocyst pattern emergence coordinating cell fate specification, sorting and morphogenesis is lacking. Using reduced systems and advanced live-image analysis, I established a method to characterise cell sorting during fate segregation. Further, combined with biophysical measurements and perturbations, I investigate the interplay between cytoskeletal dynamics, cell migration, and polarity in driving fate segregation. I demonstrate that cell sorting in the inner cell mass is characterised by active migration of primitive endoderm cells towards the cavity surface. The primitive endoderm cells in the early blastocyst display autonomous acquisition of apical polarity, a defining feature essential for their proper segregation into a single epithelial layer enveloping the epiblast. Moreover, in contrast to epiblast cells, apical polarity in primitive endoderm cells facilitates the lowering of surface tension upon encountering the cavity surface, thus being sufficient for cell positioning. Lastly, I provide evidence that cell fate plasticity is lost in late blastocysts due to which the fixed lineage composition of the inner cell mass is optimal exclusively for a particular embryo size. Altogether, the findings presented in this work provide mechanistic insights into segregation of cell fates in the mammalian blastocyst and put forth a novel understanding of robust patterning during embryonic development.
Argon Trap Trace Analysis resolves 39Ar concentrations at the 10−16 level. Perfect selectivity is achieved by exploiting the isotopic shift and a high number of photon scattering processes for detection. However, good measurement precision requires high count rates of39Ar atoms. Therefore, this work presents crucial features and characterization tools for reliable routine measurements. A new instrument for absolute quantization of the metastable 40Ar flux is developed and put into operation. The magneto-optical trap loading rate is evaluated and established as a standard characterization tool. It is part of the new collimator alignment procedure that resulted in a factor of 1.5 higher 39Ar count rate. Other improved alignment procedures are presented. In addition, a new laboratory control and monitoring system for stable day-to-day measurements is developed and installed. The second part focuses on the study of sources of metastable atoms. Two radio frequency (RF) antennas and two cooling devices are presented and their main features are analyzed. A new LN2-dewar for cooling reduces the amount of liquid nitrogen consumed by a factor of 3 to 5. In combination with the established RF antenna, increasing the source pressure to 2.0 × 10−5 mbar leads to 50% higher metastable flux, while the atoms slow down longitudinally.
Interatomic or intermolecular Coulombic decay (ICD) is an efficient relaxation pathway on the femtosecond timescale, where after inner-valence ionization of an atom or molecule, the initial vacancy is filled by an outer-valence electron and the excess energy is transferred radiationlessly to its neighbor, leading to its ionization. In this thesis, ICD-related phenomena are studied. Therefore, the work is divided into two main parts. Part I treats the new decay channel double ICD (dICD), where after the relaxation of the system, the excess energy exceeds the double ionization threshold of the neighboring species, resulting in its double ionization. We derive an asymptotic and perturbative expression for the dICD decay width as an indicator of efficiency. In Part II, nuclear dynamics during ICD and pre-ICD in NeKr and ArAr dimers are investigated focusing on different aspects. In pre-ICD, the excess energy is only sufficient for the excitation of the neighboring atom or molecule and additional energy has to be provided for its ionization. While the study about NeKr dimers undergoing ICD mostly concentrates on interference effects, the main goal of the pre-ICD in ArAr dimers study is to understand the nuclear motions and their appearance in the corresponding spectrum.
Der Epidermale Wachstumsfaktor-Rezeptor (EGFR) ist ein wichtiges Target für die Bildgebung und Therapie bei einer Vielzahl von malignen Veränderungen. Derzeit sind jedoch noch keine Bildgebungsagenzien auf der Basis von Peptiden für diesen Rezeptor vorhanden. Im Rahmen dieser Arbeit wurden Multimere (Homodi- und Homotetramer) des literaturbekannten Peptidliganden GE11 hinsichtlich ihrer EGFR-spezifischen Interaktion hergestellt und überprüft. Da sie meist überlegene Bindungseigenschaften verglichen mit denen der jeweiligen Monomeren aufweisen und aufgrund unterschiedlicher Liganden an verschiedene Rezeptoren binden können, wurde zusätzlich ein Heterodimer bestehend aus αVβ3-spezifischem c(RGDfK) und GE11 synthetisiert und ebenfalls untersucht. Des Weiteren wurden die literaturbekannten Peptide CPP, D4, EGBP, GE11, P1, P2, Pep11 und QRH sowie drei neue Peptide, Teilsequenzen des hEGF, EGF5-21, EGF13-32, EGF32 43, auf ihre Eignung als Basis für potentielle monomere Radiotracer für die PET/CT (Positronen-Emissions-Tomographie/Computer Tomographie) untersucht. Die Peptide wurden mit einem PEG5 Linker und dem Chelator NODA-GA funktionalisiert, um die Vergleichbarkeit der Ergebnisse untereinander zu garantieren, wonach die Substanzen mit [68Ga]Ga3+ markiert und in vitro bezüglich ihrer Serumstabilität, ihres 1 Octanol/WasserpH7.4-Verteilungskoeffizienten (logD(7.4)), Internalisierung in und EGFR-Bindungsaffinität an EGFR-überexprimierenden humanen Epithelkarzinomzellen A431 charakterisiert wurden. Die Peptidsynthese erfolgte an fester Phase nach Fmoc-Strategie, wobei die Vorteile einer ultraschallunterstützten oder mechanischen Kupplung anhand der Synthese des Heterodimers überprüft wurden. Die Markierungen mit [68Ga]Ga3+ wurden mittels analytischer Radio-HPLC verifiziert und ergaben nicht-optimierte molare Aktivitäten von 25 – 111 GBq/mymol mit radiochemische Reinheiten von ≥ 97%. Die radiomarkierten Peptide wiesen für die PET/CT geeignete logD(7.4)-Werte zwischen 2.17 ± 0.21 und 4.01 ± 0.13 und Serumsstabilitäten mit Halbwertszeiten von 62 bis zu 8078 min auf. In den in vitro-Evaluierungen an A431-Zellen konnte für keine der Substanzen eine EGFR-spezifische Internalisierung nachgewiesen werden. Ferner konnte in Konzentrationen bis zu 1 mM keine der Substanzen [125I]I-hEGF in kompetitiven Bindungsstudien von dem EGFR verdrängen. Unter den gleichen Bedingungen demonstrierten [125I]I-hEGF und hEGF eine hohe EGFR-spezifische Aufnahme in und Bindung an die A431-Zellen (33.6 ± 0.9% nach 1 h, reduzierbar auf 1.9 ± 0.2% durch Blockierung) und Affinität an dieselbe Zellinie (IC50-Wert: 15.2 ± 3.3 nM). Die Ergebnisse zeigen, dass 68Ga-markierte GE11-basierte Mono-, Homodi-, Homotetra- und Heterodimere wie auch 68Ga-markierte Monomere basierend auf den aktuell literaturbekannten Liganden zur EGFR-spezifischen Bildgebung nicht aussichtsreich ist und eine Entwicklung oder Identifizierung besser geeigneter Peptide, welche eine ausreichend starke Interaktion mit dem EGFR demonstrieren, erfolgen muss.
Only a few gamma-ray sources have been established as proton accelerators over the last decades, among them two extraordinary binary systems, the massive colliding wind binary Eta Carinae (eta Car) and the recurrent nova RS Ophiuchi (RS Oph). In this thesis, the nature of acceleration processes up to TeV energies are probed in these systems using very-high energy (VHE; ≥ 100 GeV) gamma-ray data from the High Energy Stereoscopic System (H.E.S.S.) in conjunction with data from the Fermi Large Area Telescope (LAT). To obtain reliable results from Imaging Atmospheric Cherenkov Telescopes like H.E.S.S., an accurate match between simulations and actual observations is crucial. Thus, in the first part of this thesis the successful validation of the simulations of the full 5-telescope H.E.S.S. array is presented. Based on this, the scientific verification of the monoscopic analysis was achieved using data from the large 28 m-telescope recently upgraded with a FlashCam prototype. The resulting spectrum of the Crab Nebula, the standard candle in the VHE regime, is found to be in good agreement with previous measurements by H.E.S.S. and other instruments. Using this verified analysis configuration, the nova RS Oph was successfully detected during its 2021 outburst, making it the first nova with confirmed VHE emission. A detailed light curve was derived from the highly statistically significant gamma-ray signal observed with the full H.E.S.S. array. The combined properties of the H.E.S.S. measurements with simultaneous Fermi-LAT data clearly favor a common origin of the whole gamma-ray emission, implying efficient acceleration of hadrons at the external shock caused by the eruption. eta Car has been firmly established as a source of gamma-rays by Fermi-LAT and H.E.S.S. over the last decade. With its highly eccentric orbit lasting 5.5 years, the periastron passage of the two stars is extremely close, making it a particularly interesting phase range. The 2020 periastron passage was the first such event to be extensively monitored by H.E.S.S. In this thesis, the detection of a VHE signal from eta Car during the 2020 periastron is presented, making use of a novel time-based image cleaning technique for the monoscopic analysis. In combination with simultaneous Fermi-LAT data, its spectral properties are characterized and together with previous and follow-up observations, for the first time, a VHE light curve spanning a full orbit is derived. At least some fraction of the accelerated particles traced by the gamma-ray emission likely escape from eta Car, potentially interacting with target material on different spatial scales. With the detection of Fermi-LAT excess emission associated to molecular clouds in the Carina Nebula, this hypothesis is tested. Whereas the cosmic ray density profile is indicative of an origin of the interacting cosmic rays from eta Car, a larger escaping flux than predicted by models or a contribution from other cosmic ray sources is needed to match the measured flux.
Neurodevelopmental disorders (NDDs) represent a huge global health burden. However, the aetiology of most psychiatric disorders remains enigmatic, making it very difficult to find adequate treatment options. While many transcription factors are associated with mental disorders, MYT1L stands out as one of very few life-long-expressed and neuronspecific transcriptional regulators. MYT1L mutations are closely linked to various neurodevelopmental disorders, like autism spectrum disorder and schizophrenia. In this work, I used embryonic stem cell-derived human neurons and mice to study how MYT1L regulates brain development, whether mutations are sufficient to cause mental disease and, if so, whether there is potential for therapeutic intervention. I found that MYT1L deficiency caused upregulation of its target genes, including members of the WNT and NOTCH signalling pathways. This resulted in neurodevelopmental delays that could be partially rescued by chemical pathway inhibition. MYT1L-deficient mice also presented with abnormal brain morphology and behavioural deficits. I found that MYT1L loss caused upregulation of non-neuronal genes, including the main cardiac sodium channel SCN5A, which might explain the unexpected neuronal network hyperactivity observed in mouse and human neurons. Supporting this hypothesis, I was able to normalise electrophysiological hyperactivity by Myt1l overexpression and Scn5a knockdown. Excitingly, the FDA-approved sodium channel blocker lamotrigine rescued electrophysiological abnormalities in vitro and behavioural deficits in vivo. The findings in this study show an important role of MYT1L as a transcriptional repressor not only during development but also after neurogenesis. Failure to silence non-neuronal gene expression in neurons might represent a novel mechanism that, at least in part, can contribute to NDD aetiology. The rescue of MYT1L deficiency-associated phenotypes in post-mitotic cells and adult mice opens up the possibility of therapeutic intervention for patients with MYT1L syndrome, including later in life.
Breast cancer is the most common malignancy in women worldwide and roughly two-thirds of breast tumors are characterized by estrogen receptor (ER) α expression allowing for a targeted treatment approach by suppressing estrogen signaling. Clinically, suppression of estrogen signaling is achieved by Tamoxifen or Fulvestrant which prevent the receptor’s activation or aromatase inhibitors which prevent the formation of (peripheral) estrogen. Unfortunately, relapses are observed in up to 41% of cases and tumor heterogeneity has recently been implicated in therapeutic resistance. To analyze endocrine therapy resistance on a clonal level, I used a previously developed barcoded in vitro model. There, two endocrine therapy sensitive ER+ breast cancer cell lines had been transduced with a barcode library. These cells had then been rendered resistant to Tamoxifen treatment (TAMR) or long-term estrogen deprivation (LTED), the latter to mimic clinically used aromatase inhibitors. Barcode analysis of complex cell pools suggested that endocrine therapy resistance arose either due to the selection of pre-existing clones or the rewiring of initially treatment-persisting cells. In the next step, I isolated and characterized endocrine therapy sensitive and resistant clones from biological replicates using phenotypic assays, RNA-Sequencing and Mass spectrometry-based (phospho-)proteomics. Phenotypically, endocrine therapy resistant clones showed either weak or strong proliferative capacity. On pathway, transcription factor and kinase activity level, I observed heterogeneity between the cell lines utilized resembling inter-tumor heterogeneity and between endocrine therapy resistant clones isolated from each cell line resembling intra-tumor heterogeneity. Activation of the unfolded protein response (UPR) was a private event, which was only observed in a single TAMR population, and correlated with sensitivity to the proteasome inhibitor Bortezomib. Conversely, TAMR and LTED populations shared the activation of multiple protein kinase C (PKC) isoforms, however to different degrees. Treatment with the pan-PKC inhibitor Sotrastaurin preferentially reduced cellular viability of endocrine therapy resistant populations showing stronger PKC activation. Finally, my in vitro findings were supported by clinical findings from the CPTAC-BRCA cohort. Generally, strong heterogeneity between individual patients was evident on pathway, transcription factor and kinase activity levels. After I had deconvoluted the cohort on a per patient basis, I identified patients with estrogen independent tumors showing UPR and PKC activation, closely resembling my in vitro models. Taken together, in the presented PhD thesis I could identify private and shared clonal endocrine therapy resistance drivers with clinical importance.
Individuals of one species share the bulk of their genetic material, yet no two genomes are the same. Aside from displaying classical variation such as deletions, insertions, or substitutions of base pairs, two DNA segments can also differ in their orientation relative to the rest of their chromosomes. Such inversions are known for a range of biological implications and contribute critically to genome evolution and disease. However, inversions are notoriously challenging to detect, a fact which still impedes comprehensive analysis of their specific properties. This thesis describes several highly inter-connected projects aimed at identifying and functionally characterizing inversions present in the human population and related great ape species. First, inversions between human and four great ape species were assessed for their potential to disrupt topologically associating domains (TADs), potentially prompting gene misregulation. TAD boundaries co-located with breakpoints of long inversions, and while disrupted TADs displayed elevated rates of differen- tially expressed genes, this effect could be attributed the vicinity to inversion breakpoints, suggesting overall robustness of gene expression in response to TAD disruption. The second part of this thesis describes contributions to a collaborative project aimed at characterizing the full spectrum of inversions in 43 humans. In this study, I co-developed a novel inversion genotyping algorithm based on Strand- specific DNA sequencing and contributed to the description of 398 inversion polymorphisms. Inversions exhibited various underlying formation mechanisms, promotion of gene dysregulation, widespread recurrence, and association with genomic disease. These results suggest that long inversions are much more prominent in humans than previously thought, with at least 0.6% of the genome subject to inversion recurrence and, sometimes, the associated risk of subsequent deleterious mutation. With a focus on the link between inversions and disease-causing copy num- ber variations, the last project describes a novel algorithm to identify loci hit sequentially by several overlapping mutation events. This algorithm enabled the description of detailed mutation sequences in 20 highly dynamic regions in the human genome, and additional complex variants on chromosome Y. Six complex loci associate directly with a genomic disease, thereby highlighting in detail the intrinsic link between inversions and CNVs. In summary, these projects provide novel insights into the landscape of in- versions in humans and primates, which are much more frequent, and often more complex than previously thought. These findings provide a basis for future inversion studies and highlight the crucial contribution of this class of mutation to genome variation.
Studying the dynamics of sub-cellular structures such as receptors, filaments, and vesicles is a prerequisite for investigating cellular processes at the molecular level. In addition, it is important to characterize the dynamic behavior of virus structures to gain a better understanding of infection mechanisms and to develop novel drugs. To investigate the dynamics of fluorescently labeled sub-cellular and viral structures, time-lapse fluorescence microscopy is the most often used imaging technique. Due to the limited spatial resolution of microscopes caused by diffraction, these very small structures appear as bright, blurred spots, denoted as particles, in microscopy images. To draw statistically meaningful biological conclusions, a large number of such particles need to be analyzed. However, since manual analysis of fluorescent particles is very time consuming, fully automated computer-based methods are indispensable.
We introduce novel deep learning methods for detection and tracking of multiple particles in fluorescence microscopy images. We propose a particle detection method based on a convolutional neural network which performs image-to-image mapping by density map regression and uses the adaptive wing loss. For particle tracking, we present a recurrent neural network that exploits past and future information in both forward and backward direction. Assignment probabilities across multiple detections as well as the probabilities for missing detections are computed jointly. To resolve tracking ambiguities using future information, several track hypotheses are propagated to later time points. In addition, we developed a novel probabilistic deep learning method for particle tracking, which is based on a recurrent neural network mimicking classical Bayesian filtering. The method includes both aleatoric and epistemic uncertainty, and provides valuable information about the reliability of the computed trajectories. Short and long-term temporal dependencies of individual object dynamics are exploited for state prediction, and assigned detections are used to update the predicted states. Moreover, we developed a convolutional Long Short-Term Memory neural network for combined particle tracking and colocalization analysis in two-channel microscopy image sequences. The network determines colocalization probabilities, and colocalization information is exploited to improve tracking. Short and long-term temporal dependencies of object motion as well as image intensities are taken into account to compute assignment probabilities jointly across multiple detections. We also introduce a deep learning method for probabilistic particle detection and tracking. For particle detection, temporal information is integrated to regress a density map and determine sub-pixel particle positions. For tracking, a fully Bayesian neural network is presented that mimics classical Bayesian filtering and takes into account both aleatoric and epistemic uncertainty. Uncertainty information of individual particle detections is considered. Network training for the developed deep learning-based particle tracking methods relies only on synthetic data, avoiding the need of time-consuming manual annotation. We performed an extensive evaluation of our methods based on image data of the Particle Tracking Challenge as well as on fluorescence microscopy images displaying virus proteins of HCV and HIV, chromatin structures, and cell-surface receptors. It turned out that the methods outperform previous methods.
The human body is made of trillions of cells that are the building blocks of all living things. Hematopoietic cells are a set of such building blocks. They are made in the bone marrow and carry out vital tasks, including fighting off infections, facilitating wound healing and carrying oxygen through the body. In health, hematopoietic cells are crucial for the maintenance of normal blood cell production which is necessary for a healthy life. In Acute Myeloid Leukemia (AML), errors in DNA repair result in the acquisition of genetic variations that drive malignant changes and growth of abnormal myeloid cells. These malignant cells fail to perform their vital functions and further hamper the production of normal blood cells.
AML is driven by a group of cells in the bone marrow with self-renewing capacity that give rise to a diverse progeny of abnormal myeloid cells. AML remains lethal due its complex and plastic cellular nature characterized by a high degree of intra-tumor heterogeneity. With the introduction of single-cell technologies, advancements in characterizing the genetic and non-genetic landscape of AML has improved. However, attempts to connect the different levels of heterogeneity, identify and target the disease-driving leukemic stem cells (LSCs) and assess the resulting functional outcomes are largely still lacking. Moreover, identifying the patients who best benefit from novel targeted therapies compared to standard cytotoxic therapies remains a challenge.
In this thesis, I first investigated the intra-patient heterogeneity of complex karyotype AML using an integrated single-cell multi-omics framework that combines structural variant discovery and nucleosome occupancy profiling (scNOVA) with concurrent immunophenotypic and transcriptomic profiling (CITE-seq). Using this framework, I revealed complex structural variant landscapes in single CK-AML cells, marked by ongoing karyotype evolution with frequent involvement of chromothripsis along with linear and circular breakage-fusion-bridge events mediating genomic remodeling. I further unveiled extensive cell-to-cell karyotype instability, exemplified by instable chromosome intermediates, like complex marker and ring chromosomes. Next, I characterized the intra-patient heterogeneity and revealed the existence of genetically distinct subclones with unique nucleosome occupancy, and transcriptomic and immunophenotypic features. By transplanting these cells into immunocompromised mice, I observed predominantly monoclonal expansion of subclones with high genomic complexity that were enriched for stemness-associated phenotypes, including high 17-gene stemness scores and expression of stem cell markers such as CD49F and CD90. Furthermore, I showed that these disease-driving LSCs showed resistance to standard chemotherapy ex vivo but could be targeted by BH3 mimetics. Finally, in an index patient, I showed that the patient-derived xenograft system recapitulated the subclone-specific evolution also during disease progression in the patient, offering a promising model to study relapse. Together, these data provide unique insights into the ongoing genetic and phenotypic complexity of CK-AML, highlight the clinical relevance of intra-patient heterogeneity in tumor evolution, and offer promising avenues to functionally explore and target the disease-driving LSCs.
Next, I explored the clinical relevance of disease-driving LSCs in AML by taking part in investigating how they can be used to predict response to a newly-approved targeted therapy comprising the BCL-2 inhibitor venetoclax in combination with azacytidine. By integrating transcriptomic, functional and clinical data we aimed to identify predictors of clinical response to this combination therapy. We revealed that while more differentiated monocytic cells had high MCL-1 expression and showed resistance to venetoclax and azacytidine, they consistently lacked disease-initiating potential and thus did not fuel leukemogenesis. In contrast, the cells with consistent LSC potential expressed high levels of BCL-2 and could be efficiently targeted ex vivo. We further showed that combining BCL-2, BCL-xL and MCL-1 protein expression ratios in these disease-driving LSCs, could be used to determine the clinical response to venetoclax and azacytidine. This flow cytometry-based “Mediators-of-Apoptosis-Combinatorial-Score” (MAC-Score) predicted initial response with a positive predictive-value of >97% and was associated with increased event-free survival. These data show that the combinatorial levels of BCL-2-family members in the disease-driving LSCs are a key determinate of response to venetoclax and azacytidine and that affordable techniques can be used to reliably predict response to this therapy.
In summary, I investigated different levels of intra-patient heterogeneity in CK-AML patient samples using an integrated single-cell multi-omics framework and explored the resulting functional outcomes. I also took part in predicting clinical response to the newly-approved therapy of venetoclax in combination with azacytidine by establishing a flow cytometry-based response score. Collectively the thesis emphasizes the importance of better identifying and characterizing the disease-driving LSCs to improve our understanding of AML as a dynamic disease entity and to offer effective ways to assess and target the disease-driving LSCs.
Pediatric brain tumors are a leading cause of cancer mortality among children and adolescents (age 0-19) because of the paucity of effective treatment regimens. Especially for ependymoma, surgical intervention combined with focal radiotherapy is the current standard of care in routine clinical practice while this regimen very often induces irreversible damage on the developing brain and patients frequently still suffer from tumor recurrence. Thus, generating de novo representative tumor models to decipher the underlying molecular mechanisms of tumorigenesis is imminent and crucial to provide more precise and mechanism-of-action based treatment plans. In my thesis, I applied various techniques to create in vivo models for several brain tumor types and identified potential therapeutic vulnerabilities. Chapter 2 focuses on dissecting the role of oncogenic fusion genes in C11orf95 fusion- positive supratentorial ependymoma (ST-EPN), a type of pediatric brain tumor with poor prognosis. C11orf95 is a zinc finger protein that binds to DNA but has not yet been well characterized. I performed in-utero electroporation in mouse embryos and found all tested C11orf95 fusion genes were able to drive malignant transformation in the cerebral cortex. The tumors faithfully recapitulated molecular characteristics of their human counterparts. The zinc finger domain and the fusion partners were essential for tumor formation. Cross-species genomic analyses demonstrated that C11orf95-related fusions can increase the expression of a sonic hedgehog mediator gene, GLI2. Targeting GLI2 with arsenic trioxide prolonged survival in mouse models, providing a basis for further preclinical studies for C11orf95 fusion-positive tumors. Based on these findings, C11orf95 is now officially designated as zinc finger translocation associated (ZFTA) by the HUGO Gene Nomenclature Committee. In the latest edition of the WHO classification of central nervous tumors, the group of ST-EPN with ZFTA fusion genes is now named as Supratentorial ependymoma, ZFTA fusion-positive (ST- EPN-ZFTA). In Chapter 3, I investigated on a novel group of neuroepithelial tumors harboring PLAGL1 fusion (NET_PLAGL1) that has been identified in 2021 only. Mouse model generation via in-utero electroporation unfortunately failed. However, after I had performed substantial methodological optimization, overexpression of PLAGL1 fusion gene through a doxycycline-mediated system in human induced pluripotent stem cell-derived neural stem cells, followed by in vivo orthotopic transplantation successfully led to brain tumor formation in mice. This inducible in vivo system offers a reliable model to study NET_PLAGL1 tumors as well as a versatile tool to answer various biological questions behind brain tumorigenesis.Array-based DNA methylation analysis to accurately classify tumors has been developed as a routine diagnostic tool for brain tumors and sarcomas. Since mouse models are the most widely used in vivo systems in pediatric cancer research, it is important to assess the molecular similarity across species based on the methylome. In Chapter 4, I describe the approach of generating a mouse model biobank for pediatric cancers. I collected and profiled 86 murine tumor models and 40 normal tissue controls. DNA methylation-based clustering showed that samples from the same model clustered together and the copy number alteration pattern of ependymoma and glioma (e.g TFG-MET fusion-driven) mouse models recapitulate their human counterparts. This validated biobank will serve as a beneficial resource for future developmental studies such as identifying cellular origin of the tumor and decoding the composition of tumor immune microenvironment.
We give an introduction to the theory of pseudorepresentations of Taylor, Rouquier, Chenevier and Lafforgue. We refer to Taylor’s and Rouquier’s pseudorepresentations as pseudocharacters. They are very closely related, the main difference being that Taylor’s pseudocharacters are defined for a group, where as Rouquier’s pseudocharacters are defined for algebras. Chenevier’s pseudorepresentations are so-called polynomial laws and will be called determinant laws. Lafforgue’s pseudorepresentations are a generalization of Taylor’s pseudocharacters to other reductive groups G, in that the corresponding notion of representation is that of a G-valued representation of a group. We refer to them as G-pseudocharacters.
We survey the known comparison theorems, notably Emerson’s bijection between Chenevier’s determinant laws and Lafforgue’s GL(n)-pseudocharacters and the bijection with Taylor’s pseudocharacters away from small characteristics.
We show, that duals of determinant laws exist and are compatible with duals of representations. Analogously, we obtain that tensor products of determinant laws exist and are compatible with tensor products of representations. Further the tensor product of Lafforgue’s pseudocharacters agrees with the tensor product of Taylor’s pseudocharacters.
We generalize some of the results of [Che14] to general reductive groups, in particular we show that the (pseudo)deformation space of a continuous Lafforgue G-pseudocharacter of a topologically finitely generated profinite group Γ with values in a finite field (of characteristic p) is noetherian. We also show, that for specific groups G it is sufficient, that Γ satisfies Mazur’s condition Φ_p.
One further goal of this thesis was to generalize parts of [BIP21] to other reductive groups. Let F/Qp be a finite extension. In order to carry this out for the symplectic groups Sp2d, we obtain a simple and concrete stratification of the special fiber of the pseudodeformation space of a residual G-pseudocharater of Gal(F) into obstructed subloci Xdec(Θ), Xpair(Θ), Xspcl(Θ) of dimension smaller than the expected dimension n(2n + 1)[F : Qp].
We also prove that Lafforgue’s G-pseudocharacters over algebraically closed fields for possibly nonconnected reductive groups G come from a semisimple representation. We introduce a formal scheme and a rigid analytic space of all G-pseudocharacters by a functorial description and show, building on our results of noetherianity of pseudodeformation spaces, that both are representable and admit a decomposition as a disjoint sum indexed by continuous pseudocharacters with values in a finite field up to conjugacy and Frobenius automorphisms.
At last, in joint work with Mohamed Moakher, we give a new definition of determinant laws for symplectic groups, which is based on adding a ’Pfaffian polynomial law’ to a determinant law which is invariant under an involution. We prove the expected basic properties in that we show that symplectic determinant laws over algebraically closed fields are in bijection with conjugacy classes of semisimple representation and that Cayley-Hamilton lifts of absolutely irreducible symplectic determinant laws to henselian local rings are in bijection with conjugacy classes of representations. We also give a comparison map with Lafforgue’s pseudocharacters and show that it is an isomorphism over reduced rings.
Cavity-enhanced frequency combs are a powerful tool for studying highly nonlinear light-matter interactions, such as multiphoton ionization (MPI) and high-harmonic generation (HHG), with promising prospects for precision spectroscopy beyond the optical spectral range. In this work, a metrology-capable extreme ultraviolet (XUV) frequency comb is produced by transferring a near-infrared comb at 1039 nm to the XUV, using intra-cavity HHG. Intensities of ∼1E14 W/cm² are reached in the cavity focus, producing XUV radiation up to 42 eV (30 nm) and tens of microwatts of outcoupled power. A high-pressure closed-loop noble gas recycling and compression system enables long-term measurements. Additionally, a novel polarization-insensitive cavity with an integrated velocity-map imaging spectrometer was developed. 3D photoelectron angular distributions from xenon MPI are tomographically reconstructed, revealing resonant Rydberg states during ionization. Furthermore, polarization-shaped pulse pairs with a variable time delay are provided for pump-probe experiments. Intense femtosecond standing waves, produced by counter-propagating pulses colliding at the focus, are probed at the nanometer scale using photoemission from a nano-tip. The coherence of the frequency comb is imprinted on the photoelectrons, allowing future precision measurements with coherent matter waves. This work broadens the scope of cavity-enhanced frequency combs and enables strong-field studies at 100 MHz repetition rate.
Psychedelics have the fascinating potential to induce altered states of consciousness. First promising research for their therapeutical application stopped in the early 70’s when psychedelics were criminalized. Since the 90’s psychedelic research is regaining revival. On the one hand new psychopharmacological entities are urgently needed because the number of patients suffering from psychiatric disorders is constantly increasing and novel drug release is declining. Justified one the other hand by the broad therapeutical potential in psychiatric disorders and the rapid and long-lasting effects, often described after just a single administration. Psychedelics might act here as “psychoplastogens”, which means that they have neuroplasticity-promoting effects. They might restore synaptic and neuronal dysfunctions, associated with psychiatric disorders, by generating a plastic cellular state in which remodulations and “brain network resetting” are likely. Support for that theory comes until now from animal research. One key candidate amongst the class of serotonergic psychedelics, named because they target the 5-HT2A serotonin receptor, is psilocybin. Psilocybin, the compound of hallucinogenic fungi, often referred as “magic mushrooms”, is intensively investigated in the treatment of depression, anxiety, obsessive-compulsive disorder and substance abuse disorder. In this doctoral thesis I first employed human induced pluripotent stem cell-derived cortical neurons for deciphering the effects and therapeutic mode of action of psilocybin. I can show that psilocin (the active metabolite of psilocybin) leads to neuroplastic, morphological and functional changes that start shortly after administration and become manifested in time, such as synaptogenesis. As one of many findings, I can first show a subacute downregulation of the 5-HT2A receptor after psilocin administration, a mechanism well-known to prevent an overshooting of the serotonergic signaling. A normalization provides an explanation for the antidepressant mode of action in depression, where an increased 5-HT2A receptor density is described. Remarkably, psilocin further directly targets main components and associates of the plasmin-generating annexin A2 – S100A10 complex which are on their own key candidates in antidepressant drug response. Not only the core proteins of the complex annexin A2 and p11 as docking station for plasminogen, the precursor of plasmin but also plasminogen-cleaving enzymes were upregulated. But moreover, this complex mediates the cleavage of precursor (pro)-BDNF into mature (m)-BDNF in the brain. In line with that I can first replicate that psilocin increases neurotrophic key factor BDNF and its associated downstream pathways which are linked to neuronal survival and plasticity in a human in vitro system. Second, I can first show that psilocin leads to an elevated cleavage of pro-BDNF in m-BDNF. That finding is of paramount importance because recent studies show that actually an imbalance of the pro-BDNF into m-BDNF cleavage, resulting in reduced m-BDNF levels might be more important in depression pathophysiology than total BDNF level reductions. My doctoral thesis suggests that exposure of human neurons to psilocin provokes a dynamic cascade which induces a state of enhanced neuronal plasticity. That neuroplasticity booster alone could explain why psilocin is effective in the treatment of neuropsychiatric disorders. But moreover, I can directly provide a novel explanation for the antidepressant mode of action of psilocybin. I first describe with the annexin A2 – S100A10 complex a new highly potent drug target for the treatment of psychiatric disorders that paves the way for the development of new antidepressant entities.
Cancer is one of the leading causes of disease-related death worldwide. In recent years, large amounts of data on cancer genetics and molecular characteristics have become available and accumulated with increasing speed. However, the current understanding of cancer as a disease is still limited by the lack of suitable models that allow interpreting these data in proper ways. Thus, the highly interdisciplinary research field of mathematical oncology has evolved to use mathematics, modeling, and simulations to study cancer with the overall goal to improve clinical patient care.
This dissertation aims at developing mathematical models and tools for different spatial scales of cancer development at the example of colorectal cancer in Lynch syndrome, the most common inherited colorectal cancer predisposition syndrome. We derive model-driven approaches for carcinogenesis at the DNA, cell, and crypt level, as well as data-driven methods for cancer-immune interactions at the DNA level and for the evaluation of diagnostic procedures at the Lynch syndrome population level. The developed models present an important step toward an improved understanding of hereditary cancer as a disease aiming at rapid implementation into clinical management guidelines and into the development of novel, innovative approaches for prevention and treatment.
SUMMARY Plasmodium falciparum malaria, a mosquito-borne, unicellular parasitic disease, remains a major health concern, particularly in the sub-Saharan Africa. Despite many attempts to develop effective malaria vaccines in recent decades, only one vaccine (RTS,S/AS01) targeting the pre- erythrocytic stage of the Plasmodium falciparum life cycle, has passed the Phase III clinical trial. However, the protective efficacy of this vaccine is low, and the protection is not long-lasting. Thus, a deeper understanding of the immune responses elicited by currently available vaccines and the identification of protective and non-protective B-cell epitopes are essential for the development of next-generation vaccine candidates that will elicit a robust and long-lasting humoral immune response against protective epitopes while avoiding the induction of non-protective antibodies. The vast majority of pre-erythrocytic malaria vaccines elicit antibodies that primarily target the Plasmodium falciparum circumsporozoite protein (PfCSP), a major protein on the surface of sporozoites with a basic structure consisting of the N-terminus (N-CSP), the junction (N-Junc), the central repeat motifs, and the C-terminus (C-CSP), which comprises a linker (C-linker) and a structured α-TSR subdomain that hosts almost all PfCSP T cell epitopes. While antibodies targeting the central repeat with or without cross-reactivity to the N-Junc or C-CSP have attracted much interest due to their promising protective capacity, little is known about the molecular dynamics, fine epitope specificity and the overall protective efficacy of antibodies specific for the N-Junc or the C-CSP domain.
To address these questions, I analyzed a large collection of human monoclonal antibodies (mAbs) against the N-Junc and C-CSP domains using a high-throughput single-cell immunoglobulin (Ig) gene repertoire analysis, following a three-dose immunization with Plasmodium falciparum radiation-attenuated sporozoites (Pf RAS). The naïve human B cell repertoire contains numerous anti-N-Junc and ant-C-CSP antibodies that undergo efficient affinity maturation and IgG class-switching, providing an explanation for the high immunogenicity of the N-Junc and C-CSP domains. Specificities for the N-Junc and C-CSP epitopes were found in both germline and mutated mAbs, and were frequently encoded by IGHV3-23/IGLV1-47 and IGHV3-21/IGLV3-1 or IGLV3-21 gene combinations, respectively. With the exception of one mAb that recognized the C-linker subdomain, all C-CSP specific mAbs targeted the α-TSR subdomain, demonstrating the immunodominance of the α-TSR subdomain. However, while the N-Junc- specific mAbs showed a lower Plasmodium falciparum parasite inhibitory capacity than the cross- reactive mAbs, none of the C-CSP-specific mAbs showed parasite inhibition, regardless of their affinity, gene usage, and epitope specificity. Direct comparison of these antibodies in mice showed that protection from blood stage parasitemia was limited only to C-CSP reactive mAb that cross-reacted with the central repeat domain and N-Junc. Taken together, these findings highlight the molecular features associated with N-Junc and C-CSP specific antibodies at B cell repertoire level and provide supportive evidence that antibodies elicited specifically against the C-CSP domain, whether through natural malaria exposure or vaccine immunization, will not contribute to protection.
Hence, to develop a broadly effective PfCSP-based subunit vaccine against Plasmodium falciparum malaria, careful consideration must be given to whether integrating the C-CSP domain into PfCSP-based immunogens is the optimal means of delivering T-cell epitopes. Instead, vaccine design should focus on the N-Junc and the conserved central repeat domains to elicit robust and cross-neutralizing antibodies.
The Epstein-Barr Virus (EBV) is a γ-herpesvirus that establishes a lifelong infection in human hosts. This infection can manifest into cancer. This affliction is attributed to the latent encoded EBV proteins. However, lytic proteins have recently been demonstrated to contribute to tumour development, with notable examples being tegument proteins BNRF1 and BPLF1. In my thesis, I studied BPLF1 in its full form to identify novel regions involved in the EBV life cycle and in carcinogenesis. These goals are achieved through expression in vitro expression studies and in the context of EBV virions that infect primary B cells ex vivo. I used co-immunoprecipitation in tandem with mass spectrometric analysis to identify novel host BPLF1 binding partner SENP6, a deSUMOylase responsible for maintaining genomic integrity. I proceeded to study the effects that BPLF1 has on SENP6 activity and the physiological consequences. I produced domain knockouts of the BPLF1 protein to map the region responsible for interaction and activity of BPLF1 on SENP6. I found that not only does BPLF1 bind to SENP6; it also effectively suppresses SENP6 activity. Downstream effects on SENP6 inhibition are the reduction of Centromeric Protein A (CENP-A) constituency at the centromeres, leading to improper chromosomal segregation during anaphase. This leads to the accumulation of genomic abnormalities such as increased rates of aneuploidy and polyploidy. I found this phenotype occurs independently from the catalytic region of BPLF1 and is mapped to the BPLF1765-1327 stretch of amino acids. B cells exposed to virus particles devoid of BPLF1, showed reduced nuclear abnormalities when compared to virus particles containing BPLF1. I observed increased SUMO2/3 conjugation and loss of CENP-A at the centromeric regions in B cells exposed to virus particles possessing BPLF1 in contrast to virus devoid of BPLF1, showing BPLF1’s interference in chromosomal stability.
Cells have evolved elaborate protein quality control systems (PQS), which include molecular chaperones and proteolytic machineries. However, when the occurrence of misfolded proteins exceeds the PQS’s capacity, they accumulate and can form aggregates. More and more evidence suggests that the accumulation of misfolded protein species into specific spatially separated deposition sites is a cytoprotective response of the cell. The yeast S. cerevisiae has at least three different such protein quality control sites: the JUxtaNuclear Quality control (JUNQ)/IntraNuclear Quality control site (INQ) and the Cyto-Q harbours unstructured, amorphously misfolded proteins, while the perivacuolar Insoluble PrOtein Deposit (IPOD) has been initially described as a deposition site for amyloid aggregates. However, more recently it has been suggested that the IPOD may also harbour other types of substrates, such as oxidatively damaged proteins and inactive/damaged proteasomes or subunits thereof. Interestingly, many of these potential substrate classes can form high molecular weight aggregates or represent large protein complexes, respectively. Because the IPOD lies directly adjacent to the phagophore assembly site at the vacuole, it was hypothesized that the perivacuolar IPOD may represent a sorting center for aggregates and larger protein complexes destined for autophagic turnover.
This study focuses on the enrichment of IPODs visualized with the model substrate PrD-GFP under different conditions, including oxidative stress, to characterize other IPOD substrates through an unbiased mass spectrometry approach. This strategy identified several proteins that co-enriched with the IPOD, mainly after oxidative stress. Among these was Pyruvate decarboxylase 1 (Pdc1), a protein susceptible to carbonylation which has been previously hypothesized to be present at the IPOD after oxidative insult.
For a Pdc1-mCh fusion protein, it was observed that the number of cells which formed Pdc1-mCh foci was increased after different forms of oxidative stress such as H2O2 or menadione treatment. The majority of these stress-induced foci colocalized with PrD-GFP marked IPODs. Other proteins found enriched at the IPOD after oxidative stress include Enolase 2 (Eno2) and Glyceraldehyde-3-phosphate dehydrogenase isozyme 3 (Tdh3). Along these lines, by staining for carbonylated proteins it was found that the overall levels of carbonylated proteins co-enriching with IPODs were much higher after application of oxidative stress. This supports the hypothesis that aggregates of oxidatively damaged proteins are another substrate group for the IPOD.
Furthermore, it has been shown that aberrant stress granules transiently associate with the aggresome on their way to autophagic degradation in mammalian cells. I hypothesized that the IPOD may play a similar role to the aggresome in yeast in this regard and indeed, in a dCuz1 background that hinders proteasomal degradation of stress granules and makes them more persistent, a proportion of aberrant arsenite-induced stress granules marked by Pab1mCh colocalized with the IPOD after arsenite stress.
In a world of increasing energy consumption and man-made global warming, the knowledge of electronic and structural properties of organic materials is crucial for their implementation and use in innovative (smart) devices since those determine device performance. Herein, the electronic and structural properties of new promising materials are studied in detail utilising advanced laser spectroscopic techniques. Considering optoelectronic devices such as organic field effect transistors and solar cells, the metal/organic interface plays an important role for device performance. Therefore, the electronic properties of an n-type semiconducting N-substituted pentacene derivative and its change at higher layer thicknesses in contact with a metal electrode is studied via two-photon photoemission spectroscopy as well as the adsorption and desorption properties via temperature-programmed desorption. Several molecular electronic states such as the highest occupied and the lowest unoccupied molecular orbital as well as the optical gap are thereby determined quantitatively. Developing optoelectronic devices further, smart devices covering more complex needs can be achieved by implementing multiresponsive mixtures reacting in differentiated ways to light, which acts as sustainable external stimulus with high spatio-temporal resolution. Hence, the switching behaviour of a mixture of two individually as well as simultaneously addressable photoswitches is researched in detail. Furthermore, the investigated mixture is 3D printable allowing fast and easy implementation in 3D structures. The photoswitches are a push-pull azobenzene derivative and a first generation donor-acceptor Stenhouse adduct absorbing longer wavelengths in the visible region of the spectrum compared to the azobenzene derivative. All steps of the trans-to-cis isomerisation of the former and the linear-to-closed isomerisation of the latter photoresponsive molecule are studied as well as the respective back reactions with special emphasis on the environmental influence and the impact of the photoswitches on each other. Therefore, several techniques, such as femtosecond transient absorption, temperature dependent kinetic visible absorption and kinetic infrared absorption spectroscopy, are used to gain a full picture. Dynamic effects of the environment and the photoswitches on each other are thereby found such that individual switching of both molecules takes place with minor impacts on each other in the mixture. Other 3D printable materials are diblock copolymers exhibiting microphase separation such as a derivative of a standard diblock copolymer for high-resolution nanolithography with additional functional groups allowing 3D printing. The lamellar structure and its regularity is studied by means of a scattering-scanning near-field optical microscope before and after 3D printing upon which a polymeric network is formed. Before printing, lamellae are thereby found whereas the printing process needs to be optimised to maintain this structure after 3D printing.
Despite the global efforts, malaria is still a serious public health concern with approximately 241 million cases and 627000 deaths globally in 2020 (WHO report 2021) with the majority of deaths occurring in young children living in Sub-Saharan Africa. This underscores the urgent need of highly effective and durable malaria vaccines which are not yet available. The Plasmodium falciparum merozoite surface protein 1 (MSP1) is the most abundant surface antigen of merozoites and has long been considered as a key antigen for naturally acquired immunity (NAI) and a promising blood stage vaccine candidate for malaria; however, sero-epidemiological studies and clinical trials in humans could not confirm protective effects of anti-MSP1 immune responses. Notably, previous studies focussed only on small fragments of the whole protein, particularly the conserved C-terminal subunit and might therefore have missed important B and T cell epitopes that are relevant for a protective immune response. Using samples from a controlled human malaria infection study in semi-immune Kenyan adults (CHMI-SIKA), I showed that pre-challenge antibodies target conserved epitopes distributed across the full-length MSP1 (MSP1FL) protein and induced a range of distinct Fc-mediated effector functions: complement fixation (AbC’), opsonic phagocytosis (OPA), respiratory burst of neutrophils (ADRB), degranulation and IFN expressionof natural killer cells (Ab-NK) that were significantly associated with protection from malaria. Notably, the breadth of effector functions was the strongest correlate of protection. The magnitude of effector functions of antibodies directed against MSP1FL was the strongest compared to other merozoite antigens highlighting MSP1FL as a major driver of anti-merozoite immune responses. Furthermore, I showed that vaccination of malaria-naïve adults from Germany with MSP1FL formulated with the GLA-SE adjuvant (SumayaVac1) elicited highly functional IgG and IgM that exert the same range of Fc-mediated effector functions that were observed for CHMI volunteers. Notably, functions reached similar or even higher levels to that of semi-immune Kenyan adults and remained over baseline levels even 6 months after immunization. Functional antibodies from protected CHMI volunteers as well as vaccinees preferably targeted the C-terminal p42 as well as the N-terminal p83 subunit which has never been included in previous MSP1-based vaccines. My study suggests that full-length MSP1 is an important target of naturally acquired and vaccine-induced functional antibodies which might be strong contributors to protection from malaria. An upcoming phase Ib study with SumayaVac1 is currently under development and expected to be tested in malaria-exposed adults in Tanzania followed by CHMI.
The binding and elution behavior of two therapeutic bispecific monoclonal antibodies (bsAbs) on the strong cation exchange resin POROS™ XS is investigated and modeled over broad ranges of pH, salt concentrations, and column loadings. One of the two bsAbs exhibits common Langmuir elution behavior under high loading and column overloading conditions, whilst the other bsAb exhibits uncommon anti-Langmuir elution behavior as a consequence of multi-layer binding on the stationary phase surface. The frequently used Steric Mass Action (SMA) model modified with an activity coefficient for the salt in solution is used to simulate the Langmuirian elution behavior. A Self-Association Steric Mass Action (SAS-SMA) model extended with two activity coefficients for the protein and salt in solution is applied to describe the anti-Langmuir elution behavior. The SAS-SMA model is able to describe self-dimerization on the resin surface and thus can predict anti-Langmuir elution behavior. The binding models are each combined with a lumped rate model to describe mass transfer inside the chromatography column. To apply these models for describing protein elution over wide ranges of pH, the pH-dependences of all model parameters, including the linear and especially the non-linear model parameters, are investigated, described, and implemented into the binding models. Therefore, extensive data sets were generated that consist of linear gradient elution experiments comprising a pH range from pH 4.5 to 8.9 and column loadings from 0.5 to 90.0 mgbsAb/mLresin. The modeling results of both antibodies show that the pH of the mobile phase has a strong influence on the non-linear model parameters, thus valuable process insights can be gained by interpretation of these results. An increasing buffer pH leads to an increase in binding sites shielded by the antibodies, whilst self-dimerization on the resin surface becomes less with increasing pH. Empirical correlations describing the non-linear model parameters as functions of pH are established and implemented into the SMA and SAS-SMA formalisms. The functionality of these modified pH-dependent binding models is verified with linear salt, pH and dual gradient elution experiments using single-component simulations. Most of these experiments can be accurately predicted under high loading and overloading conditions, whereby especially the peak shapes are well-described. Slight discrepancies between the simulated and experimental data can be observed for some of these experiments, especially when they were performed under overloading conditions. In this dissertation, it is clearly shown that these discrepancies are not primarily a consequence of limitations of the SMA and SAS-SMA models. At lower pH values (pH ≤ 5.3), overloading phenomena such as protein breakthrough during the loading phase, additional peaks, and peak-shoulders occur. The outcomes of additional experiments in which the antibodies were loaded onto the column with different counterion concentrations and loading times show that intraparticle diffusion effects and conformational changes of the bsAbs are responsible for these overloading phenomena at low pH. The applied lumped rate mass transfer model is not adequate here since it cannot describe hindered intraparticle transport and should be extended to consider these effects. Additional peaks and peak shoulders due to bsAb conformations can only be predicted by describing multi-state binding, which is shown in this dissertation for one case by a simple extension to a multi-component simulation. Furthermore, it is shown that the description of complex peak shapes arising due to competitive binding and multi-component elution of the antibodies' charge variants cannot be adequately predicted using single-component simulations. However, an extension of the model to a simple multi-component system consisting of two charge variants enables accurate prediction of some of these complex elution profiles.
Most resources of modern computer clusters are locked behind the need for a high degree of parallel efficiency, having millions of processing units on heterogeneous hardware, e.g. having both CPUs and GPUs. Post-HF wave function-based methods offer a high accuracy while having high computational scaling and memory requirements. Their need for storing, transforming, manipulating, and communicating their underlying high-dimensional quantities makes them a bad fit for modern computer architecture.
The second-order Møller-Plesset perturbation theory (MP2) is a commonly used method to recover the electron correlation energy that the Hartree-Fock method is missing. Its computational scaling is O(N^5) (N representing the number of atomic orbitals), as its two-electron integrals have to be transformed from their atomic orbital representation to their molecular orbital representation, and its memory requirements scale with O(N^4).
This thesis applies the ideas of a quadrature scheme to the MP2 method to arrive at a lower scaling form that is embarrassingly parallel. The full Q-MP2 energy integral scales with O(P^2OV) (spatial grid points P, occupied molecular orbitals O, virtual molecular orbitals V) with its largest entity needing O(PN^2) memory. In this thesis, an efficient implementation in the form of the open-source libqqc library is presented. The setup of the integration grids is the deciding factor of the accuracy of the Q-MP2 method. To investigate this a benchmark of small molecules is shown, consisting of seven molecules, three basis sets, and 28 different grid combinations. All but the smallest grid combinations are shown to be within the magnitude of the target chemical accuracy, with only four points on the one-dimensional integration grid being necessary. The error of the spatial grid falls asymptotically with the number of grid points, with the third smallest grid of 20 radial and 38 angular points being chosen for further tests as it is the smallest well-behaved one. The total single-node performance, measured as the number of floating point operations performed per second compared to the theoretical maximum, of the different variants of the algorithm is found to be below 15%. The algorithm is memory bound. The parallelisation strategy shows near-perfect load balancing over the computational nodes. The single-node parallel efficiency is shown to be superlinear for large systems, as a higher percentage of memory can be stored in low-level and fast memory caches with an increasing number of cores. This trend is followed at the multi-node level, which was investigated for up to 960 cores/20 nodes on the JUSTUS 2 computer cluster.
Future optimisation strategies will be focused on optimising the integration grids to lower the number of necessary integration grid points, integral screening, better utilisation of temporal locality, and exploitation of matrix sparsity. Finally, the quadrature scheme was extended to coupled-cluster theory (Q-CC2) and the algebraic-diagrammatic construction scheme for the polarisation propagator (Q-ADC(2)). For the latter method, the computational scaling associated with the solution of the particle-hole state was lowered from originally O(N^5) to O(P^2OV^2 ) and the memory requirement can be additionally lowered from O(N^4) to O(PN^2) by folding of the doubles space into the singles space. Compared to the performance of Q-MP2, a future implementation is expected to have a better single-node performance as more computational work needs to be done per memory transaction, and to have similar parallel efficiency as little additional node-to-node communication is necessary.
All chemical elements in the Universe, except the very few lightest species, are produced in a nuclear fusion inside the stars. Following the stellar life cycle, these chemical elements are expelled into the interstellar medium where they proceed to contribute to the chemical enrich- ment of their surroundings. Spectroscopic observations are currently the only way to infer the chemical make-up of the stars. Combining those with the physical modelling of the radiation in the stellar plasma allows us to detect and measure the number of chemical elements we all are made of. The approach is generally applied to individual stars constituting the larger scale populations, from clusters to galaxies including our Milky Way and beyond. In this thesis I focus on the non-equilibrium modelling of stellar radiation and its influence on the measured chemical abundances of various elements. I provide a general overview of the methods and necessary information used to infer the stellar chemical composition. I then present developments in the non-equilibrium modelling and apply it to the analysis of a star cluster, our host star – the Sun, and eventually a broader Galactic population. I focus on the opportunities our modelling approach presents to observationally constrain the stellar evolution and consequential enrichment of the Galactic populations.
Treatment-refractory tumors such as pancreatic ductal adenocarcinoma (PDAC) or head and neck squamous cell carcinoma (HNSCC) remain a major challenge in medical oncology and translational cancer research. Modern radiotherapy techniques are able to achieve maximal tumor damage while trying to limit off-target toxicity. Nevertheless, refractory tumors often show radioresistance. To achieve superior anti-tumor efficacy, this study investigates radiotherapy combined with virotherapy using oncolytic vaccine strain measles virus (MeV). Oncolytic MeVs feature an excellent safety profile and have shown first signs of anti-tumor efficacy in clinical trials. No cross-resistances of MeV with current therapeutic agents have been detected. They infect malignant cells as well as spread within tumors, leading to oncolysis of cancerous cells and induction of an immune response. Thus, MeV represents a promising modality for combination treatments. I hypothesize that the combination of radiotherapy and oncolytic MeV in a radiovirotherapy (RVTx) synergizes in terms of tumoricidal effects and induces a sustained anti-tumor immune response. I tested different dose regimens of radiation as well as oncolytic MeV in a panel of human HNSCC and PDAC cell lines in vitro. I assessed cell viability after RVTx treatment and could confirm synergism of RVTx in both tumor entities for specific treatment conditions. Further, I investigated possible mechanisms of action, focusing on the induction of immunogenic cell death (ICD), markers known for DNA damage and viral RNA sensing as well as downstream effects of innate immune activation. The combination treatment induced higher levels of the ICD markers high mobility group box 1 (HMGB1) and calreticulin (CALR) compared to each monotherapy. Further, interferon signaling was induced in RVTx, driven by virotherapy. I established an immunocompetent murine HNSCC model that is susceptible to MeV infection. In an in vivo pilot study using this model, I detected increased tumor infiltration of CD8+ T cells as well as virus-driven immunoreactivity in co-cultures of splenocytes and tumor cells after RVTx treatment. In two human ex vivo PDAC tumor models, I analyzed the therapeutic efficacy of RVTx. I could show viral spread in 3D spheroids. Further, RVTx induced virus-driven ICD and interferon-β release in patient-derived PDAC cultures. This study identified synergistic therapeutic regimens and provides the proof-of-concept for the induction of an anti-tumor immune response by RVTx. Insights into mechanisms of anti-tumor efficacy were gained in relevant preclinical models. Therefore, this project constitutes important steps towards the development and clinical translation of a state-of-the-art combination treatment modality to achieve sustained improvements for the therapy of refractory tumors.
Recently, a unicentric model of the observable universe was proposed. Accordingly, big bangs are frequent events in our infinitely large, flat, homogeneous and isotropic parent universe. Their progenitors are clusters of cosmically dead and massive neutron stars that merged after reaching the ultimate lowest quantum energy state, where the matter is in an incompressible superconducting gluon-quark superfluid state and zero-entropy. The resulting progenitors are of measurable sizes and immune to collapsing into black holes. Our big bang's progenitor accidentally happened to take off in our neighbourhood. As the enclosed mass of the progenitor was finite, the dynamically expanding curved spacetimes embedded the fireball started flattening to finally diffuse into the flat spacetime of the parent universe.
By means of GR-numerical hydrodynamical calculations, we use the H−metric to follow the time-evolution of the spacetime embedding the progenitor during the hadronization phase and thereafter. Based thereon, we find that the kinetic energy of newly created normal matter increases with distance in a self-similar manner, imitating thereby outflows of nearly non-interacting particles.
On cosmic time scales, this behaviour yields a Hubble parameter, H(t), which decreases slowly with the distance from the big bang event. Given the sensitivity of the CMB-Planck data to the underlying cosmological model, we conclude that our unicentric model of the universe is a viable alternative to ΛCMD-cosmologies.
Tropospheric glyoxal mixing ratios and vertical column densities were measured during 72 research flights with the German research aircraft DLR HALO over different regions etween 2014 and 2019. Over the Amazon rainforest, the bservations are complemented by simultaneous formaldehyde and methylglyoxal measurements. The glyoxal measurements are confirmed by same-day observations of the TROPOMI satellite instrument and compared to simulations of the photochemical transport model EMAC. Deviations of air- and spaceborne glyoxal are found for spatially small pollution plumes and those located near the surface. This causes smaller satellite glyoxal measurements around urban centres. Unexplained glyoxal enhancements are detected repeatedly in aged biomass burning plumes and the tropical marine boundary layer. Over the rainforest, emission factors are estimated for tropical forest fires (0.11–0.52 gglyoxal kg−1 fuel, 0.50–8.64 gmethylglyoxal kg−1 fuel) and isoprene is identified as a potential glyoxal and methylglyoxal precursor above the boundary layer. The comparison to EMAC shows an underestimation of modelled glyoxal in most regions, especially in the boundary layer and pollution plumes. This is indicative of an underestimation of glyoxal and its precursors by EMAC, with consequences for the tropospheric oxidative capacity as well as the formation of ozone and secondary organic aerosols and hence for the radiative forcing.
Immune checkpoint blockade has revolutionized immunotherapy against cancer with tremendous clinical benefits for patients. Despite these achievements, tumors utilize a plethora of suppressive mechanisms to evade immune destruction which are yet to be understood and matched by today’s immunotherapy. Our group developed a high-throughput RNAi screening to unravel the arsenal of immune checkpoints of cancer. We screened a siRNA library (around 2880 kinases and surface-associated genes) with patient-derived tumor cells and HLA-matched tumor-infiltrating lymphocytes (TILs). The library was reverse-transfected into M579-A2 melanoma cells and these were co-cultured with MART1- and gp100-specific TILs to determine TIL-mediated lysis. We identified 75 genes in tumor cells that impaired TIL-mediated cytotoxicity. Interestingly, we found that several genes and their associated pathways were found in pancreatic adenocarcinoma and multiple myeloma as well. This suggests that different cancer entities might share inhibitory modes of action. In order to distinguish between genes altering tumor susceptibility towards TIL-mediated killing and those impairing TIL activity, we established a secondary screening assaying multiple T cell activation marker, including effector cytokines. The olfactory receptor OR10H1 was one of the strongest candidates from our primary screening as its knockdown increased TIL-mediated killing in melanoma, PDAC and colorectal carcinoma. Furthermore, TILs were activated stronger after interaction with OR10H1-deficient cells as sensed by the increased secretion of type 1-associated cytokines and a reduced T cell apoptosis. We confirmed the role of OR10H1 as an immune checkpoint in vivo using a xenograft mouse model in combination with adoptive T cell transfer. We performed mode of action analyses in order to understand how OR10H1 affects T cell activity. These analyses revealed that tumor-associated OR10H1 controls cAMP-dependent signaling inside T cells. Inside TILs, cAMP activates protein kinase alpha (PKA) and PKA in turn activates C-terminal Src kinase (Csk. Csk phosphorylates an inhibitory tyrosine residue of Lck impairing its activity and shutting down TCR-associated signaling. Furthermore, PKA activates CREB and thus induces an anergy-associated gene expression profile in TILs. Our data suggest that OR10H1 alters the balance between the inhibitory (GαI) and the stimulatory/olfactory G-Protein alpha (GαS/Olf) inside tumor cells depending on the encounter of TILs. This results into increased production of cAMP in tumor cells and its subsequent transport into T cells. In summary, we established a discovery platform aiding the search for immune checkpoints in cancer. We identified OR10H1 and its associated olfactory receptor signaling as a novel pathway inhibiting TIL responses by inducing cAMP-dependent Lck inhibition.
The dynamics of adult neurogenesis in the ventricular-subventricular zone are yet to be fully understood. In particular, how the pool of neural stem cells (NSCs), in active and quiescent states, is maintained while undergoing ageing effects. Mathematical modelling of biological systems can help to gain insight into such dynamics, to discover dependencies that may not be obvious purely from the data. This thesis evaluates different models of ordinary differential equations to describe those dynamics. First, a simplified, macroscopic model of neurogenesis based on an existing model of active and quiescent NSCs has been developed via a steady-state for the fraction of active NSCs among all NSCs. It was used to relate the parameters of the two former models via the macroscopic model, that does not inherently distinguish between active and quiescent cell states. Secondly, two neurogenesis model versions with constant and time-dependent self-renewal are compared and parameter estimation is applied to the macroscopic model. Although the NSC dynamics can be observed with all models, the macroscopic lense results in non-identifiable microscopic parameters. Furthermore, the model cannot capture the fraction of active NSCs among NSCs, as expected, due to the single equation it is defined by.
More deaths can be attributed to lung cancer, than to any other cancer type. Evidence collected over the last 10 years, from randomized trials in the USA and Europe, indicates that screening by means of low-dose computed tomography (LDCT) could reduce the number of lung cancer (LC) deaths by about 20%-24%. While these findings have led to the implementation of screening programs in the USA, South Korea and Poland, discussions on their optimal design and execution are still ongoing in various countries, including Germany. Optimizing screening means finding the right balance between mortality reduction and risks, harms, and monetary costs. LDCT-scans are expensive, expose participants to radiation and put them at risk for overdiagnosis, as well as at risk for unnecessary invasive and expensive confirmatory procedures triggered by false positive (FP) results. Minimizing the number of unnecessary screening and confirmatory examinations should be prioritized. While risk-based eligibility has been shown to best target candidates, questions regarding optimal screening frequency, accurate nodule evaluation, stop-screening criteria to reduce overdiagnosis, and the use of complementary non-invasive diagnostic methods, remain open. Statistical models and biomarkers have been developed to help answer these questions. However, there is limited evidence of their validity in data from screening contexts and populations other than those in which they were developed. The analyses presented in this thesis are based on data collected as part of the German Lung Cancer Screening Intervention (LUSI) trial in order to validate models that address the questions: 1) can candidates for biennial vs annual screening be identified on the basis of their LC risk? 2) can the number of FP test results be reduced by accurately estimating the malignancy of LDCT-detected nodules? 3) What was the extent of overdiagnosis in the LUSI trial and how does overdiagnosis risk relate to the age and remaining lifetime of participants? Additionally, blood samples from participants of the LUSI were measured to evaluate: 4) whether the well-validated diagnostic biomarker test EarlyCDT®-Lung is sensitive enough to detect tumors seen in LDCT images. The LCRAT+CT and Polynomial models predict LC risk based on subject characteristics and LDCT imaging findings. Results of this first external validation confirmed their ability to identify participants with LC detected within 1-2 years after first screening. Discrimination was higher compared to a criterion based on nodule size and, to a lesser degree, compared to a model based on smoking and subject characteristics (LCRAT). This suggested that while LDCT findings can enhance models, most of their performance can could be attributed to information on smoking. Skipping 50% of annual LDCT examinations (i.e., for participants with estimated risks <5th decile) would have caused <10% delayed diagnoses, indicating that candidates for biennial screening could be identified based on their predicted LC risks without compromising on early detection. Absolute risk estimates were, on average, below the observed LC rates, indicating poor calibration. Models developed using data from the Canadian screening study PanCan showed excellent ability to differentiate between tumors and non-malignant nodules seen on LDCT scans taken at first screening participation and to accurately predict absolute malignancy risk. However, they showed lower performance when applied on data of nodules detected in later rounds. In contrast, a model developed on data from the UKLS trial and models developed on data from clinical settings did not perform as well in any screening round. Excess incidence of screen-detected lung tumors, an estimator of overdiagnosis, was within the range of values reported by other trials after similar post-screening follow-up (ca. 5-6 years). Estimates of mean pre-clinical sojourn time (MPST) and LDCT detection sensitivity were obtained via mathematical modeling. The highest excess incidence and longest MPST estimates were found among adenocarcinomas. The proportion of tumors with long lead times predicted based on MPST estimates (e.g., 23% with lead times ≥8 years) suggested a substantial overdiagnosis risk for individuals with residual life expectancies shorter than these hypothetical lead times, for example for heavy smokers over the age of 75. The tumor autoantibody panel measured by EarlyCDT®-Lung, a test widely validated as a diagnostic tool in clinical settings and recently tested as a pre-screening tool in a large randomized Scottish trial (ECLS), was found to have insufficient sensitivity for the identification of lung tumors detected via LDCT and of participants with screen-detected pulmonary nodules for whom more invasive diagnostic procedures should be recommended. Overall, the findings presented in this thesis indicate that risk prediction models can help optimize LC screening by assigning participants to appropriate screening intervals, and by increasing the accuracy of nodule evaluation. However, there is a need for further external model validation and re-calibration. Additionally, while excess incidence can provide estimates of overdiagnosis risk at a population-level, a better approach would be to obtain model-based personalized estimates of tumor lead and residual lifetime. Better individualized decisions about whether to start or stop screening could be taken on the basis of the relationship between these estimates and the risk of overdiagnosis. Finally, although there is evidence for the potential of biomarkers to complement LC screening, the so far most promising candidate (EarlyCDT®-Lung) cannot be recommended as a pre-screening tool given its poor sensitivity for the identification of lung tumors detected via LDCT. In conclusion, while steps have been taken in the right direction, more research is required in order to answer all open questions regarding the optimal design of lung cancer screening programs.
The performances of sprinters and long jumpers with below the knee amputation (BKA) have improved continuously since the development of prostheses specifically for athletic movements. In the last years, a number of athletes with BKA have attempted to compete in non-amputee competitions. Due to the specific shape and material properties of the running-specific prosthesis (RSP), concerns exist that it may give athletes an advantage over non-amputee athletes. In this work, we investigate and compare sprinting and long jump movements of athletes with and without unilateral BKA using accurate computer models. In this context, the aim of the work is to describe similarities and differences between the athletes’ movements and to show that the employed model- and optimization-based computations are useful for this purpose. We created subject-specific multi-body models for five different athletes (four non-amputee athletes, one athlete with unilateral BKA) in order to be able to investigate the different movements. Depending on the research question, the models vary in the number of degrees of freedom (DOFs), from 16 DOFs for a two-dimensional model in the sagittal plane to 31 DOFs for a three-dimensional model. For the athlete with BKA, we created a three-segment model of the RSP with one rotational DOF in the sagittal plane. The respective motion is described by a sequence of several phases, which differ by the type of ground contact. Each of these phases is described by its own set of ordinary differential equations (ODEs) or differential algebraic equations (DAEs). We use multi-phase optimal control problems (OCPs) with discontinuities to generate sprint and long jump motions. Three different formulations of OCPs are adopted in this work. (1) We formulate a least squares OCP to reconstruct the dynamics of sprint and long jump motion capture recordings of the individual athletes. (2) For the generation of realistic motions, which can be used for prediction, we formulate a synthesis OCP; this optimizes an objective function consisting of a weighted combination of chosen optimization criteria. (3) Last, in the study of sprint movements, we use an inverse optimal control problem (IOCP): this consists of an inner loop, in which a synthesis OCP is solved, and an outer loop, which adjusts the weights of the individual optimization criteria such that the distance between the inner loop solution and a reference movement becomes minimal. We have successfully applied these three optimization problem formulations to the computation of two sprint steps of three athletes without and one athlete with unilateral transtibial amputation. Here, the movements of the non-amputee athletes differ from that of the amputee athlete in a large number of variables. In particular, the athletes use different actuation strategies for running with and without a RSP. We have observed lower torques in the amputee athlete in the leg affected by the amputation than in the non-amputee control group. In contrast, significantly larger torques occurred in the joints of the upper extremity in the amputee athlete. Furthermore, the comparison has shown that the asymmetry created by the RSP is reflected throughout the body and affects the entire movement. Using the OCPs for motion reconstruction (1) and synthesis (2), we have successfully computed the last three steps of the approach and the jump of a long jump for an athlete without and an athlete with unilateral amputation. In the reconstructed solutions, the amputee athlete achieves a greater jump distance compared to the non-amputee athlete, despite a slower approach velocity, because his take-off is more efficient. In the synthesis solutions, on the other hand, the non-amputee athlete achieves the greater jump distance because he generates a greater vertical force during the take-off and achieves a better ratio of gain of vertical to loss of horizontal velocity. Finally, we have presented our idea of a simulator tool to compare the amputee athlete with himself without amputation and have demonstrated it using the sprint and long jump movements. For this purpose, we have kept the model of the athlete with unilateral transtibial amputation from the previous studies and have created a non-amputee version of the same model by mirroring the biological leg. We have selected one objective function each for sprinting and for long jump and have solved the OCP for motion synthesis (2) for both model versions. Using the differences to the solutions based on the models of two real athletes, we have highlighted the importance of the simulator tool in the evaluation of advantages and disadvantages due to the use of the RSP.
Building precise, robust patterns and structures from an initially homogeneous state is fundamental to developmental biology. Digit patterning is a representative example of a periodic pattern in development. Previous studies have shown that a reaction–diffusion (Turing) system, in which diffusible activators and inhibitors interact, is the most likely explanation of how the spatial pattern of the digits is formed. Although self-organisation mechanisms such as the Turing system successfully recapitulate many aspects of digit patterning, critical questions remain regarding its timing and behaviour. First I addressed the question of timing, or how long reaction-diffusion plays a role in the developing digits. I perturbed the digit patterning process of embryonic limbs by inserting beads that contain morphogens involved in the reaction-diffusion mechanism. Then I quantified the degree of pattern change, or plasticity of the patterning, from limbs harvested at different developmental timing throughout the digit patterning stage. For quantification, I developed a custom image analytic pipeline that extracts relevant topology and represents the difference between perturbed and unperturbed patterns. Modelling the plasticity profile over the digit patterning process, through extensive interplay of experiments and modelling, revealed that plasticity during digit patterning decreases in a sigmoidal manner. Transcriptomics analysis that matches with the sigmoidal decrease observed in expression patterns further identified gene candidates that could be critical to the digit patterning. Further, the timing of reaction-diffusion is discussed in the context of the tissue movements, revealing that Sox9 digit patterning happens significantly earlier than cell density changes. The second part aims at improving our understanding about which pathways and components of the pathways are involved in the digit forming Turing network. Previously identified digit patterning Turing network, such as BSW model, abstracts the entire Wnt and Bmp signalling pathways’ activities into each node. Thus there is insufficient knowledge on the mechanistic role of Wnt signalling mediated Sox9 repression. To further clarify detailed mechanisms of the Turing network, I used an unbiased screening approach to systematically perturb digit patterning using small molecule inhibitors, ligands, and peptides at different doses in systems such as limb culture and micromass. Out of multiple steps critical to Wnt signalling, including Wnt production, Wnt receptor interaction, Wnt canonical pathway cytosolic interactions, and Wnt canonical pathway transcriptional interactions, I identified that inhibition of Wnt production and Wnt transcriptional component inhibition category most effectively disrupt digit patterning. I also identified candidate ligands such as sFRP1 and Dkk1 as potential extracellular Wnt inhibitors that effectively change digit patterning upon application. These results provide the first quantitative insight into the duration of the reaction-diffusion based mechanism in a biological system, and how a screening approach complemented with data driven modelling can complement and clarify workings of a reaction diffusion based system. Further work in improving our knowledge on the Turing system with tissue growth, cell movements, and ectodermal-mesenchymal interaction will eventually allow generation of a complete organogenesis simulation model.
Um globale Klimaziele zu erreichen ist Partizipation auf der individuellen Ebene erforderlich. Dabei machen die Treibhausgasemissionen des Personenverkehrs einen großen Bestandteil des persönlichen Anteils am Klimaschutz aus. Somit werden innerhalb dieser Arbeit persönliche Emissionsbudgets anhand verschiedener Zielvorgaben wie dem Pariser Klimaabkommen berechnet und Methoden zusammengestellt, mit welchen die Klimawirksamkeit häufig genutzter Verkehrsmittel berechnet werden kann. Innerhalb des Budgets sind dann Überlegungen möglich, wie konkrete Mobilitätsentscheidungen darin aussehen könnten. Anhand dieser Berechnungsgrundlagen wurden Mobilitätsprofile beziehungsweise Indikatoren für "klimafreundliches" Verhalten ermittelt, mit denen durch Abfragen weniger Informationen schon ein guter Überblick über die Emissionen einer Person gewonnen wird. Ziel ist es eine möglichst hohe Transparenz für die Person, die eine Mobilitätsentscheidung für sich selbst trifft, zu schaffen. Damit kann der Handlungsspielraum aufgezeigt werden, in welchem sich das eigene Verhalten abspielen muss, um gewisse nationale oder globale Ziele zu erreichen.
The fluorescence-based nanoscopy methods MINFLUX and MINSTED are currently revolutionizing the field of imaging and single molecule tracking by achieving molecular spatial precision with low photon numbers. Using a polymer-based in vitro assay with strongly reduced fluorescence background, I identified MINFLUX-compatible spontaneously blinking fluorophores by quantifying their blinking properties. Due to the spontaneous blinkers being live-cell compatible and having few milliseconds short on-events, I expect them to advance the imaging field by drastically accelerating MINFLUX measurements. The main focus of this work, however, is the application of the nanoscopy methods MINFLUX and MINSTED for tracking of the motor protein kinesin-1. Requiring an only ~1 nm small fluorophore as label, they are inherently less artefact-prone than established techniques which require the attachment of comparatively large beads for a similar spatio-temporal resolution. With an improved interferometric MINFLUX approach, we successfully resolved regular steps and substeps of the kinesin-1 stalk and heads. By discovering that ATP binds to the motor in the one-head-bound state and is hydrolyzed in the two-head-bound state, we aim to solve a long-standing controversy in the field. Furthermore, we deduced that when the rear head of kinesin-1 detaches from the microtubule, it rotates around its front into a rightward-displaced unbound state. In conjunction with an observed stalk rotation, I concluded the motor to walk in a symmetric hand-over-hand fashion. Finally, we successfully resolved the stepping of kinesin-1 with MINSTED, confirming many findings from the MINFLUX experiments and observing motor sidestepping and protofilament switching. These findings will prove helpful in developing treatments for diseases linked to malfunction of kinesins. Beyond that, this thesis establishes MINFLUX and MINSTED for the tracking of dynamic biological processes on the single molecule level.
We study some problems and develop some theory related to persistent homology, separated into two lines of investigation. In the first part, we introduce lifespan functors, which are endofunctors on the category of persistence modules that filter out intervals from barcodes according to their boundedness properties. They can be used to classify injective and projective objects in the category of barcodes and the category of pointwise finite-dimensional persistence modules. They also naturally appear in duality results for absolute and relative versions of persistent (co)homology, generalizing previous results in terms of barcodes by de Silva, Morozov, and Vejdemo-Johansson. Due to their functoriality, we can apply these results to morphisms in persistent homology that are induced by morphisms between filtrations. This lays the groundwork for an efficient algorithm to compute barcodes of images and induced matchings of such morphisms, which performs computations in terms of relative cohomology and then translates to absolute homology via the aforementioned dualities. Our method is based on a previous algorithm by Cohen-Steiner, Edelsbrunner, Harer, and Morozov that did not make use of relative cohomology. Using it is crucial, however, because our algorithm applies the clearing optimization introduced by Chen and Kerber, which works particularly well in the context of relative cohomology. We provide an implementation of our algorithm for inclusions of filtrations of Vietoris–Rips complexes in the framework of the software Ripser by Ulrich Bauer. In the second part, we introduce local connectedness conditions on a broad class of functionals that ensure that the persistent homology of their associated sublevel set filtration is q-tame, which, in particular, implies that they satisfy generalized Morse inequalities. We illustrate the applicability of these results by recasting the original proof of the unstable minimal surface theorem given by Morse and Tompkins in terms of persistent Čech homology in a modern and rigorous framework. Moreover, we show that the interleaving distance between the persistent singular homology and the persistent Čech homology of a filtration consisting of paracompact Hausdorff spaces is 0 if it satisfies a similar local connectedness condition to the one used to ensure q-tameness, generalizing a result by Mardešić for locally connected spaces to the setting of filtrations. In contrast to singular homology, the persistent Čech homology of a compact filtration is always upper semi-continuous, which has structural implications in the q-tame case: using a result by Chazal, Crawley-Boevey, and de Silva concerning radicals of persistence modules, we show that every lower semi-continuous q-tame persistence module can be decomposed as a direct sum of interval modules and that every upper semi-continuous q-tame persistence module can be decomposed as a product of interval modules.
In this work we focus on Bose-Einstein condensates for two particular purposes. First, as a system for which the analysis of spatial entanglement is worthwhile, given its two regimes (relativistic/nonrelativistic) regarding the dispersion rela- tion of Bogoliubov quasiparticles. It is therefore a promising scenario to put to test the formalism around entanglement entropies computation from a quantum field theoretical approach, providing a setting to look further into questions re- lated to its divergences in a relativistic quantum field theory. We put forward results for condensates of one- and two-dimensional spatial geometries, to find a self-regularised theory in the ultraviolet, and an agreement with the literature in all the expected results. Furthermore, we give new results regarding the crossover behaviour from nonrelativistic to relativistic regimes. Our calculations are done considering a condensate of infinite extent. The second aim is to implement the condensate as a quantum simulator for relativistic fields in curved spacetimes, building background geometries which can be both, spatially curved and time- dependent, being able to simulate the class of FLRW universes. We provide the theoretical construct of such a simulator, and its successful experimental implementation, with results related to the detection of particle production in curved spacetimes. Both topics of inquiry are interesting in and of themselves, while taken together they present the opportunity to look into the question of entanglement in different spacetime geometries, including causally disconnected regions, in an experimental context.
In dieser Arbeit wird untersucht, wie Nanopartikel mit Zellmembranmodellen interagieren und welche Faktoren Einfluss auf die Nanotoxizität nehmen. Hierfür werden Zellmembransysteme sowie die zugehörigen Modelle für deren Beschreibung präsentiert, wobei die Komplexität im Laufe der Arbeit zunimmt. Dabei stehen die Zellmembranen von Eukaryoten im Mittelpunkt, aber auch die Membranen von Prokaryoten werden thematisiert. Das Ausgangssystem ist aus DMPC aufgebaut, wobei eukaryotische Zellmembranen durch das Einbauen von Cholesterol modelliert und für die Simulation von prokaryotischen Zellmembranen geladene Lipide verwendet werden. Neben den Auswirkungen der Zellmembranzusammensetzung wird die Beeinflussung der Systeme durch die Änderung der Temperatur, des verwendeten Salzes und der Ionenstärke sowie durch die Zugabe von Nanopartikeln untersucht. Bereits an Luft können verschiedene Charakteristika des Systems, wie beispielsweise die Oberflächenstruktur, Bilagenanzahl und Bilagendicke der Oligolipidbilagensysteme, untersucht werden. In wässriger Umgebung werden die lösungsmittelinduzierten Veränderungen der Struktur und Stabilität des Gesamtsystems genauso untersucht wie der Einfluss von Temperaturänderungen. Zudem wird in der wässrigen Umgebung die Interaktion zwischen den Oligolipidbilagen und Nanopartikeln untersucht. Durch die wässrige Umgebung können Salze in die Lösung eingebracht werden, wodurch die biologische Relevanz des Systems zunimmt und der Einfluss der Ionenstärke auf die Charakteristika des Systems untersucht werden kann. Zudem ist von besonderem Interesse, in welcher Form die Zugabe von Salzen die Interaktion von verschiedenen Zellmembranmodellen mit Nanopartikeln beeinflusst. Neutronenreflexions- und Röntgenreflexionsmessungen stehen bei dieser Arbeit als experimentelle Methoden im Vordergrund. Diese werden jedoch durch eine Vielzahl weiterer Messmethoden, wie dynamische Differenzkalorimetrie, Rasterkraftmikroskopie und Ellipsometrie, ergänzt, sodass ein differenziertes Gesamtbild der Systeme entsteht.
Cardiac diseases result in altered protein and gene expression levels due to altered signal transduction pathways. RNA binding proteins (RBPs) are an emerging group of post-transcriptional regulators that control RNA between protein and RNA levels. Analysing the role of RNA binding proteins in cardiac dysfunction would be vital in understanding the coordination of multiple post-transcriptional events during such diseases. However, the specific role of RBPs in controlling protein expression in the diseased myocardium is still not completely understood. Ribosomal sequencing and RNA sequencing was used to identify mTOR-dependent and translationally regulated transcripts in response to TAC surgery. Ybx-1 showed up to be one RNA binding protein that is upregulated only during pathological hypertrophy in our screen. Experiments in isolated cardiomyocytes in vitro showed that Ybx-1 depletion prevents cellular growth by inhibiting protein translation. Furthermore, Ybx-1 expression depends on mTOR signalling and is independent of mRNA transcription. Ybx-1 knockdown in vivo preserves heart function during pathological cardiac hypertrophy. eEF2 mRNA was identified as a potential mRNA that binds to Ybx-1 and is upregulated during cardiac hypertrophy. Cardiac hypertrophy involves upregulation of protein synthesis, and elongation factors such as eEF2 regulate it. Identifying the crosstalk between Ybx-1 and eEF2 can help understand how these factors contribute to cardiac dysfunction.
Data generation is rapidly progressing and data interpretation is hardly keeping up. New tools and approaches are needed to assess, filter and decide upon the impact of variants on biological systems. This work first presents a novel method of interpreting variants found to be exclusively heterozygous in public datasets (1000 Genomes Project and gnomAD). Variant pathogenicity is scored by a benchmarked, Bayesian integration of a diversity of gene, protein, sequence and structural features. A new 3D clustering method was developed to identify and understand pathogenic variant mechanism. This method uses known functional knowledge from a protein of interest and is homologs, and intramolecular distances from known/predicted structures to group and map functional information. This puts candidate variants into context by according to previously known functions of residues within the same group. Experimental validation of putative pathogenic variants is important for understanding variant mechanism. Several variants of RHOA, a small GTPase relevant to many crucial signalling pathways, were subjected to a battery of laboratory experiments. Variants affecting sites involved in interactions with other proteins are known to typically cause a loss-of-function phenotypes in cancer. However, it became evident that variants showed unique mechanisms in terms of observed phenotypes. Overall it can be argued that there is no single mechanism related to RHOA dysregulation in cancer. Lastly, a new method was developed with the aim of assisting diagnostics via clinical genetics. The method is able to produce a biological triplicate of laboratory results within four to five weeks, which would be a clinically useful timeframe in which to act on decisions related to dysregulated proteins. Modified HEK cells are transfected with tetracycline-controlled plasmids, containing the wild-type and mutated genes of interest. After selection and tetracycline induction the cells are harvested and total cell RNA is used to study gene expression via microarrays. Gene expression patterns can then be used to assess whether a given protein mutation is changing the enzyme activity, which can additionally be tested by rapid follow-up experiments such as phosphoantibodies. This hope is that the results can then be used to adjust treatment regimens, for instance, by highlighting putative oncogenes.
Genome sequencing efforts, coupled with technological advances and cost reductions, have led to the discovery of an increasing number of disease-related genetic variants. For the vast majority of these variants there is no known molecular mechanism for how they are related to the disease. This problem is particularly evident for diseases with complex genotype-phenotype relationships, such as cancer. Fortunately, the parallel growth of data on protein families, structures, interactions, modifications, and other aspects of function, in addition to the development of new computational methods provide the means to predict or identify disease variant mechanism. In this thesis, I first present a systematic analysis of a large dataset of pan-cancer missense mutations to investigate whether positive selection of certain types of amino acid substitutions can reveal interaction-disrupting cancer driver mutations. Hundreds of mechanistically interesting variants were identified in both potentially novel cancer-associated proteins and well-established cancer driver genes. I discuss new insights and for some instances, attempt functional interpretations by integrating information on protein structure and interactions that suggest putative novel mechanisms that question the classical oncogene/tumour suppressor paradigm. There is a wealth of publicly available resources that already provide valuable information on all aspects that define gene and protein function. This information has been collected from thousands of experiments or publications and has usually been manually verified or predicted using new approaches. This means that interpreting variants can be a tedious process of manually consulting and integrating the different functional data from multiple databases. Mechnetor was developed to aid this process: a freely available web tool that helps users understand the mechanism of protein variants. With a simple input from the user, Mechnetor automatically collects and integrates various relevant functional data and presents them in an interactive network that allows easy visualisation and interpretation of the results. Many databases are created from the individual efforts of hundreds of labs conducting similar experiments, combining their results to build and increase the confidence of biological knowledge. I had the opportunity to collaborate with the group of Prof. Dr. Felix iv Wieland (Heidelberg University Biochemistry Center) in analysing and interpreting the results of one such experiment: a proteome-wide study of S-palmitoylation in Drosophila melanogaster. S-palmitoylation is an important reversible post-translational modification that controls protein membrane location and trafficking and is thus linked to many cellular processes. In contrast to humans, palmitoylation target proteins and responsible enzymes are largely unknown in invertebrates. Here, we identified and characterised the most complete set of S-palmitoylated proteins in Drosophila to date, as well as the putative substrate profiles of 10 Drosophila palmitoyl acyl transferases. Our results provide new insights and reveal many functional similarities of palmitoylation between Drosophila and humans.
Optimality is a cornerstone of biology, as evolutionary forces drive biological systems towards optimal performance. In this work, I develop theoretical models to reveal optimality principles in two biological systems: ligand discrimination by immune receptors and nuclear multiplication by parasites.
In multicellular organisms, antiviral defense is mediated by signaling molecules. They are usually characterized by highly inhomogeneous distributions due to scarcity of producer cells, diffusion and localized degradation. And yet, a molecular hub of antiviral response, the type I interferon receptor (IFNAR), discriminates between ligand types by their affinity regardless of concentration. In the first part of this work, I address the long-standing question of how a single receptor can robustly decode different ligand types. I frame ligand discrimination as an information-theoretic problem and systematically compare the major classes of receptor architectures: allosteric, homodimerizing, and heterodimerizing. As a result, the architecture of IFNAR---namely asymmetric heterodimers---achieve the best discrimination power over the entire physiological range of local ligand concentrations, enabling sensing of ligand presence and type. Here, receptor turnover, which drives the receptor system out of thermodynamic equilibrium, enables buffering against even high concentration fluctuation. Overall, these findings suggest that IFNAR is optimized for detecting and separating the presence of different ligand types in a noisy environment.
The malaria-causing pathogen Plasmodium falciparum is a eukaryotic parasite with a complex life cycle that includes proliferation within red blood cells. During the blood stage, the parasite invades a red blood cell, undergoes several rounds of asynchronous nuclear division, becoming multinucleated, and eventually forms and releases around 20 daughter parasites. Although clinical symptoms of malaria are manifest during this stage, a true understanding of the nuclear multiplication and its asynchrony remains missing. In the second part of this work, I address this topic by modeling the nuclear multiplication with various concepts of theoretical physics. The theoretical models are complemented by live-cell microscopy experiments, tracking nuclei and DNA replication. Our findings suggest that Plasmodium falciparum has evolved optimal resource utilization by exploiting a sequential sharing of replication machinery, a general mechanism for efficient and fast proliferation. This result was achieved by first investigating nuclear multiplication, showing that the number of daughter parasites is regulated by a counter mechanism. Second, we demonstrate that the nuclei are coupled by a shared resource that limits DNA replication and thereby actively generates asynchrony. In order to address the question in what way this asynchrony might be beneficial for the parasite, I introduce a minimal biophysical model for allocation of a shared enzyme to individual nuclei. The model captures parallel and sequential DNA replication mode, the latter being able to describe the observed asynchrony of the parasite. When the shared enzyme is limiting, a sequential replication utilizes resources more efficiently, resulting in faster completion of nuclear multiplication.
The malaria parasite Plasmodium falciparum has a unique, complex life cycle and exports a variety of proteins into the erythrocyte to alter the physiological properties of its host cell. These modifications are crucial for survival and render the parasite a master of immune evasion, which is one of the main reasons why there has not been developed an efficient vaccine to eradicate this devastating disease. Many exported proteins are thought to play a crucial role in protein export and host cell modifications. However, scientific breakthroughs in this field were hampered due to a lack of efficient genetic systems for the blood stages of the parasite. Over the last years, several new genetic tools were established for P. falciparum that allow the investigation of essential genes. In this study, four predicted essential proteins were analyzed using the glmS ribozyme system, an inducible knockdown method on mRNA level that is activated by glucosamine-6-phosphate. Coupled with selection-linked integration (SLI), it allows faster generation of transgenic cell lines than conventional subcloning methods. The glmS system worked efficiently in all cell lines studied with significant downregulation of the proteins of interest with 2.5 mM glucosamine. Concerning the development and morphology of the parasites, there was no growth defect observed via microscopy or SybrGreen-based growth assays. PF3D7_0220300 and PF3D7_0220600 are predicted to be membrane-bound proteins partly co-localizing with the Maurer’s clefts marker SBP1, and the erythrocyte membrane, respectively. PF3D7_0113300 and PF3D7_0301600 are predicted to be soluble proteins with the second partly co-localizing with SBP1. Using an in vitro static cytoadherence assay, I could show that binding to chondroitin sulfate A was significantly reduced in all cell lines except CS2PF3D7_0113300glmS with the strongest effect observed upon downregulation of PF3D7_0301600 and PF3D7_0220300. Quantification of the mean fluorescence intensities of PfEMP1 VAR2CSA via flow cytometry only showed significantly less signal after downregulation of PF3D7_0220300, further demonstrating the importance of the studied exported proteins in host cell modification, specifically cytoadherence and presentation of PfEMP1 on knobs. These results should be further investigated to get a more comprehensive understanding of the parasites’ biology. Moreover, inducible knockout systems using a dimerizable Cre recombinase could circumvent some disadvantages of the glmS system resulting from remaining protein levels or the cytotoxicity of glucosamine and should be considered in further studies.
Thermoresponsive hydrogels such as poly(N-isopropylacrylamide) (pNIPAM) are highly interesting materials for generating soft actuator systems. Whereas the material has so far mostly been used in macroscopic systems, we here demonstrate that pNIPAM is an excellent material for generating actuator systems at the micrometer scale. Two-Photon Direct Laser Writing was used to precisely structure thermoresponsive pNIPAM hydrogels at the micrometer scale based on a photosensitive resist. We systematically show that the surface- to-volume ratio of the microactuators is decisive to their actuation efficiency. The phase transition of the pNIPAM was also demonstrated by nanoindentation experiments. We observed that the mechanical properties of the material can easily be adjusted by the writing process. Finally, we found that not only the total size and surface structure of the microactuator plays an important role, but also the crosslinking of the polymer itself. Our results demonstrate for the first time a systematic study of pNIPAM-based microactuators, which can easily be extended to systems of microactuators that act cooperatively, e.g., in microvalves.
We present a new analytic model describing gravitational wave emission in the post-merger phase of binary neutron star mergers. The model is determined by a number of physical parameters that are related to various oscillation modes, combination tones or non-linear features. The time evolution of the main post-merger frequency is incorporated. The model achieves high fitting factors for a sequence of equal-mass simulations of varying mass. All parameters of the model correlate with the total binary mass. For high binary masses, we identify new spectral features originating from the non-linear coupling between the quasi-radial oscillation and an antipodal tidal deformation of the remnant, the inclusion of which enhances the corresponding fitting factors. We find that subdominant frequency components are crucial for the construction of faithful gravitational wave templates. Because of the high fitting factors our model is particularly suitable for searches with upgraded detectors of the current generation (aLIGO+, aVirgo+) or future detectors. We find a quasi-universal relation for the proximity of a binary configuration to black-hole formation. We then develop a method for determining the threshold mass for prompt black-hole formation and the maximum mass of non-rotating neutron stars. This procedure relies on one precise measurement of the inspiral and post-merger phase.
In my PhD project, I used the pseudostratified epithelium of the wing imaginal disc of Drosophila melanogaster as a model system to study the significance of cellular metabolism and nuclear positioning for tissue development in vivo. The wing disc is a larval precursor tissue that mainly gives rise to the adult wing. It is surrounded by the larval hemolymph, providing metabolites, such as lipoprotein-bound lipids. As a read-out for the metabolic state of the wing disc, I investigated the disc epigenome as most histone modifying and demodifying enzymes require metabolic co-factors or substrates. In this study, I identified a nonuniform distribution of histone acetylation in the wing disc with high acetylation levels specifically in nuclei facing the tissue surface. This rim pattern is not defined by cell cycle or cellular identity but by the position of the nucleus within the tissue. Accordingly, genetic ablation of the disc rim causes the formation of a new rim with high histone acetylation in the remaining, now outward facing, tissue region. High acetylation levels in nuclei in the disc rim correlate with hemolymph-exposure as indicated by accumulation of lipoproteins on outwardfacing membranes. The acetylation pattern persists during wing disc development suggesting a continuous regulatory role in tissue growth. The non-uniform distribution of histone acetylation is achieved by local activity of the acetyltransferase nejire in the rim of the disc. Spatially restricted nejire activity in the rim is not determined by non-uniform intrinsic activity or expression of the enzyme but by local availability of its metabolic substrate acetyl-CoA. Required acetyl-CoA is specifically generated in outward-facing nuclei due to increased levels of nuclear acetyl-CoA synthase which converts acetate to acetyl-CoA. Metabolically, this rim acetylation pattern depends on acetate derived from fatty acid β-oxidation, which is also nonuniform and increased in the rim of the wing disc, as evidenced by a high mitochondrial membrane potential in this tissue region. Rim histone acetylation epigenetically impacts expression of genes in the wing disc implicated in developmentally important signaling pathways, including Hippo, Notch, and Hedgehog. Jointly, my findings suggest a novel role for nuclear positioning and a concordantly aligned metabolism in determining cell fate and, consequently, wing disc development.
We discuss the extension of the, originally Newtonian, moving-mesh hydrodynamics AREPO code to study general relativistic systems. This includes the implementation of general relativistic hydrodynamics and coupling AREPO to a solver for the Einstein field equations, which adopts the conformal flatness approximation. We validate the implementation by evolving static neutron stars and comparing to independent calculations. We present the first general relativistic moving-mesh simulation of a neutron star merger. We find that the general dynamics and features of the post-merger gravitational wave emission agree with independent simulations performed with smoothed particle hydrodynamics and static-mesh tools. We observe that dynamical features in the post-merger phase, such as the quasi-radial oscillation mode and the double-core structure, survive longer in our moving-mesh simulation. Similarly, the post-merger gravitational wave signal is damped very slowly. These features suggest that the moving-mesh simulation has lower numerical viscosity and highlights that the moving-mesh approach can be very beneficial in simulations of neutron star mergers. As another part of this thesis, we examine relations between gravitational wave frequencies from isolated stars or merger remnants and stellar properties, such as the radius. We show that the scatter of points in such relations encodes information about the equation of state.
The engulfment and cell motility (ELMO) proteins are cytoplasmic adaptor proteins which are evolutionary conserved in several organisms. In higher vertebrates the ELMO protein family consists of the three paralogs ELMO1, ELMO2 and ELMO3. All three ELMO proteins regulate the activity of the Rac family small GTPase 1 (RAC1). As RAC1 regulators, individual ELMO proteins facilitate basic and major cell functions such as cytoskeleton organization, cell migration, phagocytosis, the engulfment of apoptotic cells and myoblast fusion. Several studies have shown that the ELMO proteins are involved in a variety of cellular and developmental processes and linked to multiple diseases, such as cancer, diabetes mellitus, inflammatory bowel disease and developmental disorders. However, the individual functions of ELMO1, ELMO2 and ELMO3 are hardly understood and since barely any studies cover comparative data about all three ELMO proteins, it is mostly unknown whether the Elmo proteins have similar functions and act redundantly or not. To address this question, elmo1-/-, elmo2-/- and elmo3-/- knockout zebrafish were generated using the CRISPR/Cas9 technique and with these mutants a comprehensive comparison of the phenotypic changes in organ morphology, transcriptome and metabolome was performed. The main results were decreased fasting and increased postprandial blood glucose levels in adult elmo1-/-, as well as a decreased vascular formation in the adult retina of elmo1-/-, but an increased vascular formation in the adult retina of elmo3-/- zebrafish. The comparison provided few similarities between the knockout lines, as increased Bowman space areas in adult elmo1-/- and elmo2-/- kidneys, an increased hyaloid vessel diameter in elmo1-/- and elmo3-/- and a transcriptional downregulation of the vascular development in elmo1-/-, elmo2-/-, and elmo3-/- zebrafish larvae. Despite that, elmo1-/-, elmo2-/-, and elmo3-/- zebrafish exhibited several distinct changes in the vascular and glomerular structure, as well as in the metabolome and the transcriptome. Especially, elmo3-/- zebrafish showed extensive differences due to a strong transcriptional dysregulation in larvae and an impaired survivability. Together, the data demonstrate that the three zebrafish Elmo proteins regulate not only similar but many divergent biological processes and mechanisms and show a low functional redundancy.
Clathrin-mediated endocytosis (CME) is one of the main uptake processes in cells. The biomechanical study of the formation of clathrin-coated structures has been attracting more attention since the first discovery of the clathrin protein more than 50 years ago. For CME to occur, adhesion energy between the cell and cargo must overcome an energy barrier produced by turgor pressure, membrane tension, and bending energy. While the molecular force mechanism of CME is relatively better understood, the role of cellular forces by rigidity sensing in supporting CME from the ventral side is less studied. My doctoral thesis aimed to elucidate how the cellular adhesion forces to the extracellular matrix (ECM) contribute to the CME of nanoparticles at the cellular ventral side. The experimental setup entails the immobilization of nanoparticles on substrates coated with an ECM protein such as fibronectin. Next, cells expressing CME adaptor protein 2 (AP2) tagged with e-GFP (i.e., MEF, HeLa) were seeded on the substrate and were subsequently analyzed at around 4 hours post-seeding. Traction force microscopy (TFM) was used to quantify the ability of cells to generate traction forces. As TFM requires a substrate that can be deformed by adherent cells, I described in the first part the preparation of elastic polyacrylamide (PA)/viscoelastic polydimethylsiloxane (PDMS) substrates and their mechanical characterization using nanoindentation experiments. Additionally, I investigated the response of materials upon the exposure to ultraviolet light (both PA and PDMS) and oxygen plasma (only PDMS elastomer). Next, I described the surface patterning on the substrate, including micropatterning (both PA and PDMS) using the maskless photolithography method and nanopatterning (only PA hydrogel) using the block-copolymer micellar nanolithography (BCMN) method. Finally, I described the development of an integrated bioengineering toolbox that includes a novel method called ”Local Ultraviolet Illumination Traction Force Microscopy (LUVI-TFM)” that could be combined with micro- and nanopatterned sub- strates to perform 2D- and 2.5D-TFM on a single cell or multicellular clusters. This toolbox was devised so users could choose a single tool or a combination of multiple tools to study cell-matrix interaction. In the second part, I described the use of the bioengineering tools established in the previous part to study the role of traction forces in initiating CME of nanoparticles at the cellular ventral side. First, I observed that electrostatically immobilized nanoparticles were removed under cellular traction forces by using live 2D-LUVI-TFM. However, it was unclear if traction forces caused the particle removal. To elucidate it, I employed micropatterning to control the size and shape, thereby the traction force hotspots of a single cell. I could also immobilize nanoparticles in regions where the cell exerted high traction forces. On micropatterned cells, I observed a low spatial correlation between the clathrin adaptor protein 2 (AP2) clusters and focal adhesion marker clusters (paxillin, β1 integrin, and β3 integrin). Furthermore, I observed that traction forces alone were insufficient to remove nanoparticles from the surface. Despite no removal, I observed immobilized nanoparticles initiated CME in high traction force regions. Concerning the role of traction force in initiating CME of nanoparticles at the cellular ventral side, I focused on cells that were transitioning between the early adhesion phase and the initial contractility at around 4 hours post-seeding. In this phase, cells exhibited the shape of a symmetric ”bulls-eye egg” and were easier to be compared. I observed that AP2 clusters were highly distributed at the cell periphery, irrespective of the presence of covalently immobilized nanoparticles and substrate rigidity. Furthermore, I observed no significant difference between cells growing on the 3 kPa substrate and the glass concerning the cluster area and the signal lifetime of AP2. Lastly, I observed that the cell pushed down the substrate at the cell periphery, and AP2 clusters were found highly distributed inside the indentation area. Eventually, AP2 clusters were recruited above covalently immobilized nanoparticles in the indentation area. This result strongly suggested that the normal component of traction forces plays an undeniably important role in initiating CME of nanoparticles at the cellular ventral side.
Monoclonal antibodies are biomolecules that are able to bind to a specific antigen. Antibodies are important drugs for the targeted treatment of various types of cancer. Due to their complex molecular structure, monoclonal antibodies are usually produced through the cultivation of genetically modified mammalian cells. At the production scale, the cultivation is typically carried out using sparged stirred tank bioreactors and the product quality as well as the yield depend, among other factors, on the hydrodynamic conditions inside the utilized bioreactor. The optimization of cell cultivation for the production of monoclonal antibodies is of high economical importance for the pharmaceutical industry. The simulation of the flow field inside the utilized bioreactors with computational fluid dynamics enables the prediction of relevant process characteristics, which must be considered during the scale-up of cell culture processes. The focus of the present study is on the hydrodynamic characterization and the selection of the operating conditions during scale-up of the cell culture processes of four single-use bioreactors with varying sizes ranging from the lab-scale to the production scale, the Mobius® CellReady 3 L, the XcellerexTM XDR-10, the XcellerexTM XDR-200, and the XcellerexTM XDR-2000. Additionally, the hydrodynamic characteristics of a miniaturized stirred tank bioreactor, the Ambr®250, and another of the XcellerexTM bioreactors, the XcellerexTM XDR-500, are investigated. The simulations have been carried out with the Euler-Euler and the Euler-Lagrange approaches with the open source software OpenFOAM and the commercial software MixIT. The considered process characteristics include the mixing time, the hydrodynamic stress, the average strain rate in the impeller zone, and the volumetric oxygen mass transfer coefficient. These are representing the homogenization in the liquid phase, the mechanical stress acting on the cultivated cells and the availability of oxygen, which is essential for aerobic organisms. Only through the hydrodynamic characterization of the different bioreactors can the causal relationship of the bioreactor operating conditions like the impeller speed, the working volume, and the sparging strategy with the process performance of the cell cultivation be understood, which is required for the optimization of operating conditions for the different bioreactors. For larger biroeactor volumes an increase in the mixing time cannot be avoided, whereas a similar maximum hydrodynamic stress, a similar average strain rate of the impeller zone, and a similar volumetric oxygen mass transfer coefficient are observed for all investigated bioreactors. To optimize mixing without risking cell damage, the maximum tolerable average strain rate of the impeller zone is selected as the scale-up criterion for the impeller speed. Experimental cell culture results provided by Yuichi Aki from Daiichi-Sankyo Japan support the suitability of this criterion through a successful scale-up of the cell cultivation from the Mobius® CellReady 3 L to the XcellerexTM XDR-200. Other typical scale-up criteria like the volumetric power input and the impeller tip speed result in lower impeller speeds than the with presented strategy, therefore appearing less suitable to optimize the mixing time during scale-up. This emphasizes the advantages of a detailed hydrodynamic analysis over classical scale-up parameters.
Hintergrund Vorhofflimmern ist eine häufige Ursache für thrombembolische Ereignisse. Der interventionelle Verschluss des linken Vorhofohrs (LAA) stellt eine mechanische Option zur Prävention von mit Vorhofflimmern assoziierten Thromboembolien bei Patienten mit erhöhtem Blutungsgefahr. In Anbetracht der hämodynamischen und homöostatischen Rolle des linken Vorhofohrs könnte jede Intervention, die auf diese Struktur abzielt, entsprechend das systemische Metabolom beeinflussen. Das Ziel dieser Arbeit liegt darin, der Einfluss des LAA-Verschlusses auf den Metabolismus der essentiellen Aminosäuren, des Kreatinins und von Kynurenin anhand der Metabolom-Analyse zu untersuchen. Methoden In der vorliegenden monozentrischen, prospektiven, nicht-randomisierten Beobachtungsstudie wurden insgesamt 44 Patienten mit erfolgreichem LAA-Verschluss eingeschlossen. Alle Blutproben wurden vor dem Eingriff (T0) und am Tage des mittelfristigen Follow-ups nach sechs Monaten (T1) abgenommen. Die gezielte metabolische Analyse erfolgte mit der Elektrospray-Ionisierung Flüssigchromatographie-Massenspektrometrie mit Fokus auf dem Metabolismus von essentiellen Aminosäuren, Kreatinin und Kynurenin. Ergebnisse Es zeigte sich signifikante Anstiege von drei Aminosäuren innerhalb von 6 Monaten nach LAA-Verschluss: Phenylalanin (8%, P = 0,006), Tryptophan (20%, P = 0,0006), und Tyrosin (20%, P = 0,0001). Darüber hinaus war die Kynurenin-Konzentration signifikant um 8% (P = 0,0239) steigend. Entsprechend war die Kynurenin/Tryptophan-Ratio fallend. Des Weiteren war die Fischer-Ratio um 7% (P = 0,0009) fallend und die Tyrosin/Phenylalanin-Ratio um 4% (P = 0,0159) steigend. Schlussfolgerung Die vorliegende Studie zeigte, dass der interventionelle LAA-Verschluss das menschliche Metabolom beeinflussen kann. Die Studie unterstützt die Hypothese, dass insbesondere bestimmte essentielle Aminosäuren und Kynurenin als klinische Biomarker dienen könnten, die pathophysiologischen Veränderungen innerhalb eines halben Jahres nach erfolgreichem LAA-Verschluss abzubilden.
Despite decades-long’ efforts to combat malaria, it is still responsible for the death of over half a million people each year. All clinical symptoms of malaria are caused by the rapid asexual proliferation of parasites of the genus Plasmodium in the blood of patients. Here, Plasmodium replicates via schizogony: an atypical form of replication, where nuclei multiply asynchronously. Only once approximately twenty nuclei are formed, daughter cells assemble. However, the coordination of DNA replication and nuclear division as well as the molecular determinants of asynchronous nuclear multiplication are unknown.
In my thesis, I investigated the organization and regulation of asynchronous nuclear multiplication during schizogony in Plasmodium falciparum (P. falciparum), which causes the most severe form of human malaria. I first determined that P. falciparum proliferates via alternating rounds of DNA replication and nuclear division, with nuclei seemingly acting as ‘cells-within-cells’. By showing that the episomally expressed replication fork protein PCNA1::GFP transiently accumulates only in those nuclei that undergo DNA replication, I established a marker for DNA replication compatible with life-cell imaging. In combination with the marker 3xNLS::mCherry for the nuclei, imaging of PCNA1::GFP allowed me to track DNA replication and nuclear division in nuclei of single parasites as they underwent schizogony. I then quantified the overall dynamics of nuclear multiplication as well as the dynamics and organization of the individual DNA replication and nuclear division events. To establish a mathematical model of nuclear multiplication, I collaborated with P. Binder, showing that DNA replication is influenced by a limiting factor and that asynchronous nuclear divisions enable rapid parasite proliferation. I also investigated the molecular basis of the transient accumulation of PCNA1::GFP in nuclei during S-phase and found that this is most likely caused by association of PCNA1 with the DNA during replication. As PCNA1 shuttles between the cytoplasm and the nucleus, I also analyzed sequence motifs potentially important for the nucleo-cytoplasmic transport of PCNA1 and found that PCNA1 may contain a classical nuclear export signal (NES), which should facilitate nuclear export via the export receptor exportin-1. Yet, this NES may not be functional, as chemical inhibition of exportin-1 in an engineered cell line which is sensitive to the exportin-1 inhibitor leptomycin B (LMB), did not affect nuclear accumulation or export of PCNA1::GFP.
Together, I characterized the spatiotemporal organization of nuclear multiplication, defining the basic organization and regulation of the cell cycle in P. falciparum in the blood stage of infection. This may not only help to uncover novel targets for malaria intervention but also to expand our understanding of the unusual cell cycle biology of an early-branching eukaryote.
Thermoregulation is a dynamic homeostatic process, tightly regulated by the autonomic nervous system. How the brain coordinates maintenance of the body temperature within a narrow range of 37˚C, a condition that is needed for the survival of most of the species, remains unclear. Among the brain regions implicated in thermoregulation is the anterior portion of the hypothalamus, the preoptic area (POA). In this region neurons that respond to direct temperature stimuli and to the skin and spinal cord warming were found, suggesting that these warm-sensitive neurons (WSNs), are the cells that detect deep brain temperature and integrate it with temperature information from the periphery. The limiting factor in studying the WSNs of the POA and their role in thermoregulation is the lack of specific molecular markers that identify them. Therefore, the goal of this thesis work was to characterize WSNs of the POA at the molecular level and to find their genetic marker(s). To achieve this goal I used a primary POA cell culture and performed calcium imaging while applying a temperature stimulus of 45˚C in the presence and the absence of the TRPM2 antagonist, 2-Aminoethoxydiphenyl borate (2-APB). I identified and hand-picked temperature responding cells and temperature non-responding cells for the single-cell RNA-sequencing (scRNA-seq). Analysis of the scRNA-seq data pointed to the limitations of the P9 cell culture used. The majority of the temperature non-responding cells expressed glial marker genes together with neuronal markers, a combination not found in vivo. I concluded that the genetic heterogeneity of the sequenced cells was too large and putative WSNs’ molecular markers identified from cultured neurons would be ambiguous. In addition, to find markers of WNSs one has to take another approach, such as Patch-seq to analyze these neurons in more natural conditions. One of the POA neuronal populations activated by a change in ambient temperature is expressing leptin receptor (POALepR). These neurons also exhibit an increase in action potential firing frequency (AP FF) during the process of chronic heat exposure to 36˚C that also leads to an increased heat endurance (at 39˚C). in mice. This intrinsic property of neurons (not affected by synaptic blockers), seems to be needed for a mouse to endure heat, as the animals in which the firing of POALepR is abolished fail to do so. To learn more about the role of the POALepR neurons in the heat acclimation process I used FACS (Fluorescence Activated Cell Sorting) to isolate the POALepR neurons from POA of non-acclimated and acclimated ( 5 and 30 days at 36 ˚C) LeprCreHTB mice and performed RNA sequencing. I identified three genes Kcnq2, Kcnn2, and Kcnh2, all three coding for potassium ion channels, whose expression level changed with the course of heat acclimation. I have tested the functionality of these ion channels in AP viii firing of the POALepR neurons, by employing electrophysiology and pharmacology in acute POA slices. Ion channels Kv7.2 and Kv11.1, coded by Kcnq2, and Kcnh2, respectively, exhibited a role in shaping the AP firing of POALepR neurons. Applying the antagonist of Kv7.2 disrupted harmonious AP firing of POALepR neurons coming from acclimated mice, rendering their membrane potential unsteady and their firing bursty. In addition, the application of the Kv11.1 antagonist increased the AP FF of POALepR neurons even further in the long-term acclimated condition. Heat acclimation is a naturally occurring process happening across mammalian species, including humans. It is important for enduring physical burdens in hotter climates as it leads to the improved function of the thermoregulatory system. It is forthright to hypothesize that the POA, the central regulator of temperature homeostasis, plays a role in heat acclimation. However, knowledge about it is scarce. Knowing which molecules change in POALepR neurons transcriptome to increase firing, and to which other thermoregulatory relays these neurons project will help us understand their role and the role of POA in heat acclimation.
We use methods from symplectic geometry to study periodic solutions of differential delay equations (DDEs, also known as retarded functional differential equations, RFDEs). Using polyfold theory, we prove that near a given non-degenerate 1-periodic orbit of a vector field in R^n , there is a 1-dimensional family of 1-periodic delay orbits smoothly parametrized by delay. Then we generalize this result in several ways. Moreover, we prove an abstract compactness theorem for perturbed non-local unregularized gradient flow lines in R^2n , which is one step towards the construction of Floer theory for Hamiltonian delay equations.
From the genome to the proteome, each molecular step is tightly controlled. Advances in RNA-sequencing technologies show that a system of RNA modifiers extensively and dynamically regulates the transcriptome. The posttranscriptional changes alter mRNA stability, localisation, and translational efficiency. However, the effects and dynamics of one of the least studied RNA modifiers, Apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1 (APOBEC1), are unknown. APOBEC1 catalyses cytosine deamination to uracil in a process known as RNA editing. APOBEC1 has a vital role in lipid metabolism, modifies phagocytosis in mouse macrophages, and its activity contributes to the heterogenic nature of macrophages. This work aimed to study how editing modifies macrophage phenotype, transcript-level effects, and dynamics during macrophage activation. To determine whether APOBEC1 editing is temporally regulated after activation, I analysed RNA-sequencing data from polarised mouse bone-marrow-derived macrophages and the macrophage cell line RAW264.7. I show that global editing levels are altered over time with an early increase followed by a drastic decrease. I report a striking distinction in the pattern of transcript level editing changes after proinflammatory stimulation; transcripts involved in phagosome maturation (processes that acidify phagosomes) have decreased editing while transcripts essential for antigen presentation and processing (processes that increase or require higher pH levels) have increased editing. I find that APOBEC1 increases antigen presentation and processing machinery protein levels and decreases factors involved in acidification and antigen digestion. I demonstrate that the lack of editing decreases the translational efficiency of Cytochrome B-245 Beta Chain (CYBB). CYBB is part of an enzyme complex that produces reactive oxygen species in phagosomes consuming protons during the process, which is critical for preventing antigen over-digestion. The loss of APOBEC1 causes dysfunction in the regulation of phagosomal pH resulting in lower-than-normal pH, which in part is responsible for the previous observations of increased phagocytosis in knockout macrophages. Overall, my work highlights a novel role of APOBEC1 editing in regulating transcript translational efficiency and control of the primary functions of macrophages in phagocytosis and antigen presentation.
We study on Restricted Additive Schwarz Preconditioned Exact Newton method (RASPEN), a nonlinear preconditioning of Newton's method for solving the nonlinear algebraic systems of equations which result from the discretisation of partial differential equations (PDEs). The preconditioned system is created by the help of additive Schwarz method to enable the parallel computation and is supposed to be more suitable for Newton’s method. We also propose the coarse grid correction for RASPEN due to the fact that the one-level scheme has a scalability concern when doing a large-scale computation. Adding the second level would remedy this drawback. Our coarse space is based on the idea of Nicolaides coarse space with some extensions. It does not need an explicit coarse mesh and can be constructed in the purely algebraic manner. Furthermore, the setup of the coarse problem can be done in parallel. We apply RASPEN on various scenarios in order to investigate the flexibility of RASPEN and the effectiveness of the two-level approach.
In acute myeloid leukemia (AML), initiation of tumorigenesis via multiple oncogenic mutations occurs throughout various stages of hematopoiesis that disrupt the corresponding transcriptomic and epigenetic profiles. The cancer cells that emerge are referred to as blasts and share biological features from these disease-specific alterations and patterns associated with differentiation and the tumor cell of origin. The resulting blasts show a large inter- and intra-tumor heterogeneity within molecularly defined AML subgroups that are highly relevant for risk stratification and personalized treatment strategies. Sequencing methods that analyze the transcriptome (scRNA-seq) and epigenome (scATAC-seq) are ideally suited to resolve tumor cell heterogeneity as well as non-malignant cell types in the microenvironment. Additionally, scATAC-seq allows to map the binding of transcription factors (TF) and infer cell-specific regulatory networks. Here, I dissected inter- and intra-tumor heterogeneity in patients with different genetic aberrations representing major subgroups in AML, namely MLL fusions, IDH mutated, and FLT3-ITD rearranged AMLs. I established and adjusted the experimental and bioinformatic procedures to generate reproducible and scalable data by scRNA/ATAC-seq of peripheral blood and bone marrow biopsies from AML patients. I could demonstrate that leukemic cells could be successfully distinguished from the microenvironment based on marker gene annotation from the human cell atlas and ploidy inference. Furthermore, I used the experimental and data analysis framework to analyze specific molecular features of the three AML subgroups. First, I characterized changes in the transcriptome and classified developmental stages of leukemic cells carrying MLL-EDC4 fusions along the hematopoietic stem cell to the myeloid trajectory compared to other MLL fusions. Cell type prediction revealed extensive malignant cell diversity and a phenotype skewed towards stem- and progenitor-like populations in MLL-EDC4 leukemic cells. To further elucidate transcriptomic properties of MLL-EDC4 cells, TF activity was inferred. The results agreed with differential gene expression highlighting many TFs that play a critical role in hematopoiesis, endothelial-to-hematopoietic transition, or leukemic stem cell activation. Second, I developed an approach to resolve the subclone- specific response during FLT3 inhibition with midostaurin. Analysis from scRNA and scATAC V showed different FLT3 activity/chromatin signatures within clusters of leukemic cells in the relapse that could be explained by midostaurin resistance and the emergence of distinct subclones as detected by scDNA-seq. Third, I characterized how the chromatin accessibility landscape was influenced by IDH1 mutated cells treated ex vivo with targeted therapy compared to IDH1 wild-type cells. Treatment with the IDH1 inhibitor revealed a partially reversible pattern of accessibility while other mutation-induced epigenetic modifications could not be reverted. The scRNA-seq data acquired for the three different AML subgroups were then exploited to perform a cell type prediction analysis. The relative abundance of different malignant cell types discovered varied amongst tumors, with some having just two identities and others having a wide range of malignant cells. MLL fusions, except for MLL-EDC4, generally conferred a more differentiated phenotype predominantly consisting of monocytes/macrophage CD14-like and promonocyte-like cells. Both tumor entities harboring FLT3-ITDs or IDH1 mutations showed a more complex composition of cell types along the myeloid differentiation trajectory than MLL fusions. The composition of cell types was generally more skewed to early progenitors at the point of diagnosis when compared to their matching relapse sample. This indicates a partial differentiation of AML cells that treatment might induce. In summary, this thesis provides novel insights into the tumorigenesis process in AML by using a systematic and functional analysis approach of the transcriptome and open chromatin in single cells for three major genetically defined AML subgroups. A better comprehension of cellular hierarchies, epigenetic effects, clonal evolution, and their impact on gene regulation might help to understand disease progression, stratify patient risk, and help to improve the treatment of hematopoietic malignancies in the future.
In Saccharomyces cerevisiae, the position of the mitotic spindle is surveyed by the spindle position checkpoint (SPOC). SPOC ensures that cells do not exit mitosis if their anaphase spindle is misaligned thereby ensuring faithful chromosomal segregation. Defects in SPOC can cause multiploidy and decrease cell survival. The SPOC kinase, Kin4, activates the Bfa1-Bub2 GTPase activating protein (GAP) complex via Bfa1 phosphorylation which in turn inhibits the mitotic exit network (MEN), a pathway that contributes to mitotic exit. Recent work has prompted Kin4- independent mechanisms that engage SPOC. Here, I have investigated the role of glycogen synthase kinase (GSK)-3 homolog, Mck1, as a novel SPOC component that activates SPOC independently of the Kin4 pathway. I show that, Mck1 and Kin4 work in parallel to stop the Cdc14 early anaphase release (FEAR) dependent activation of MEN to promote SPOC in cells with misaligned spindles. The data also indicates that Mck1 prohibits cells with compromised MEN from exiting mitosis. I illustrate that Mck1 executes its SPOC function by targeting Cdc6, a core component of the pre-replicative complex and a mitotic cyclin-dependent kinase (MCdk) inhibitor, for degradation before the cells enter mitosis. I also uncover that the cells overexpressing CDC6 cannot hold the SPOC arrest and exit mitosis. Moreover, this effect of overproduced Cdc6 was not evident when its N-terminal domain, which inhibits the M-Cdk activity, was lacking. In line with this, overexpression of the N terminal of Cdc6 could prompt SPOC deficiency. Additionally, the elevated levels of Cdc6 in mck1Δ cells capture more Clb2 (M phase cyclin) molecules. Altogether this denotes that the association of Cdc6 with Clb2 via the N-terminus seems to be crucial in regulating SPOC and mitotic exit. As cells enter the mitotic phase of the cell cycle, M-Cdk complexes phosphorylate MEN components like Mob1. I observe that the cells lacking Mck1, fail to adequately phosphorylate Mob1, which is an indication 11 of lower M-Cdk activity. Overall, this suggests that mck1Δ cells enter mitosis with higher Cdc6 levels and therefore, lower M-Cdk activity leading to SPOC deficiency and mitotic exit. This work has contributed to the understanding of Kin4 independent mechanisms that regulate SPOC and govern mitotic exit in the absence of FEAR. I also uncovered a novel function of GSK-3 kinase, Mck1. Given that GSK-3 kinases are highly conserved between organisms, the results obtained using budding yeast may open new directions of investigation into how spindle alignment and mitotic exit are regulated in higher eukaryotes.
The 2016 WHO Classification of Tumors of the Central Nervous System separates IDH-mutant gliomas into two distinct subtypes depending on the preservation or deletion of the chromosome arms of 1p and 19q termed “astrocytomas, IDH-mutant” and “oligodendrogliomas, IDH-mutant and 1p/19q-codeleted”. Currently, the assessment of the chromosomal copy number profile of 1p and 19q are essential to distinguish between the two tumor entities. An additional challenge is the WHO grading of astrocytomas which shows low correlation with survival. Biomarkers for facilitating the differential diagnosis of IDH-mutant glioma are of high clinical relevance. Therefore, there is a need for new markers and screening methods to effectively detect tumor malignancy and predict prognosis. Over the past decades mass spectrometry has become the method of choice for proteomic investigations in medical scientific areas. This study integrates a proteomic workflow into a clinical environment and explores the proteome of IDH-mutant gliomas with the aim of finding novel diagnostic and prognostic biomarkers for astrocytoma and oligodendroglioma. For this, sample preparation techniques and protocols for fresh frozen as well as formalin-fixed-paraffin-embedded tissues were established and tumor samples analyzed using different mass spectrometry platforms. A dual surrogate biomarker for 1p/19q status in IDH-mutant gliomas was discovered which could simplify the diagnosis of these tumors, making it less dependent on elaborative genetic analyses. Furthermore, it was observed that protein abundances correlate in sum with copy number of their respective chromosomes in mutant gliomas. Potential prognostic biomarkers were found which could not only improve the grading of IDH-mutant astrocytomas, but also help us further understand how these tumors overcome oxidative stress. Taken together, this study shows the potential impact of proteomic pipelines in a clinical environment and how it can complement already existing diagnostic infrastructures in the neuropathology. Importantly, it shows how a novel technology can help reveal novel potential biomarkers, which could be used by institutions where no genetic or proteomic analyses are available.
Breast carcinoma is the leading cause of cancer-related death in women worldwide. The most common breast cancer subtype is Luminal A. Most patients with this subtype initially respond well to standard endocrine therapies. However, many of them develop metastasis years later. It is well-known that the tumor microenvironment (TME) plays an important role in supporting tumor progression. A major component of the TME are the cancer-associated fibroblasts (CAFs), which have been reported to have multiple tumor-promoting functions. The exact mechanisms though are still under investigation, and no successful anti-CAF therapies have been discovered yet. In my PhD studies, presented in this thesis, I investigated the role of CAFs in Luminal A breast cancer progression. To this end, I utilized CAFs isolated from Luminal A patient biopsies, as well as the classical in vitro models of Luminal A breast cancer – the MCF7 and T47D cell lines. In the TME, CAFs are exposed to many factors that activate them and drive them into a tumor-promoting phenotype. Previously, it has been shown that the combination of TNFα and TGFβ1 was sufficient to confer CAF-like properties on CAF precursor cells. Therefore, to mimic CAF activation in vitro, I stimulated the CAFs with these two cytokines. To study how the cytokine-activated CAFs could support breast cancer progression, the effect of CAF-conditioned medium (CM) on migration, autophagy and recovery post chemotherapy was examined in MCF7 and T47D cells. The goal was to identify novel CAF-secreted factors and/or signaling pathways that they activate in the breast cancer cells to support Luminal A relapse. To this aim, via mass spectrometry analysis, I studied the effect of the stimulation on the CAF secretome and full proteome. In addition, utilizing RNA sequencing, I investigated the effect of the cytokine-activated CAF CM on the MCF7 cells, and compared it to the effect of the CM from unstimulated CAFs. Finally, I compared the results from both analyses to better understand the complex crosstalk between CAFs and Luminal A breast cancer cells and how it could contribute to Luminal A breast cancer progression. First, I demonstrated that the stimulation with TNFα was sufficient to induce the CAFs to secrete factors with pro-migratory effect on the Luminal A cells. Second, I was able to identify upregulation of STAT/IFNβ1 signaling in the CAFs in response to the TNFα stimulation. In addition, the CAFs were able to relay the STAT signaling to the breast cancer cells via secretion of IFNβ1. Third, I identified STAT2 to work in a STAT1-independent manner as the transcription factor necessary for the TNFα-stimulated induction of the ISG (interferon-stimulated gene) signature in the CAFs. Although I could not show a clear connection between the CAF interferon signaling and the breast cancer migratory phenotype, the stimulation with TNFα appeared to potentially impede with the CAFs ability to support Luminal A recovery post chemotherapy. Thus, TNFα seemed to play a double role in the complex CAF-Luminal A crosstalk. While TNFα-stimulated CAFs secreted factors, which increased Luminal A breast cancer migration, some of these factors and/or even TNFα itself seemed to prevent the recovery of the tumor cells after chemotherapy. Further investigation of the complex CAF-Lum A crosstalk and the role of TNFα in it is necessary. Meanwhile the role of TNFα as a double-edge sword in the TME-cancer crosstalk is something to consider when deciding on the next best therapeutic option for treatment of refractory Luminal A breast cancer.
In contrast to the majority of animals, mammalian embryonic development is highly regulative, beginning from one or several functionally-identical cells and sequentially acquiring increasing complexity over a relatively short period of time. During this period, the embryo also undergoes significant changes in morphology so as to accommodate the increase in complexity, especially during the landmark developmental event of gastrulation. How these morphological changes are enacted, coordinated, and controlled at the supra-cellular scale is yet unclear. The study of this question, and indeed this scientifically interesting period of development, has been historically difficult. This is partially due to the fact that unlike in other animals such as the zebrafish or the fruit fly, most mammalian embryos – including that of mice, the most common model organism in which such studies are conducted – are undergoing or have already undergone implantation into the uterine tissues during this period. To facilitate the study the peri-implantation development of mouse embryos, I have developed a 3D ex vivo culture system that is compatible with long-term light-sheet imaging, jointly with colleagues and collaborators. I characterised the development of embryos in this culture system and optimised it so that it supported the physiological growth and morphogenesis of embryonic and several extra-embryonic lineages. After validating this culture system, I used it to investigate the cell- and tissue-scale changes that occurred in the embryonic tissues during the peri-implantation period. In the mouse embryo, this period is associated with significant growth and differentiation of both embryonic and extra-embryonic tissues. For example, the epiblast (an embryonic cell lineage) adopts a pseudo-stratified epithelium organisation, while a subpopulation of the visceral endoderm (an extra-embryonic cell lineage) specialises into a distinct lineage that patterns the underlying embryonic ectoderm to lay down the first embryonic body axis. I demonstrated that the 3D ex vivo culture system supports live imaging at sufficient spatial and temporal resolution to visualise these processes. In addition, using an automatic 3D segmentation pipeline developed by colleagues and collaborators, I showed that the culture system can be used to study cell- and tissue-scale dynamics. The regulative nature of early mammalian development is also evident in the fact that the early mammalian embryo can tolerate drastic deviations in tissue size and cell number during development and correct these deviations so that at birth, embryos are once again within the stereotypical range of sizes for the species. The early mouse embryo can tolerate at least 2 four-fold changes in cell number, brought about by removal or addition of blastomeres; however, these size deviations are reportedly resolved by the time gastrulation is initiated, with embryos all undergoing gastrulation with the epiblast cell number at a certain threshold. I observed discrepancies between my findings and those previously reported, demonstrating that this process is still poorly understood despite decades of study, and identify cell- and tissue-scale parameters that may play a role in the sensing and correction of size deviations in the embryo.
Cells are subjected to mechanical forces and must sense and adequately react to them in order to develop and survive – a process known as mechanotransduction. This conversion of mechanical into biochemical signals is clustered at mechanotransduction hubs, i.e. protein complexes specialized for this purpose. Two examples of such hubs are on the one hand focal adhesions at the plasma membrane, which mediate signaling of the cellular inside with the outside matrix, and on the other hand the kinetochores, which control the proper segregation of chromosomes during cell division. In this work, I primarily used molecular dynamics simulations to investigate one protein from each of these two mechanotransduction hubs to further decipher their mechanisms for transducing mechanical signals. For the crucial focal adhesion component Integrin-linked Kinase (ILK) I elucidated a non-conventional function of ATP-binding to the pseudokinase ILK. ATP promotes the structural stability of ILK and allosterically influences the interaction between ILK and its binding partner parvin, which leads to enhanced mechanoresistance of the ILK:parvin complex. Cell-level experiments from collaborators demonstrated that these features result in focal adhesion stabilization and proper traction force buildup, whichmanifests itself in efficient cell migration. Combined, these results suggest that ILK, stabilized and altered by the presence of ATP, might be capable to function as an active mechanotransducer. The partially disordered inner centromere protein (INCENP), on the other hand, is a passive participant in mechanotransduction at kinetochores. I detected that its disordered region transitions from globular to coil states in response to phosphorylation, which considerably tunes its length and may influence its phase separation properties. These features would allow INCENP to act as length-variable tether to regulate the activity of the chromosome segregation kinase Aurora B by controlling Aurora B’s access to targets in response to kinetochore tension. My work thus sheds light on two widely different mechanisms by which non-enzymatic scaffold proteins are involved in mechanotransduction. In this way, we are expanding our palette of the manifold principles of mechanical signaling and thereby coming closer to grasping the complexity of cells.
The advantages of carbon-ion pencil beam radiotherapy imply an increased sensitivity of the dose distribution in the patient to any changes in the patient geometry, such as internal anatomical changes or patient misalignment. This can lead to a deterioration of the dose distribution within the patient. Monitoring methods of the internal patient’s dose distribution for carbon-ion beam radiotherapy are therefore of great importance to early detect possible under- or over-dosage in the patient, eventually, reduce the tumor safety margins applied around targeted tumor volumes and thus decrease the delivered dose in healthy tissues. Up to now, several non-invasive in-vivo ion-beam monitoring methods have been developed. These are mostly based on the detection of different kinds of secondary radiation, such as annihilation-photons from β+ emitters, prompt photons, or prompt charged nuclear fragments, emitted from a patient during the treatment delivery. These secondary radiations are the results of nuclear interactions of the primary treatment beam with the irradiated tissue. They potentially carry valuable information about the primary treatment beam range, position, or intensity in the patient. However, so far none of the monitoring methods has reached sufficient maturity for a wide application in clinical routine. This thesis aimed to develop methods for detection and localization of therapy-relevant geometry variations of 2 mm in head models, mimicking possible inter-fractional changes on the surface or inside patients’ heads. In contrast to previous research which concentrated on single stationary pencil beams, this thesis was focused on entire therapy-like treatment plans composed of thousands of single pencil beams with low numbers of primary-ions and irradiated under clinic-like conditions in terms of dose, dose rate, and tumor volume. In this thesis, methods were based on the detection and tracking of charged secondary nuclear fragments (secondary ions) emitted from the patient during carbon ion radiotherapy delivery. Subsequently, methods for analysis and interpretation of the measured secondary-ion paths (tracks) were developed. The developed radiation detection methods exploited the capabilities of a novel mini-tracker, based on the Timepix3 technology developed at CERN and positioned behind the patient. The deadtime-free data acquisition enabled a gapless recording of all impacting secondary ion tracks. Moreover, it enabled synchronization of the data with the beam application monitoring system, and thus assign each measured secondary ion with its respective pencil beam, opening entirely new research possibilities. The experiments were performed at the Heidelberg Ion-Beam Therapy Center (HIT), closely mimicking clinic-like conditions. Single fields of carbon-ion treatment plans with a prescribed fraction dose of 3 Gy (RBE) were used to simulate treatments of spherical tumor volumes in the used head models. Two types of head models were used: a homogeneous plastic cylinder and an anthropomorphic head phantom composed of real bones and tissue-equivalent materials. Secondary ions exiting the head models during irradiation were detected with a mini-tracker composed of two small (2cm²) parallel Timepix3 detectors placed downstream of the head with a certain angle with respect to the beam axis. Inter-fractional changes were modeled by adding or removing 2-mm-thick slabs positioned in front or inside the targeted head models. Within the thesis, it was demonstrated that the developed method for the analysis of the measured track distributions, taking into account the actual time-dependent position of the pencil beam, approximated the measured position of the secondary ion creation in the head model significantly better than the methods developed up to now. By using this method, surface changes down to 1 mm were found to be detectable even for the anatomical head phantom. Internal changes of 2-mm-thickness extending over the whole lateral tumor dimension (wide changes) were found to be detectable for all investigated positions between the dose plateau and the distal end of the tumor. The significance was at least 3 standard deviations for a single mini-tracker and of at least 9 standard deviations when using 8 mini-trackers at 30°, as it is planned for the future. Correct localization of all the studied changes was achieved within 6.3 mm of their actual position. This is sufficient to provide information to the clinicians about the part of the dose distribution which is affected. The detection of 2-mm-thick changes affecting only a part of the tumor (narrow changes), required the development of a new method based on the additional information on the lateral pencil beam positions. With this technique, internal 2-mm-thick changes as small as 10 mm in diameter placed in front of the tumor, were demonstrated to be detectable with a significance of almost 2 standard deviations. This technique makes the developed monitoring method sensitive to the lateral position of the cavity and thus reaches the third dimension. Positions of the mini-tracker closer to the beam axis were found to provide higher detection efficiencies due to the larger amount of data, but also lead to larger geometrical uncertainties and lower localization accuracies. At larger angles, the accuracy of the change localization was found to be better. For future measurements, multi-angle detection systems are recommended to maximize both detectability and localization accuracy. Finally, the applicability of the monitoring of carbon-ion pencil beam delivery in a real patient treatment was demonstrated by designing a patient-friendly measurement system that was shown to be safely used in a clinical environment. After investigating the influence of the developed system on the beam delivery, and with the fulfillment of all clinical and safety requirements, the integration of this system into the clinical workflow of the HIT facility was achieved. With this detection system, the first measurement of a real patient irradiation fraction was performed. The amount of measured data was sufficient to determine a secondary-ion emission profile along the depth of the patient’s head. And a differentiation between pencil beams with a 1 cm range difference was demonstrated. In conclusion, this thesis presents novel methods for carbon ion treatment monitoring of external and internal patient geometry changes in the head based on secondary ion tracking, allowing detection changes down to the clinically desired 2 mm. The designed monitoring system was proven to be well incorporable into a clinical workflow. Thus, the presented work paves the way towards monitoring inter-fractional changes along the beam direction during carbon-ion beam therapy and builds the basis for the upcoming clinical trial at the HIT facility.
With the rapid development of modern computational techniques, more complex systems have been found to have their global organization principles. In this thesis, we aim to establish a method to systematically unravel chromosome organization principles, which can serve as a general framework for the analysis of 3D genome architecture and other systems.
We start the analysis with crucial physical properties. We compute the contact probability curve for different polymer models and conclude that the asymptotic behavior of the contact probability curve does not depend on the definition of contact. Moreover, the effect of bending rigidity and compartmentalization is examined. The persistence lengths for homogeneous and heterogeneous semi-flexible self-avoiding walks are computed, and it is observed that the persistence length in the heterogeneous case is systematically smaller than in the homogeneous case.
To access genome-wide organizational patterns, experimental nucleosome positioning data for Candida albicans are investigated. Specifically, by performing hierarchical clustering on the auto-correlation function of the data, repeated patterns are observed across the entire genome, which supports a classification beyond the typical categories of heterochromatin and euchromatin.
In addition to observing the patterns, we successfully develop a quantitative characterization of intra-chromosomal organizational structure by extracting the inter-nucleosomal potential. These effective potentials capture the interaction between nucleosomes that incorporates the dynamics of related complexes.Moreover, an essential thermodynamic property, namely isothermal compressibility, is computed from the potential. By applying k-means clustering to potential parameters and thermodynamic compressibility, genome-wide clustering result is obtained, and information that leads to the genomic mechanical code is collected.
Finally, we focus on patterns of local structures. The organization principles of the CTCF (abbreviation for nucleotide sequence CCCTC-binding factor) are revealed. The averaged nucleosome frequency near CTCF binding sites is computed, and the corresponding spatial structure is observed for the first time.
Super-resolution techniques have enabled fluorescence microscopy to surpass the diffraction limit of light and resolve nanoscale biological structures with molecular specificity. As with other microscopy data, quantitative analyses of super-resolution images have enabled great insight into the underlying architecture of many macromolecular structures. However, this is still a challenging process, especially in the field of single-molecule localization microscopy (SMLM). Unlike pixelated images yielded by most microscopy techniques, SMLM data is composed of a list of fluorophore coordinates and their specific positional uncertainties. Therefore, applying pixel-based approaches to SMLM data requires image rendering, which can cause loss of information and can complicate the analysis. These drawbacks can be mitigated by using coordinate-based approaches. However, currently available coordinate-based approaches in SMLM are typically only applicable to simple geometries or require identical structures. These approaches do not support a basic task in structural analysis: postulating a model with a suitable underlying geometry to probe key parameters in a biological structure. Here, I present LocMoFit (Localization Model Fit), a new framework for fitting a parameterized geometric model to SMLM data at the level of localizations. Based on maximum likelihood estimation, LocMoFit extracts meaningful parameters from individual structures and can select the most suitable model. Using the nuclear pore complex, microtubules, and clathrin-mediated endocytosis (CME) as examples, I demonstrate the application of LocMoFit in in situ structural biology for extracting descriptive parameters of complex, heterogeneous and even dynamic structures. Beyond that, I further showcase applications including assembling multi-protein distribution maps of six nuclear pore components, calculating single-particle averages without any structural prior, and reconstructing the progression of endocytosis - a highly dynamic process - from static snapshots. On the one hand, the quantitative analysis allowed to address a long-standing controversy of how the clathrin coat is rearranged during CME in mammalian cells. On the other hand, the dynamic reconstruction of representative endocytic proteins over the endocytic progression shows the potential of revealing previously unknown nanoscale features that may be associated with force generation during CME in yeast. To ensure all these functionalities are accessible, I implemented LocMoFit as open-source and provided instructions and model templates. A simulation engine and visualization routines are also supplied for users to examine the plausibility of their own analysis, which is also what I used to validate the robustness of the framework in this work. With these, I believe LocMoFit will enable any user to extend the information that can be faithfully extracted from SMLM data.
One major challenge of modern-day theoretical physics concerns the extension of our standard models for particle physics and cosmology. A motivation therefor arises from several problems and tensions that these models face and which suggest their modification. In this thesis we utilise distinct approaches to tackle some of these issues from different perspectives. In a classical field-theoretic approach, we first consider an extension to the \textLambda CDM model called coupled dark energy. Characteristic for this scalar-tensor theory, which has been shown to possess the ability to alleviate the (in-)famous Hubble tension, is an intrinsic coupling within the dark sector of the universe. We will demonstrate that under certain conditions this model can give rise to a novel transient regime of weak gravity. This may help to solve or at least alleviate the $\sigma_8$ tension of the \textLambda CDM model. We then shift to the paradigm of string theory, which presumably provides the required ultraviolet completion of gravity including the other fundamental forces, and assess its consequences on two extensions of standard-model physics. The first is constituted by the postulation of a new particle, the QCD axion, which arguably represents the most prominent way to solve the strong CP problem. In string theory, there are many candidates for this new particle and we investigate phenomenological consequences of such a stringy realisation of the axion. In particular, we find a novel way to seemingly solve the notorious issue of too much dark radiation, which is a generic prediction of these constructions, via a fast decay channel of the internal volume into standard-model Higgses. Even though we ascertain that eventually the dark radiation problem presumably re-appears due to the altered cosmological setting, we are confident that our results will prove to be helpful for future constructions. The second extension to standard-model physics we consider is a cosmological one, namely quintessence. With regard to the recently postulated de Sitter-swampland conjecture, we analyse the realisability of such a dynamical form of dark energy in a stringy context. Taking into account several phenomenological requirements, we identify two major challenges that need to be overcome: a so-called light-volume problem implying a very light internal-volume modulus, that would give rise to inadmissible fifth forces, and a novel F-term problem, which emerges from the fact that the required supersymmetry breaking scale raises the resulting scalar potential and hence the effective vacuum energy to a value that is parametrically above the observed one.
Volcanic gases are part of the fundamental geochemical cycles on Earth. They provide information on the planet’s interior and influence the climate and the oxidation state of the atmosphere. However, there remain severe inconsistencies between field observations and models within the field of volcanic gas analysis. This cumulative thesis aims to improve the understanding of volcanic degassing processes by combining three different but related approaches: (1) A model for the chemical kinetics within the early turbulent mixing process of hot magmatic gases with atmospheric air is developed. It questions conventional approaches that assume thermodynamic equilibrium during the gas emission phase and, hence, has severe implications for current interpretations of volcanic gas measurements. (2) A high-resolution spectrograph is conceptualised and developed. The resolving power of ca. 100000 exceeds that of conventional field-deployable instruments by more than two orders of magnitude. Its high light throughput and mobility enables a range of new volcanic measurements, such as the quantification of the hydroxyl radical, which is an important intermediate species in hot volcanic gases. (3) A novel imaging technique for volcanic trace gases is developed. It significantly enhances the accuracy of volcanic volatile flux quantification and shows great potential for spatially resolving the still poorly constrained halogen conversion processes within volcanic plumes. Prototypes of both instrument developments demonstrate their anticipated performance in field measurements. The techniques introduced in this thesis also exhibit extensive potential for further atmospheric remote sensing applications including improved measurements of greenhouse gases, air pollutants, atmospheric oxidants, or plant fluorescence.
Despite global efforts for its elimination, malaria continues to be a major infectious disease worldwide. In 2020, more than 240 million cases and more than 600 000 deaths were reported. This represents a setback in the progress observed in the past decades, which had also reached a plateau in the past few years. This data also shows that the milestones set in the World Health Organization’s Global Technical Strategy for Malaria 2016-2030, a plan adopted by the United Nations, have not been met. With an increase in mosquito resistance to insecticides and parasite resistance to antimalarial therapies as main biological threats to malaria control, there is a dire need of new medicines with novel modes of action that are refractory to resistance development. SC83288 is a promising clinical candidate for the treatment of severe malaria that has a novel structure. With high in vitro activity against multiple P. falciparum strains and no evidence of cross-resistance with other known antiplasmodial drugs, the compound appears to have a novel mode of action as well. The main objective of this doctoral thesis was to gain a better understanding of the antimalarial mechanism of SC83288 and to identify its molecular targets. To this end, a wide range of methodologies were employed. The analysis of the potential relationship of the compound with antifolates led to the observation that it has an affinity for ATP-binding sites, such as those present in kinases. An untargeted metabolomics study revealed that upon treatment with SC83288, parasites accumulated products of the metabolism of AdoMet through a pathway that has not been reported before in P. falciparum. In addition, treated parasites presented lower levels of phosphatidylserine lipids and intermediates of the Kennedy pathway for the synthesis of phosphatidylethanolamine. Live cell imaging showed that early-treated parasites were not able to undergo DNA replication and displayed membrane disruptions accompanied by cell death. A label-free proteomic approach revealed that SC83288 interacts with a number of parasite proteins, from which four were identified as potential targets: fumarate hydratase, protein kinase CK2, DNA replication licensing factor MCM2 and cytosolic [Fe-S] protein assembly protein CIA1. An analysis of the transcriptional response of P. falciparum to SC83288 revealed a major downregulation of transcription, and a correlation to the gene transcription profile elicited by methylene blue. Taken together, these results led to the formulation of the hypothesis that the main target of SC83288 is the plasmodial enzyme fumarate hydratase. It is additionally proposed that the compound has multiple targets, which may include CK2, MCM2 and CIA1. Although further experimental evidence is needed to validate this hypothesis, the results represent a substantial leap towards the elucidation of the mode of action of SC83288.
We study a compact family of totally elliptic representations of the fundamental group of a punctured sphere into PSL(2,R), discovered by Deroin and Tholozan and named after them. We describe a polygonal model that parametrizes the relative character variety of Deroin–Tholozan representations in terms of chains of triangles in the hyperbolic plane. We extract action-angle coordinates from our polygonal model as geometric quantities associated to chains of triangles. The coordinates give an explicit isomorphism between the space of representations and the complex projective space. We prove that they are almost global Darboux coordinates for the Goldman symplectic form.
This work also investigates the dynamics of the mapping class group action on the rela- tive character variety of Deroin–Tholozan representations. We apply symplectic methods developed by Goldman and Xia to prove that the action is ergodic.
Contact sites between membranes of different cellular organelles are involved in a wide variety of biological processes. While many of the key factors involved in their formation have been identified and many proteins that act primarily at these contact sites have been studied extensively, a comprehensive picture of both the architecture but also the function of these contacts is still missing. This lack of knowledge is to a large degree due to the small dimensions of these interfacing membranes, which make their identification and analysis using conventional methods rather challenging. This work establishes a proximity-labelling assay based on a split biotin ligase connected to known protein tether-pairs at three distinct contact sites of the endoplasmic reticulum (ER) with lysosomes, mitochondria and endosomes, respectively. Proteins that were biotinylated at these membrane contact sites were then identified by mass spectrometry based proteomics. The resulting list of identified hits contained both novel proteins, as well as proteins that had previously been reported to fulfill a role at membrane contact sites, highlighting the efficacy of the assay. The function of one such protein, GRAMD1B, was investigated further. This protein is essential for non-vesicular cholesterol import to the ER from the plasma membrane, but was also found to locate to contact sites with lysosomes and interact with the major lysosomal cholesterol exporting protein, NPC1. Results of the split biotin ligase assay confirmed a localization at lysosome-ER contact site, which led us to further study its function at this contact. Using a lysosomally prelocalized, modified cholesterol probe in GRAMD1B-overexpressing cells, pulse-chase measurements of cholesterol esterification suggested an involvement of GRAMD1B in two separate lysosome-to-ER transport processes with different temporal scales: A fast and direct cholesterol transport at lysosome-ER contacts dependent on NPC1 as well as an import from the plasma membrane to the ER at slower timescales. This was substantiated by the fact that the impact of GRAMD1B-overexpression on the faster transport process was reduced by NPC1-deficiency, while trapping of cholesterol at the plasma membrane only reduced the effect at later time points. In addition, live-cell microscopic analysis showed GRAMD1B’s ability to recruit ER tubules to cholesterol-laden lysosomes and confirmed a dependency on the presence of NPC1 in the lysosomal membrane. This further strengthens the hypothesis of a dual role of GRAMD1B in the transport of cholesterol to the ER, with spatial coupling of two cholesterol transporters at the lysosome-ER interface. Overall, this work shows that proximity labelling using split biotin ligase fragments fused to known contact site tether pairs is a powerful tool to identify proteins acting at organelle contacts. One such protein, GRAMD1B, acts in multiple routes of subcellular cholesterol trafficking, where this work focusses on its function in direct lysosome-to-ER transport of sterols. The applied methods, particularly the use of modified cholesterol probes prelocalized to lysosomes to observe lysosomal egress, as well as the insights gained from studying GRAMD1B’s actions at lysosome-ER contacts, will contribute to a more comprehensive understanding of subcellular cholesterol homeostasis, which, in turn, is relevant for a wide variety of physiological and pathophysiological processes.
The important role of RNA binding proteins (RBPs) in regulating the fate and functions of RNAs has led to the development of transcript-specific as well as transcriptome-wide techniques allowing an unbiased and comprehensive identification of RBPs. These methods have extended our knowledge of the extent of RBPs in a cell, and studying the roles of these newly identified RBPs in cellular processes has provided us with novel insights into the RNA binding mechanisms, functions and regulation of RNA binding proteins.
For my PhD work, I assessed the RNA binding functions of two proteins identified in high-throughput screens. The first protein is the Fragile X Mental Retardation protein (FMR1), identified in a transcript-specific pulldown targeted at the Drosophila maternal mRNA oskar. I show that FMR1 is a bona fide component of the oskar RNA-protein complexes that interacts with the oskar 3’UTR in vivo. FMR1 positively regulates Oskar protein levels in the oocyte, without any effect on oskar RNA levels. Oskar protein nucleates germ plasm assembly and germ cell formation in the embryo, and the reduction in Oskar protein levels leads to a reduction in the number of pole cells formed in embryos knocked down for FMR1. Finally, I tried to determine how FMR1 regulates translation, with roles identified as both a repressor and activator of translation. FMR1 contains two types of RNA binding domains: two KH domains and a C-terminal RGG box. I show that, in vitro, FMR1 activates translation through the KH domains and requires the C-terminal RGG box for repression of translation. I have thus identified a new role of FMR1 in germline development in Drosophila melanogaster, and also a putative mechanism of how FMR1 performs antagonistic functions in translation regulation. The second protein I studied is the microtubule binding protein EB1, identified as a putative RNA binding protein in a transcriptome-wide RNA interactome capture study performed in Drosophila embryos. Preliminary data showed that EB1 binds to polyU25 RNA in vitro, and uses the same binding surface for interacting with microtubules and RNA. I show that EB1 binds to microtubules and RNA in a mutually exclusive manner in vitro. Furthermore, I performed a RIP-seq experiment to identify the in vivo targets of EB1, but failed to validate the interaction of any of the top candidates with EB1 in vivo. This does not, however, negate a role of EB1 as an RNA binding protein altogether, as RNA might be regulating the functions of the protein, and this would require further investigation.
Lactic acid bacteria are a group of bacteria that share the characteristic of lactate fermentation, and are in particular focus of microbiological research not only because of their involvement in human health, but also due to their role in the food industry. On the one hand, they can be used as probiotics, contributing to healthy micro flora in the human body. On the other hand, they can take part in the production of fermented foods and flavour development. En- terococcus faecalis and Streptococcus pyogenes are two lactic acid bacteria that cause several infections in the human body. Therefore, they have been in the focus of clinical studies for the past few decades. The rising trend of resistance to multiple antibiotics makes the treatment of the infections caused by theses two pathogens very hard. To overcome this progressive trend of resistance, it is important to find novel drug targets in these pathogens. In the present study, I investigated the metabolic characteristics of these two pathogens using an integrative method, comprising multi-omics data integrated with the respective genome-scale metabolic models un- der the conditions comparable to different tracts in the human body. First, I investigated the effect of glutamine auxotrophy on the metabolic adjustments of E. faecalis (in the case of a ∆glnA mutant) in response to a change in environmental pH, using an integrative approach combining metabolic and proteome data with genome-scale modelling. The result suggested that the higher energy demand in the ∆glnA mutant of E. faecalis is most likely due to the lack of control on glutamine transport system as a result of the absence of glnA in the mutant. In the next part, I developed a method for functional analysis of the solution space of the genome-scale metabolic models. This method employs random perturbation to discover the reliability of flux distribution in the network. Additionally, it allows to find out which type of experimental data is most effective in limiting the solution space when the data are used as constraints. Finally, I generated tract-specific genome-scale metabolic models for E. faecalis and S. pyogenes in or- der to find tract-specific drug targets in their metabolic networks. I used multi-omics profiles (metabolic, transcriptome and proteome data) obtained under the conditions comparable to nat- ural physiological condition in the human body, namely root canal, unrinary tract and plasma, and used the data to constraint the respective genome-scale metabolic models. The models were used to find potential drug targets using different levels of threshold for metabolic flux values and growth rate of the bacteria. The results suggested that there exist potential drug targets in different subsystems in the metabolic network, from central carbon metabolism to transport system. The presented profiles of drug targets have to be validated experimentally in order to be used for the development of new treatment approaches.
A plants capacity to continuously generate new organs and tissues is fueled by pluripotent stem cells, embedded in specialized niche tissues, called meristems, which provide strict regulation of stem cell fate to balance the need for cell proliferation and cell differentiation. In the Arabidopsis shoot apical meristem (SAM), maintenance of a stable stem cell population largely depends on the non-cell autonomous activity of the homeodomain transcription factor WUSCHEL (WUS). WUS mRNA is expressed in the organizing center (OC) within the L3 of the shoot meristem. From here, WUS protein moves several cell layers upwards towards the overlying L2 and L1 and into the stem cells in the central zone (CZ). While it has been shown that short-ranged cell-to-cell movement of the WUS protein, via cytoplasmic bridges called plasmodesmata, is essential for WUS function and shoot stem cell maintenance, many aspects of WUS mobility, including its mechanism and regulation, remain unclear or under debate. Here, I chose a quantitative approach to characterize WUS mobility: For this, I have developed a semi-automated analysis pipeline for layer-specific quantification of fluorescence intensity along the apical-basal axis of the SAM, taking into account meristem curvature. I have then used this analysis in combination with high-resolution live-cell imaging on dissected, unfixed shoot apices to systematically characterize the mobility of differently tagged WUS fusion proteins in complementation lines as well as transcriptionally inactivated mutant alleles in wildtype meristems. From these data, I hypothesize that the mechanism for WUS mobility in the SAM involves a component of active transport that is not directional but regulated via protein retention in the L3. Considering previous studies, my data further indicates that specificity of active WUS movement may be mediated by the WUS homeodomain (HD) and suggests the presence of regulatory sequences not only in the unstructured region between WUS HD and WUS box, but also at the N- and C-terminus.
Proteases have evolved in all kingdoms of life with the capability to catalyze irreversible and highly regulated hydrolysis of peptide bonds. Intramembrane proteases share common features as such enzymes are polytopic membrane proteins with their active sites buried several Ångstrom deep within the lipid bilayer. Although the list of physiological substrates is steadily growing, an important remaining question is whether these proteases have a conserved substrate recognition mechanism. In the present thesis, I focused on the human mitochondrial rhomboid protease PARL and its substrate recognition and cleavage mechanism on the example of PGAM5. Dysfunctional mitochondrial quality control disturbs cellular energy metabolism and programmed cell death, triggering disruptive diseases such like neurodegeneration. Genetic deficiency of PGAM5 causes a Parkinson’s-like movement disorder in mice. PARL serves as a safeguard of mitochondrial homeostasis and is processing PGAM5 when the mitochondrial membrane potential is disrupted. Until today, PGAM5 substrate determinants have not been rigorously investigated. Here, I characterize for the first time several cleavage determinants in PGAM5 on basis of mutational studies in human tissue culture, in vitro proteolytic assays with purified recombinant proteins and in a collaborative project using CD spectroscopy and liquid-state NMR. I can show that the N-terminal portion of the PGAM5 TM domain plays a special role and is a critical determinant for PARL-catalyzed processing. Interestingly, besides cleavage resistant forms, I obtained PGAM5 mutants with highly increased cleavage by PARL uncoupling it from its native regulation. NMR analysis revealed that the PGAM5 TM domain harbors two split helices zoned by a hinge-like loop and mutations within the N- or C-terminal helix suggest an altered interaction with PARL or bending into the PARL active site with subsequent modified intramembrane cleavage. Moreover, I found that a balanced net charge in the C-terminal juxtamembrane region prevents premature PGAM5 from PARL-catalyzed cleavage so that cleavage-resistant PGAM5 oligomers can assemble upon mitochondrial import. Under mitochondrial stress after disruption of the membrane potential with CCCP, I propose a model in which PGAM5 oligomers at the inner mitochondrial membrane disassemble into monomers by an unknown mechanism leading to efficient cleavage by PARL in order to trigger PGAM5’s downstream activities. Taken together, my findings indicate that the substrate recognition mechanism of PARL relies on a membrane-potential-dependent oligomeric switch and different substrate features with hierarchical importance.
Wnt signaling pathways are a set of signal transduction cascades which are activated through the interaction of Wnt proteins with so-called Frizzled receptors [1-3]. These pathways are critically involved in many biological processes such as embryonic development, regeneration, organogenesis, cell division, cellular and tissue homeostasis, among many others [3, 4]. In addition, alterations of these signaling pathways have been linked to various types of diseases such as cancer [5-7], familial tooth agenesis [8], bipolar disease [9], Alzheimer's disease [10], and cardiac valve formation [11]. Wnt signaling components are accordingly promising drug targets to treat these diseases. Wnt pathways are probably among the best characterized receptor-ligand signaling pathways. Wnt proteins are therefore key players in biological signaling and promising drug targets to treat a plethora of diseases. Although several proteins involved in Wnt trafficking and secretion have been identified over the past years, little is known about the contribution of different lipid species into these processes. The trafficking and secretion of Wnts could be modulated by the type and number of acyl species covalently linked to Wnt proteins. Currently, the best described acyl modification is the palmiteoylation of a serine residue located around amino acids 205-215 mediated by the ER-resident O-acyltransferase Porcupine is responsible for this process. This lipid modification has been described for Wnt3a, Wnt5, xWnt8, and Wnt1, and it has been assumed to also take place in other members of the family of Wnt proteins. Despite the extensive data available, the debate around the lipid-modified amino acids in Wnt proteins has not yet reached a consensus. Recent results from O. Voloshanenko (M. Boutros group, DKFZ, Heidelberg, Germany) suggested that there may be other amino acid residues in Wnts that are lipidated, apart from this canonical serine residue. Furthermore, the specific saturation of the acylated chain that binds to Wnt remains inconclusive.
In this thesis, I aimed to define other putative acylation types and lipid-modified sites in Wnt proteins and to determine the role of these alternative lipidations in Wnt secretion and signaling. Furthermore, I evaluated the impact of Wnt signaling and Wnt secretion on the lipidome of HEK293T and HCT116 cells. To achieve this, I employed a combination of chemical biology tools, mutagenesis experiments, and mass spectrometric measurements. In particular, I focused on Wnt11 as a working model. I studied its acylation using clickable lipids such as palmitic acid alkyne (cC16:0) and palmitoleic acid alkyne (cC16:1n-7). One of the early observations was that palmitoylation and secretion of Wnt11 were not wholly abolished in Porcupine knockout cells or some mutant variants of Wnt11. However, these observations seem to depend on the type of clickable fatty acid used. Our results suggest a lipid modification of Wnt11 at serine 215 via the monounsaturated fatty acid cC16:1n-7, consistent with the previously predicted models. However, lipid modification with the saturated fatty acid cC16:0 showed variations in the experimental replicates, which did not fully resolve whether Wnt11 contains another modification site. Importantly, our experiments stress that unsaturation is a key feature for Wnt acylation. The relevance of covalent lipid binding for the secretion and signaling activity of Wnts has also been assessed. It was demonstrated that lipidation is essential for the signaling activity of Wnt11 but is not strictly necessary for its secretion. In addition, an impact of Wnt protein expression on the overall cellular lipidome of HEK293T and HCT116 cells has been tested, yielding preliminary observations on the crosstalk between the Wnt signaling and the overall cellular lipid homeostasis. This study is expected to contribute to our understanding of how post-translational lipid modifications influence Wnt cellular secretion, signaling and, conversely, how proteins of the Wnt signaling pathway affect the lipid composition of cells.
This work introduces two experimental approaches to control quantum dynamics in molecules, employing core electrons as messengers. A laser source providing ultrashort pulses has been developed to access the timescale of electronic and structural dynamics inside molecules. Pulses of few-cycle durations in the 1 μm to 2 μm short-wavelength infrared (SWIR) spectral region provide intensities up to 10^15 W/cm^2. In combination with a vacuum beamline, this experimental setup allows for ultrafast laser control of molecular dynamics probed by core-electron transitions via x-ray absorption spectroscopy (XAS). The first experiment investigates the manipulation of molecular electronic structure. Here, a soft x-ray (SXR) pulse probes simultaneously to an SWIR pulse of variable intensity. The measured intensity dependent absorbance changes in SF6 reveal an increased effective electronic-exchange energy. This demonstrates the alteration of this purely quantum-mechanical component of the electron-electron interaction for the first time. In a second experiment, an SWIR pulse induces coherent molecular vibrations with amplitudes of ten times the diameter of the nucleus. Subsequently, a time-delayed SXR pulse probes the bond-length changes via core-level transitions. This enables an unprecedented 14 femtometer precision which paves the way for site-specific vibrational metrology in gas-phase molecules. Overall, these results enable ultrafast chemical control on a quantum level.
Keeping balance is essential for the survival of mammals. When facing challenges, these animals rely on a neural system that promptly coordinates physiological and behavioral adaptations that are triggered to maintain homeostasis. In such context, neuroactive peptides, including neuropeptides and some peptide hormones, have been proposed to play an important role in the modulation of neural activity. These molecules have been implicated in the control of several processes, such as sleep, reproduction, and feeding, and are also believed to influence thermoregulation. The mammalian core body temperature (Tcore) is maintained by the thermoregulatory circuitry, which is orchestrated by the preoptic area (POA) of the hypothalamus. Different lines of evidence have suggested a role for neuroactive peptides in thermal balance, especially acting at the POA. Despite this, the molecular mechanisms that modulate Tcore during thermal challenges remain unclear.
Here, to investigate which neuroactive peptides are released at the level of the POA during thermal challenges, an unbiased screening of samples collected in vivo from male mice was performed. For this, first, to enrich neuroactive peptide detection, an optimized protocol was established for the in vivo sampling and the sample preparation for liquid chromatography and tandem mass spectrometry (LC/MS-MS) analysis. Next, the optimized protocol was applied on samples collected from the POA of freely behaving adult male mice during three different types of thermal stimuli. These included the chemogenetic activation of a subpopulation of preoptic thermoregulatory neurons expressing the vesicular transporter VGLUT2 (POAVGLUT2), which induced hypothermia, and the ambient warm and cold temperature stimulation. The results suggested that, in total, peptides deriving from 18 neuroactive peptide precursors were detected to be differentially released at the mouse POA during thermal challenges. Among these precursors and respective peptides, somatostatin and secretogranin 1 were differentially detected in samples collected during the stimulation of POAVGLUT2 neurons compared to the control group. Additionally, in comparison to control conditions, the results suggest a differential release of peptides from the precursors of orexins, nociceptin, and nucleobindin 1 in warm-exposed mice, as well as, thyrotropin-releasing hormone (TRH), and diazepam binding inhibitor (DBI) in animals peripherally stimulated with cold. Moreover, the verification of the unbiased screening focused on two candidates, orexin and adiponectin. For both, in vivo samples collected either during warm or cold peripheral stimulation were evaluated with ELISA. In addition, further experiments investigated a potential role of orexins in thermoregulation, by focusing on the POA distribution of orexin receptor 2, the influence of orexin-A on preoptic neurons, and the injection of orexin receptor antagonists into the POA. Results of these experiments may have provided further evidence for the engagement of pro-orexin derived peptides in Tcore control. Additional studies are required to define these and the other peptides as modulators of preoptic thermoregulation. Nonetheless, the list of candidates of thermoregulatory peptides provided by this thesis could contribute to a better comprehension of the molecular control of POA during adaptation to thermal challenges. Given the vital importance of maintaining the Tcore in mammals, such candidates could potentially also be implicated in health and disease.
Trypanosoma brucei (T. brucei) is an extracellular unicellular pathogen, which is the causative agent of African sleeping sickness in sub-Saharan Africa. It has a two-host life cycle in its vector tsetse fly (Glossina sp.) and its mammalian hosts. The work of this thesis focuses on the metacyclic stage of the parasite, which is the mammalian infectious form injected by the fly, and the metacyclic Variant Surface Glycoprotein (mVSG). VSG is expressed in the metacyclic and bloodstream forms. The latter differentiate from metacyclic in the mammalian host and are capable of evading the host immune system by constantly changing the expression of its surface proteome makeup. Despite a vast number of VSG genes present in the genome repertoire (~2000 genes and pseudogenes), only a specific subset of five VSGs, called metacyclic VSGs (mVSGs), are expressed in the metacyclic stage of the Lister 427 strain. In this study, we are comparing mVSGs and bloodstream VSGs at the protein structure level. Using structure-based prediction by protein threading, we showed that VSGs in T. brucei are divided into two different structure classes, A and B. We have determined the structure of mVSG 531 and mVSG1954, classified in Class A and B, respectively, by X-ray crystallography. The homodimer structure of mVSG531's Nterminal domain shows the conservation of the three-helix bundle core and the dumbbell-like shape observed in the previously determined VSGs structures in Class A, VSG2 (MITat 1.2) and VSG1 (MITat 1.1), which are expressed in the bloodstream form. The structure of mVSG1954 also shows similarities to VSG3 (MITat 1.3), classified in Class B and expressed in the bloodstream form. Like VSG3, it exists as a monomer in the crystal asymmetric unit, but also and forms non-crystallographic trimers. While the VSG3 is O-glycosylated, there is no evidence of O-glycosylation in the mVSG1954 structure. The results address a long-standing question of whether the mVSGs were similar to or different from the bloodstream VSG coat proteins, showing that structurally and biochemically, the two life-stages classes of VSGs are very similar.
The notion of harnessing the patient’s intrinsic immune system to target tumor cells has sparked the development of immunotherapy as a promising new approach to treat cancer. However, only a minority of patients benefit from currently available immunotherapeutic approaches, as many tumors escape immune attacks and develop immune evasion mechanisms. Hence, it is crucial to identify cellular factors that influence the success of immune cell-mediated tumor clearance.
In this project, I developed a genetically defined autochthonous liver cancer mouse model with conditional neoantigen expression for the investigation of neoantigen-mediated immune responses and immunotherapeutic treatments. In addition to that, I used primary neoantigen-expressing tumor cells and antigen-specific T cells to establish a co-culture assay in order to study mechanisms of cytotoxic T cell-mediated killing. This assay was further used to perform a lentiviral CRISPR/Cas9 screen, which identified new tumor cell-specific mediators of T cell-dependent killing, among others Activin A receptor type I (Acvr1). Additional experiments validated the role of Acvr1, a type I receptor serine kinase of the bone morphogenetic protein (BMP) pathway, in T cell-mediated killing. Moreover, I could show that Acvr1 knock-out reduced T cell killing efficiency by downregulating the expression of the death receptor Fas, thus reducing FAS ligand (FASLG)-mediated apoptosis induction.
In summary, this study included the establishment of new in vivo and in vitro model systems for the investigation of neoantigen-specific immune responses and tumor cell clearance. With that, I was able to identify Acvr1 as new mediator of T cell-dependent tumor cell killing and prospective drug target. These findings offer the opportunity to further explore and improve immunotherapeutic approaches to potentially enhance the success rate of immunotherapies in the future.
Cells resize and reshape their organelles in response to changing physiological demands. This thesis focuses on the endoplasmic reticulum (ER), an organelle which, depending on cell type and physiological conditions, displays remarkable adaptability in both size and shape. Although much is known about the role of proteins in ER morphogenesis, the mechanisms controlling the biogenesis of its membrane, and hence size, are unclear. In this work, I build upon a genetic screen performed to identify genes involved in ER expansion in the budding yeast, Saccharomyces cerevisiae, and I focus on the poorly characterised ER membrane protein Ice2p. I show that Ice2p is required for and promotes ER expansion, and that it does so independently of the central determinant of ER homeostasis, the unfolded protein response (UPR). To further uncover the molecular role of Ice2p, I explore known genetic interactions and discover that Ice2p opposes the activity of Pah1p, which is a conserved phosphatidic acid phosphatase with a central role in the regulation of lipid synthesis in yeast. Specifically, I show that Ice2p interacts with and inhibits the conserved Spo7p-Nem1p complex, which normally dephosphorylates and activates Pah1p. By showing that Ice2p cooperates with pathways transcriptionally controlling lipid synthesis, and with the UPR to maintain cell homeostasis, I place Ice2 into a broader cellular context. Additionally, I present preliminary data approaching the physiological conditions during which cells use Ice2p-Pah1p to control the size of their ER. Finally, in the last part of the thesis, I follow up on the unexpected observation that the reticulon proteins, which are membrane proteins with a central and conserved role in ER morphogenesis, form cytosolic puncta after prolonged ER stress, a phenotype especially pronounced in cells lacking Ice2p. These data suggest that there are mechanisms controlling the membrane association of reticulon proteins, and as an extension ER shape. Overall, this thesis extends the understanding of mechanisms regulating the biogenesis of ER membrane as well as its morphogenesis and provides the ground for future work not only in yeast but also in higher eukaryotes.
The dynamics of quantum fields in curved spacetime give rise to various intriguing phenomena. Among them is the production of particles in an expanding spacetime. This process is likely responsible for seeding the Universe's large-scale structure, which, in turn, causes the temperature fluctuations in the cosmic microwave background and grows into the distribution of galaxies and galaxy clusters we observed today. In this work, we simulate this process in an ultracold quantum gas. The simulation is based on a novel and particularly straightforward mapping between a mass-less, free, relativistic scalar field in a curved spacetime and the phononic excitations of a Bose-Einstein condensate. Here, the density distribution and speed of sound of the background condensate determine the geometry of the spacetime.
Additionally, this thesis introduces a new ultracold atom machine that creates and controls a quasi-two-dimensional Bose-Einstein condensate of potassium-39. This experimental system combines a high control over the condensate's density with the possibility to dynamically adjust the atomic interaction -- and thus the speed of sound -- via a broad Feshbach resonance. We use this control to implement the two aspects of a Friedmann-Lemaître-Robertson-Walker (FLRW) metric: spatial curvature and the expansion of space.
To demonstrate spatial curvature, we probe wave packet dynamics and show that a harmonically trapped Bose-Einstein condensate approximates a hyperbolically curved space.
For the expansion of space, we perform a global change of the speed of sound. We realize three different power-law expansions, corresponding to accelerated, uniform, and decelerated expansion. For all three, we observe the emergence of fluctuations equivalent to cosmological particle production. To characterize these fluctuations, we compute their correlation function and power spectrum. In the time evolution of these quantities after the expansion, we identify an intriguing feature. It is connected to a complex phase of the produced quantum state and shows a clear dependence on the expansion history. Understanding if and how such a feature can be used in real cosmological observations is an intriguing prospect for future research.
Additionally, a good agreement between our experimental results and analytical predictions confirms that our experimental system simulates the dynamics of a quantum field in a curved and expanding space. This is the starting point for the future investigation of more complex spacetime geometries.
Although type Ia supernovae are widely applied as cosmological distance indicators and contribute significantly to the enrichment of the Universe with iron group elements, their physi- cal nature is not clear yet. The heterogeneous class of type Ia supernovae is divided into several subtypes according to their observational properties, and a whole variety of possible explosion scenarios has been put forward in order to explain these extraordinarily bright events. The focus of this work lies on the numerical modeling of explosions in Chandrasekhar mass carbon-oxygen white dwarfs as well as on the investigation of the nucleosynthesis yields of several explosion chan- nels. To this end, numerical simulations of the explosion, the propagation of radiation through the expanding debris, and the galactic chemical evolution of the Milky Way were conducted. It was found that, in line with earlier studies, deflagrations in Chandrasekhar mass white dwarfs are a viable model for type Iax supernovae, a subluminous subclass of type Ia supernovae. However, the parameter study employing different ignition conditions, central densities, metallicities, com- position and rigid rotation revealed that the very faint end of the subclass can not be reproduced. Furthermore, a set of gravitationally confined detonation simulations has been carried out. In this scenario an initial deflagration is followed by a detonation initiated near the surface of the white dwarf core. It could be shown that the synthetic observables do not agree with either normal or subluminous type Ia supernovae but that objects similar to SN 1991T might be explained by this explosion mechanism. Finally, the nucleosynthesis yields of various different explosion models were analyzed. This study shows that explosions in sub-Chandrasekhar mass white dwarfs with an accreted helium shell can contribute significantly to the abundance of manganese, zinc, and copper in the Universe and should be included in future galactic chemical evolution studies.
In the last 20 years the Proteolysis Targeting Chimera (PROTAC) technology has revolutionized the field of drug discovery. PORTACs are heterobifunctional molecules consisting of a chemical ligand, named in this context warhead, and an E3 ligase recruiter united by a linker. These molecules bring the Ubiquitin Proteasome System machinery into the proximity of a protein of interest through for temporal and reversible degradation without the need of genetic manipulation. In comparison to classical chemical inhibitors, PROTACs provide a new approach to repress the activity of a pathogenic protein, which is independent of its catalytic or enzymatic activity. Because of lack of chemical binders, just a small fraction of the proteome has been reported to be targeted by PROTACs.
To increase the number of proteins prone to be targeted by PROTACs, two strategies are followed in this work, exemplified by two new PROTACs. First, the incorporation of a perfluoroaromatic moiety within the structure of the degrader, which can target the sulfhydryl motif in the cysteine residue of the sequence Phe-Cys-Pro-Phe in a nucleophilic aromatic substitution reaction. This tetrapeptide was inserted into the C-terminus of Cas9 (Cas9FCPF), while the perfluoroaromatic moiety was tether to Lenalidomide, a broadly used ligand and recruiter of the E3 ligase cereblon. The resulting PROTAC-FCPF successfully degraded Cas9FCPF in a time- and concentration-dependent manner, with a Ubiquitin Proteasome System (UPS)-driven proved mechanism with a half-degradation concentration (DC50) of around 150 nM over 24 hours treatment. PROTAC-FCPF was also effective against genetically engineered related proteins dCas9FCPF, Cas12FCPF, and Cas13FCPF, all modified to have the Phe-Cys-Pro-Phe sequence inserted in their C-termini. The second strategy was High-Throughput Screening, out of which the compound O4I2 was identified to chemically induce the expression of the Yamanaka factor Oct4 and, thus, supported the induction of somatic cell reprogramming and maintenance of human induced Pluripotent Stem cells. In a proteomics analysis of cells treated with O4I2, it was observed that the Splicing Factor 3 Subunit 1 (SF3B1) was the main hit, a protein that has been associated to several tumors. To target it, O4I2 was bound to another cereblon recruiter, Thalidomide. The resulting PROTAC-O4I2 successfully degraded SF3B1 in a time- and concentration-dependent manner, driven by the UPS with a DC50 of 244 nM over 24 hours of treatment. Furthermore, the degrader spared other proteins that were also hits during the proteomics analysis, and successfully degraded the mutant SF3B1K700E, present in 55% of patients with myelodysplastic syndromes.
The aim of this work is to establish an overview of the tin isotope ratios of cassiterite and stannite from various mineralized regions in Europe, the Mediterranean and Central Asia in order to assess the possibility to geochemically discriminate tin ore deposits, which could have been exploited in ancient times. The motivation for this study was to eventually relate the tin found in ancient bronze objects to specific tin ore deposits and thus to clarify the origin and distribution of the tin bronze technology in the Bronze Age of the so-called Old World. For this purpose, we determined 413 primary and secondary cassiterite and stannite samples from the major tin provinces in SW England and Ireland, the Saxonian-Bohemian province, the Iberian Peninsula, France, Italy, Serbia, Egypt and Central Asia. The tin isotope compositions were analysed in solution with a multi-collector inductive-coupled plasma mass spectrometer (MC-ICP-MS) in the Curt-Engelhorn-Zentrum Archäometrie in Mannheim. The samples mainly derive from granitic pegmatites and hydrothermal vein mineralizations of tin ore deposits associated with granite complexes in the Variscan and Asian fold belts. Overall, the isotope ratios in primary and secondary cassiterites are highly variable and range from δ124Sn/120Sn -0.82 to 0.85 ‰. This variation is observed in the tin ore samples from SW England which have an average δ124Sn/120Sn of 0.10 ± 0.59 ‰ (2SD). Among the tin provinces of the Variscan fold belt in Europe those of SW England and the Saxonian-Bohemian province (δ124Sn/120Sn = 0.12 ‰ ± 0.37) show the largest variations but the ranges of isotope ratios in both regions overlap to a large extent. Despite the large overlap, cassiterite from Spain (δ124Sn/120Sn = -0.07 ‰ ± 0.35) and France (δ124Sn/120Sn = -0.005 ‰ ± 0.31) tend to have on average lighter isotopic compositions than SW England, the Saxonian-Bohemian province or Portugal (δ124Sn/120Sn = 0.07 ‰ ± 0.40). However, the stannite samples from SW England and the Saxonian-Bohemian province have significantly lighter isotope ratios than the associated cassiterites. The tin ores from Central Asia exhibit the largest total variation of 1.94 ‰ ranging from -1.27 to 0.67 ‰ for δ124Sn/120Sn. This extent of fractionation is observed in cassiterites from Afghanistan and Uzbekistan. Afghanistan with its pegmatitic cassiterite has the lightest isotopic composition of all investigated areas with -0.38 ± 0.84 ‰ for δ124Sn/120Sn and, therefore, stands out as an identifiable source. Similar to the European stannites, the Asian stannites also have significantly lighter isotope compositions. Because of the large overlap and the highly variable isotope composition of cassiterites from all tin provinces a clearcut discrimination based on tin isotope ratios is difficult. But on a more detailed scale within each tin province it is possible to distinguish several mining or granite areas by their Sn isotope composition. However, it is also difficult to distinguish between different mineralization types.
Cancers are often riddled with areas of low oxygen concentration (i.e. hypoxic regions) and these are, in fact, more resistant to treatments such as photon radiation which primarily relies on oxygen for the production of reactive oxygen species (ROS) to induce damage on the DNA. Cancer cells adapt to hypoxia by activating complex changes in their cellular processes, for example, in the oxidant environment and DNA damage response (DDR) as well as modifying their metabolism. The present study showed that hypoxic tumour cells from various organs have strong, albeit different, reducing capabilities and can adequately alter their energy production processes for enhanced survival and resistance to irradiation (IR). Hypoxic lung cancer cells have reduced DNA damages which correlated with their enhanced clonogenic survival. In addition, their antioxidant capacity was upregulated, resulting in a reduced cytosolic H2O2 content. Moreover, nuclear H2O2 induction of DNA double strand breaks (DSBs) was oxygen dependent, highlighting the reliance of photon IR on oxygen to generate ROS for indirect DNA damage. In conclusion, our findings highlight common and/or distinctive traits in different tumours with regions of hypoxia and elucidate the resistant behaviour of these cells, and can therefore possibly provide insight into improving radiation sensitivity
Nematostella Vectensis belongs to the phylum cnidaria which is the closest relative of bilaterians and has become an important model organism for evolutionary development biology. However, because of its high light sensitivity, large size, and constant need to move freely in order to properly develop and survive, in toto live imaging of Nematostella is a major challenge in microscopy. Several important questions, e.g. how animal behavior during muscle hydraulics guides the animal morphology and how neuronal dynamics control muscle movements and body deformations that are necessary for animal development remain unanswered. In the field of microscopy, light-sheet fluorescence microscopy (LSFM) has emerged as a preferred tool to image light-sensitive large samples due to optical sectioning and high-speed functional imaging capabilities. In a conventional light microscope, the specimen needs to be embedded in agarose and placed within a narrow space between orthogonally lying illumination and detection objectives. These constraints however make it impossible to use on Nematostella or other freely moving animals. On the contrary, recently developed, so-called single objective light-sheet microscopes are capable of imaging freely moving animals, but the lateral field of view (FOV) in such existing techniques is limited to 1*1mm2 . To overcome these challenges, in this thesis I developed an oblique plane microscope for imaging freely moving specimens at mesoscopic scales (MesoOPM) that provides light-sheet scanning based rapid volumetric imaging capability with cellular resolution and over 2mm FOV. By utilizing a long working distance illumination objective and placing it at 65 degrees angle to the optic axis of the detection objective, we can maintain the open-top configuration that allows easy sample mounting. Furthermore, the rapid tilt-invariant light-sheet scanning along the image plane of the detection objective is achieved by precisely synchronizing an electrically tunable lens with the galvo scanner in the illumination arm. I performed meticulous optical design optimization of the microscope to maximize the spatial resolution under these conditions. The experimental PSF of the MesoOPM was then measured with diffraction limited beads and found to be 1.62*2.81µm2 in lateral dimensions and the 5.27µm in axial dimension. Finally, the imaging capabilities of the microscope are showcased by imaging the muscle structure and the nervous system of a freely moving live Nematostella at 300 frames per second, covering the entirety of the animal over a FOV of 1.56mm*0.78mm*240µm in two seconds. This is the first-ever report of imaging 3d volume of a live freely moving Nematostella in its entirety using a fluorescence microscope. This technology opens up a whole new direction of imaging an entire freely moving specimen which will allow us to study the interactions between animal behavior and the environment by visualizing the underlying cellular structures, which so far has been a challenge.
Plants dynamically adjust their development and growth pattern to maximize their chances to reproduce even under challenging conditions. The cellular basis for this remarkable phenotypic plasticity are stem cell populations that are maintained throughout the whole life of a plant. Intricate sensing and signaling mechanisms are required to instruct stem cell behavior according to current needs. Consequently, light- and nutrient signals are integrated and modulate the core molecular circuits underlying regulation of the shoot apical meristem (SAM). We know that expression of the stem cell master regulator WUSCHEL (WUS) is modulated by photoreceptor mediated light signaling pathways together with sugar and nitrate. Activity of the evolutionary conserved TOR kinase is required to integrate and relay these signals, but we do not understand how TOR activity is conveyed to modulate WUS expression. In this work, I identified CK signaling in an RNAseq approach as the major downstream effector of TOR activity controlling WUS expression and shoot development. I demonstrate that TOR activity stabilizes trans-Zeatin which is known to be one of the major determinants influencing shoot growth and meristem maintenance. Mechanistically, this is achieved by TOR dependent translational repression of CKX catabolic enzymes, allowing plants to swiftly adjust their growth factor milieu in response to dynamic environments. This study is the first example for TOR mediated translational repression of a catabolic enzyme and thus provides a mechanistic framework integrating the contributions of central metabolism and core developmental regulators towards stemness.
In recent years, the topic of sex differences has rightfully become a focus of scientific research. Current findings suggest sexual dimorphism in the neurophysiological brain pathways that are crucial for drug-seeking and addictive behavior, but how this affects the underlying neurochemical processes, is still widely unexplored. Nevertheless, female subjects have been systemically ignored for decades in the field of microdialysis experiments and the few existing studies using female animals provide only small numbers. Moreover, there is increasing evidence that single preclinical studies often lack reproducibility. Therefore, a hypothesis-free meta-analysis approach was used that provides adequate statistical power for this subject area. The main question of this thesis was whether data of microdialysis experiments indicate a difference in the dopaminergic overflow in reaction to drugs of abuse in male and female rats. Thereby, systematic data mining was performed on the PubMed online library (https://www.ncbi.nlm.nih.gov/pubmed/) focusing on studies measuring extracellular dopamine concentrations in the striatal complex. The focused lied on six drugs of abuse (alcohol, amphetamine, cocaine, nicotine, morphine and tetrahydrocannabinol) and two brain regions (caudate putamen and nucleus accumbens). Data from 45 microdialysis experiments on female rats (number of animals = 842) were extracted and statistically compared with data from 6402 male rats. Overall, 291 studies were included, providing averages of the peak percentage baseline value of dopamine for 103 different dosages. All drugs under investigation notably increased the dopaminergic transmission in the striatal complex. For some drugs, a positive dose-response relationship was detected. Regarding the entity of dose groups, no sex differences in the dopaminergic response to drugs of abuse were found, but for some small subgroups. Neither did the rats’ age, strain, stage of consciousness or the route of administration have an impact on the overall peak percentage baseline values, suggesting robustness of these parameters. Attempts were also made to extract the rats’ estrous cycle as a variable, but only one study monitored it. Overall, the neglect of female subjects in basic research, which had lasted for decades and is far from defeated, was a phenomenon well reflected in the results of the search query in this thesis. What can be therefore concluded, is that future research should intensify its efforts to include female subjects and to close the sex-gap in preclinical as well as in clinical research. This will provide more data that are crucial to get valid results about sex similarities or differences, as this thesis only shed light on a small subdivision.
Die vorliegende Dissertation befasst sich mit der Synthese und Charakterisierung neuartiger Azaacene und Aza-acenoacene aus Acridonen. Dabei steht der Einfluss der Regioisomerie auf die Kristallpackung und die potentielle Anwendung als Halbleitermaterial in organischen Feldeffekttransistoren im Fokus. Im ersten Kapitel der Arbeit wurden zunächst zwei Typen von Diazapentacen-Regioisomeren 1 und 2 miteinander verglichen (Abbildung 1, links). Die cis-trans-Isomerie verändert die optoelektronischen Eigenschaften, Stabilität und Kristallpackung entscheidend. Die Größe des Silylsubstituenten (triisopropylsilyl TIPS, triethylsilyl TES) beeinflusst die Kristallpackung des cis-Derivats. Ein dargestelltes, zum instabilen trans-Isomer (Abbildung 1, links) verwandtes Tetraazaacen 3 wird durch das Einführen von Stickstoff in den zentralen aromatischen Ring stabilisiert. Eine Verlängerung des aromatischen Systems zum trans-Diazahexacen führte zum Schmetterlings-Dimer 4; das Monomer konnte nicht isoliert werden. Weiterhin wurden Aza-Acenoacene 5 und 6 synthetisiert und die erste Anwendung solcher Moleküle in organischen Dünnfilmtransistoren (OTFT) realisiert (Abbildung 1, Mitte). Schlüssel zum Erhalt einer geeigneten Festkörperpackung war die verringerte Anzahl an TIPS-Ethinylsubstituenten im Vergleich zu ihren verwandten, literaturbekannten Verbindungen. Auch hier konnte eine Abhängigkeit der optoelektronischen Eigenschaften und auch der Performanz in elektrischen Bauteilen von der Regioisomerie festgestellt werden. 6 erreicht maximale Mobilitäten von 2.88·10-2 cm2 (Vs) 1, etwa einem Zehntel der Mobilität, die für das unter gleichen Bedingungen verarbeitete Vergleichsmolekül TIPS-Pentacen gefunden wurde. Im letzten Kapitel wurde die Charakterisierung von organischen Dünnfilmtransistoren der vielversprechenden TIPS-TAP-Derivate Br4TAP und I4TAP untersucht (Abbildung 1, rechts). Mit Cl4TAP wurden von Miao et al. Mobilitäten von bis zu 27.8 cm2 (Vs) 1 gefunden.1 Für die Homologe mit Brom und Iod wurden nach quantenchemischen Rechnungen höhere Mobilitäten erwartet.2 Diese Erwartungen wurden für Br4TAP mit Maximalwerten von 54.4 cm2 (Vs) 1 erfüllt. Für I4TAP konnte aufgrund von Defekten in der Dünnfilmstruktur eine geringere Mobilität von bis zu 9.24 cm2 (Vs) 1 gemessen werden.
Targeted therapies improve the prognosis of advanced anaplastic lymphoma kinase-driven non-small cell lung cancer (ALK+ NSCLC) patients. However, clinical courses vary considerably due to acquired drug resistance. Thus, timely detection of treatment failure is crucial to guide subsequent therapies and optimize patient outcome. The analysis of tumor alterations in cell-free DNA (cfDNA) represents a novel approach to monitor cancer dynamics during therapy in longitudinal plasma samples. Besides mutations and copy number alterations, cancer-specific epigenomic changes have emerged as promising biomarkers for cfDNA-based tumor assessment. This thesis aimed to identify tumor-derived methylation (5mC) and hydroxymethylation (5hmC) alterations in cfDNA of metastatic ALK+ NSCLC patients, associate these epigenetic biomarkers to gene expression in lung cancer and assess their suitability for monitoring of tyrosine kinase inhibitor therapy in serial plasma samples. To this end, 79 longitudinal plasma samples from 31 patients were collected alongside plasma of 14 healthy individuals. Genome-wide 5mC and 5hmC profiles were generated by cell-free methylation immunoprecipitation and 5hmC selective chemical labeling, followed by sequencing. Additionally, 5(h)mC profiles of primary monocytes, neutrophils and erythroid progenitor cells were prepared using the same methods. These hematopoietic cells constitute the major non-tumor contributors (72.2%) to cfDNA of cancer patients. A technical novelty of this study was the enrichment for tumor-derived 5(h)mC signals in cfDNA by excluding genomic regions highly (hydroxy-)methylated in the reference blood cell types. Of 9,603,454 300-bp genomic loci, 577,701 (5mC; 6.0%) and 499,681 (5hmC; 5.2%) exhibited low (or no) signal in the profiled blood cells. The blood cell signal-reduced 5mC regions demonstrated an increased correlation to lung cancer tissue methylation (Spearman, r = 0.26), compared to the entire dataset (r = 0.11), and revealed cancer- as well as tissue-specific 5mC signals. Cancer versus control analysis at the remaining genomic regions identified 5,499 differentially methylated (DMRs) and 495 differentially hydroxymethylated regions (DhMRs). Hierarchical clustering analysis based on the D(h)MRs cleanly separated patient from control samples and clustered patient cfDNA according to the inferred tumor burden within the samples. This suggests that sample separation is primarily driven by tumor-derived signals and confirms that the identified D(h)MRs are enriched for cancer 5(h)mC alterations. DMRs proximal to transcription start sites were enriched at genes downregulated in lung cancer tissue, demonstrating that cancer-specific gene regulatory 5mC marks can be retrieved from cfDNA. Many of these genes (e.g. GATA4 and HOXA9) were previously described to confer tumor suppressive functions in NSCLC. 5(h)mC levels in cfDNA correlated with tumor-derived genomic alterations (e.g. EML4-ALK fusion and global chromosomal instability [t-MAD score]) determined in matched plasma samples. The highest correlation was observed between PTGER4 methylation and t-MAD scores (Pearson, r = 0.86). Four 5mC (SOX9-AS1, HOXA10-AS, PRAC1, and PTGER4) and three 5hmC biomarkers (IL1RAP, KPNA7 and KIF25) were employed for therapy monitoring in ten patients with available longitudinal samples (³2). In particular 5mC biomarkers mirrored cancer dynamics found by radiologic imaging and genomic tumor alterations in cfDNA. At four instances, cfDNA 5mC levels anticipated therapy relapse in advance of imaging with a maximum lead time of 481 days. In one patient, both 5mC and 5hmC biomarkers detected disease progression ahead of imaging and genomic alterations in cfDNA, highlighting the sensitivity of 5(h)mC-based tumor assessment. In conclusion, 5mC and 5hmC profiling from cfDNA provides an opportunity for sensitive cancer detection and therapy monitoring. The tissue-specificity and the regulatory functions of these DNA modifications provide data about the tumors that currently cannot be obtained by copy number or single nucleotide variation profiling.
In this work, I describe technical and biological aspects of a High-Content Screening experiment conducted to identify pharmacological modifiers of human cystic kidney disease in zebrafish larvae. Human cystic nephropathy was mimicked in zebrafish larvae with the help of morpholino-mediated knockdown of the ift172 gene. IFT172 is part of the ciliary intra flagellar transport complex; its deficiency impairs the function of the primary cilia, resulting in impaired urine flow and the development of fluid filled cysts. An automated pipeline was developed to optimise large scale sample handling, image acquisition, quantification and analysis. Dedicated hardware and software tools were developed to solve issues associated with the spatial orientation of larvae in microtitre plates, automated Image capture, data pre-processing and quantification. After successful implementation of the high-content screening platform, I screened 1,280 known small-molecular pharmacological compounds from the Prestwick library in 12 larvae per compound. The initial screen yielded 60 candidate compounds with potential cyst suppressive activity. The hits were further validated in dose response studies, which led to the final confirmation of 17 target compounds. These compounds share several interesting putative modes of action, including estrogen antagonism, enkephalinase and proton pump inhibition, anti-tyrosinase activity, calcium channel regulation, retionoic acid receptor interactions and topoisomerase blockade. In conclusion, the establishment and execution of an automated High-Content Screening in a transgenic zebrafish model yielded several promising novel pharmacological targets that can be further explored with regards to their cyst-suppressing activity and suitability for use in human cystic kidney diseases.
Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and lethal disease with a miserable prognosis. Chemotherapeutic regimens, like FOLFIRINOX or gemcitabine plus nab-paclitaxel remain the standard treatment of care in patients diagnosed with PDAC while only achieving a modest increase in overall survival. Transcriptional subtyping of PDAC discerns tumors into two broad lineages which provides the opportunity to improve patient stratification and treatment, but has not been translated into clinical practice yet. Meanwhile, resistance to chemotherapy continues to be the limiting factor that prevents a patient’s cure from cancer and finding improved therapeutic options could overcome resistance of PDAC to current treatment regimens. With our previously established culturing model for patient-derived PDAC cells, we could investigate the development of resistance to paclitaxel and/or gemcitabine in the different subtypes. We developed a long-term treatment regime that enabled the generation of drug-resistant cells which were analyzed in a multi-omics approach. ATP-binding cassette (ABC) transporter B1 (ABCB1), a membrane-bound glycoprotein that is predominantly expressed in excretory tissue and a common resistance mechanism against paclitaxel in various tumor entities, was found to be overexpressed in all paclitaxel-resistant cells. CRISPR/Cas9-guided knockout and pharmacological inhibition re-sensitized these drug resistant cancer cells to paclitaxel. We found ABCB1 to be heterogeneously expressed in the paclitaxel resistant cell population and expression was lost after prolonged absence of paclitaxel treatment. Short-term drug treatment dynamically increased the proportion of ABCB1-expressing cells in former paclitaxel resistant cell population, re-acquiring paclitaxel-resistance. In of the paclitaxel resistant cell lines, expression of ABCB1 was further enhanced by de novo generation of extrachromosomal DNA (ecDNA), carrying the ABCB1 gene. Similar to ABCB1 gene expression, number ecDNA inside paclitaxel resistant cells was dynamically increased upon paclitaxel treatment. These findings describe a dual mechanism for acquired ABCB1 expression that is dependent on paclitaxel treatment and leads to the induction of ABCB1 expression and amplification of ABCB1-carrying ecDNA.
Radiomarkierte Fluoreszenzfarbstoffe erfahren aktuell eine stetig wachsende Nachfrage als Medikamente für die multimodale Bildgebung (z.B. PET/OI oder SPECT/OI) und Behandlung von Krebs. Sie versprechen somit ein hohes Potential für klinische Anwendungen. Die radiomarkierten Farbstoffe können zur präoperativen Planung (Prestaging) durch Zuhilfenahme der PET/SPECT-Bildgebung zur präzisen Differenzierung zwischen gesundem Gewebe und Tumorgewebe eingesetzt werden. Die anschließende Fluoreszenz-gestützte intraoperative Chirurgie ist eine äußerst effiziente Methode zur akkuraten R0-Tumorentfernung. In der vorliegenden Arbeit wurden neuartige Nahinfrarot(NIR) absorbierende und emittierende Farbstoffe hergestellt, welche für die bimodale PET/SPECT- und optische Bildgebung geeignet sind. Bisher wurden in der Literatur noch nicht über radiomarkierte Si-Rhodamine berichtet. Die nicht-radioaktiven Farbstoffe, welche zur Familie der Si-Rhodamine gehören, wurden über mehrere organische Syntheseschritte erhalten und anschließend vollständig charakterisiert. Außerdem wurden die photophysikalischen Eigenschaften der Si-Rhodamine in wässriger Lösung mit klinisch zugelassenen Farbstoffen (z.B. Indocyaningrün oder Protoporphyrin IX) verglichen, um die erforderlichen Voraussetzungen für die Fluoreszenz-gestützte Chirurgie zu gewährleisten. Dabei zeigen die nicht-radioaktiven Fluoreszenzfarbstoffe im wässrigen Medium Quantenausbeuten bis zu 10% und hohe Photostabilitäten nach Bestrahlung mit NIR-Licht. In radiochemischen Experimenten wurden die Si-Rhodamine mit den am häufigsten genutzten klinischen Radionukliden Technetium-99m und Iod-123 für die SPECT-Bildgebung und mit Fluor-18 als Positronenemitter für die PET-Bildgebung markiert. So zeigte die Radiomarkierung des Si-Rhodamin-Grundgerüsts mit Technetium-99m eine radiochemische Ausbeute von 59% mit einem Verteilungskoeffizienten von log DpH=7.4=1.11 des isolierten Komplexes 78. Die ausgezeichnete in vitro Stabilität des nicht-radioaktiven Rhenium-Komplexes 77 und des Technetium-Komplexes 78 in Dekomplexierungsexperimenten mit L-Histidin in phosphatgepufferter Salzlösung oder humanem Serum zeigen vielversprechende Resultate der Metallkomplexe für in vivo Experimente. Nach ausführlicher Optimierung der Radiomarkierungsexperimente am Si-Rhodamin mit Fluor-18 wurden radiochemische Ausbeuten von bis zu 54% erhalten. Zusätzlich zeigen die radiofluorierten Si-Rhodamine nach zwei Stunden Stabilitäten über 74% in humanem Serum. Außerdem weisen die kationischen und lipophilen nicht-radioaktiven Si-Rhodamine eine beachtliche Aufnahme in Mitochondrien von PC3-Zellen auf, sodass diese Farbstoffe als Perfusionsmarker für die PET- und optische Bildgebung geeignet wären. Ein wesentlicher Vorteil ist, dass der für die Radiofluorierung genutzte Vorläufer auch für die Radioiodierung mit Iod-123 genutzt werden kann. Somit konnte das radioiodierte Si-Rhodamin mit einer radiochemischen Ausbeute von 54% hergestellt werden und zeigt zudem den höchsten Wert der molaren Aktivität von 7.64 TBq/µmol für NIR-Farbstoffe. Des Weiteren wurde ein für die SPECT-Bildgebung kompatibles Si-Rhodamin exemplarisch an bekannte Tumorvektoren wie z.B. das PSMA-1007-Bindungsmotiv geknüpft. Anschließend wurde in vitro die Affinität des nicht-radioaktiven PSMA-1007-funktionalisierten Si-Rhodamins zu LNCaP-Tumorzellen untersucht. Zusätzlich wurde in vivo die Bioverteilung des Konjugats in Nacktmäusen mit Prostatatumor-Xenograft evaluiert. Die neuentwickelten Si-Rhodamine stellen eine neue Klasse radiomarkierter Fluoreszenzfarbstoffe dar, die vielversprechende Eigenschaften für die multimodale Bildgebung zeigen.
Metabolic Syndrome is a multifactorial metabolic disorder characterized by obesity in association with altered lipid profiles, elevated glucose levels, and/or increased blood pressure, forming a cluster of risk factors for cardiovascular diseases and type 2 diabetes. Changes in lifestyle and diet are powerful strategies in preventing the development of the metabolic syndrome. An evolving research field linked with preventable diseases is the consumption of bioactive-enriched foods since for some bioactives the dose for healthpromoting effects can only be achieved by enrichment. Docosahexaenoic acid (DHA) is an n-3 fatty acid displaying lipid-lowering activity. In the liver, DHA inhibits lipogenesis thereby promoting an improved lipid profile in the blood. However, the impact of DHA on preadipocytes, the source for new adipocytes generated during adipogenesis for additional lipid storage, remains insufficiently characterized. Cell differentiation processes are controlled by epigenetic mechanisms including DNA hydroxymethylation and are associated with cell-type specific transcriptional regulation. To profile epigenetic gene regulation of our model system, I first characterized human adipocyte differentiation of Simpson-Golabi-Behmel Syndrome (SGBS) cells at the level of DNA hydroxymethylation (5hmC) using two whole-genome sequencing techniques. I observed gain in the 5hmC mark during adipocyte differentiation, particularly enriched at enhancer regions, around adipogenic transcription factor (TF) binding sites. Based on cluster analyses, I was able to describe clusters with different dynamics of hydroxymethylation associated with the binding of specific TFs. Early hydroxymethylation clusters were enriched with TF binding motifs involved, e.g., in clonal expansion at an early stage of adipogenesis. On the other hand, hydroxymethylation emerging during late adipogenesis was associated with TF binding in the second phase of chromatin remodeling, such as of the peroxisome proliferator-activated receptor gamma (PPARG) and of CCAAT/enhancer binding protein alpha (CEBPA). Integrated analysis of hydroxymethylation and gene expression recapitulated the involvement of hydroxymethylated enhancer regions in the regulation of gene expression programs characteristic of mature adipocytes. By maintaining mature adipocytes in culture for 14 days after completion of adipocyte differentiation, I could provide evidence for 5hmC as a stable epigenetic mark. In vivo relevance of these findings was confirmed by a high degree of overlap with hydroxymethylation in white adipose tissue (WAT). As 5hmC is often considered as an intermediated mark generated during DNA demethylation, I investigated potential mechanisms involved in 5hmC stabilization. I detected that acetylated nei like DNA glycosylase 1 (acNEIL1), reported previously as a hydroxymethylation binding protein, was enriched in WAT promoter and enhancer regions, but its binding was independent of the hydroxymethylation mark, especially in promoter regions. Based on metabolome data, I rather hypothesize that declining α-ketoglutarate (αKG) levels during adipogenesis could compromise ten-eleven translocation (TET)-mediated demethylation and thereby contribute to stabilization of the 5hmC mark in nonproliferating mature adipocytes. In the second part of my thesis, I investigated the effect of long-term cultivation (3 - 4 weeks) of SGBS in the presence of low-dose DHA on genome-wide DNA methylation (EPIC array) and gene expression levels (RNA-seq). DHA-treatment resulted in massive methylation differences. However, these methylation differences mainly overlapped with gradual cultureassociated methylation changes. A subset of the differentially methylated CpGs was located in partially methylated domains (PMDs) associated with the nuclear lamina and characterized by late replication timing. These sites displayed gradual loss in methylation, correlating with a methylation-based mitotic score (EpiCMIT.hypo) and attributed to an impairment of DNA methylation maintenance during replication. At the gene expression level, I could separate effects directly attributable to DHA from culture-associated gene expression changes. Notably, I detected anti-inflammatory activity by reduction of tumor necrosis factor alpha (TNFα) signaling and repression of sterol regulatory element-binding protein 1/2 (SREBP1/2) signaling, possibly contributing to reduced lipogenesis and increased insulin sensitivity – two crucial mechanisms in the prevention of the metabolic syndrome. To sum up, molecular understanding of the interplay of cellular metabolism and epigenetics in gene regulation of human adipocyte differentiation might be crucial for the deeper understanding of the development of metabolic syndrome – a disease with altered energy metabolism. Furthermore, deciphering the molecular mechanisms of DHA in human preadipocytes might help to understand how to reverse some of the adverse effects of obesity and its associated metabolic complications, showing us a path towards improved public health.
Maps combine different types of data, from a number of different sources, into the uniform grid of their coordinate system. They are invaluable resources as they allow the connections between data to be understood in their spatial context. We have been making biological maps for decades of all kinds of systems - for example, model organisms like Drosophila melanogaster and Caenorhabditis elegans have large atlases of neuronal cell types available.
These large multimodal atlases (i.e. datasets that combine multiple data sources into one coordinate system) tend to undergo a similar ‘lifecycle’. First, a standard coordinate system is formed, followed by collection of the required datasets. These are then integrated into the common coordinate system, before they are analysed, and finally shared as a resource to the biological community.
Electron microscopy (EM) offers many advantages when integrated into atlases of this kind. Firstly, it can achieve very high resolution, making it ideal for visualising small features like the fine structure of different organelles. It also provides the full spatial context of a tissue - allowing the locations and orientations of cells to be understood in a comprehensive manner. This comes at the cost of a number of challenges though. For one, EM is quite low throughput, meaning that often only a single sample can be imaged, or only small regions of a sample. Also, EM data can be extremely large (many terabytes in size) making it difficult to store, analyse, and share with other researchers.
In my thesis, I aim to ease the integration of large-scale volume EM into multimodal atlases at various points within this lifecycle. First, at the acquisition stage, I look at increasing the speed, ease and precision of targeted EM acquisition. I develop two different methods - one focused on trimming resin-embedded blocks with an ultramicrotome, and the other with a Serial Block Face Scanning Electron Microscope (SBEM). Both these methods are provided as user-friendly Fiji plugins, making them accessible to those with no programming experience.
Second, at the analysis stage, I create an analysis pipeline for comparing morphology (from EM) with gene expression of cell types in a large multimodal atlas. This focuses on a large atlas of the organism Platynereis dumerilii that combines volume EM with gene expression patterns for over 200 genes. This analysis provides full body clustering of cells based on morphology and gene expression, and shows a clear correspondence between gene expression and morphological tissue boundaries from the EM.
Finally, at the resource sharing stage, I look at improving the ease of sharing and exploration of these massive EM datasets. To do this, I contribute to the software MoBIE that allows interactive browsing of very large, remotely stored image data. I focus on making the creation of MoBIE projects accessible to those with no programming experience, adding a user interface for creating projects via the Fiji plugin.
Die Analyse medizinischer und biologischer Bilddaten erfordert häufig die Isolierung einzelner Strukturen aus einem 3D-Volumen durch Segmentierung. Trotz einer Vielzahl halb- und vollautomatischer Verfahren erfolgt die Bildsegmentierung oft noch manuell und gilt nach wie vor für viele Szenarien als die arbeitsintensivste und zeitaufwendigste Aufgabe innerhalb der 3D-Bildanalyse. Die herkömmliche manuelle Segmentierung vieler Schichten, gefolgt von einer linearen Interpolation und einer manuellen Korrektur der Ergebnisse verhindert jedoch in vielen Fällen die Analyse einer großen Anzahl von Proben. In dieser Arbeit wird darum ein neues parameterfreies Verfahren zur halbautomatischen Segmentierung großer komplexer 3D-Bilddaten entwickelt, das auf einer Interpolation von wenigen manuell vorsegmentierten Schichten basiert, wobei es den gesamten zugrunde liegenden volumetrischen Bilddatensatz berücksichtigt. Die Interpolation erfolgt durch gewichtete Random Walks, deren Unabhängigkeit voneinander eine Berechnung durch massiv parallele Hardware, wie den Grafikprozessoren (GPUs), ermöglicht. Anhand einer vielfältigen Auswahl von Beispieldatensätzen wird gezeigt, dass dieses GPU-basierte Verfahren, insbesondere bei der Segmentierung sehr großer komplexer Bilddaten, wie sie typischerweise in der Mikro-Computertomographie (µCT) entstehen, dazu in der Lage ist, sowohl den Zeit- und Arbeitsaufwand erheblich zu reduzieren als auch die Qualität der Ergebnisse deutlich zu steigern. Dabei erreicht es eine deutliche Beschleunigung und höhere Genauigkeit gegenüber dem konventionellen Ansatz der fast ausschließlich manuellen Segmentierung und auch gegenüber den am weitesten verbreiteten Segmentierungsalgorithmen. Die GPU-basierten Random Walks sind insbesondere dann geeignet, wenn wenig Vorwissen über das zu segmentierende Objekt vorhanden ist, zum Beispiel bei der Beschreibung einer neu entdeckten Art, oder zum Erstellen von Trainingsdaten für ein anschließendes maschinelles Lernen. Um darüber hinaus bei der Segmentierung vieler ähnlicher Proben eine weitgehend automatische Segmentierung zu ermöglichen, wird hier zusätzlich ein auf künstlichen neuronalen Netzen basierendes Verfahren vorgestellt und anhand von 110 µCT-Scans von Honigbienengehirnen evaluiert. Ergänzend wird auf Basis der entwickelten Algorithmen die neue Online-Segmentierungsplattform Biomedisa präsentiert. Die Plattform ist über einen Webbrowser zugänglich und erfordert keine komplexe und langwierige Konfiguration von Software- und Modellparametern. Sie richtet sich gezielt an die Bedürfnisse von Wissenschaftlern/-innen, die nicht über umfangreiche Computer- und Softwarekenntnisse verfügen. Die integrierten GPU-basierten Methoden ermöglichen eine intuitive Anwendung für verschiedene bildgebende Verfahren innerhalb eines breiten Spektrums wissenschaftlicher Disziplinen. Die Plattform wurde bereits in mehreren Studien erfolgreich eingesetzt. Darüber hinaus ermöglicht ihr modularer Aufbau eine leichte Erweiterung der hier vorgestellten Kernfunktionen um weitere benutzerspezifische Funktionalitäten.
Multicellular organisms rely on the concerted interaction of a multitude of cells, which are often highly specialised and give rise to complex tissues. As vastly different cellular phenotypes emerge from the same genotype that is shared across all cells of an organism, the transcriptome represents a key mediator driving different cell types and cell states that give rise to functional tissues. These are also subject to environmental factors or intrinsic changes that may disrupt homeostasis and lead to disease. In the human lung, the effects of tobacco smoke exposure, still the greatest risk factor for lung cancer, have not been fully resolved at the cellular level. Moreover, cellular heterogeneity may be significant for the emergence of lung cancer in never smokers, a growing proportion of global cases. A focused investigation of cellular heterogeneity in the healthy lung and lung cancers is therefore highly warranted. During the last decade, technological advancements have made it possible to interrogate the transcriptome of single cells by novel next generation sequencing approaches. While previous studies were limited to averaging transcriptome information over many cells, single cell RNA sequencing (scRNA-seq) technologies are now enabling the investigation of cellular phenotypes in healthy and diseased tissues at unprecedented resolution. In this thesis, I adapt different scRNA-seq technologies to process fresh or biobanked samples from different tissues and species, thus enabling comparisons across diverse origins. We identify specific advantages, limitations and experimental challenges associated with each technology. I then perform a comprehensive single-cell transcriptomics study of healthy lung and lung adenocarcinoma (LADC). Based on twelve healthy lung samples, we generate a reference cell atlas that provides a rich resource for investigating cellular diversity in the human alveolar lung. Its utility is demonstrated by probing the expression of genes that are implicated in host cell entry of SARS-CoV-2 virus, thereby vi contributing to our understanding of coronavirus infections. By comparing single cell profiles from smokers and never smokers, we also resolve the involvement of distinct cell types in the maintenance of an inflammatory state in smoker lungs, and we identify key mediators of inflammatory processes induced by tobacco smoke exposure in fibroblasts and endothelial cells. To investigate cell type diversity and microenvironment interactions in LADC, I analyse 26 tumour tissue samples and resolve functional malignant cell subpopulations linked by a differentiation hierarchy in both smokers and never smokers. They comprise proliferating and intermediate undifferentiated cells as well as two differentiated tumour cell states implicated in cancer progression and invasiveness. Distinct macrophage and fibroblast subpopulations which contribute to a tumourigenic environment are also detected. A subset of proliferating tumour cells show differential immune modulating activity dependent on smoking status, with implications for future treatment approaches. Taken together, these results provide a comparison of rapidly developing scRNA-seq technologies for use in further studies and demonstrate their utility to dissect cellular heterogeneity and identify transcriptional programmes in the healthy and diseased lung. By applying these technologies, I add to our understanding of SARS-CoV-2 entry into human lung cells, define the alveolar lung cell types affected by tobacco smoke exposure, and provide deeper insight into cellular heterogeneity of LADC and the tumour microenvironment. These findings represent a valuable reference for future translational studies.
Economic land concessions are commonly believed to increase employment in rural areas, to offer new economic opportunities in the countryside, and to encourage local economic diversification through investments upstream and downstream of the concessions. ELCs are also considered to generate state revenues at national and sub-national level and reduce poverty in rural areas.
However, this possible progress is bought by massive land-use conflicts, social problems and land degradation. Natural resource-based communities face severe challenges.
My research deals with the problems of rural communities in Ratanakiri Province in Cambodia. Former common resources have been granted to private investors (mainly from Vietnam) to develop large-scale agricultural enterprises. The study uses concepts of resilience in livelihood framework to analyze these impacts caused by state development plans. The theoretical framework helps to explain how the economic land concession evolves and affects the resources and communities. The rationale of my contribution originally comes from the main idea of sustainable development which aims to balance different needs of the environment, society and economy. Communities in the research area lost their sense of commonality in resources protection and push on the degradation of existing resources.
In the study the resilience of each community is analyzed based on its buffer capacity, self-organization, and capacity for learning. And, it aims to disclose the unpredicted impacts of state development plans on indigenous communities.
While it is known that animal behavior depends on morphology, it remains poorly understood whether and how organismal behavior impacts morphogenesis. During development, behavior emerges at different scales when cells differentiate and form specialized tissues and organs. Such emerging behaviors may include movement or electrical activity taking place on scales ranging from the cellular to the organismal level. Since development is a continuous process, these emerging behaviors have the potential to feed back on development. Indeed, some behaviors that emerge at the tissue and organ scale, in particular contractile behaviors, such as the beating of the developing heart, have been shown to generate mechanical forces that contribute to morphogenesis. Thus, morphology and behavior develop together, and may impact one another. However, this potential two-way relationship has not received much attention at the organismal scale. This is largely because simultaneously studying behavior and morphogenesis is complicated by the different spatiotemporal scales at which these processes take place. Furthermore, while in most species morphogenesis largely takes place during embryonic development, most organismal behaviors are expressed post-embryonically, when they primarily serve animal survival through interaction with the external environment. For these reasons, studying the link between organismal behavior and morphogenesis requires a system in which both processes take place at the same time, and at spatiotemporal scales that are not too far apart. In this thesis, I use the sea anemone Nematostella vectensis as a model organism to study the link between organismal behavior and morphogenesis. During a process called larva-polyp transition, Nematostella undergoes a simple morphogenetic change from a roughly ellipsoidal larva to a cylindrical polyp with oral tentacles, while simultaneously expressing distinct organismal behaviors such as cilia-based swimming, settlement, and muscle-driven body contractions. Using an imaging setup that allows tracking and monitoring of morphogenetic and behavioral changes at the organismal scale, I characterize the dynamics of larva-polyp transition in wild-type animals. Here, I find that the animals can change their size and shape both separately and simultaneously, and that animal settlement behavior correlates with elongation dynamics. While increase of organismal size largely depends on body cavity inflation by water uptake, change of animal shape requires tissue remodeling during which cells change shape and rearrange. Furthermore, pharmacological, mechanical, and genetic perturbations suggest an important role for muscular-hydraulics in both animal behavior and development. These findings suggest a mechanism in which short-term behavioral contractions generate mechanical forces that induce tissue remodeling on long time scales, thus indicating a potential role for animal behavior in driving morphogenesis.
Carbon ion therapy is a promising treatment modality that is not widely accessible to patients due to limited resources and a high cost of treatment. Therefore, it is necessary to consider mixed modality treatments where carbon ions are utilized in combination with the more widely available and accessible, photon therapy. In contemporary clinical combined treatments, photon fractions and carbon ion fractions are separately optimized and simply accumulated based on the RBE weighted dose. Such a “naive” combination does not fully exploit physical and radiobiological advantages emerging from the interplay of both modalities. Carbon ions excel at delivering high RBE conformal dose to the target volume and avoid delivering dose to distal healthy tissue. Photons, besides generally larger integral dose, have a lower RBE and are desirable to irradiate target subvolumes that are adjacent to healthy tissue or have healthy tissue infiltrated by tumour tissue, due to the greater fractionation potential. This thesis presents a novel method to exploit these differences by simultaneously optimizing photon and carbon ion fluence contributions in order to answer the question: what is the ideal combined photon-carbon ion fluence distribution given a specific fraction allocation between photons and carbon ions? The joint optimization framework allows for the synergistic optimization of photon–carbon ion treatments based on the cumulative biological effect, incorporating both the variable RBE of carbon ions and the fractionation effect within the linear quadratic (LQ) model. As a part of this study, the joint optimization workflow was implemented within the open source treatment planning toolkit matRad. Joint optimization strategies yield individually non-conformal photon and carbon ion dose distributions that cumulatively deliver a homogeneous conformal biological effect distribution in the target volume. Compared to conventional combined treatments, joint optimized treatments exhibit better conformity and better sparing of critical structures through a spatial redistribution of dose between modalities and a non-uniform fractionation schedule within the target volume. Depending on the fraction allocation between modalities, there exists an optimized temporal distribution of biological effect where parts of the target volume are hypofractionated while areas around dose limiting critical structures are spared through fractionation. The additional degrees of freedom from the spatial and temporal redistribution of fluence enables the exploration of a new spectrum of plans that can better address physical and radiobiological treatment planning challenges. Apart from a proof of concept, the impact of key underlying treatment parameters were also investigated. With regards to fraction allocation for photon–carbon ion treatments, the joint optimized treatments were shown to benefit from a reduction in carbon ion fractions due to their limited fractionation capacity. The choice of LQ model parameters and an assumed fractionation benefit drives the biological motivation to fractionate dose, without it the joint optimization was purely driven by the physical characteristics and beam angles selected for treatment. Furthermore, the choice of LEM version for carbon ion RBE estimation predicts the fractionation capacity of carbon ions. The clinically used LEM I predicts a higher effectiveness of carbon ions in the entrance region and fragmentation tail as compared to LEM IV. Therefore, the use of LEM I in joint optimization results in a lowering of carbon ion contributions in order to spare healthy tissue located at the entrance channel and fragmentation tail. Finally, the method was demonstrated for six glioblastoma patients, where the CTV contains tumour infiltrated healthy tissue that would benefit from a fractionated treatment. In comparison to the current clinical standard of independently optimized photon–carbon ion plans, the optimal plan dose to CTV was primarily delivered by photons while carbon ions are restricted to the GTV with variations depending on tumour size and location. The joint optimization approach results in a targeted application of carbon ions that (1) reduces dose in normal tissues within the target volume which can only be protected through fractionation and (2) boosts central target volume regions in order to reduce integral dose. In conclusion, this thesis presents the first joint optimization framework that allows for an evidence based and mathematically optimal allocation of photons and carbon ions in mixed modality treatments.
Postoperative death within 30 days after surgical intervention is the third largest contributor to mortality globally. Causes of postoperative mortality are manifold but also comprise challenging perception and the inability to estimate physiological tissue parameters during interventions. To capture data emanating from underlying physiological tissue properties, hyperspectral imaging (HSI) together with machine learning-based analyses has been proposed as a solution in recent literature. However, HSI data in the clinical setting is sparse, as its acquisition is crucially limited by a small number of approved devices and the need for clinical trials. Therefore, the present work investigates common deep learning frameworks for HSI and proposes a two-step image generation pipeline to synthesize hyperspectral tissue images. To validate the image generation pipeline, spectral correctness and textural realism were assessed both qualitatively and quantitatively. Results of the textural Kernel Inception Distance (KID) exhibited state of the art (SOTA) performance for both paired and random generated HSI patches. Furthermore, the feasibility of using the synthetic, unlabelled data for an image segmentation task was tested and found to not lead to improvement. From the conducted experiments it can be concluded that RGB image synthesis can be adapted to the HSI domain, while synthetic additional data has to be tailored for individual tasks.
Today many biological studies rely on high-throughput techniques that yield data on thousands of samples or cells with tens of thousands of measured features. Exploring such an amount of data poses a visualisation challenge, that can be solved by switching from static plots to interactive ones. This provides a way of intuitive navigation through large datasets in a manner that helps the user to grasp the bigger picture visually. The field of interactive visualisation of biological data is an actively developing one. However, it is more often used only for data presentation and is still not so common during a research project's early, exploratory stages. This project is aimed to explore and propose solutions to fill this “visualisation gap”.
I investigate the possible benefits for biological studies of the combination of JavaScript and R programming languages. R is one of the most common tools in biostatistics and provides a wide variety of implemented libraries for processing omics data. JavaScript is a language for enabling user's interaction with a web page and nowadays is used by most available web resources. Thus, the two languages are very effective in their own application fields, and interactive visualisation of omics data lies precisely in their combination. As an outcome of the project, I present three R packages (one of which also provides a purely JavaScript interface) for data visualisation.
The first one, "jrc", is intended for package developers and serves as a foothold for further project steps. "jrc" provides direct communication between a web page and a running R session. It allows the user to run R code from a web page and execute JavaScript code from an R session. In addition, it provides a basis for publicly available interactive apps deployed on a server. With this, "jrc" can be used as a foundation for the packages that use JavaScript to visualise data stored and processed in an R session.
The second one is "sleepwalk". It is a simple but effective tool to explore distortions introduced by dimensionality reduction techniques when visualising biological data. These approaches (such as MDS, t-SNE, UMAP, etc.) are particularly but not exclusively popular in single-cell studies. There, researchers commonly visualise cells as points on 2D embedding and then study the obtained clusters and trajectories. "sleepwalk" helps one explore the underlying patterns of such plots by interactively comparing the displayed neighbourhoods to the original distances in the high-dimensional feature space.
Finally, "rlc" (or LinkedCharts) is a plotting library that allows one to construct one's interactive app with minimal effort and coding skills. It is designed in a way that does not require users to learn special complex syntax. Instead, I adjusted it to routinely used practices in the omics data exploration. I also left a broad space for customisation so that users could adapt the apps to their particular tasks rather than trying to fit their data into the predefined templates. With all this, the "rlc" can be a powerful tool to facilitate exploratory data analysis by interactive visualisation. It centers on but is not limited to the idea of linking multiple charts when a user's manipulation with one plot affect another one (for example, a click on a point shows more specific information on the thus selected sample).
Overall, the packages presented here can be helpful when applied in everyday analyses and serve as a basis and inspiration for new solutions in the interactive visualisation of biological data.
How cells coordinate their dynamic gene expression to produce patterns that transcend to the tissue scale is a fundamental question in biology. In the context of somitogenesis, the timing of segmentation is controlled by a molecular oscillator, the segmentation clock. The segmentation clock oscillations, particularly those regulated by Notch signaling, exhibit traveling wave patterns in the presomitic mesoderm (PSM) due to phase shifted oscillations along the AP axis. How their spatiotemporal dynamics are coordinated to produce such patterns is not fully understood. I first describe the origin of the traveling waves, with data obtained by imaging the onset of segmentation clock dynamics, at the gastrulating stages of the mouse embryo. Detailed analysis of the oscillations revealed that they are initially synchronous across space at its onset, but accumulate spatial phase shift over the first 6+ cycles, thereby leading to the emergence of traveling wave patterns. Such an accumulation of spatial phase shift agrees with the presence of a period gradient, where period mismatches between adjacent tissue increase the lag in oscillation phase over time. The existence of an oscillation period gradient has been reported in the later stages of the embryo across species, but not much is known about its regulation. FGF and Wnt signaling gradients present in the PSM have been nominated to control oscillation dynamics. However, past studies have reported that FGF signaling does not affect the segmentation clock, as segmentation pace was unaltered when FGF signaling was perturbed. Using an in vitro model of the PSM also showing an emergence of traveling waves, I characterized the effect of exogenous FGF signal on the wave dynamics in detail. The series of spatially and temporally designed FGF addition experiments indicate that FGF signaling governs the rate of change in oscillation period, or biologically, the rate of cellular maturation towards differentiation. Taken together, I uncover a novel role of FGF signaling in regulating spatiotemporal dynamics of segmentation clock oscillations in the mouse presomitic mesoderm.
Deregulation of oncogene expression is one of the main drivers in tumorigenesis. Genetic alterations, such as gene amplification and structural variation, or epigenetic mechanisms based on the chemical modification of DNA or histones, facilitate the activation of proto-oncogenes that convey growth and survival advantages to the cells. Previously, our group identified focal amplification of the chromosome arm 12q in 14 of 60 glioblastoma patients (23.3 %) of which 4 patients harboured fusion genes with the oncogene GLI Family Zinc Finger 1 (GLI1). In this study, I investigated the frequency and structure of GLI1 fusion genes, mechanisms of GLI1 transcriptional activation, GLI1-dependent tumour cell phenotype, and the potential value of GLI1 as a therapeutic target in precision-oncology in glioblastoma and liposarcoma. Initially, I identified GLI1 fusion genes linked with focal amplification on chromosome arm 12q in three independent glioblastoma cohorts (HIPO016, HIPO043, and TCGA-GB). GLI1 fusion genes were associated with high expression of GLI1 and its target genes, such as HHIP, PTCH1, and FOXS1. The boundary of the 12q amplification region often coincided with the GLI1 locus, presumably causing the breakage within the gene and the formation of fusion transcripts. The analysis of sarcoma tumours of the NCT MASTER study revealed high GLI1 expression in subtypes of osteosarcoma and soft tissue sarcoma. In addition, GLI1 fusion genes were found in liposarcoma and leiomyosarcoma. Furthermore, the disruption of a CTCF binding site upstream of the GLI1 locus upregulated the RNA expression of GLI1 and its target genes and increased cell proliferation. These data suggest that fusion-related genetic and epigenetic mechanisms regulate GLI1 expression. To explore its oncogenic function, I conducted phenotypic assays with and without GLI1 suppression and observed a reduction in tumour cell proliferation, anchorage-independent growth and increased apoptosis upon shRNA depletion or inhibition with the GLI1 inhibitor GlaB. The downregulation of several DNA repair pathways upon GLI1 depletion suggested that patients with aberrant GLI1 expression might benefit from combined GLI1 and DNA repair inhibitor therapy. To address this question, I performed a pre-clinical drug combination screen of GLI1 and DNA repair/cell cycle checkpoint inhibitors in glioblastoma and liposarcoma cell lines. In the primary screen, I tested inhibitors individually to identify effective and selective drugs of which the most promising candidates were tested in combination in the subsequent secondary screen. Both glioblastoma and liposarcoma showed high sensitivities to the SHH inhibitor JK184 and the GLI1 inhibitor GlaB. Synergistic effects were observed when GLI1 inhibitors were combined with inhibitors of the ATR/CHK1 axis, i.e., the CHK1 inhibitor LY2606368 or the ATR inhibitor Berzosertib. The independent validation of the screening results in cellular assays showed an increased effect of the combination treatment compared to the single agents on short- and long-term tumour cell proliferation. I furthermore confirmed the reduction in tumour growth upon treatment with GlaB and LY2606368 in a glioblastoma cerebral organoid model. In conclusion, these data suggest that concurrent targeting of the SHH/GLI1 and ATR/CHK1 axes provides a possible precision-therapy approach for tumours with high GLI1 expression.
Living cells are densely populated with macromolecules (Fulton 1982). To understand how proteins and nucleic acids dynamically inhabit and function in cellular volumes, knowledge of molecular crowding is critical to appreciate their modes of interactions and their spatio-temporal distributions. However, none of the existing approaches to measure molecular crowding allow for label-free and spatially-resolved analyses at the molecular scale. In this thesis, I quantitatively and structurally described molecular crowding inside cells utilizing recent advances in cryo-electron tomography (cryo-ET) (Koning et al. 2018, Schaffer et al. 2017, Turk and Baumeister 2020). Specifically, I investigated intracellular crowding in yeast cells under varying nutritional conditions. As their cytosol undergoes a dramatic transition from a liquid- to a solid-like state upon starvation (Joyner et al. 2016, Munder et al. 2016), I mapped changes in local molecular concentrations of ribosomes and fatty acid synthase (FAS) complexes, as well as structural rearrangements of these macromolecules, and other meso-scale protein assemblies. For this purpose, I co-developed methods for automated, high-throughput cryo-sample preparations, in particular cryo-focused ion beam (FIB) milling, and automated data mining utilizing deep-learning algorithms. These workflows allow for the analysis of large datasets which take stochastic cell-to-cell variations into account and are also applicable to other cell types. The automated methods will increase throughput and enable exploration of new biological questions in the long term. In this thesis, I showed that energy-depletion leads to large-scale reorganization of the wild-type yeast cytosol, including variations in particle distributions, conformational changes of specific macromolecular species and the formation of various higher-order assemblies. Determination of their structures provided novel insights into local alterations of macromolecules within the cellular context under different physiologically-relevant conditions. In particular, for both ribosomes and FAS distinct structural conformations were observed upon energy depletion which hint at stationary states, possibly protecting these molecular machines during stress. Future structural analysis of all visualized macromolecular assemblies in combination with coarse-grain and molecular dynamics modeling, will ultimately enable a more holistic understanding of cytosolic phase transitions at a molecular level.
Imaging Atmospheric Cherenkov Telescopes (IACTs) are complex instruments for ground-based γ-ray astronomy and require sophisticated software for the handling of the measured data. In part one of this work, a modular and efficient software framework is presented that allows to run the complete chain from reading the raw data from the telescopes, over calibration, background reduction and reconstruction, to the sky maps. Several new methods and fast algorithms have been developed and are presented. Furthermore, it was found that the currently used file formats in IACT experiments are not optimal in terms of flexibility and I/O speed. Therefore, in part two a new file format was developed, which allows to store the camera and subsystem data in all its complexity. It offers fast lossy and lossless compression optimized for the high data rates of IACT experiments. Since many other scientific experiments also struggle with enormous data rates, the compression algorithm was further optimized and generalized, and is now able to efficiently compress the data of other experiments as well. Finally, for those who prefer to store their data as ASCII text, a fast I/O scheme is presented, including the necessary compression and conversion routines. Although the second part of this thesis is very technical, it might still be interesting for scientists designing an experiment with high data rates.
Transition metal dichalcogenides (TMDs) are an intensively investigated class of layered materials and are regarded as promising candidates for various applications based on their exotic, layer-dependent optical and electronic properties. When applications are envisioned for a new material, control over the properties of the material is indispensable for efficient integration. Hence, the functionalization of layered nanomaterials is an ever-growing field with countless possibilities for tailoring these properties. However, literature focuses mostly on novel functionalization approaches and proof-of-principle applications, with fundamental questions of heterogeneous reactions at the nanosheet-solution interface rarely being tackled. In the first part of this thesis, insights into the influence of the surface chemistry on heterogeneous functionalization reactions at surfactant stabilized transition metal dichalcogenide nanosheets will be presented. A significant dependence of the heterogeneous gold nanoparticle functionalization of group VI TMDs, namely WS2 and MoS2, will be presented and a simple model is developed to explain observed regioselectivities based on the choice of surfactant and material. Preferential adsorption of representatives of commonly employed surfactants to distinguishable sites on the nanosheets is demonstrated and linked to the chemical structure of the respective surfactant.
Based on this demonstration, precise surfactant-mediated control of heterogeneous functionalization reactions can be envisioned and a generalization of the model for other material-surfactant systems is plausible.
The layer dependence and sensitivity of optical properties of transition metal dichalocgenides towards their environment is one particularly fascinating characteristic of this class of materials. However, a major drawback of the same characteristic is, that reproducibility and comparability of experimental results cannot be guaranteed where perfect control over the environment is not maintained. Especially for film deposition, where individual nanosheets come in close proximity, film morphology has a severe impact on optical properties and can lead to uncertainties in data interpretation. In the second part of this thesis, a promising method for thin-film production from liquid dispersions of nanosheets is presented, capable of largely alleviating these problems, at least on the laboratory scale. A custom deposition setup was developed to enable reproducible formation and transfer of films after preassembly of the layered materials at the interface between two immiscible solvents. These films are extremely thin and smooth, both on the order of 10^0 to 10^1 nm, and nanosheets are aligned over a sizeable area. A proof-of-principle experiment is presented that demonstrates non-covalent solid-state functionalization of WS2 thin-films with organic dyes and indications for electronic interactions between films and dyes are discussed based on changes in photoluminescence of both dyes and films. This well-defined deposition method is further compared to a complementary deposition approach producing porous films with randomized nanosheet orientation and the influence of morphology on the electrocatalytic activity of WS2 thin-film electrodes towards the hydrogen evolution reaction is discussed.
This deposition method should enable experimental designs previously inaccessible to layered nanomaterials produced from liquid phase exfoliation and improve reliability of both film production and data interpretation.
Every cell division relies on functional centromeres to guarantee correct chromosome segregation. They are particularly important in the germline to ensure successful reproduction and embryonic development. Centromeres are epigenetically defined by the incorporation of the histone H3 variant CENP-A and are embedded into pericentromeric chromatin. Non-coding RNAs generated by transcription from centromeric and pericentromeric regions propagate centromere function and specification. Yet, little is known about how (peri-) centromeric RNAs are regulated and which interaction partners they have.
The primary goal of my doctoral thesis was to identify factors that are associated with and influence the function of the lncRNA SatIII from the (peri-) centromeric regions of the X chromosome of Drosophila melanogaster. In this thesis, I identified RNA-binding proteins (RBPs) present at the centromere by re-analyzing a published mass spectrometry dataset that was produced by a crosslinked CENP-A pulldown approach. In doing so, I found out that the RBP Fmr1 is one of the top enriched factors and demonstrated that it binds to SatIII RNA in vitro. Additionally, I performed Fmr1 CLIP-Seq and showed that Fmr1 binds to (peri-)centromeric RNAs in the female germline. Moreover, Fmr1 associates with a subset of mRNAs including some that encode proteins which are important for centromere function, and also positively affects their translation. In order to find novel interactors of Fmr1 mass spectrometry was used. Thereby, I was able to identify the RBP Rump, which interacts with Fmr1 in an RNA-dependent manner, indicating their co-binding to common target RNAs. In fact, there is an overlap between Fmr1 and Rump CLIPSeq data in the non-coding RNAs that both proteins target. Additionally, I produced RNA-Seq data and showed that both proteins regulate the levels of non-coding RNAs that emerge from the centromere and elsewhere in the genome (in the male germline). Finally, Rump was shown to be required in maintaining CENP-A levels in mature sperm.
Overall, this thesis demonstrates an implication of the RBPs Fmr1 and Rump in centromere function and regulation in the germline of Drosophila melanogaster.
In this thesis, we develop new mathematical models and methods for the Optimal Control of constrained biomechanical Multi-Body Systems (MBSs) for problems appearing in therapy design of Cerebral Palsy (CP). We model the human body while walking as a constrained rigid MBS, and the gait as a solution of an Optimal Control Problem (OCP) which is constrained by the dynamics of this MBS. Here, changing foot-ground contact configurations lead to jumps in the differential states. Assuming that it is possible to provide a patient-specifically calibrated OCP whose (selected) solution models the gait of a patient, such kind of Optimal Control model can be employed to predict the effect of medical treatments on the gait pattern. In this setting, we focus on three aspects: possibly changing sequences of foot-ground contact configurations due to medical interventions, worst-case scenarios in presence of uncertainties, e. g., in the applied medical treatments, and a suitable translation of interventions into changes of the employed Optimal Control model. For the case that the sequence of foot-ground contact configurations after a medical treatment is unknown, we develop an approach for the numerical solution of OCPs with switches, switching costs, and jumps in the differential states, which can occur at switching. For this, we consider a Mixed-Integer Optimal Control Problem and extend the Partial Outer Convexification approach. We develop two types of so-called switching indicators which are utilized on the one hand as a trigger for events that are associated with certain types of switches, and on the other hand for the computation of switching costs. In the considered setting, medical interventions can be seen as changes of parameters that enter the gait modeling OCP. However, in medical practice unavoidable inaccuracies can occur in the implementation of an intervention. Therefore, we study worst-case scenarios for parametric OCPs with parameter uncertainties. We develop and examine an approach for the determination of worst-possible parameter realizations and the according OCP solutions which is suited for model-based treatment planning of CP. Here, we deal with a bilevel optimization problem with an OCP on the lower level. In order to apply our approach for worst-case treatment planning, we provide a suitable model for medical treatments. Since many interventions in CP management eventually aim at extending the ranges of motion of joints, we present a modeling approach that translates treatments of this kind into changes of parameters which enter the dynamics of the gait modeling OCP. The usefulness of the developed approaches is demonstrated in two case studies.
Magnetic resonance (MR)-guided radiotherapy (MRgRT) enables new treatment procedures such as online treatment plan adaption based on daily imaging of the patient. This allows for the delivery of high radiation doses to the tumor while optimally sparing surrounding healthy tissue. However, such complex workflows require thorough quality assurance including dedicated end-to-end tests to validate the feasibility and overall accuracy of each specific treatment workflow. In this thesis, end-to-end tests for online adaptive MRgRT in presence of inter-fractional anatomy changes were developed. For this, a standardized protocol for 3D polymer gel (PG) dosimetry and new anthropomorphic materials simulating the image contrast of real patients in computed tomography and MR imaging were developed. These methods were validated resulting in uncertainties < 5 % for PG dose measurements and < 3.5 % for the imaging parameters of the phantom materials. Finally, two end-to-end tests were developed and implemented at a 0.35 T MRgRT device using (i) a highly reproducible geometric phantom and (ii) an anthropomorphic and deformable pelvis phantom. These tests resulted in Gamma-index passing rates (3 %⁄ 3 mm) of 93.1 % (i) and 98.9 % (ii) for the PG-filled targets and demonstrated the feasibility of online adapted MRgRT in presence of inter-fractional anatomical changes.
In relativistic heavy-ion collisions, it is expected that the quark-gluon plasma (QGP) forms at an extremely high temperature and/or high baryon density. Hard-scattered partons (quarks and gluons), resulting in sprays of particles called jets, are crucial probes to investigate the characteristics of the QGP as they strongly interact with the QGP while traversing through the medium. The energy loss of jets due to the interactions with the QGP has been experimentally observed, and the theoretical prediction of Mach shock waves induced by jets as one of the possible interactions between jets and the QGP was suggested. In this work, an analysis of the angular correlations of particles (inclusive primary hadrons and identified protons) with respect to the axes of reconstructed jets is performed, to search for possible signs of a Mach shock wave induced by jet-medium interactions. This analysis was performed with the data measured in central \PbPb collisions at \sNN = 5.02 TeV in ALICE, using the \pt range for associated particles, \ptrange{2}{4} and for background-subtracted jets above 25 \GeVc. The analysis of the jet-hadron and jet-proton correlations did not show signs of the Mach shock waves in the QGP. Additionally, hadron-hadron correlations in pseudorapidity (\deta) with the tracks belong to $\pi - \pi/4 < \Delta\phi < \pi + \pi/4$ in the jet-hadron correlations were studied in a toy Monte-Carlo simulation and in the data. The expected signals created by the Mach shock waves were observed well via the hadron-hadron correlation method in the simulation, however, the signals were not observed in the experimental data and we cannot draw a conclusion from the result due to large statistical fluctuations. This observation can imply that the actual signal might be smaller than the signal in the simulation or washed out during the QGP medium evolution. Therefore, more statistics are require for more precise measurements. Though the current statistics in the results are too limited to make a conclusive statement, it will be interesting to examine these correlation functions considering the event-plane dependence and \pt-differential results with the increased statistics, planned in future ALICE measurements in 2022.
Melanoma is a devastating disease with a growing incidence, which represents a challenge for scientists and physicians worldwide. Due to its high metastatic potential and the rapid development of resistances to established therapies, this cancer still accounts for the majority of skin cancer-related deaths. In my thesis, I could show that ADCK2 plays a role in melanoma cells, especially affecting their metastatic capacity. After knocking down ADCK2 I observed a lower cell viability, but better migration and invasion of melanoma cells. Moreover, I noticed a reduced pigmentation of the pellets of melanoma cell lines, which could be explained by lower expression of melanocyte and higher expression of neural crest cell (NCC) markers. These results indicate a phenotype switch of melanoma cells to a more de-differentiated state, which is often associated with a higher metastatic potential. Additional analyses revealed a positive correlation between ADCK2 and Sox5, FoxD3, MYL6 and RAB2A expression in melanoma cells. Since the ADCK2 knockdown (KD) most significantly affected the migrative capacity of melanoma cells, I continued investigating the connection with MYL6, a protein involved in cell motility. I could demonstrate that overexpression (OE) of ADCK2 in melanoma cells had the opposite effect on cell viability and motility compared to the KD. The effect of ADCK2 OE on cell viability and migration could be reversed by a KD of MYL6, which suggests that MYL6 could be a downstream effector of ADCK2. Immunofluorescence stainings of ADCK2 KD cells revealed that these cells showed an altered actin cytoskeleton compared to control cells. One can assume that MYL6 could influence the cell motility of melanoma cells due to its function within non-muscle myosin 2, a known binding partner of actin filaments. Since ADCK2 negatively affected the metastatic potential of melanoma cells, it could be considered a tumor suppressor. As tumorigenesis and development share many similarities, I further investigated the role of ADCK2 (and other kinases) in the differentiation process of human induced pluripotent stem cells (hiPSCs) towards neural crest cells (NCCs). First to mention, I generated and validated two hiPSC lines that were stably maintained on Matrigel, one by reprogramming fibroblasts and another one by subcloning an existing cell line. Both cell lines were then successfully differentiated into NCCs in a xeno-free environment in ten days. Moreover, I could detect a downregulation of stem cell and upregulation of NCC marker expression upon KD of ADCK2 and DBF4 on day 0 followed by ten days of differentiation. Due to this result, one can speculate, that a KD of ADCK2 and DBF4 could lead to a more efficient differentiation of hiPSCs into NCCs. However, additional experiments are necessary to test this hypothesis. Moreover, it needs to be examined if these NCCs are functional and resemble naturally occurring NCCs. Additionally, I could demonstrate that a KD of TYK2 or EPHA4 resulted in increased stem cell marker and decreased NCC marker expression, which indicates that these kinases might be necessary for NCC development. Further experiments are needed to clarify this assumption. To conclude, I found that ADCK2 plays a role in melanoma cell behavior, but no strong role in the development of NCCs. A KD of ADCK2 induced a phenotype switch of melanoma cells to a more NC-like identity, validated by an upregulation of NCC markers and an increased motility. It would be interesting to further study the involvement of ADCK2 in melanoma and NCC development, which could yield more insights into melanoma development and thereby could open up new possibilities for melanoma therapy or diagnosis.
Studying the rapid neutron capture process (r-process) in stellar environment, that leads to the creation of about half of elements heavier than Fe, remains one of the fundamental questions of modern physics and therefore an active field of interdisciplinary research within nuclear structure, atomic and plasma physics, particle physics as well as nuclear astrophysics. Apart from other key measurable like neutron capture cross sections and decay lifetimes, nuclear masses are of utmost importance for pinpointing the r-process using theoretical and experimen- tal approaches. Exotic nuclides which participate in the r-process typically have extremely low production yields and short half-lifes. Those, accessible today at radioactive-ion beam facilities can be efficiently investigated in a storage ring. In such facilities non-destructive methods of particle detection are often used for in-flight measurements based on a frequency analysis. Due to low particle number and thus inevitably the low signal level, the detectors should be very sensitive and fast. While there are many sensitive detectors available to provide the information on particle’s revolution frequency, the main idea of this work is to design a position-sensitive cavity doublet for the Rare-Ri storage ring in RIKEN, Japan. Additionally, a new data acquisition system based on the software defined radio (SDR) was developed and tested with an existing resonant Schottky pickup during an experimental campaign in the ESR at GSI Darmstadt. SDR based data acquisition systems are essential for use in future distributed pickup systems such as those planned in the collector ring (CR) of the FAIR project. Finally a fully automated measurement system has been developed that can be used to measure the field profiles of such cavities in conjunction with the above mentioned data acquisition system. A toy model of such a position-sensitive resonant Schottky cavity doublet has been designed, constructed, manufactured and tested using the automated measurement system in the lab as well as at the linear accelerator S-DALINAC at University of Darmstadt.
In this thesis, I attempt to gain a deeper insight into the details of the human mirror neuron system by finding the effective connectivity of its central regions and simulating them with computational modeling. To achieve this aim, I have used the measured functional magnetic resonance imaging (fMRI) data for healthy participants while performing key tasks of social cognition (imitation of emotional faces, theory of mind, and empathy). Using a self-developed firing-rate-based extension of the statistical analysis procedure dynamic causal modeling (DCM), I was able to determine the effective network structure of the human mirror neuron system from the fMRI data and compare it between the different tasks of social cognition. In particular, far more complex processing occurs in imitation than in the other two tasks, which seems plausible given that imitation involves matching observed and self-performed emotional expression. Furthermore, we were able to show that the extended DCM procedure allows for significantly better model evidence, both for our novel data and for previously established datasets from other research groups. Thus, in addition to the substantive insight, this project has provided an important methodological advance for all users of the widely used DCM procedure.
Furthermore, the main regions of the mirror neuron system are modeled in detail by a modification of an existing, completely data-driven spiking network model of the prefrontal cortex. Here, I use the estimated parameters of the modified DCM to match the time series of the simulated and observed data. This two-stage approach allows both to account for the neural mass signals measured by fMRI and assess the fine-scale temporal dynamics of the local dynamics, and thus derive predictions about the physiological details that cannot be obtained from non-invasive recordings alone.
A local, active thermographic measurement method was advanced and used for measuring the mean viscous shear stress in the water-sided boundary layer of a wind driven air-water interface in the presence of water waves, at low wind speeds up to u 10 = (4.8 ± 0.3) m/s. Higher wind speeds of up to u 10 = (10.7 ± 0.7) m/s were considered to explore the boundaries of the application of the method. The measurements were conducted at the annular wind-wave facility Aeolotron in Heidelberg, Germany. The measurement technique utilizes a thin line which is heated onto the surface perpendicular to the wind direction. The broadening of the line is enhanced by Taylor dispersion due to shear flow in the boundary layer. The temporal development of the line width is monitored by an infrared camera. The broadening is compared to numerically simulated line widths, enabling the determination of the viscous shear stress. This initial study with a rough surface showed promising results for both the evaluation of longer time intervals, and temporally resolved measurements on the order of few seconds. Stationary conditions with the air and water compartments in dynamical equilibrium, corresponding to a quasi- infinite fetch, and non-stationary conditions after turning the wind on were considered. The stationary results were compared to results for the viscous shear stress obtained by water-sided particle streak velocimetry, with results deviating by less than 15%. The non-stationary measurements indicated an overshoot in the viscous shear stress shortly after turning the wind on.
In this thesis, we study the infrared regime of QCD with functional methods. To this end, we present the Mathematica package QMeS-Derivation. It allows for the derivation of symbolic functional equations from a given master equation. We apply this tool to Landau gauge Yang-Mills theory, where gauge invariance is implemented via BRST symmetry. We solve a self-consistent set of momentum-dependent functional equations within a vertex expansion and compare the correlation functions obtained from different functional approaches. We find good agreement of the results, hinting at gauge consistency of our setup. We proceed by using the obtained Euclidean results to compute four-gluon correlation functions, from which we extract the scalar and pseudo-scalar glueball mass by finding spectral representations of the dressings. The obtained results agree well with the masses computed from other methods. Next, we consider QCD at finite temperature and finite chemical potential and provide a setup for thermal correlation functions, where we introduce a thermal split in the quark-gluon vertex. This setup allows for a study of the phase diagram of QCD, where special emphasis is put on the investigation of the chiral phase transition.
The J/ψ mesons is a bound state of a charm quark and its anti-quark. The production of J/ψ mesons serves as an important probe of the quark-gluon plasma (QGP) formed in heavy-ion collisions. The presence of the deconfined strongly interacting medium results in the color screening of the potential between charm quarks and anti-quarks created during hard scattering processes. At sufficiently high collision energies such as the LHC energy, the large abundance of produced charm quarks leads to the (re)generation of J/ψ mesons during the QGP evolution and/or at the phase boundary. In this work, the J/ψ production yield in Pb–Pb collisions at √s_NN = 5.02 TeV recorded by ALICE is measured at midrapidity via the dielectron decay channel J/ψ → e+e− as a function of centrality and as a function of transverse momentum down to p_T = 0.15 GeV/c in the most central and semi-central collisions. The J/ψ nuclear modification factor R_AA increases slightly from semi-central to the most central collisions. The p_T -differential J/ψ R_AA shows a large suppression of the J/ψ yield at high p_T and the J/ψ R_AA increases with decreasing p_T. The measurement is compared to other experimental results and to model calculations. The presented results strongly support picture of (re)generation as an important J/ψ production mechanism at the LHC especially significant at low p T and at midrapidity.
The present thesis deals with the application of techniques developed in the field of quantum optics to the exotic atom positronium (Ps), for the purpose of the preparation of cold ensembles of Ps atoms in a magnetic field. The positronium atom, which describes the bound state between an electron and its own antiparticle, the positron, shows numerous peculiarities setting it apart from all other atoms which have been subject to laser cooling so far. In particular, its antimatter character as well as the extraordinarily small mass are accompanied by several unusual phenomena and, together with magnetic-field-induced effects, entail a highly complex laser cooling scheme. Due to the exotic properties, combined with the purely leptonic composition, cold and dense clouds of Ps atoms would represent an ideal testing ground for several fundamental theories and pave the way for many further fascinating applications, such as positronium Bose-Einstein condensation. The conducted numerical simulations, which consider the full complexity of the scheme, reveal that a high cooling efficiency can be achieved with appropriate laser radiation in weak (|B| < 50 mT) and strong fields (|B| > 0.7 T) for many realistic experimental configurations. Based on these results, Ps laser cooling has been realised experimentally within the AEgIS experiment at CERN. The measurements clearly demonstrate successful exertion of a symmetric optical force on the Ps ensemble in a field of |B| = 180 G, namely in the form of a significant population enhancement in the centre of the velocity distribution, which represents a key feature of laser cooling as it is unambiguous evidence for laser-induced recoil effects.
Multicellular organisms require specialized cell types in order to function. While a widely accepted definition does not exist, cell types are regarded as groups of cells with similar properties, such as RNA expression, protein abundance and epigenetic modification. Single-cell RNA sequencing (scRNAseq) is a recent breakthrough for explor- ing cell types, providing expression estimates for all genes in thousands of individual cells. Using data-driven algorithms, such as unsupervised clus- tering, scRNAseq has discovered new cell types and created large reference data sets, next to other exploratory achievements. More recently, scRNA- seq was applied to patient cohorts that include different groups, for example disease and healthy or disease subtypes. These multi-sample multi-condition data sets enable statistical inferences between groups, such as differential ex- pression testing. In contrast to projects exploring unknown tissues or species, patient cohorts often study known cell types defined by specific marker genes. Here, I present Pooled Count Poisson Classification (PCPC), a novel cell type classification approach designed for inference with multi-sample multi- condition scRNAseq data sets. PCPC implements a statistical model that allows researchers to distinguish cells according to marker-based cell type definitions, enabling reproducible and comparable analysis between data sets and technologies (e.g. scRNAseq and flow cytometry). Specifically, PCPC pools marker gene counts across related cells to overcome technical noise, and compares them to a user-defined threshold using the Poisson model. In this work, I apply PCPC to three different data sets to demonstrate its utility. The first application shows it is able to annotate all lineages in data from human cord blood mononuclear cells (CBMCs), with a single marker gene per cell type. The second application shows PCPC is able to discriminate fine cell type sub- sets, using data from a human tumor of mucosa-associated lymphoid tissue (MALT). Many cell types in the MALT tumor microenvironment, and T cell subsets in particular, are transcriptionally related, making their classification difficult. In spite of this challenging complexity, PCPC can even use lowly expressed marker genes, such as FOXP3 marking CD3E + CD4 + FOXP3 + reg- ulatory T (T reg ) cells. Furthermore, I find T reg cells isolated from the MALT tumor can further be subdivided into CCR7 + and ICOS + subsets, indicating a mixture of naive-like and activated T reg cells. In comparison to unsuper- vised clustering and the marker-based tool Garnett, classification with PCPC has more flexibility and fewer misclassifications, respectively. Thus, PCPC removes obstacles in studying complex tissues with scRNAseq, such as the microenvironment in human tumors. Furthermore, I demonstrate a multi-sample multi-condition comparison using data from a patient cohort of aggressive and indolent lymphoma subtypes. PCPC is applied to classify CD3E + CD8B + cytotoxic T cells, followed by differential expression testing between the aggressive and indolent subtypes. This uncovers significantly lower LGALS1 expression in indolent tumors, further implicating this gene in tumor aggressiveness and T cell inhibition. Currently, PCPC requires data generated with unique molecular identifiers (UMI), as well as substantial manual work. Due to its ability to resolve com- plex tissues with few marker genes, PCPC may bring clarity to transcrip- tomic cell type definitions and prove useful for multi-sample multi-condition comparisons in scRNAseq data.
The major goals of the thesis are design and characterization of functional self-assembled monolayers (SAMs) in context of electrostatic interfacial engineering and molecular electronics as well as a study of their thermal stability. The issue of electrostatic engineering can be addressed using custom-designed SAMs with either terminal dipolar groups or dipolar groups embedded into the molecular backbone. As for the first task, the novel concept of embedded dipole was successfully applied to the oxide substrates, which are highly important for photovoltaic applications. A variation of the work function of indium tin oxide (ITO) by 0.5 eV as compared to the reference non-polar functionalization was achieved at the invariable character of the SAM-ambient interface, allowing, thus, to decouple electrostatic engineering from the interface chemistry. The extremely low work function value for one of the tested monolayers expands a rather limited selection of SAMs capable of significantly lowering the work function of ITO. As a further task, electrostatic effects in charge transport across monomolecular films were studied, which is currently one of the most intensely discussed topics in molecular electronics. The tuning of the electrostatic properties was achieved by the fabrication of binary SAMs of biphenylthiolates (BPT) on Au(111), namely by mixing of BPT with fluorine-substituted-BPT (F-BPT) and 4-methyl-4′-BPT (CH3-BPT) with 4-trifluoromethyl-4′-BPT (CF3-BPT). The charge tunneling rate across the binary SAMs was found to vary progressively with their composition between the values for the single-component monolayers, and could, consequently, be fine-tuned and correlated with the work function. The observed behavior was tentatively explained by the appearance of an internal electrostatic field in the SAMs, leading to a change of the energy-level alignments within the junction upon contact of the SAMs to the top eutectic GaIn electrode. The height of the respective injection barrier is, however, unaffected by such a field, corresponding to the values of the transition voltage, which do not change notably with the SAM composition. Analysis of the presented and literature data suggests that the position of a dipolar group in SAM-forming molecules has significant impact on the charge transport behavior of the respective SAMs in the context of molecular electronics. As the next sub-project in the latter context, custom-designed SAMs of ferrocene/ruthenocene-substituted biphenylthiolates and fluorenethiolates on Au(111) were studied. The novel element of these SAMs was the fully conjugated molecular backbone, in contrast to the previous studies utilizing alkyl linkers as elements of molecular diodes. The designed SAMs exhibited a highly exceptional charge transport behavior showing conductance switching triggered by the applied bias. The extent of this switching, described by a maximum rectification ratio (RR) higher than 1000, was comparable to the best performing molecular diodes but in contrast to these “devices” was maintained at very low bias, close to zero volts. The observed behavior could be tentatively explained by a non-reversible redox process affecting the electronic structure of the molecules and their coupling to the top electrode. The above results are particularly promising to create novel molecular devices for potential applications in electronic circuits, molecular memory, or as an electrochemical sensor. Finally, the issue of thermal stability of functional SAMs on coinage metal and oxide substrates was addressed. This issue is of a crucial importance for applications, defining the temperature range of SAM-based devices and framing the preparation routes involving high temperature steps. Several representative SAMs with thiol anchoring group on Au(111) substrates and phosphonic acid (PA) anchoring group on Al2O3 substrates were studied by high resolution X-ray photoelectron spectroscopy chosen as the most suitable experimental tool. The range of the thermal stability and the degradation pathways were found to depend on the chemical composition of the SAM-forming molecules and the character of the substrates, with such crucial parameters as the strength of substrate-anchoring group bond and the presence of a backbone-specific “weak links”. In general, PA monolayers on oxide substrates were found to have higher robustness and better thermal stability compared to thiolates SAMs on coinage metal substrates. My results show, however, that is always advisable to test thermal stability of a specifically designed functional SAM in context of possible “weak links” as far as this stability is important for a particular application.
A central question in the life sciences is how an observed phenotype is realized by a given biological system. One explanatory factor is certainly the genotype of such a system which can easily be characterized due to the development of high-throughput next-generation sequencing (NGS) technologies. This allows to generate many hypotheses which are best tested by manipulating genotypes and observing the resulting change in phenotype. However, respective high-throughput technologies for genotype manipulation are lagging behind.
This thesis presents technical advancements in the field of genome engineering and next-generation sequencing which allow to construct and characterize collections of mutants with tagged genes.
First, an improved version of a targeted NGS strategy is introduced, which is termed Tn5-Anchor-Seq. This protocol for genome walking sequencing allowed to characterize unknown sequences adjacent to known genomic sites so that for example tag integrations could be mapped. It builds upon the concept of tagmentation and was designed with scalability, efficiency and sensitivity in mind.
Second, CRISPR-Cas12a-assisted tag library engineering (CASTLING) is presented as a high-throughput pooled strategy for gene tagging in the yeast Saccharomyces cerevisiae. It was implemented and validated using a set of 215 genes which were simultaneously targeted. Furthermore, genome-wide targeting was explored revealing that ~50% of yeast genes can be covered within one single experiment. Factors important for further application of this technology were identified and are discussed.
Third, the insights gained during the development of the CASTLING strategy motivated the application of their concepts for single gene tagging in mammalian cells. Usually, mammalian gene targeting is relatively inefficient and laborious. Therefore, a convenient CRISPR-Cas12a-assisted PCR tagging strategy was developed. Several targeted NGS approaches including Tn5-Anchor-Seq supported the validation of this technology. Furthermore, these analyses allowed the characterization of an experimental artifact associated with mammalian PCR tagging which can most likely be explained by aberrant tag expression.
Finally, Tn5-Anchor-Seq was applied to the characterization of a diagnostic RT-LAMP assay for SARS-CoV-2 detection. The high scalability of the resulting LAMP-sequencing protocol allowed to sequence RT-LAMP reactions from 768 patient samples and by that helped to validate the sensitivity and specificity of this assay. This was important for deploying additional testing capacity during the COVID-19 pandemic.
In conclusion, this thesis introduces and showcases scalable approaches for pooled and single gene taggings in yeast and mammalian cells. In this context also an improved genome walking procedure was implemented which furthermore supported the establishment of a diagnostic assay for SARS-CoV-2 detection.
Understanding biology across various spatial scales requires appropriate tools that allow the collection and analysis of information about specific events throughout the organism of interest. Imaging techniques are one of the most powerful tools of modern biology enabling direct visualisation of structures and processes with high spatial resolution and often in real time. Especially potent imaging modalities were developed based on light microscopy that allow high resolution imaging in the cellular and subcellular regimes. Unfortunately, these techniques are not suitable for imaging in highly scattering and thick samples such as mammalian tissues. While those can be imaged by biomedical imaging techniques such as MRI or CT, they typically achieve much lower lowered spatial and temporal resolution as well as are incompatible with the large toolkit of molecular probes and approaches used in life sciences (e.g. fluorescent proteins).
Here, emerging technologies based on photoacoustic imaging (where light is used to excite acoustic waves inside the tissue) enable bridging the biomedical and biological techniques by combining light based excitation (that is compatible with the optical toolkit of life sciences) with deep penetration capabilities characteristic for the biomedical regime. Several techniques of photoacoustic imaging were developed with photoacoustic tomography emerging as a particularly interesting modality for large-volume, high-quality imaging in mammalian tissues, due to it's ability to simultaneously record information from the whole volume of interest. In particular, there is a rising interest in developing all-optical photoacoustic approaches which use light both for exciting and detecting the acoustic waves. These bring promise of simplification and miniaturisation of the detector elements which would allow for easier animal handling and enable combination with other light based modalities such as multiphoton microscopy.
The aim of this thesis was to establish a high-performance state-of-the-art all-optical photoacoustic system based on a Fabry-P\'erot pressure sensor that could be used in life sciences for imaging applications in mouse biology. In particular, I explored possible improvements in speed and sensitivity with the long-term goal to enable whole-brain calcium based neuroimaging in mice. In this thesis, I describe my work towards the realization of this system including the detailed discussion of the system design and operating principles. I show preliminary imaging experiments in fish and mice to validate the capabilities of our photoacoustic tomography setup to perform high resolution \textit{in vivo} imaging.
The main part of the thesis is then concerned with the description of various approaches for increasing photoacoustic tomography sensitivity, including the use of adaptive optics for enhanced cavity coupling, passive photodiode amplification as well as deep learning based denoising. Furthermore, a full theoretical framework is presented for explaining the effects of interactions between optical aberrations and Fabry-P\'erot cavity modes, which is then extended to explain fundamental optical processes in adaptive optics. Moreover, two frameworks are described for increasing the volume rate of Fabry-P\'erot based systems including optimising the scanning trajectory as well as using optical multiplexing for parallel readout from the sensor. Finally, further directions of work are discussed including tackling the effects of skull induced acoustic aberrations on the image resolution and the choice of possible candidates for photoacoustic calcium sensors.
High-throughput techniques such as microarrays and RNA-sequencing enable the relatively easy and inexpensive collection of bulk gene expression profiles from any biological condition. Recently, also the transcriptome of single cells can be efficiently captured via novel single-cell RNA-sequencing technologies. Functional analysis of bulk or single-cell gene expression data has been proven to be a powerful approach as they summarize the large and noisy gene expression space into a smaller number of biologically meaningful features such as pathway and transcription factor activities. In the first part of this thesis, I expanded the scope on the pathway analysis tool PROGENy and the transcription factor analysis tool DoRothEA through thorough benchmarking pipelines. First I transferred their regulatory knowledge from human to mouse to enable the functional characterization of gene expression profiles from mice. Moreover, I demonstrated the robustness and applicability of both tools on human single-cell RNA-sequencing data. In the second part of this thesis, I focussed on the analysis of gene expression profiles from mice and humans in the context of acute and chronic liver diseases. Finally, I identified and functionally characterized exclusively and commonly regulated genes of chronic and acute liver damage in mice and a set of genes that were consistently altered in a novel chronic mouse model and patients of chronic liver disease. Especially the latter demonstrates that, although major interspecies differences remain, there is a common and consistent transcriptomic response to chronic liver damage in mice and humans. This set of genes could be further investigated to study the pathophysiology of the liver in in-vitro and in-vivo studies.
Glioblastoma (GBM) is the most aggressive tumor of the central nervous system. Median survival time is 15 months from diagnosis. Standard of care for GBM patients includes surgery, radiation and chemotherapy. All known therapies for GBM patients do not lead to prolonged survival. Some targeted therapies can lead to a prolonged progression free survival. Overall survival of patients however is not increased by any available therapy option. MEOX2 is a homeobox-transcription factor. It is highly upregulated during embryogenesis and responsible for the development of muscles, angiogenesis and limb development. MEOX2 can regulate the epithelial-mesenchymal transition of cells via cyclin dependent kinase signaling. MEOX2 is not expressed in the adult brain tissue however is significantly upregulated in GBM and correlates with tumor grade. High MEOX2 expression in gliomas is generally correlated with a worse clinical prognosis for patients, underlining the potential role of MEOX2 in tumorigenesis. As current therapy options can not provide a cure for GBM, it is crucial to understand the role of MEOX2 and reveal candidates connected to the malignant transformation of gliomas for further investigations. In a lower grade progression free state, glioma could be treated like a chronic disease. Therefore, I identified MEOX2 regulatory effect on cancer associated pathways and unraveled the target genes of MEOX2 in two GBM patient-derived tumorsphere lines. I also identified an ERK phosphorylation site in MEOX2 in the GBM tumorsphere line (S155) and elucidated the nuclear MEOX2 protein distribution depending on its phosphorylation status. I was also able to identify MEOX2 as an oncogene in vivo, using a xenograft mouse model where I observed increased proliferation upon MEOX2 overexpression in a GBM tumorsphere line that exhibits slower growth kinetics. Lastly, I observed MEOX2 knockdown in one of two GBM tumorsphere lines reduced sensitivity to EGF activated ERK phosphorylation that was reversed by a MEK inhibitor. All results indicate a role of MEOX2 as an oncogene in gliomas, and MEOX2 may serve as a future prognostic marker in gliomas. It is favourable to identify GBM in an early state which is currently not possible due to diagnosis based on MRI scans, this way GBM has a lower amount of lesions and accumulated mutations, which are gained in tumor development.
Single crystals of organic semiconductors are chemically pristine and exhibit nearly perfect long-range structural order. As such, they provide an ideal platform to investigate intrinsic properties. Vibrational spectroscopy techniques, such as Raman and Fourier-transform infrared spectroscopy (FT-IR), are widely employed techniques for the characterization of organic materials. They are versatile tools that can be used to study molecular packing and polymorphism in crystalline organic semiconductors, albeit with poor spatial resolution. Two fundamentally different scanning probe techniques with infrared spectroscopy and imaging capabilities offer a spatial resolution below 100 nm - atomic force microscopy-infrared spectroscopy (AFM-IR) and scattering-type infrared scanning near-field optical microscopy (IR-SNOM). This thesis compares the AFM-IR and the IR-SNOM with each other and to the conventional FT-IR spectroscopy with regard to their applicability to small-molecule organic semiconductors. To this end, single crystals of TIPS-pentacene, TIPS-tetraazapentacene, rubrene and per uorobutyldicyanoperylene carboxydiimide (PDIF-CN2) are used as the testbed. Significant differences are observed in the spectra of the crystals depending on the technique and polarization of incident light that are associated with the intrinsic molecular structure and packing as well as the different working principles of the applied methods. Furthermore, the imaging mode of the AFM-IR and the IR-SNOM is tested on solution-deposited microcrystals of PDIF-CN2. Micro- and nanostructures of layered organic materials can also be created by liquid-phase exfoliation (LPE), a popular technique used to produce two-dimensional nanosheets from layered inorganic crystals. The orthorhombic and the triclinic polymorphs of rubrene are dispersed in aqueous surfactant solution by ultrasonication. Distinct nanostructures of rubrene, referred to as nanorods and nanobelts, are formed that are isolated via liquid cascade centrifugation. Their crystalline nature is confirmed through electron diffraction measurements and Raman spectroscopy. Absorbance and photoluminescence (PL) of the dispersions are found to be similar to rubrene solutions due to random orientations of the nanostructures, however, their PL lifetimes are comparable to the macroscopic crystals. The likely arrangement of rubrene molecules within the nanorods and the nanobelts is deduced from AFM images, electron diffraction patterns, and IR-SNOM spectra.
This dissertation reports the design, development and benchmarking of novel research software inspired by the field of computer vision and data-science. The aim was to create versatile and robust solutions tailored to the requirements of microscopy-based phenotypic screening studies in small model organisms. The resulting software tools address various steps of the screening workflow, from manual ground-truth annotations, automated detection of regions of interest, targeted imaging of specific tissues or organs using feedback microscopy, image-classification, and interactive data exploration. Importantly, the tools are generic by design and were benchmarked on several microscopy datasets of zebrafish larvae and medaka embryos. They are particularly suitable for phenotypic screening studies at the tissue- and organ specific level. The software is easy-to-use and readily accessible to biomedical researchers with little to no prior knowledge of computer vision or image-processing. The tools are integrated in common scientific image-analysis packages and accompanied by extensive documentation in the form of articles in academic journals, readme files accompanying the source codes and online video tutorials. To foster their distribution and the inspiration of derived work, most of the underlying source code is available online in open-source repositories.
Ribosomes are conserved in the three domains of life, Archaea, Bacteria and Eukarya. They are essential for protein synthesis in all living cells. Formation of ribosomes in eukaryotic cells occurs through an energy-consuming multi-step process, involving a myriad of ribosome assembly factors and snoRNAs. This complex process starts in the nucleolus by the synthesis of a large ribosomal RNA precursor, the 35S pre-rRNA in yeast, which is co-transcriptionally cleaved to separate the pre-40S and the pre-60S pathways. The first intermediate formed during the early phase of ribosome assembly is the 90S pre-ribosome, the precursor to the small subunit. While the 90S to pre-40S route proceeds with its own set of assembly factors and snoRNAs including U3 and U14, little is known about the formation of the earliest pre-60S intermediates and whether snoRNAs have a role in this process. To gain insight into these early steps of large subunit synthesis, I isolated and studied a pre-60S assembly intermediate at the beginning of its construction, where specific snoRNAs also come into play. This analysis revealed a network of assembly factors and snoRNAs participating in the building of this primordial pre-60S particle. This particle is characterized by two large α-solenoid scaffold modules Rrp5 and Urb1, SPOUT methyltransferases Upa1 (YGR283C) and Upa2 (YMR310C), several RNA helicases including Mak5 and two prominent snoRNAs, C/D box snR190 and H/ACA box snR37. My findings suggest that snR190 and snR37 play a structural role in pre-60S assembly and their disruption caused changes in the primordial pre-60S composition. Moreover, I used the uncharacterized Upa1 and Upa2 as single baits to isolate the primordial pre-60S, which helped to unravel its overall shape by negative stain EM. By using a dominant mak5 helicase mutant, I showed that this nascent pre-60S intermediate does not efficiently separate from the 90S pre-ribosome, which enabled for the first time the visualization of a 70 nm super 90S–pre-60S bipartite particle by electron microscopy. These findings are relevant for human ribosome biogenesis, which occurs predominantly via the post-transcriptional mechanism and, thus, is expected to also involve a pre-ribosomal intermediate carrying both 90S and pre-60S moieties before pre-rRNA cleavage. Altogether, this work sheds further light on the earliest steps of pre-60S assembly and its connection to the upstream 90S pathway.
The recent success of targeted anticancer therapeutics has propelled cancer genomics to the forefront of clinical oncology. Patients with actionable mutations can now be treated with compounds that target cancer-specific pathways while minimizing damage to healthy tissues. Precision oncology aims to match treatment to a tumor’s mutational composition. Beyond genomic sequencing technologies, drug sensitivity assays play a crucial role in functional profiling of cancers. Tumor sample accessibility makes blood cancers particularly amenable to ex-vivo drug screening. Given the number of functional assays established in leukemias and lymphomas, critics often question the diagnostic value of ex-vivo drug sensitivities. One limitation of compound screening techniques in liquid cancers is the omission of microenvironment signals secreted by the bone marrow in vivo. To assess the impact of the microenvironment on drug response, I analyzed the high-throughput imaging data obtained from a compound screen conducted in primary leukemias and lymphomas (n = 108). In this study, patient-derived cancer cells were exposed to compound perturbations both alone and in coculture with a bone marrow stromal cell line. In total, 50 compounds were probed at 3 different concentrations across 2 culture conditions. I developed an automated analysis workflow, which was applied to > 700,000 confocal microscopy images. To extract multivariate phenotypes, I implemented a Python package (bioimg) for single-cell morphological profiling and performed the statistical analysis of compound effects in mono- and coculture. One of the key findings of the leukemia-stroma coculture study was that about 50% of the probed compounds were less effective in coculture compared to monoculture. Stratifying by compound class, I found that the efficacy of chemotherapeutics, BET and proteasome inhibitors was diminished by stroma-mediated protection. JAK inhibitors were the only compounds in the screen that reduced stromal protection. However, pro-survival effects of stroma were not uniform and stroma-induced morphology changes observed in cancer cells varied among samples. To understand this variability, I explored drug-gene associations in the presence of microenvironment signals and found that IGHVunmutated and trisomy-12-positive samples gained stronger stromal protection when treated with BCR inhibitors. In addition to precision medicine, antibiotic resistance research makes common use of highthroughput screening techniques. Misuse of antibiotics is driving the evolution and expansion of antibiotic-resistant pathogens, posing a significant risk to global public health. The current drug development efforts to combat this urgent threat are inadequate outside of academia. To address this gap, researchers have developed drug combination profiling systems to identify synergistic and antagonistic drug pairs. Species and strain specificity of synergies and antagonisms necessitates high-throughput investigations in multiple bacterial strains. To tackle these challenges, I analyzed the largest antibiotic combination screen in Gram-positive bacteria that exists to date. In this study, about 2000 drug pairs were probed in B. subtilis, S. pneumoniae, and two S. aureus strains. I developed a computational analysis pipeline that processed high-throughput bacterial growth data. My analysis revealed the landscape of drug interactions in Gram-positive species. I observed high within-class synergistic rates, especially for cell wall targeting compounds and inhibitors of protein and DNA synthesis. Compared with drug interactions observed in Gram-negatives, both abundance and interspecies conservation rates of synergies and antagonisms were lower in the Gram-positive organisms. Currently, high-throughput screening remains the only feasible method of uncovering drug-drug interactions on a large scale, as only a minority of synergy and antagonism mechanisms have been fully elucidated. To facilitate the rational design of combinatorial therapies, I analyzed drug-drug and druggene interaction data in E. coli and S. typhimurium with the goal of identifying genetic determinants of drug-drug interactions. Consistent with the previous findings in E. coli, my analysis revealed that ATP and lipopolysaccharide biosynthesis were among the most important biological processes for drug-drug interactions. In E. coli I found that ATP synthesis was particularly important for synergistic interactions among cell wall targeting compounds. Furthermore, I could show that it is possible to predict novel drug-drug interactions using chemogenomic data in E. coli and S. typhimurium. Finally, using a trained machine learning model I was able to identify genes predictive of drug-drug interactions within specific drug classes and calculate feature importance for individual predictions.
In the last years, our laboratory has developed and refined a novel platform of episomal self-sustaining S/MAR DNA vectors for gene and cell therapy. Their non-viral and non-integrative nature avoids integrational genotoxicity problems, a known issue with currently used gene therapy vectors. Previous work demonstrated their ability to provide persistent expression in cell lines and primary T-Cells. Further work successfully applied them to genetically modify mouse stem cells. Without altering their pluripotency capabilities, sustained maintenance and expression during reprogramming, differentiation and chimaera formation were shown. In this project, we aimed to extend our knowledge and understanding of these episomal vectors and developed and applied the technology towards human induced pluripotent stem cells (hiPSCs).
As a first step, we demonstrate the potential of our generated S/MAR DNA vectors in cancer cell lines and implemented vector establishment protocols with antibiotic selection or by purification of expressing cells via FACS sorting. We then provided proof of principle evidence that we can genetically modify hiPSCs using our S/MAR DNA vector system and demonstrated their isogeneity and unaltered capabilities to act as genetically modified cell source for gene and cell therapy. Besides implementing xeno- free hiPSC culturing which can easily be transferred to a GMP conforming protocol, we used cells isolated from urine as a novel, non-invasive cellular source for the generation of hiPSCs.
For the first time, we then moved from using the stable expression of GFP as an easily trackable reporter gene towards the restoration of a functional transgene in these cells. The potential of our S/MAR DNA vectors for use in advanced cell models and as prophylactic gene therapy for Birt-Hogg-Dubé syndrome (BHD) was then investigated. BHD patients harbour germline mutations in the gene for Folliculin (FLCN). After second hit mutations, functional FLCN is lost which leads to the development of kidney cancer. Currently, the only treatment available is the surgical resection of these tumours. However, this treatment does not restrict the development of further second hit mutations and tumours. Previous work by our group and others indicates that the pathways in which FLCN is involved and the tumorigenesis can be avoided by introducing a functional copy of FLCN. Thus, one potential treatment for this disease would be the introduction of mutation-proof copies of Folliculin into cells before the tumorigenic event takes place.
Utilising CRISPR/Cas9 we generated FLCN-knock-out urinary derived hiPSC cell lines. By establishing GFP or FLCN encoding S/MAR DNA vectors in both WT and KO cells, we generated important cell models for the study of FLCN. Characterisation of the cell lines as well as single-cell RNA sequencing suggested little impact of the S/MAR DNA vector on the cells. Also, FLCN expression levels were shown to be not required for the exit of pluripotency of these hiPSCs, rendering them a developmentally earliest cell model to study FLCN expression in a variety of cell models in the future. We finally demonstrated their unaltered nature and persistent transgene expression by implementing a kidney organoid differentiation protocol as an advanced cell model to investigate BHD.
Together, our data demonstrate the versatile application possibilities of the combination of our S/MAR DNA vector platform in combination with urinary and fibroblast derived hiPSCs for functional pathway analysis, disease modelling, patient- specific drug screenings or future cell therapies with optimised, non-viral gene therapy vectors.
The mechanical properties of the cell surface are master regulators of various cell processes, ranging from shape determination, to migration, to fate acquisition. The surface of animal cells consists of the plasma membrane, the actomyosin cell cortex, and Membrane-to-Cortex Attachment (MCA), defined as the protein-mediated tethering of the plasma membrane to the cell cortex beneath. MCA has been shown to contribute to cell surface mechanics and to be involved in the regulation of different biological processes at the cell surface. However, MCA remains the most elusive element of the animal cell surface and there is a clear gap in our understanding of its roles and regulation. This is mainly due to the lack of proper methods to specifically perturb MCA in cells. In this thesis, I am going to describe my PhD work on MCA. In the first part of my PhD, I engineered and validated a molecular tool, named iMC linker, which allows to increase specifically MCA in cellular model systems. Next, With iMC linker at hand, we studied MCA from both a biological and a biophysical viewpoint. First, we focused on cell differentiation, a process already shown to be regulated by mechanical properties of the extracellular matrix and the cell surface. Using mouse embryonic stem cells as a model system, we found that cells need to reduce their MCA in order to differentiate. Preventing this reduction by expressing iMC linker, locks the cells in a state of naïve pluripotency. Therefore, we uncovered a novel role for MCA in regulating cell differentiation. Second, I will describe our current efforts in deciphering the biophysical contribution of MCA to the mechanical properties of the cell surface. Strikingly, we found that MCA regulates cell cortex mechanics. Specifically, an iMC linker- mediated increase in MCA is coupled with a reduction in cell cortex stiffness and cortical tension. The implication of these findings may be relevant for various cellular processes regulated by cortex mechanics, such as cytokinesis and cell fate acquisition.
RNA editing is an epitranscriptomic modification of rising prominence in health and disease. It is catalyzed by enzymes from the families of 'Adenosine Deaminases Acting on RNA’ (ADAR) or ‘Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like’ (APOBEC). Multiple RNA editing deaminases, however, not only can they edit RNA, but also mutate DNA. ADARs particularly, are naturally capable of editing dsRNA co-transcriptionally, as well as mutating DNA in DNA/RNA hybrids. Although, the mutagenic role of ADARs is well-studied in vitro, its relevance with in vivo models has yet to be explored. DNA/RNA hybrids (or R-loops) form co-transcriptionally in the human genome between the nascent RNA and the template DNA strand, and I hypothesized that ADARs can access them to mutate the DNA strand in the hybrid, after losing touch with the nascent RNA-target. Here, I focus on ADAR1, which is overexpressed in Multiple Myeloma (MM) leading to aberrant editing activity and poor disease outcomes. RNA-seq and Whole-Exome Sequencing (WES) matched datasets from 23 MM patients pre- and post-relapse revealed acquisition of unique mutations post-relapse, enriched in the vicinity of RNA editing events pre-relapse. For proof-of-concept experiments in cell lines, I employed site-directed mRNA editing tools to target ADARs to specific transcripts, and evaluated whether ADAR-mediated DNA mutation was generated in their cognate genes. I found that ADARs may mutate genomic DNA in a rate of 1 in 25 000. Last, I explored the evolutionary impact of mutagenesis mediated by RNA editing enzymes (ADARs and APOBECs) in single-stranded RNA viral genomes from SARS-CoV-2 and showed that RNA editing enzymes may drive genome evolution by gradually accumulating co-occurring mutations, which similarly in cancer biology would translate to clonal expansion for tumor adaptation. Overall, my findings, suggest that DNA mutations may arise as collateral genomic damage by RNA editing deaminases, the initial job of which was to edit the cognate transcript in situ.
Accompanied by the fast evolution of graphical processing units, there is a rapid development of deep learning methods with applications in almost all natural and applied sciences. Simultaneously, a growing interest is emerging around alternative, more energy- and time-efficient computing devices. Driven by these developments, we propose in this thesis several possible directions in the field of quantum chromodynamics and beyond. We start with the exploration of novel frontiers to perform scientific computing tasks on the spike-based BrainScaleS neuromorphic device. This includes numerical computations in statistical physics and the representation of entangled quantum states. We continue by establishing a new mathematical framework to tackle the so-called sign problem impeding a statistical analysis of many physical systems, including quantum chromodynamics at finite density. Dealing with the same problem, a machine learning-driven algorithm is proposed in the subsequent part of the thesis. Utilizing deep neural networks to recognize undiscovered structures and to get novel insights into physical data is a further direction pursued by employing methods from explainable machine learning and by proposing a new unsupervised training algorithm for generating lower-dimensional representations. The thesis concludes with a supervised learning framework for approaching the inverse problem of reconstructing spectral functions from Euclidean propagators.
Mathematical models are becoming increasingly important for describing, explaining, and predicting human behavior in terms of underlying mechanisms and systems of mechanisms. Although the ontology of such mechanisms remains largely unknown, their epistemic value and inferential power are now widely acknowledged throughout the behavioral sciences. Broadly speaking, whenever an assumed mechanism transforms information into behavior, it is referred to as a cognitive process. Cognitive processes are the conceptual fabric used to fill the explanatory gap between the mysterious firing of neurons and the mundane recognition of a long-forgotten acquaintance in the morning train. Consequently, modelers of cognitive processes earn their livelihood in an attempt to make the “ghost in a machine” tractable by replacing the ghost with hidden parameters embedded in an abstract functional framework. The purpose of such parametric models is twofold. On the one hand, they can be viewed as formal expedients for understanding the messy and noisy human data in much the same way as the models physicists employ to make sense of the data coming from spiral galaxies and interstellar clouds. On the other hand, parametric models can be viewed as behavioral simulators and used to mimic the output of cognitive processes by generating synthetic behavior. Interestingly, there is a strange asymmetry in the challenges surrounding these two goals. Simulating behavior requires only specifying a cognitive model as a computer program and running the program with a desired parameter configuration. It is thus a generative process mainly constrained by the creativity and imagination of individual modelers. Differently, reverse engineering human data to recover hidden parameters is hampered by two external factors: the resolution and abundance of data and the availability of universal and efficient inferential methods. As for the latter, behavioral scientists have often sacrificed fidelity and complexity in order to adjust their models not to reality but to the limitations of existing inferential methods. Such a strategy is definitely viable in the early (often linear and beguilingly clear) stages of scientific inquiry, but it does not live up to the challenges and questions posed by later (often non-linear and disconcertingly fuzzy) stages. The main argument of this thesis is that questions of inferential tractability are of secondary importance for enhancing our understanding of the processes under study. Accordingly, the core purpose of this thesis is to develop frameworks which leave such questions to specialized ``black-box'' artificial neural networks and enable researchers to focus on developing and validating faithful ``white-box'' models of cognition. Instead of a ready-made solution, the thesis explores a beginning of a solution. It presents a potentially fruitful coupling between human and artificial intelligence, an approach which is expected to gain more and more momentum as the world fills with artificial agents. Ultimately, this thesis strives to increase creativity by embracing complexity.
Graft-versus-host disease (GVHD) remains a major complication following hematopoietic stem cell transplantation (HCT), affecting the skin as the earliest and most common target. Skin infiltrating alloreactive lymphocytes causing keratinocyte cell death are pathophysiological hallmarks of GVHD. In search for a therapeutic approach targeting skin localized alloreactive lymphocytes in acute GVHD (aGVHD), near-infrared photoimmunotherapy (NIR-PIT) was established as a strategy that has not been yet introduced in the field of dermatology. NIR-PIT combines the systemic application of a monoclonal antibody labelled with a photosensitizer. Upon exposure to near-infrared light, the conjugated agent causes rapid cell death along with its fluorescence emission. Initially, the selectivity of this approach was demonstrated in vitro when targeting CD8 T cells. For studying the therapeutic value of NIR-PIT in vivo, a humanized mouse model of cutaneous aGVHD was established. Here, CD8 specific NIR-PIT caused the entire and selective ablation of CD8 T cells from human skin grafts in which aGVHD was induced. At the same time, inhibition of histopathological signs and expression of the disease specific biomarker anti-leukoprotease elafin were observed. These studies assigned important effector functions to CD8 T cells in the context of cutaneous aGVHD. Hence, NIR-PIT can be emphasized to serve as a novel skin-selective therapeutic approach for clinical use. Up to this point keratinocyte cell death that occurs downstream to allorecognition was believed to result from either apoptosis or unspecific necrosis. As an extension of the first part of these studies, the exact mode of keratinocyte cell death in aGVHD was examined. To this end, we compared lesional and non-lesional skin obtained from aGVHD patients and observed a strong upregulation of molecules of the necroptotic signaling pathway: nucleic-acid (NA) sensor Z-DNA-binding protein 1 (ZBP1), phosphorylated receptor interacting protein kinase 3 (RIP3) and phosphorylated mixed-lineage kinase like transcription profile (MLKL). The chief role of interferon gamma (IFNγ) for inducing the activation and de novo expression of the necroptotic signaling pathway was initially demonstrated in keratinocyte cultures. These findings were both confirmed in ex vivo skin organ cultures and in vivo in a humanized mouse model of experimental human aGVHD. Finally, inhibition of STAT1 and RIP3 activities using tofacitinib and GSK’872, respectively, could prevent necroptosis, thus confirming the results. These studies uncovered how IFNγ-mediated necroptosis and the necroptotic signaling pathway play a central role in aGVHD related keratinocyte cell death. While in the first part of these studies the important role of alloreactive CD8 T cells in aGVHD and a novel therapeutic approach for a targeted therapy of skin diseases was introduced, subsequent studies identified necroptosis as a novel cell death pathway executed by IFNγ producing alloreactive T cells.
The question how hierarchically modularised structures arise from simpler ones is of central importance when desiring to understand our world. To that end, I present the Evolution Mechanics framework which aims to find a concise description of the mechanisms by which evolutionary systems unfold into hierarchically organised modules. While inspired by the evolution of biological life, Evolution Mechanics is abstracted from it and takes a more general perspective, providing a consistent language to address the fundamental processes giving rise to the complexity we observe all around and within us. In a second part, I study the evolution and behaviour of ecological interaction networks. Using an evolutionary food web model, I investigate the structures that arise within it, its response to local and global perturbations, and its capacity to be resilient against these perturbations. These studies not only illustrate aspects of Evolution Mechanics, but stress the importance of taking into account evolutionary processes when aiming to understand these systems.
39Ar Atom Trap Trace Analysis (ATTA) is a new dating method for the environmental sciences. With its half-life of 269 years and a dating range of 50 to 1000 years, it closes a gap in the set of the previously available dating methods. This thesis provides the first application of 39Ar ATTA on a lake system, i.e. Lake Kivu in Rwanda, including an elaborate data analysis routine using a 1D simulation tool. With 39Ar ATTA, extreme concentrations of 39Ar were found in some distinctly localised water layers in Lake Kivu and using the 1D simulation analysis, this effect could be traced to groundwater inflow springs featuring the highest 39Ar concentrations described so far. Several methodological improvements are presented as well which were necessary to achieve feasibility for this 39Ar ATTA study on Lake Kivu. Additionally, the potential of a total dissolved gas sensor (TDG) is explored with the aim of expanding its capabilities to measure the dissolved gasses in Lake Kivu and their partial pressures. An experimental setup to this end that simulates a pressure environment comparable to Lake Kivu is presented. Data from this experiment show the complexity of the coupled system of dissolved CH4 and CO2, water and a PTFE membrane. Moreover, a method to achieve a good partial pressure resolution from the TDG measurements of Lake Kivu is presented. Finally, a brief summary of the application of 39Ar ATTA on several environmental systems further demonstrates the versatility of 39Ar dating with ATTA for different environmental research contexts.
Chromothripsis is a form of genomic instability that begins with a shattering event leading to clustered chromosomal aberrations. Chromothriptic cells acquire multiple genomic alterations in confined genomic regions restricted to one or a few chromosomes. Due to the very high prevalence of chromothripsis in different types of cancers especially Sonic Hedgehog medulloblastomas, osteosarcomas and neuroblastomas and due to the link between chromothripsis and poor prognosis for cancer patients, finding therapeutic options to specifically target tumor cells with chromothripsis is highly relevant. Chromothripsis is linked to homologous recombination (HR) repair deficiency in a number of tumor types. Therefore, synthetic lethality may potentially be used, whereby HR repair deficiencies of cancer cells are exploited by applying a PARP inhibitor, which spares repair-proficient normal cells. In the primary screen, I searched for potential additive or synergistic partners of a PARP inhibitor (BGB290) and also searched for drugs that are potent as single agents. The analysis of the primary screen done in 15 cell lines identified romidepsin and nanaomycin A as promising candidates to eliminate tumor cells with chromothripsis. To confirm the potential additivity or synergism of these drugs with the PARP inhibitor, a secondary screen was done where drugs were titrated against each other in 8×8 matrix settings. The secondary screen revealed a strong synergistic interaction between BGB290 and romidepsin in chromothriptic Sonic Hedgehog (SHH) medulloblastoma patient derived xenograft spheroid models but antagonism in non-chromothriptic control cells. Functional assays revealed that the synergistic effect between BGB290 and romidepsin was not TP53 dependent. Romidepsin and BGB290 combination treatment caused G2 cell cycle arrest and apoptosis in chromothriptic DAOY cells. The mitotic rate was decreased by romidepsin and BGB290 combination treatment in DAOY cells as shown by acetyl-αtubulin and phospho-histone H3 immunostaining. Gene expression analysis revealed that the romidepsin and BGB290 combination treatment was associated to the regulation of MYC target genes. To further analyze the link between MYC expression and the effects of the combination treatment, I used an inducible MYC ON / OFF system. When MYC was overexpressed, romidepsin and BGB290 showed additivity, while in the MYC OFF state the additive effect was replaced by antagonism. This indicates that MYC overexpression plays a role in the interaction between BGB290 and romidepsin. Lastly, the in vivo efficacy of BGB290 and romidepsin combination treatment was confirmed in patient-derived xenograft mouse models.
How does biology innovate? Ever since Darwin’s famous visit of the Galapagos Islands and his studies beak shapes and food preferences of finches has this question been asked, addressed, and revisited. The study of biological innovation is at the core of all our attempts to explain morphological differences throughout different stages of animal development. Today, the availability of modern molecular techniques makes it possible to study biological innovations in great depth at the organismal, cellular, or genomic level. And still, it remains unclear how these different levels are linked to eventually produce something entirely new. In my thesis I identified two ways of how biology can create something new in the early stages of fly development, one at the cellular level and one at the tissue level. My discoveries are based on a comparison of the first hours of embryo development in the midge Chironomus riparius and the fruit fly Drosophila melanogaster. This comparison allowed me to identify and characterize cause and consequence of biological innovation, from new genes to new cell biology and putative adaptive benefits. The first part of the results section comprises my comparative work on the formation of the first epithelium in insect embryos, the blastoderm. Here I identified tall blastoderm cells as a feature of presumably higher fly species and small blastoderm cells as a feature of more basal flies. To characterize the function of tall cells and how they emerged I used a comparative approach to distinguish tall from small blastoderm cells and their formation using the fruit fly Drosophila melanogaster as a representative of tall cells and the midge Chironomus riparius as a representative of small cells. By moving from tissue- to cell-level organization, I identified slam, as the first of a set of new genes, that act as headmaster of blastoderm columnarization in flies. By experimental engineering the blastoderm of Chironomus from a cuboidal to a columnar blastoderm I show that this novel Rho/F-actin regulator controls epithelial cell lengthening by an extension of E-cadherin based adhesion along the basolateral membrane. These experimentally columnized cells were less affected by desiccation suggesting an advantage by an increased barrier function of the epithelium. The second part of my thesis focused on a previously identified diversity in extraembryonic tissue development within Diptera, where higher flies show a reduction in extraembryonic tissue development. More basal flies develop extraembryonic tissues that spreads over and covers the entire embryo. By studying interactions of yolk sac membrane with overlaying extraembryonic serosa cells in the scuttle fly Megaselia abdita I revealed decoupling of both tissues was necessary to ensure free spreading of the serosa to cover and protect the embryo. When interfering with the mechanism for decoupling, by interfering with yolk cortical actin or kock-down of Megaselia-Matrix metalloprotease 1 (Mab-Mmp1), coupling of both tissues was prolonged and serosa stayed at a dorsal domain similar to a reduced extraembryonic tissue. The reduction of extraembryonic tissue here pretty much coincides with the transition from small to tall cells.
Nanomaterials play an important role in the flourishing field of nanoscience. Size reduction of materials results in a broad range of outstanding physical and chemical properties as well as a wealth of potential applications. A particularly interesting class of low-dimensional nanostructures are two-dimensional (2D) materials, i.e. individual layers of so-called van der Waals crystals. The research was triggered in 2014 by Geim and Novoselov through the isolation and characterization of graphene, a single layer of two-dimensionally arranged sp2 hybridised carbon atoms. 2D nanomaterials can be obtained by various methods including bottom-up approaches such as chemical vapour deposition and top-down approaches such as liquid phase exfoliation (LPE) and mechanical exfoliation. In recent years, LPE has gained increasing attention due to the high production rates and broad applicability to a range of structures beyond graphene including transition metal dichalcogenides (TMDs), hexagonal boron nitride, metal phosphorus trisulfides and many more. In LPE, high energy and shear forces (e.g. through sonication) are applied to reduce the dimensions of the crystal and the resulting nanosheets are stabilized in the liquid medium through appropriate solvents and surfactant systems. The resultant nanosheets are extremely polydisperse in lateral size and thickness so that LPE is typically coupled with size selection, for example through centrifugation. Due to this additional processing step, it is difficult to assess the impact of the stabilizer on for example the optical properties of the nanosheets which will be a function of both size and stabilizer. In addition, the number of pure organic solvents suitable to prevent reaggregation is very limited which is a bottleneck for further processing and deposition. The goal of the work conducted within the scope of this thesis is to establish protocols to make high quality 2D nanosheets from LPE accessible in a range of liquid media and to achieve a deeper understanding of the impact of the stabilizer on the optical properties of the nanomaterial. To this end, tungsten disulphide (WS2), a semiconducting transition metal dichalcogenide was chosen as model substance due to unique optical fingerprints of the monolayers (e.g. narrow linewidth photoluminescence from exciton only in WS2 monolayers). Throughout this thesis, monolayer-rich dispersions of WS2 nanosheets were prepared by sonication-assisted LPE in a common detergent solution in combination with liquid cascade centrifugation for size selection. In the first part, a protocol was developed to transfer these nanosheets into a range of additive/solvent systems. The advantage over a direct exfoliation in this systems is that dispersions containing nanosheets of the same size/thickness can be compared. This allowed to assess the impact of various chemical environments on the optical properties and to study effects associated with the dielectric screening of excitons (e.g. changes in exciton energy and width). With this foundation established, the nanosheets were transferred into a range of common pure organic solvents using a modified protocol. This is more challenging due to aggregation taking place. Nonetheless, this broad screening made it possible to relate the changes in exciton response to physical parameters such as refractive index and dielectric constant. Importantly, it was confirmed that monolayers can be stable in solvents that are not suitable for the exfoliation itself greatly expanding the choice of solvent for further processing. The third part focuses on precise deposition of the nanosheets on substrates using spin coating. Experimental difficulties such as aggregation and restacking of nanosheets in solvents are addressed in detail together with solutions to improve the colloidal stability of the nanomaterials. In the optimized samples, monolayer properties, such as exciton photoluminescence, are retained after deposition. At last, a new route for transferring nanosheets from water-based WS2 dispersions into different media is introduced which greatly facilitates deposition. In this approach, water-insoluble polymers are added to the aqueous surfactant solution prior to sonication. Through hydrophpobic interaction, the polymer is driven to the interface between the hydrophobic part of the detergent and the nanomaterial. This polymer coating on the nanomaterial reduces aggregation after transfer to hydrophobic organic solvents, suitable for thin-film processing. Such techniques for nanomaterial processing are highly demanded for the integration of these materials into functional devices.
Keratinocyte cancers (KC) are the most common malignancy in fair-skinned populations, with millions of cases diagnosed yearly. Although historically overlooked by cancer registries, their continuously rising incidence and the economic burden they pose on health systems have put KC in the spotlight. KC arise from epidermal keratinocytes and comprise two main tumor types: basal cell carcinoma (BCC) and cutaneous squamous cell carcinoma (cSCC). The latter mostly originates from pre-cancerous dysplasias named actinic keratosis (AK) or in situ carcinomas known as Bowen’s disease (BD). Two subclasses of AK/cSCC were previously identified based on their DNA methylation profiles, reflecting two distinct cells-of-origin. This notion was further explored in this thesis by epigenomically characterizing 102 epidermal samples using Infinium MethylationEPIC BeadChips. This cohort included healthy controls, AK, cSCC, BD, BCC, and non-cancerous senile warts (seborrheic keratosis; SK), thus comprising the full range of malignancy in epidermal tumors. Methylation patterns at keratinocyte-specific enhancers stratified the samples into two subclasses with epidermal stem cell-like or keratinocyte-like profiles. While SK and BCC samples predominantly displayed keratinocyte-like profiles, cSCC and its precursors displayed both, indicating potential clinical implications. Further analyses indicated distinct cell division rates and invasive features between cell-of-origin subclasses. Lastly, single-cell methylomics and transcriptomics validated the cell-of-origin-based stratification of epidermal tumors. Dermal fibroblasts are a heterogeneous cell population with essential roles in maintaining skin architecture and function. However, their diversity in human skin and its functional significance are still relatively unclear. In this work, fibroblast heterogeneity was systematically analyzed using the single-cell transcriptomes of more than 5,000 fibroblasts from sun-protected healthy human skin. Four distinct subpopulations with specific localization and primed to exert differential secretory, mesenchymal, and pro-inflammatory roles were identified. Importantly, this priming was substantially reduced upon intrinsic aging. Collectively, this work provides important insight into the cellular origin of KC, establishes new opportunities for robust patient risk assessment, and represents a detailed analysis of human dermal fibroblast heterogeneity and their intrinsic aging.
Semiconducting, single-walled carbon nanotubes (SWCNTs) have mechanical and electronic properties that render them a promising material for solution-processable, stretchable and flexible electronics. However, their strong tendency to form aggregates in dispersion constitutes a large obstacle to realize the film uniformity necessary for the transition of devices from laboratory to commercial scale. The resulting inhomogeneities in film morphology lead to an undesired spread in device performance. Based on the tailored formulation of colloidal inks via suitable solvents and additives the first part of this thesis presents a simple yet effective method to slow down aggregation of polymer-wrapped SWCNTs in organic solvents. This effect on aggregation by 1,10- phenanthroline as a stabilizing additive can be monitored with time-dependent absorption spectroscopy. The improved homogeneity of the SWCNT networks deposited from stabilized dispersions after several days of ink storage lead to higher charge carrier mobilities with strongly reduced device-to-device variations compared to inks without additive. The intrinsic ambipolarity of SWCNTs is a great disadvantage for their use in electronic circuits as it leads to large power dissipation. While pure hole conduction can be achieved relatively easily by doping with, for example, ambient oxygen, facilitating exclusive electron conduction represents a large challenge. A solution-processable n-dopant from the family of guanidino-functionalized aromatics (GFAs) is introduced to overcome this limitation. The resulting SWCNT network field-effect transistors (FETs) exhibit pure electron transport with high mobility while hole transport is fully suppressed, excellent switching behavior and good operational stability. Their application potential (combined with a doped p-type FET) is highlighted by complementary inverters with very low power dissipation. This modification of the charge transport behavior is applied to another promising solution-processable semiconductor, i.e., donor-acceptor-polymers. Doping of these polymers with two GFA compounds under various processing conditions improves electron injection and transport while hole transport is suppressed. Again, these transistors display good environmental stability under operating conditions. The extended applicability of the newly introduced GFA dopants to different semiconductors emphasizes their potential for transistors based on solution-processable semiconductor
Understanding cell signaling is probably one of the biggest challenges in modern biology. Thanks to the advance in new technologies, like next generation sequencing and other high throughput techniques, commonly referred as omics technologies, researches can generate great amounts of comprehensive biological data. More recently, these technologies have advanced to the point where one can analyze genes or proteins in single cells, even with spatial resolution. The ability of these approaches to generate great amounts of data requires of complementary techniques to analyze it. Analyzing and contextualizing this amounts of data has provided great insight and development in our current understanding and treatment of many diseases. Current research on disease mechanisms focuses mainly on molecular processes in order to understand the underlying systems driving them. Many approaches have so far focused on intracellular signaling and it has not been until recent years that the role of cell-to-cell communication in health disorders has gained importance. With this goal in mind, I will be presenting approaches to model and analyze biological data accounting for intra- and intercellular communication. The models and analyses presented combine omics data with prior biological knowledge. For this, I rely and our in-house resource OmniPath to extract the relevant intra- and intercellular interactions.The analytical approaches presented in this thesis range from the more classical differential expression and gene set enrichment analysis to more advanced and recent machine learning methods. Thanks to the different strategies applied across different settings, I was able to extract relevant biological insights with applications in clinical and biological research. Despite the approaches presented in this thesis being mainly focused on cancer, these surely can be further extended and applicable in many other contexts.
The mean global ocean temperature increase since the last glacial maximum was recently estimated to be about 2.6 °C (Bereiter et al., 2018). This temperature increase is not homogeneously distributed throughout the ocean. While the surface and deep ocean are well studied, the dynamic thermocline ocean was long neglected. As cold-water corals (CWCs) inhabit these highly dynamic water depths, they are valuable archives for paleoceanographic studies. In this thesis, CWCs are used to reconstruct thermocline water temperatures using their Li/Mg ratio. A first study focuses on the Angolan margin. Here, glacial cooling of about 6 °C indicating a shoaling of the thermocline was found. Furthermore, CWCs from six locations throughout the Atlantic were studied. Similar temperature patterns over the last 40 ka were found at all locations. Radiocarbon data was used to estimate reservoir ages and changes in water mass pathways. An aging of thermocline waters south of 35 °N in the Atlantic is observed. During the LGM, extremely high Li/Mg ratios were found in regions with volcanic activity. These might be influenced through increased Li concentrations in the seawater. Thus, in the second part of the thesis, the hypothesis of locally increased Li input through hydrothermal fluids was tested. Therefore, the lithium isotopic composition of six CWCs was analysed. The isotopic composition of the CWCs does not vary, excluding an influence of hydrothermal fluids on the Li/Mg ratio of the CWCs. Hence, the origin of unusal high Li/Mg ratios of glacial corals remains unknown.
During the development of an organism, chemokine signaling provides long range guidance for migrating cells and tissues. Using an intricate system of promiscuous receptor-ligand interactions, the embryo coordinates the robust positioning of its future organs in space and time. Cells both sense and actively shape a highly dynamical and diverse signaling environment, thus making it challenging to systematically study cell signaling regulation and function in vivo.
To approach this problem, I combined novel methods from the fields of microscopy, cell biology and image analysis. Specifically, this includes multiple-view light sheet microscopy for imaging developmental processes simultaneously at the cell and organism scales and tandem fluorescent lifetime reporters as an emerging tool to visualize signaling receptor turnover in vivo. To quantitatively compare three dimensional embryo acquisitions across genetic conditions and multiplex fluorescent readouts, I used nonlinear image registration to perform spatial normalization. Further, the so obtained sample deformation maps were exploited to perform morphometric phenotype analysis. Together, these tools led to the development of a computational analysis framework enabling quantification of chemokine signaling activity and function during embryonic development.
This framework was then applied to search for novel interactions between the chemokine scavenger CXCR7 (or ACKR3) and the signaling receptor CXCR4 in the early zebrafish embryo. In recent years, the competition of these receptors for their shared ligands has been shown to be implicated in several developmental processes as well as tumor progression. In my analysis, loss of the scavenger was revealed to exert a strong activating effect on CXCR4 expressing tissues across the entire embryo. The remarkable up-regulation of signaling activity in the absence of the scavenger was however found to be aphenotypic in many target tissues. Therefore, I proceeded to investigate at which level the phenotypic impact of this genetic perturbation is compensated for and found receptor desensitization to likely be dispensable in this context.
In conclusion, this work provides an example of how spatial normalization and multiplexing of in toto light sheet images can be used to investigate the general logic and functional output of chemokine-scavenger interactions during embryonic development.
Quantum chemical simulations of molecular properties are crucial to obtain in-depth insight into a multitude of chemical and biological phenomena. In particular for investigating light-driven systems, modeling of electronic excitations by computational means is indispensable for supporting, complementing, and extending experimental findings. The complexity in terms of electronic structure, intermolecular interactions, and dynamics of the involved molecular systems, however, pushes the limits of computational feasibility. Hybrid quantum-classical environment schemes tackle this complexity by splitting the system into a quantum region and its environment. Thus, they retain the high-level quantum chemical description for the part of interest without neglecting the pivotal effects of the environment.
In this thesis, I develop methods for modeling molecular properties in complex environments. The first half of the thesis is dedicated to new combined approaches of the polarizable embedding (PE) model and the algebraic-diagrammatic construction (ADC) scheme for the polarization propagator for computational spectroscopy simulations. I derive and implement two PE-ADC coupling schemes: The first scheme – pt-PE-ADC – uses a self-consistent PE reference state with a canonical ADC procedure and is suited for computation of electronic excitation energies including a posteriori perturbative corrections. The second scheme – LR-PE-ADC – includes direct coupling to the polarizable environment in an iterative manner, making it suitable for excited electronic states and higher-order response properties. Furthermore, I derive working equations to evaluate analytic nuclear gradients using PE-ADC. To advance the availability of the PE model in general, I implement a standalone, open-source, and hybrid Python/C++ library, called CPPE, and interface it to several freely available quantum chemical host programs. The PE-ADC schemes are implemented with adcc, a toolkit for development of ADC-based methods and combinable with several Python-driven host programs. The simple and clean design of both libraries allows for extension of existing workflows and rapid prototyping. Moreover, I implement response properties using ADC and the intermediate state representation (ISR) in a new Python library, called respondo. The synergy of all three libraries enables the user to implement new features in a straightforward manner, while maintaining usability and efficiency for practical calculations. I test the individual approaches in several benchmark calculations and case studies. For example, I find that excitation energy errors using pt-PE-ADC for microsolvated p-nitroaniline are much smaller than the intrinsic error of ADC itself. Furthermore, I investigate the charge transfer (CT) state involved in the photoprotection mechanism of the flavoprotein dodecin. In addition, I conduct the first computations of higher-order response properties with ADC and a polarizable model. In these studies, I observe that LR-PE-ADC greatly improves the accuracy of the property compared to simpler coupling schemes. I further show that corrections for electron spill-out artifacts and the physically sound evaluation of PE-ADC intensities are decisive when benchmarking against supersystem calculations. With my theoretical derivations and open-source implementations, I provide, to the best of my knowledge, the most complete and unique feature set of polarizable models combined with ADC to date.
The second half of the thesis first contains a general performance improvement of PE models. I implement a PE scheme where the classical electric fields in the environment are evaluated using the fast multipole method (FMM) instead of direct summation. Consequently, the electric field evaluations as rate-limiting step of the classical part exhibit an asymptotic linear scaling in the PE-FMM scheme, making it suitable for efficient simulations of environments with over a million polarizable sites. Next, I show algorithm details for numerically stable solution of response equations in the ADC/ISR framework, and I analyze convergence behavior of different solver algorithms. These algorithms are beneficial for efficient evaluation of PE-ADC response properties, too. I present derivations and numerical case studies of complex excited state polarizabilities which extend the ADC/ISR framework beyond ground state response properties. Then, I investigate the distortion of molecules under external forces. I develop a new electronic structure method to apply hydrostatic pressure in standard quantum chemical simulations via Gaussian potentials, called GOSTSHYP. This implicit embedding scheme directly exerts pressure on a molecule via compression of the electron density, such that it becomes possible to treat atoms and molecules and to run geometry optimizations and dynamics simulations at a pre-defined pressure. This feature set is not found in any other comparable method. I use steered molecular dynamics (SMD) simulations with quantum chemical strain analysis tools to elucidate the rupture process of rubredoxin. I prove that the extremely low rupture force does not result from hydrogen bond networks to the protein as assumed so far in the literature, but that its origin is likely more intricate. Finally, I present the design of novel photocages based on fluorene derivatives. Using an efficient computational screening protocol, I propose cyclopenta-dithiophene as scaffold, leading to the next generation of fluorene-based photocages with desirable absorption and uncaging properties.
Cooperation is the cornerstone of life and human societies—its evolution is a perennial question. Evolutionary Game Theory models elucidated mechanisms promoting cooperation. However, they pay little attention to its emergence, operate with predefined states of defectors and cooperators, and presume defection as the natural state. Models need more realism. Here, I introduce ReCooDy, a model combining population dynamics, limited resources, dynamic networks, coevolution of nine parameters, and optional social dilemma interactions, all implemented in the Utopia framework, which allows performant, flexible, and reliable computer simulation. ReCooDy investigates the emergence of cooperation and defection in a generalized continuous public goods interaction that incorporates “true defection”—exploitative destruction for selfish benefits. I show that macroscopically classifying agents as selfish or selfless oversimplifies the intricacy of the emerging mesoscopic social dilemma. Simulations exhibit the emergence of cooperation, even for minute synergies if interacting is not crucial. For moderate synergy, agents evolve specializations that depend vitally on the interactions. Further, defection emerges naturally as a response to cooperation. ReCooDy exhibits recurrent dynamics patterns with history dependence and frequent strategy collapses through cascaded thresholds caused by Red Queen dynamics. Thus, simulations reveal ReCooDy's deterministically chaotic self-organized nature and the overall unintuitive phenomenology of more realistic social dynamics modeling.
Two-photon microscopy is currently the technique of choice for deep imaging in scattering, opaque specimen such as the in-vivo mouse, due to inherent optical sectioning and longer wavelength excitation light, which is generally less effected by scattering. However, the maximum penetration depth of two-photon microscopes is fundamentally limited by the on-set of out-of-focus fluorescence near the surface with increasing excitation power, which for the mammalian brain prevents imaging beyond ~1mm. Three-photon excitation fundamentally improves the depth limit due to a significantly increased signal-to-background ratio at depth and longer wavelength excitation. Unfortunately, optical aberrations stemming from the optical system and inhomogeneities within the sample lead to a degradation of resolution and contrast and loss of signal intensity at depth. However, aberrations can be corrected and near diffraction limited resolution recovered with so-called adaptive optics strategies. While two-photon microscopy has been combined with adaptive optics to correct for aberrations, very fine, sub- micron structures such as spines in the mouse brain are difficult to resolve with current methods in deep cortical or even sub-cortical brain regions. To tackle this challenge, in my PhD work I developed a custom three-photon microscope with integrated adaptive optics to increase the practical imaging depth and resolution for non-invasive in-vivo imaging of mouse tissue with the main focus on neuroscience application. In particular, I have designed and build a custom multi-photon microscope based on 1300nm excitation and shown its capability to image GFP-labeled neuron somata and even small structures such as dendritic branches up to a depth of 1.2mm in the intact mouse brain, which is among the best achievements demonstrated so far in the literature. At such large tissue depths, however, heart pulsation leads to brain motion and thus to intra-frame artefacts which prevent frame averaging to improve signal-to-noise ratio (SNR) of small structures. Therefore, we developed dedicated software and hardware to actively synchronize our image acquisition in real time to the cardiac cycle of the mouse. This improves SNR of small structure at depth without the need for sophisticated image registration techniques in postprocessing. Another main achievement of my work has been the development and integration of adaptive optics and its control software into our multi-photon microscope. Here I chose an indirect wavefront sensing approach which is more suitable for ultra-deep imaging. Together with our active motion-correction, our adaptive optics three-photon microscope enabled high, synaptic resolution imaging throughout an entire cortical column in the in-vivo mouse. In particular, we were able to improve (axial) resolution by ~3-fold and thus to visualize fine structures in the hippocampus, a sub-cortical brain region, at over 1mm depth. To further highlight potential applications of our method in the field of neuroscience, I have also performed proof-of-principle experiments in which I imaged the calcium dynamics of astrocytes, a cell type of the glia family, in the white matter of the intact mouse brain. These so-called fibrous astrocytes which are prevalent among myelinated nerve fibers in the white matter were so far, to best of our knowledge, not accessible for other non-invasive imaging methods. In summary, I have developed a motion-corrected adaptive-optics multi-photon microscope which enables intravital imaging at unprecedented depths and with near diffraction limited resolution. While most of my demonstrations were related to mouse neurobiology, I expect our new methods to find further applications in other fields such as mouse cancer and developmental biology.
Several transcription factors (TFs), such as the tumour suppressor p53, the immune response regulator NF-B, the yeast stress response regulator Msn2 and others, exhibit different nuclear accumulation patterns (dynamics) depending on the upstream activating stimulus. TF dynamics thus encode information about the type and intensity of the stimulus perceived by a eukaryotic cell. TF dynamics are believed to govern cell fate because they lead to the activation of distinct sets of target genes. Studies on how information about either internal or external stimuli is transmitted through signalling pathways into specific cell fates have shown that promoters of target genes play a critical role in decoding the information encoded in TF dynamics. Earlier studies suggested that the binding affinities of the TF for the promoters of the different target genes may orchestrate the observed differential gene expression under different TF dynamics. It was later shown that nucleosome positioning and, as a consequence, promoter accessibility determines how rapidly a promotergets activated, thus making it more or less sensitive to different TF dynamics. The distance between the core promoter and the TF binding sites as well as the core promoter itself have also been demonstrated to affect the expression of different target genes. Other studies on p53 and NF-Bmeasuring transcript levels of various target genes have revealed differences in the stability of transcripts belonging to early and late response genes. Some p53 target genes show oscillatory transcript levels in response to p53 pulses. In such studies, an external stimulus such as a cytokine, radiation or a chemical agent whose effects may not be fully understood were used to impose different TF dynamics. The lack of full clarity on the effects of the agents used to induce the TF dynamics may therefore undermine observations and the explanations given in such studies. Despite the progress made in understanding the role played by TF dynamics in gene expression regulation, it is still not clear which mammalian promoter elements contribute to decoding TF dynamics, and how they do so. In this study, I constructed a library of synthetic optogenetic circuits consisting of a library of synthetic light-responsive TFs and a library of promoters designed with well-studied elementsto investigate the relationship between TF dynamics and promoter activation in mammalian cells. Such a synthetic biology approach allows us to minimize the complexity, which is inevitable when studying endogenous pathways.I observed that there is a threshold for the time the TF must remain bound to the cognate responsive elements (REs) at the promoter (TF dwelling time) for transcription to be successfully initiated. The TF dwelling time is set by the affinity of the TATA binding protein (TBP) for the TATA-box (TB). A high-affinity TATA-box consents efficient assembly of the transcription pre-initiation complex (PIC), which reduces the TF dwelling time required for transcription initiation. The affinity of the TF for the REs defines the TF concentration (amplitude) threshold necessary to achieve the required TF dwelling time. Consequently, promoters with low-affinity REs and TATA-box filter out low-frequency pulsatile signals, but are activated by sustained TF signals. Additionally, reducing DNA looping efficiency by increasing the distance between the REs and the TATA-box, turns an otherwise TF dynamics-insensitive promoter into a promoter that can distinguish TF dynamics. I also show that the efficiency of translation initiation is critical for differential expression of target genes in response to different TF dynamics observed at the protein level. viiiFinally, I investigated a different type of synthetic TF bearing only the DNA binding domain (DBD)which interacts with a light-responsive co-regulator that bears thetransactivation domain(TAD). This scenario resembles several natural TFs, such as the TEAD/YAP pair. I found that this system is very sensitive to the interaction strength between the DBD-and TAD-bearing proteins. Furthermore, a high concentration of nuclear DBD-bearing TF impedes gene expression due to the competition for the REs between its free and TAD-bearing protein-bound fractions. These observations will help to further understand gene expression regulation by dynamics and how TEAD concentration in mammalian cells can be targeted in cancers where TEAD/YAP is dysregulated
Two problems of PT-symmetric quantum field theory are discussed: In the first part, the D-dimensional quantum field theory with the self-interaction phi^2 (i phi)^epsilon is analyzed. Techniques introduced previously in a first-order study of the perturbative nonlinearity expansion in epsilon, [Phys. Rev. D 98, 125003], are generalized for the application at higher orders and used to determine the expansion coefficients of the ground-state energy density, the p-point Green’s functions, and the effective mass of the theory. The perturbative renormalization of the two-dimensional model is discussed to second order in the nonlinearity expansion and the behavior is contrasted with that in a coupling-constant expansion through a multiple-scale analysis. In the second part, the 3 + 1 dimensional Nambu–Jona-Lasinio model is modified by PT-symmetric and anti-PT-symmetric non-Hermitian bilinears to analyze the role of PT symmetry in fermionic quantum field theory. The generated masses of the fermion as well as the scalar and pseudoscalar mesons are obtained. The study is supplemented by an analysis of the fermion mass in the similarly modified 1 + 1 dimensional chiral Gross-Neveu model.
Metastatic melanoma is one of the most aggressive skin cancers and is associated with poor prognosis. BRAF and MEK inhibitors are used to treat patients with BRAFV600E-mutated advanced melanoma. However, the development of resistances to these treatments compromises therapeutic success. Our lab previously demonstrated that forkhead box D1 (FOXD1) plays a critical role in melanoma migration and invasion. Here, I found that FOXD1 was highly expressed in melanoma cells. Immunohistochemical assessment of 105 samples from patients with metastatic melanoma revealed that high FOXD1 expression in tumors was associated with poor survival and correlated with low MITF, SOX10 and high AXL expression. Upregulation of FOXD1 expression enhanced the resistance of melanoma to vemurafenib (BRAF inhibitor) or combinatorial treatment with vemurafenib and cobimetinib (MEK inhibitor). On the other hand, loss of FOXD1 increased the sensitivity of naïve melanoma cells towards vemurafenib or combinatorial treatment with vemurafenib and cobimetinib. Furthermore, high FOXD1 expression levels were found in BRAF inhibitor (BRAFi)-resistant cells. Downregulation of FOXD1 resulted in a resensitization of BRAFi-resistant cells to vemurafenib. By using microarray analysis, connective tissue growth factor (CTGF) was found to be one of the most downregulated genes in FOXD1 knockdown (KD) cells while its expression was highly increased upon FOXD1 overexpression. Thus, CTGF was identified as a downstream factor of FOXD1. In addition, in vitro expression analysis and evaluation of clinical samples demonstrated that CTGF and FOXD1 expression were positively correlated. By using a CHIP assay and a dual reporter luciferase assay, I discovered that FOXD1 could regulate the expression of CTGF by directly binding to the CTGF promoter. This result was confirmed with RT-PCR and western blot. In addition, I found that the protein level of CTGF was highly increased in BRAFi-resistant cells. Similar to FOXD1 knockdown, the knockdown of CTGF resensitized BRAFi-resistant cells to vemurafenib. FOXD1 KD cells treated with recombinant CTGF protein were less sensitive towards vemurafenib compared to untreated FOXD1 KD cells. Based on these findings, I conclude that the transcription factor FOXD1 could promote dedifferentiation and targeted therapy-resistance in melanoma cells by regulating the expression of CTGF. Apart from the results above, I also demonstrated that cytokines such as TGF-β are regulated by FOXD1, and FOXD1 could promote EGFR-RAS-MAPK/AKT pathways activation. Taken these results together, FOXD1 might be a promising new diagnostical marker and a therapeutic target of targeted therapy resistant melanoma.
Background: Feature extraction and signature identification are two critical steps to understand diverse biological processes. Signatures are defined as groups of molecular features that are sufficient to identify certain genotype or phenotype. In particular, Non-negative Matrix Factorization (NMF) has been used to identify signatures in complex genomic datasets. However, running a basic NMF analysis is a challenging task with a steep learning curve and long computing time; furthermore, the usability of these algorithms is lessened by limited resources to interpret the results obtained from them. This creates a pressing need for the development of tools that mitigate such obstacles.
Results: In this study we developed ButchR and ShinyButchR, a fast and user-friendly toolkit to decompose datasets (slicing genomics) and learn signatures using NMF. The package can be freely installed from GitHub at https://github.com/wurst-theke/ButchRr. We used ButchR to identify a new regulatory subtype in neuroblastoma, which showed mesenchymal characteristics and was phenotypically associated to multipotent Schwann cell precursors. Additionally, we created a new workflow to infer regulatory relationships between genes and their _cis_-regulatory elements for individual cells, followed by inference of regulatory-signatures.
Conclusions: ButchR/ShinyButchR is an useful toolkit for analyzing multiple types of data, and inferring signatures that are able to capture relevant biological information. This toolkit is a new valuable resource to the scientific community, and it can be used to understand complex biological processes.
Plasmodium falciparum is the causative agent of the most devastating human malaria worldwide. The disease is transmitted when a female Anopheles mosquito injects sporozoites into human skin, which migrate and infect the liver. Upon completion of the liver stage, the parasite enters the bloodstream and infects circulating red blood cells (RBCs), thereby starting the intra-erythrocytic cycle. The environment within RBCs represents a challenging milieu for the parasite to develop and propagate normally. Therefore, to ensure its survival, P. falciparum exports over 400 proteins to the host cell involved in several host cell modifications. These parasite-induced host cell renovations are responsible for much of the pathology associated with malaria. Despite the intensive and continuous research work over the years, some fundamental questions remain unanswered, especially the role of many exported proteins. Elucidating their function is of utmost importance as a better understanding of parasite biology is needed to prioritize targets. Functional analysis of such proteins has been hampered in the past due to the lack of adequate genetic systems in this model organism. However, the recent advent of genetic tools such as the selection linked integration targeted gene disruption (SLI-TGD) strategy now allows rapid gene disruption in the P. falciparum system. Additionally, the glucosamine-6-phosphate activated ribozyme (glmS) system was developed to conditionally knockdown the expression of essential proteins, thereby enabling their functional characterization. In this project, we aimed to identify and characterize exported proteins essential for the survival and propagation of the parasite during the intra-erythrocytic development. For this purpose, we used a bioinformatics pipeline approach to prioritize our targets and select 15 genes encoding for exported proteins. Subsequently, these genes were subjected to a screening using the SLI-TGD approach. Furthermore, the glmS ribozyme system was used to analyze and characterize essential genes that could not be disrupted in the SLI-TGD screening. Of the 15 gene candidates screened in this project, 14 genes could not be disrupted, but only the pfj23 gene could be knocked out. Analyses of parasites depleted of Pfj23 revealed aberrant SBP1 distribution and segmented Maurer's clefts architecture. Also, infected erythrocytes with disrupted Pfj23 displayed deformed and worm-like elongated knobs morphologies. Moreover, the binding of infected RBCs to chondroitin sulfate A was significantly reduced upon Pfj23 inactivation. Among the 14 genes that could not be disrupted, the PF3D7_0301800 gene was selected to generate a regulatable copy of its protein using the glmS ribozyme system. Characterization of PF3D7_0301800 revealed aberrant KAHRP distribution upon its downregulation. Additionally, knockdown of PF3D7_0301800 displayed iRBCs with smooth surface without knobs referred to as the "knobless" phenotype. Further analyses could reveal the role of PF3D7_0301800 in KAHRP trafficking and knobs formation. Hence, understanding the function of PF3D7_0301800 and Pfj23 could provide essential insights into the parasite's biology.
The nucleosome consists of a core complex of two copies each of four histone proteins wrapped by about 1.65 turns of DNA. The DNA arms entering and leaving the core are known as linker-DNA (L-DNA) arms. The linker histone, H1, associates with the nucleosome at the region bounded by the two ends of the DNA leaving the core. The nucleosome in conjunction with the H1 forms the chromatosome, the smallest repeating unit of the chromatin. How does H1 associate with the nucleosome? Are there any contributions from the L-DNA stretches flanking the core to H1 association? Does the length or the sequence of the L-DNA affect the way H1 associates with the nucleosome? Does the binding mode of H1 affect the higher-order structuring of the chromatin? The work presented in this thesis aims to address these questions. Chromatosomes were reconstituted from core histone octamers (Xenopus laevis), linker histone of subtype H1.0b (X. laevis), and 226 bp DNA containing the strongly positioning Widom 601 sequence. The linker histone was labelled with the fluorophore Alexa 488 on the globular domain or the upstream end of the C-terminal tail domain (CTD), and the DNA was labelled with Alexa 594 on either one or the other L-DNA arm. Single-pair FRET (Förster Resonance Energy Transfer) spectroscopy was used to measure the proximity of the globular domain or the CTD to one or the other L-DNA. First, it was determined how the length and the sequences of the L-DNA flanking the H1 affect the location of the H1 on the nucleosome. It was observed that the structured globular domain of the H1 was proximal to L-DNA arms that contained an A-tract consisting of eleven contiguous adenines. However, the globular domain was not proximal to either a purely GC tract or a mixed sequence having a 64% AT content. The fluorophore on the disordered CTD was equidistant from both the L-DNAs, despite sequence variations. Distances extracted from the single-pair FRET measurements were used to build models of A-tract-containing nucleosomes. It was observed that the globular domain of the H1 associated in an on-dyad fashion on the nucleosomes, and two conserved arginine residues on the globular domain of H1 were proximal to the characteristically narrow minor groove of the A-tract. To experimentally check whether the A-tract recognition was mediated via the minor grooves as observed in the models, or via hydrophobic interactions with the thymine methyl groups, a nucleosome was reconstituted with two A-tracts, one complementary to thymine and the other complementary to methyl-group-lacking deoxy-uridine. The globular domain showed similar proximity to both the A-tracts, proving the role of the A-tract minor groove in its recognition. Finally, I studied how linker histones compact two types of trinucleosomes containing A-tracts flanking the first and the third nucleosome either on the inner or the outer L-DNA. The extent of compaction was measured by single-pair FRET between the two inner L-DNAs, one joining the first and second, and the other joining the second and third nucleosomes. Based on the mononucleosome models, linker histones associating with the first and the third nucleosome are expected to be oriented towards the A-tracts on the outer or the inner L-DNAs. Both the trinucleosome types were highly compacted in the presence of linker histones. However, there was no difference in the extent of compaction between the two types of trinucleosomes. Overall, this thesis shows a new mode of DNA sequence recognition by the linker histone that may affect the compaction of AT- and A-tract-rich heterochromatin. However, trinucleosome compaction by linker histones was not observed to be affected by the location of A-tracts.
Human Immunodeficiency virus type 1 (HIV-1) is a lentivirus that infects non-dividing cells of the immune system. In non-dividing cells, nuclear import of the viral genome occurs through the nuclear pore complex (NPC), a large macromolecular assembly that forms a channel of ~40 nm across the nuclear envelope. This process requires interactions between NPC components known as nucleoporins and CA (capsid) proteins of HIV-1 post-fusion complexes. In mature HIV-1 virions, multiple copies of CA assemble into a lattice of hexamers and pentamers, forming a cone-shaped capsid core of ~60 nm in width and ~110 nm in length encasing the viral genome. Because of its large size, it was generally believed that the capsid core entirely or partially disassembled prior to its translocation through the NPC. However, nuclear entry and uncoating are rare events and challenging to characterize and therefore subject to a long-standing debate.
Here, cryo-electron tomography (cryo-ET) on cryo-focused ion beam (FIB) milled T-cells was combined with subtomogram averaging (SA) to study the ultrastructure of HIV-1 capsids in the process of nuclear entry. Using a HIV-1 variant arrested at the nuclear pore due to a defect in binding of the CA lattice to the host cell protein CPSF6, snapshots of HIV-1 nuclear entry at multiple stages were captured. Reverse transcription-competent HIV-1 complexes were identified in the cytosol, docking to and within the NPC, and in the nucleoplasm of T cells. Surprisingly, in the cytosol and at the NPC, the viral complexes retained cone-shaped capsids highly resembling in size and geometry the mature capsid cores within intact virions. The density at their interior suggested that they were associated with the viral genome. The cone-shape capsids deeply enter into the NPC central channel with their narrow ends. The hexameric lattices of these capsids were intact or nearly intact. These findings argued against the current models of uncoating in the cytosol or at the NPC and rather supported translocation of intact HIV-1 capsids through the NPC. Instead, HIV-1 capsids uncoat in the nucleus of T cells. Inside the nucleus, tube-shape fragments still containing few lattice elements were observed. The lack of density at their inside suggested that the viral genome was released from these complexes. Uncoating may thus occur through the partial opening and remodeling of the hexameric lattices of HIV-1 capsids, rather than a step-wise disassembly. To address how an intact HIV-1 capsid can enter the central channel of NPC, the human NPC from T cells was structurally analyzed in cellulo by SA. The NPC overall structure was not altered upon infection but rather dilated in comparison to the structure previously obtained from purified envelopes. The diameter of the central channel of NPC in T cells corresponded to ~64 nm, suggesting that the translocation of intact HIV-1 capsid through the NPC is geometrically possible. The dilated conformation of NPCs in T cells is independent of HIV-1 infection, but represents a physiological condition of actively transporting NPCs.
Human papillomavirus (HPV)-induced malignancies have long been considered the ideal scenario for the development of a therapeutic cancer vaccine, as viral proteins can serve as immune targets. However, the attempts to develop a therapeutic vaccine against HPV-induced malignancies have not been clinically successful to date. One possible reason may be the hypoxic microenvironment present in most tumors, including cervical cancer. Recent studies have shown that hypoxia leads to decreased levels of E6 and E7 proteins in HPV-transformed cells. Hypoxia is also known to dysregulate the levels of HLA I (human leukocyte antigen class I) molecules in the context of different tumors. Furthermore, hypoxia has been reported to both enhance and suppress cytotoxic CD8+ T cell functions. Hence, the aim of this project is to investigate the effect of hypoxia on antigen presentation in HPV16-transformed cells as well as their targeting by CD8+ T cells. In this study, it was observed that hypoxia led to downregulation of the protein levels of HPV16 oncoproteins E6 and E7 in several cervical cancer cells transformed with different HPV16 variant lineages. More than 24 hours of hypoxia were needed for complete loss of expression of the E7 oncoprotein in HPV16-transformed CaSki cells. Presentation of E6 and E7-derived epitopes on the cell surface, in context of HLA class I (HLA-I) molecules, is essential for recognition and targeting of these cells by cytotoxic T cells. Peptide presentation occurs with the help of a repertoire of proteins (proteasome, transporters, chaperones and enzymes) known as the antigen processing and presentation machinery (APM). Any perturbations in the APM could result in an altered epitope repertoire on the cell surface. Thus, the effect of hypoxia on the APM was investigated in different HPV16-transformed cells, as well as two HPV negative control cells, using label free quantitation (LFQ) mass spectrometry. Next, using flow cytometry, the effect of hypoxia on the surface expression of the HLA-I (HLA-A2) molecule was assessed. Interestingly, no significant change was observed in the expression of any of the APM proteins. The surface levels of HLA-A2 were also not significantly affected by hypoxia in any of these cells. No effect of hypoxia on the APM is promising from the context of therapeutic vaccine design. However, for the success of the vaccines, it is essential that the HPV16-transformed cells are actually targeted by cytotoxic CD8+ T cells. Interestingly, enhanced killing of CaSki cells by E6 and E7-derived peptide-specific CD8+ T cells was observed in a majority of the cases, with the exception of one CD8+ T cell line, which showed suppressed killing upon hypoxia. The contradictory results are however consistent with the contradictory results published about the effect of hypoxia on CD8+ T cells, in the context of different tumors. Enhanced cytotoxicity in most cases was surprising given the results that the target proteins are decreased in hypoxia. Using hypoxia- VIII preincubated targets, it was demonstrated that the enhanced killing of CaSki cells under hypoxia is suppressed, with the likely reason being the decreased expression of the source oncoprotein under hypoxia. Thus, the inhibitory effect of hypoxia on the target cells seems to supersede the positive effect of hypoxia on the CD8+ T cells. In summary, the results obtained in this thesis provide important insights into the effect of hypoxia on cervical cancer cells and their targeting by CD8+ T cells. The study highlights the importance of considering the effect of the tumor microenvironment (TME), particularly hypoxia, while developing immunotherapies. Incorporating these insights into developing combination therapies will hopefully pave the way to making immunotherapies a mainstream standard of care for HPV-induced malignancies.
Hepatotropic viruses constitute a global health concern, as the WHO estimates over 300 million people worldwide suffering from chronic viral hepatitis. One of the causative agents is the Hepatitis C virus (HCV). In many infected, HCV is able to evade clearance by the immune system and establish a persistent infection. Here, the characteristics of a constantly activated immune response turn from antiviral to pro-inflammatory, thereby establishing a chronic inflammation of the liver, which promotes increasingly severe levels of liver damage, liver failure, and the development of liver cancer. In this study, we investigated the consequences of prolonged activation of antiviral signaling pathways in a virus-free cell culture system, using the lung adenocarcinoma cell line A549 and the immortalized hepatocyte cell line PH5CH. Ectopic expression of NS5B, the RNA-dependent RNA polymerase of HCV, produced double-stranded RNA (dsRNA) that was sensed by the pattern recognition receptor retinoic acid-inducible gene I (RIG-I). This induced the expression and secretion of interferons (IFNs) beta and lambda 1, pro-inflammatory cytokines tumor necrosis factor and interleukin 6, and chemokines CCL3, -4, -17, and CXCL10. Cells expressed interferon-stimulated genes (ISGs) and exhibited reduced proliferation, which we could attribute to a concerted effect of IFN-beta and IFN-lambda 1. We showed that IFN-lambda 1 expression correlated with NS5B expression on a single cell level, while ISG induction and impairment of proliferation was exerted through paracrine IFN signaling. NS5B expression was sufficiently high to trigger the RIG-I pathway for over 14 days, before unspecific downregulation of the transgene, likely in combination with counterselection of NS5B high-expressing cells, led to ceasing production of IFNs. We compared the supernatant from NS5B-expressing cells to a defined mix of IFN-beta and IFN-lambda 1 in elicited changes in global gene transcription of exposed cells. In A549 and in PH5CH, both treatments triggered a very similar response in the upregulation of, for the most part, well-defined ISGs. Long-term exposure of naïve A549 cells to NS5B-derived supernatant for one month showed only subtle differences in ISG gene expression, suggesting that cells do not become refractory to IFN-beta or IFN-lambda 1. Of three genes that were significantly upregulated only after treatment for one month, the tetraspanin TM4SF4 warrants further investigation due to its implication in hepatocellular carcinoma and a possible link to SARS-CoV2 infection. In summary, this study describes a cell culture system that enables to study the effects of continuous antiviral signaling for over two weeks, thereby mimicking a persistent viral infection while excluding virus-mediated interference. Furthermore, the system allows to produce a mix of secreted factors that resembles the epithelial-derived secretome present in an infected organ. Further elucidating the composition of this mix as well as long-term co-culture experiments of NS5B-expressing cells and non-parenchymal liver-resident or -infiltrating cells may help elucidate the complex intercellular dynamics that form the basis of HCV-associated chronic infection, inflammation, and liver deterioration.
Enhancers are regulatory DNA sequences that control gene spatial-temporal patterning based on their primary DNA sequence. Through the binding of proteins called Transcription Factors (TFs), enhancers turn genes “on” or “off” across fields of cells to express genes in complex patterns throughout development. To this day, we still cannot accurately and precisely synthesize an enhancer de-novo based on our best models. These findings suggest that we still have a limited understanding of how much regulatory information is encoded within the primary sequence of an enhancer. Furthermore, it is thought that enhancers are one of the primary drivers of evolution. Yet, we are far from predicting enhancer evolution due to limited technology and sparse experimental data. In this thesis, I review the field of enhancers, their evolution, and the regulation behind the shavenbaby locus. I next highlight the high-throughput technology developed to study enhancer mutants at a higher throughput with the help of a custom liquid-handling robot called Flyspresso and an adaptive-feedback confocal microscopy plugin. With this automated pipeline, I carry out a mutational scanning experiment on an enhancer at the shavenbaby locus called E3N to simulate possible paths and modes of evolution. I find that developmental enhancers are densely encoded and highly pleiotropic. I also identified new TF binding sites and examples of developmental biases that either constrain or drive evolution. I then discuss a mutational hotspot that evolves ectopic expression of shavenbaby in the developing wing and haltere, which I hypothesize is due to a transcriptional repressor. I additionally create a gene expression atlas for the late Drosophila embryo to map fragile and robust components of the E3N expression pattern and identify more TF binding sites. Finally, I summarize this thesis with an updated working model for E3N and an explanation to what extent we can predict E3N’s evolution.
Zur Untersuchung des Impulstransportes über die windbeeinflusste Luft-Wasser-Grenze wurde am linearen Wind-Wellen-Kanal der Universität Heidelberg ein neuer Versuchsaufbau realisiert. Dieser ermöglicht die zweidimensionale Bildgebung der Geschwindigkeitsfelder mithilfe der Particle-Streak-Velocimetry unter Verwendung von mit dem Fluoreszenzfarbstoff Pyranin versetzten Nebeltröpfchen als Tracern. Dabei wurde erstmals keine separate Laser-Height-Camera zur Messung der Wasserhöhe verwendet, sondern beide Techniken in einer Kameraufnahme kombiniert. Zusätzlich wurde zu jeder luftseitigen Grenzschicht simultan ein Bild der wasserseitigen aufgenommen. Die Messreihen wurden durchgeführt bei einem Fetch von 244cm und mit einer Bildrate von 300Hz. Untersucht wurden anhand dieser Datensätze zum einen die Praktikabilität der bisherigen Auswertungsalgorithmen, zum anderen mit einem neuen Ansatz der Bildauswertung die Bereiche der viskosen Grenzschicht, wasser- und luftseitig. Aus ersterem folgt die Notwendigkeit effizienterer und schnellerer Algorithmen, letztere zeigte, dass der neue Ansatz den Anforderungen der Particle-Streak-Velocimetry genügt. Darüber hinaus konnten die Geschwindigkeit der Wasseroberfläche visualisiert und Indizien für eine intermittierende Oberflächenerneuerung des Wassers gefunden werden.
Die Verbindungsklasse der Boronsäuren zeichnet sich durch ihre vielfältigen Anwendungen aus. Die prominente SUZUKI-MIYAURA-Kreuzkupplung ist heutzutage eine der am häufigsten eingesetzten chemischen Reaktionen. Boronsäuren werden nach wie vor als nützliche synthetische Intermediate zu C–C-Bindungsknüpfungen angesehen, gewinnen jedoch auch zunehmend an Bedeutung in den Materialwissenschaften, der Bioanalytik oder der Strukturbiologie. In der Medizinischen Chemie kennzeichnete die Zulassung des ersten boronsäurehaltigen Arzneistoffs, Bortezomib, durch die FDA im Jahre 2003 einen Meilenstein. Während der synthetische Zugang zu aromatischen Boronsäure-Derivaten gesichert ist, fehlt es an Syntheserouten zur routinemäßigen Darstellung aliphatischer und peptidischer Boronsäuren mit möglichst hoher Diversität. Die häufigsten Probleme bei der Synthese sind dabei der Aufbau der α-Aminoboronsäure-Partialstruktur, die Entschützung der intermediären Boronsäure-Ester sowie die Aufreinigung der finalen Verbindungen.
Im Rahmen der vorliegenden Arbeit wurden verschiedene Routen zur Darstellung von α-Aminoboronsäuren untersucht. Dabei wurde festgestellt, dass sich manche der literaturbekannten Methoden zwar zur Darstellung entsprechender Fragmente eigneten, die anschließenden Fragmentkondensationen in Lösung jedoch nur mit erhöhtem Aufwand realisierbar waren. Daher wurden innovative Ansätze etabliert, die den Aufbau größerer Molekül-Bibliotheken ermöglichen.
Um den Zugang zu komplexen Boronsäuren zu erhalten, wurde eine neuartige Methode zur effizienten und simplen Entschützung von Boronsäure-Estern entwickelt. Die Transesterifizierung mit flüchtiger Methylboronsäure in einphasigen Systemen ermöglichte dabei die quantitative Überführung aromatischer, aliphatischer und peptidischer Ester in die entsprechenden freien Boronsäuren. Die Aufreinigung konnte dabei durch simple Evaporation von Reagenz, Nebenprodukt und Lösungsmittel erfolgen. Auch die Hydrolyse von äußerst stabilen Pinandiolestern erfolgte effizient mithilfe des in dieser Arbeit entwickelten Ansatzes.
Mithilfe der monophasischen Transesterifizierung wurde der Zugang zu einer bisher unbekannten Verbindungsklasse eröffnet, den Fmoc-α-Aminoboronsäuren, welche sich zur routinemäßigen Festphasensynthese an kommerziell erhältlichem 1-Glycerol-Harz eignen. Die hier beschriebenen Synthesemethoden ermöglichen den Zugang zu einer Vielzahl an Boronsäuren nach einem Baukastenprinzip und haben daher das Potenzial, konventionelle Routen zu ergänzen oder sogar zu ersetzen.
1. Identification of dynamic RNA-binding proteins in primary cardiomyocytes uncovers Cpeb4 as a regulator of cardiac growth Mutations or decreased expression of mRNA-binding proteins (mRBPs) can lead to cardiomyopathies in humans. The present study defined the first compendium of dynamically binding mRBPs in healthy versus diseased primary cardiomyocytes at a system-wide level by RNA interactome capture. Among these mRBPs, Cytoplasmic polyadenylation element binding protein 4 (Cpeb4) was defined as a dynamic mRBP in diseased cardiomyocytes, and was found to regulate cardiac growth both in vitro and in vivo. To investigate the functions of Cpeb4 in cardiomyocytes, mRNAs bound to and regulated by Cpeb4 were identified. These data implicate that Cpeb4 regulates transcriptional activity by differential translation of transcription factors involved in cellular remodeling in response to pathological growth stimulation. Among Cpeb4 target RNAs, two Zinc finger transcription factors (Zeb1 and Zbtb20) were identified. The present study shows that Cpeb4 regulates the translation of these mRNAs and that Cpeb4 depletion increases their expression. Thus, Cpeb4 emerges as critical regulator of myocyte function by differential binding of specific mRNAs in response to pathological growth stimulation. 2. mTOR-proteasomal dysfunction following deletion of Pras40 inhibits cardiac growth but results in cardiac failure The mammalian target of Rapamycin complex 1 (mTORC1) increases cell size by initiating translation as well as by inhibiting catabolic functions such as proteolysis and autophagy. A previous study from our lab proposed Proline-rich Akt substrate 1 (Pras40) as a cardioprotective, endogenous inhibitor of mTOR-dependent protein synthesis during pathological growth. Pras40 is released from mTORC1 during growth, but other interactions are largely unknown. The present study aims at understanding the molecular mechanism of Pras40 to cardiac growth and function. To test consequences of Pras40 deletion on cardiac function in vivo, two novel Pras40 knock-out mice were subjected to pathological and physiological hypertrophy (Transverse aortic constriction, swimming). Conversely to Pras40 overexpression, growth was significantly blunted in KO animals and function reduced. mTORC1 signaling as well as proteasomal function were severely disturbed in KO animals. Mechanistically, chymotrypsin-like 26S proteasomal activity was blunted in KO hearts as well as isolated cardiomyocytes from KO animals. Disturbed proteasomal function in KO mice lead to severe alterations in metabolic functions highlighting the importance of both intact mTORC1 signaling and proper proteasomal maintenance during cardiac stress. Reactivation of proteasomal activity in vivo in KO mice restored cardiac function to WT levels, and overexpression of mutant, mTOR-released Pras40 had a similar effect. The present study provides evidence that Pras40 links anabolic protein synthesis and catabolic proteolysis in the heart: At rest, Pras40 binds and inhibits mTOR, but when released during pathological growth, Pras40 directly interacts with the 26S proteasome and modulates its activity.
This thesis is devoted to the study of extremely energetic short-timescale astrophysical events, Gamma-ray bursts (GRBs). GRBs exhibit broad-band bright non-thermal emission, which was analysed using two major experiments: the High Altitude Water Cherenkov observatory (HAWC) and the High Energy Stereoscopic System (H.E.S.S.). The two experiments are in many respects complementary for the observation of very high energy (VHE) gamma-ray emission from GRBs, and in this work the respective advantages were exploited to maximise the sensitivity to VHE signals. After the analysis of several tens of GRBs observed using H.E.S.S. until 2017, where no significant emission was detected, improvements in the observation strategy of H.E.S.S. allowed the detection of GRB~180729B and GRB~190829A. These detections are presented in context with multi-wavelength data, proposing plausible emission mechanisms, thus concluding a decade-long search for these elusive phenomena at VHE.
In the second part, novel methods to improve the accuracy of the HAWC detector simulation are presented, including better modelling of the detector efficiencies and electronics. A model that accounts for the detector response and the GRB flux evolution has been developed to estimate the optimal integration time for VHE searches with HAWC. Thanks to these improvements, it is possible to exploit the wide field of view and high duty cycle of HAWC for the search of VHE emission in several tens of GRBs. Preliminary evidence for emission is found in one of the GRBs studied, and upper limits are obtained for all the GRBs analysed and placed in context of the X-ray properties of these events. Finally, the limits and detections presented in this work are placed within the framework of the current understanding of GRBs and prospects for future and present VHE gamma-ray detectors are presented.
Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death worldwide, and often chemotherapy fails due to post-treatment cell survival and patient relapse. Although extensive qualitative research has been performed on drug resistance, the combination of experimental and computational methods offers the perspective of a quantitative prediction for tumour-specific therapeutic approaches. In this context, it is important to study molecular mechanisms involved in drug resistance in PDAC on a systems level and integrate knowledge about involved signal transduction pathways. The aim of this thesis is to investigate two of these mechanisms and quantitatively analyse their role in drug resistance.
In the first sub-project of the thesis, the ubiquitin ligase Casitas B-lineage lymphoma c (CBLc) was characterised as a subtype biomarker for drug resistance in PDAC cells by a combination of mathematical modelling and experiments. It was observed that CBLc confers drug resistance to PDAC cells by amplifying the activation of downstream effectors of the MAPK and PI3K/Akt pathways, which stands in contrast to the well-established role of CBL ubiquitin ligases as negative regulators of membrane receptor tyrosine kinases (RTKs). The observed effect of an increased Erk and Akt activation in presence of Erlotinib could be explained by mathematical modelling assuming a novel function of CBLc as a scaffold for mediators of downstream phosphorylation reactions, responsible for tuning cell response to external stimuli.
The second sub-project of the thesis addressed the spatio-temporal dynamics of drug delivery in PDAC tumour tissue depending on the heterogeneous expression of a drug-metabolizing enzyme, CYP3A5, a member of the cytochrome P450 enzyme family. Recently, it was observed that patient-derived model cell lines of the exocrine-like PDAC subtype express this enzyme, which resulted in drug resistance in cell culture experiments. Accordingly, it can be predicted for tumour tissues that CYP3A5 expression results in local drug gradients and survival of cancer cells. To quantitatively simulate this effect, an agent-based reaction-diffusion model of 3D cell cultures was created. Based on experimental data, the formation of resistant tumour niches due to CYP3A5-expressing cells was simulated. The model was used to create predictions about the selection of resistant cell populations upon treatment with oncological drugs such as erlotinib and paclitaxel.
In conclusion, quantitative descriptions of two distinct cellular mechanisms of drug resistance in the complex landscape of PDAC were established. On the one hand, a new functional role of the potentially oncogenic protein CBLc was mechanistically characterized; on the other, the effect of heterogeneously expressed drug-degrading enzymes resulting in tumour niches protected from cytotoxic drugs was characterised via mathematical modelling. In future, the integration of the developed models could be applied to optimize experimental strategies for in vitro testing of targeted cancer inhibitors and combinations of chemotherapy agents on PDAC and other tumours.
Deep Learning-based models are becoming more and more relevant for an increasing number of applications. Bayesian neural networks can serve as a principled way to model the uncertainty in such approaches and to include prior knowledge. This work tackles how to improve the training of Bayesian neural nets (BNNs) and how to apply them in practice. We first develop a variational inference-based approach to learn them without requiring samples during training using the popular rectified linear unit activation function's piecewise linear structure. We then show how we can use a second approach based on a central limit theorem argument to get a good predictive uncertainty signal for an active learning task. We further build a reinforcement learning-based approach in such an active learning setup, learning a second BNN that requests labels to support the primary model optimally. As a third variant, we then introduce a new method for learning BNNs by optimizing the marginal likelihood via a model selection based approach, relying on the concept of type-II maximum likelihood, also known as empirical Bayes. Using PAC-Bayes theory to develop a regularization structure, we show how to combine it with a popular deterministic model for out-of-distribution detection, demonstrating improved results. Using this joint combination of empirical Bayes and PAC-Bayes, we finally study how to use it to learn dynamical systems specified via stochastic differential equations in a way that allows incorporating prior knowledge of the dynamics and model uncertainty.
The production of prompt Λ+c hadrons at midrapidity |y| < 0.5 in proton–proton collisions at √s = 13 TeV as a function of charged-particle multiplicity is presented. Also analogous measurements of D0 and D+s production are currently being carried out and the D+s/D0 as well as the Λ+c/D0 production ratios are discussed. In contrast to the D+s/D0 production ratio, the Λc+/D0 production ratio shows an increase with increasing multiplicity. In addition, its ratio is significantly underestimated by predictions where fragmentation is tuned to e+e− and e−p measurements. On the other hand, comparisons with a canonical approach of a statistical hadronisation model with augmented baryon production as well as with a fragmentation model implementing colour reconnection beyond leading colour approximation are shown to qualitatively describe the shape and multiplicity dependence. The Λ+c/D0 ratio is similar in shape and magnitude compared to the light-flavour ratio Λ/KS0 measured at comparable multiplicities. Together, these results indicate (i) hadronisation mechanisms beyond pure in-vacuum fragmentation in pp collisions as well as (ii) potential common mechanisms of baryon formation in the light-flavour and heavy-flavour sector.
The Virtual Monte Carlo (VMC) detector simulation framework which is used by the ALICE and FAIR collaboration has been extended to support track partitioning among multiple different simulation engines. Before, it was only possible to use one chosen engine for the entire event simulation. Especially in view of the coming LHC Run 3 and 4, faster simulations are crucial to cope with the expected increase of experimental data. The enhanced VMC framework is now capable of running full-simulation together with fast-simulations. Based on specified user conditions, the simulation of single tracks can be transferred from full-simulations to be handled by fast-simulations when feasible to speed-up the detector simulation.
Nutrient and energy metabolism in organisms oscillates in a time-of the-day-dependent manner under the control of an endogenous timing mechanism called the circadian clock. This is a cell autonomous, self-sustained molecular mechanism, which is synchronized by a key environmental signals, notably light and food availability. There is a wealth of evidence showing a bidirectional interaction between food-regulated clocks and the rhythmic expression of metabolic genes in peripheral tissues, notably the liver. For example, genetic or environmental disruption of the circadian clock is linked with metabolic disease, such as obesity and type 2 diabetes. Furthermore, cycling changes in cellular redox potential impact on the expression of circadian clock genes and influence energy metabolism. Therefore, it is vital to understand how animals integrate input from lighting conditions and food availability to ultimately coordinate their daily metabolic rhythms. In this regard, one key issue is whether there are genetically distinct light and food regulated circadian clock mechanisms. The Foulkes group has used zebrafish and blind cavefish models to demonstrate that certain metabolic pathways cycle according to the light dark cycle and are unaffected by the timing of feeding activity, while other pathways are predominantly feeding time regulated. Based on these preliminary data, this thesis project used fish models and fish-derived cell lines to explore the genetic mechanisms linking metabolism with light and food regulated circadian clocks.
The first part of this project aimed to explore at which stage during early zebrafish development a feeding-regulated clock first appears. Due to reduced feeding activity in constant darkness it was not feasible to examine the impact of feeding on clock gene expression. However, it was revealed that regular handling and disturbance of fish larvae, under otherwise constant environmental conditions results in the emergence of circadian clock rhythmicity. Several lines of evidence indicate that stress serves as a Zeitgeber and results in the emergence of rhythmicity in clock gene expression as well as clock outputs such as the cell cycle.
The second part of this thesis explored whether genetically distinct light and food regulated clocks coexist in fish cells and which transcriptional control mechanisms link food-regulated circadian clocks with metabolism. It was demonstrated that during restricted feeding in zebrafish, rhythmic expression of core clock genes in the liver is regulated according to the timing of light-dark cycles, whereas the expression of genes involved in the control of metabolism are influenced by feeding time. However, this study was unable to confirm previous data obtained using NMR, where it was shown that circadian rhythmicity in the levels of essential amino acids is regulated by the light-dark cycle while rhythmic non-essential amino acid levels are influenced by feeding time. Instead, by UPLC-MS/MS analysis, daily changes in the concentration of both essential and non-essential amino acids were shown to be set by the phase of regular timed feeding and not by the light dark cycle. Furthermore, the NAD+ biosynthesis pathway and autophagy were affected by a clock which is set by feeding time and not by light-dark cycles. In addition, regular nocturnal feeding resulted in an increase in obesity. These findings point to the presence of at least two distinct clock mechanism in the zebrafish liver.
In order to explore in more detail, the nature of the multiple clock mechanisms in zebrafish cells, the next part of this project employed multi-omics approaches and revealed infradian rhythmicity in amino acid concentrations in cultured fish cell lines. However, neither the expression of amino acid transporters nor autophagy exhibited infradian rhythmicity, instead showing circadian rhythmicity. In order to explore the involvement of the classical circadian clock mechanism in generating infradian rhythmicity, a cell line expressing a dominant negative form of clock1 gene was examined and shown to lack infradian rhythmicity in amino acid levels. Interestingly, the mRNA expression of Asparagine synthetase (asns) shows infradian rhythmicity, which are disrupted in Δclock1 cells. These data lead to the hypothesis that asns may be involved in the regulation of infradian rhythms in amino acid levels and point to a complex interplay between circadian and infradian rhythmicity.
Increasing evidence suggests that the accumulation of misfolded protein species into specific spatially separated deposition sites is a cytoprotective response of the cell. Yeast has at least three different protein quality control sites for the deposition of aggregated proteins. The JUxtaNuclear Quality control (JUNQ)/IntraNuclear Quality control site (INQ) and the Cyto-Q harbours unstructured, amorphously misfolded proteins, while the perivacuolar Insoluble PrOtein Deposit (IPOD) is regarded as a specialized deposition site for highly ordered amyloid aggregates. Recently, it was found that targeting of amyloid aggregates to the IPOD depends on proteins that function either in actin cable-based transport processes (Myo2, Tpm1/2) or in vesicular transport and vesicle fusion events (Sec18, Sec14, Sec21, Vps1). Knockdown/deletion of either of the above-mentioned factors resulted reversibly in multiple small aggregates of the model amyloid PrD-GFP dispersed throughout the cytoplasm instead of its proper accumulation at the IPOD. These multiple aggregates, also interpreted as transport intermediates co-localized with the Atg9 vesicle marker, Atg9, and the CVT (Cytoplasm-to-Vacuole Targeting) pathway substrate preApe1. Based on these findings, it was hypothesized that the recruitment machinery for amyloid aggregates to the IPOD overlaps with that for preApe1 to the neighbouring PAS (Phagophore Assembly Site) and involved Atg9 or related vesicles that are transported along actin cables to the IPOD. In the current study, we falsified this hypothesis by evaluating the effects of in vivo gene knockout studies of ATG9 and other key components of this pathway on the recruitment machinery.
In order to identify the key molecular factors and narrow down the vesicular pathways involved in the recruitment machinery for amyloid aggregates to the IPOD, we performed an unbiased mass spectrometry approach to isolate the transport intermediates of PrD-GFP generated in a VPS1 null mutant or a SEC18 knockdown strain. Candidates tested in follow-up experiments using in vivo knockdown/knock-out, and co-localization techniques confirmed our initial hypothesis that vesicular transport is involved in amyloid recruitment to the IPOD. Furthermore, we found that proper recruitment of PrD-GFP to the IPOD is disrupted upon depletion/deletion of components involved in Golgi to endosome targeting and intra-Golgi transport processes (Mon2, Dop1, Cop1) as well as candidates mediating homotypic membrane fusion events as well as endosome to vacuole transport (Vps33, Vps45). Using fluorescence microscopy, we observed that Vps33, a Sec1/Munc18 family (SM) protein and core component of HOPS/CORVET multisubunit tethering complexes co-localized with the multiple PrD-GFP aggregates generated in MON2 null mutants. Based on these findings, it was proposed that PrD-GFP aggregates are recruited to the IPOD via endosomes/MVB (Multivesicular bodies), which are known for their role in delivering substrates to the vacuole as a part of endosomal vacuolar transport.
In this work, we substantially advance the formalism of the temporal functional renormalisation group which constitutes a non-perturbative framework for computing the dynamics of correlation functions in quantum field theories. To that end we carefully revisit the derivation of the temporal flow equation, paying particular attention to properties arising from a causal temporal regulator. We use the manifest causality of the formalism to integrate the general temporal flow analytically. The result are novel one-loop exact equations for fully dressed correlation functions. Further leveraging causality, we derive the complete Dyson-Schwinger hierarchy and the s-channel effective vertex in terms of specific truncations of the temporal flow. We solve the problem of renormalising the general causal temporal flow. We demonstrate that certain types of causal integral equations can be solved by an explicit numerical method. We numerically solve the integrated flow in a truncation involving the propagator of the $\phi^3$-theory in $1+1$ dimensions. Our results indicate the emergence of universal dynamics. Due to the high degree of flexibility of approximation schemes of the temporal flow, energy conservation in generic truncations is not guaranteed automatically but becomes a non-trivial feature instead. We explore energy-conserving truncations by deriving the causal temporal flow of the energy-momentum tensor, which we integrate analytically.
This is an invitation to play magnetic billiards. We consider a billiard table that is an n-dimensional compact Riemannian manifold with smooth boundary. This is a generalization of the classical billiard game. In particular, we study periodic orbits on a prescribed energy level in the magnetic setup. We show that for sufficiently high energy values above the Mañé critical value, there exists a periodic magnetic bounce orbit with bounded period.
The main topic of this thesis is the time-resolved study of the dynamics occurring during the strong-field ionization of atoms and its application to the advancement of attosecond science. Using attosecond transient absorption spectroscopy (ATAS) in the regime where the near-infrared pump and the extreme ultraviolet probe pulses overlap, sub-cycle structures in the buildup of xenon ion population are observed and analyzed. It is established that these structures can serve as a timing tool to track dynamics with attosecond precision, thereby opening up a new domain of measurement schemes based on ATAS. At the same time, the line shapes of the observed resonances are used to reveal the dynamics of the time-dependent dipole moment of the atoms during the strong-field ionization. The investigation of the phase of the dipole moment suggests that a strong-field induced modification of the configuration interaction and dipole couplings between bound and continuum states occurs through polarization of the system. This phenomenon calls for an extended analytical description of strong-field ionization in multi-electron systems beyond the single-active electron approximation that takes laser-induced polarization of the atom into account.
In organischen Halbleiterschichten aus Donor- und Akzeptormaterialien besteht einenger Zusammenhang zwischen der Dynamik photoelektrischer Prozesse und den vor-handenen Energiezuständen. Diese werden durch die Eigenschaften der Materialienselbst bestimmt, sind aber auch von der Wechselwirkung der Moleküle miteinanderabhängig und damit von deren relativen Orientierung sowie deren räumlichen Di-stanz. Die Energiezustände sind also direkt von der Morphologie der organischenSchicht mitbestimmt. In dieser Arbeit wird die Morphologie funktioneller Schichtenunter anderem von organischen Solarzellen visualisiert, welche aus einem Polymerund einem Nicht-Fulleren Akzeptor, im Speziellen PBDB -T und ITIC, gemischtwerden. Die Visualisierung der Morphologie erfolgt mittels Hellfeld Transmissions-elektronenmikroskopie (TEM) sowie analytischer TEM und wird mit den jeweili-gen Bauteilparametern korreliert. Durch Kryo-TEM Tomographie wurden kohären-te PBDB -T und ITIC Kristalle am Donor-Akzeptor-Übergang von PBDB -T:ITICMischschichten gefunden. Dabei dominieren ITIC Kristallstrukturen eines spezifi-schen Polymorphs. Mit diesen Resultaten demonstriert die vorliegende Arbeit Mög-lichkeiten zum visuellen Nachweis spezifischer morphologischer Eigenschaften vonorganischen Funktionsmaterialien. Diese Visualisierung ist grundlegend für die Mo-dellierung der genauen Anordnung von PBDB -T und ITIC Molekülen in der Misch-schicht und damit wegweisend für eine Simulation und Optimierung des Modellsys-tems PBDB -T:ITIC und darauf aufbauender Systeme.
The cross sections of proton-capture reactions are extremely important to model explosive nucleosynthesis, in particular for the poorly understood production of the rare p-nuclei. Taking advantage of the unique possibilities at the Experimental Storage Ring (ESR) at GSI, the so-called proton-capture campaign has been started in 2009 focusing on the study of (p,γ) reactions for hot, explosive stellar scenarios. In this thesis, the recent experiment of the campaign, performed in March 2020, is analyzed and discussed in detail. The (p,γ) and (p,n) reaction cross-sections have been successfully measured at 10 MeV/u using a stable 124Xe ion beam and for the first time also using a radioactive ion beam, namely 118Te with 6 days half-life. In addition, a novel experimental scheme to improve the sensitivity of the method has been developed. Using the stable 124Xe beam it is demonstrated that the application of this new technique enables measurement at maximum sensitivity for proton-induced reactions in inverse kinematics.
Magnetic resonance imaging (MRI) is highly versatile, offering many contrast settings inherently sensitivity to tissue microstructure at the sub-voxel scale (below the imaging resolution). Since its invention, images produced with MRI have mainly been based on classical reconstructions, with contrast determined by the signal attenuation from local tissue and MRI sequence design. In the advent of machine learning becoming practical, wide availability of computational power and high-resolution imaging such as laser scanning microscopy, new processing techniques involving MRI interpretations based on comparisons with known signals and ground-truth microstructure can be explored. Data-driven signal classifications enable model-less predictions of tissue properties on a single voxel level, offering artificial MRI contrasts. In this thesis, groundwork is laid for the exploration of such contrasts and suitable MRI sequences, with a demonstration of the feasibility of such an approach based on transverse relaxation for brain tumor detection. The thesis is focused on the role of microvascular geometry on reversible transverse relaxation in the context of tumor imaging. Comprehensive quantifications of cancer-induced vessel remodeling are provided, and the effects thereof studied with MRI simulations. Consequently, a numerical framework was developed for correlations of MRI signal properties with underlying microstructure for further exploration of artificial contrasts.
This work presents high-resolution thermal expansion and magnetostriction studies revealing magnetoelastic coupling and thermodynamic properties of single-crystals of the correlated electron systems Gd2PdSi3, Cu3Bi(SeO3)2O2Cl, and Cr2Ge2Te6. Magnetization and specific heat measurements complement the dilatometric investigations. Gd2PdSi3 is a metallic antiferromagnet with a complex phase diagram which shows three phase transitions already in zero-field. Among other magnetic orders it evolves a skyrmion lattice phase when a magnetic field is applied. This skyrmion lattice phase is strongly enhanced under the application of uniaxial pressure. New phase boundaries in the phase diagram are found and magnetoelastic coupling is quantified. The antiferromagnetic insulator Cu3Bi(SeO3)2O2Cl is a multiferroic which shows geometric frustration. Its high-temperature structural phase transition is strongly affected by uniaxial pressure, whereas pressure only has small effects on the antiferromagnetic (AFM) transition at low temperatures. The low-temperature AFM phase exhibits a metamagnetic spin-flip transition for B || c. Mixed-phase behavior and linear magnetoelastic coupling are observed in the transition region. Furthermore, the magnetic phase diagrams for the a- and b-axis of Cu3Bi(SeO3)2O2Cl are constructed for the first time. Cr2Ge2Te6 is a layered quasi-two-dimensional van der Waals material with a uniaxial magnetic anisotropy. The magnetoelastic coupling in Cr2Ge2Te6 is directly measured and correlations up to high temperature are observed. Furthermore, the critical behavior around the ferromagnetic phase transition is analyzed and a Grüneisen analysis shows that applying uniaxial pressure leads to large changes in the critical temperature.
Die "Sepsis-induzierte Immunsuppression" ist gekennzeichnet durch einen persistierenden antiinflammatorischen Status des Immunsystems postseptischer Patienten. Die Konsequenz ist eine erhöhte Anfälligkeit für das Auftreten sekundärer, häufig opportunistischer Infektionen zusammen mit einer erhöhten Mortalitätsrate dieser Patienten. Die Mechanismen, die dem immunsuppressiven Phänotyp zugrunde liegen, sind bisher noch weitgehend ungeklärt, aber die Beteiligung epigenetischer Reprogrammierung von Immunzellen oder bereits sogar der hämatopoetischen Stamm- und Vorläuferzellen der Immunzellen wird hypothetisiert. Analog kann sich eine durch verschiedene Stressoren in den Zellen der Keimbahn manifestierte epigenetische Signatur auch auf die Nachkommen übertragen. Die Sepsis, als Immunpathologie, stellt mit ihrem schweren Krankheitsverlauf einen solchen dramatischen Stressfaktor für den Organismus dar. Das Ziel der vorliegenden Arbeit war aufzuklären, auf welchem molekularen Weg sich die Spätfolgen einer Sepsis im Individuum über einen so langen Zeitraum manifestieren und aufzudecken, ob es ein epigenetisches „Erbe“ der Sepsis gibt, welches die Immunfunktion der Nachkommen verändert. Die im Rahmen dieser Arbeit erhobenen Daten zeigen erstmals, dass eine Sepsis einen Zustand trainierter Immunität auf zellulärer Ebene in naiven Knochenmarkmonozyten induziert. Dies geschieht durch eine Erhöhung der basalen Glykolyse, einhergehend mit einer transkriptionellen Reprogrammierung von Genen, die in den Metabolismus und in die Immunantwort involviert sind. Das Resultat ist die verstärkte Responsivität der Zellen auf einen zweiten Stimulus. Zusätzlich induziert eine Sepsis eine Verschiebung der Hämatopoese in Richtung der myeloiden Linie. Des Weiteren demonstrieren die Ergebnisse dieser Arbeit, dass die Sepsis Einfluss auf die männliche Keimbahn nimmt und dort zu einer epigenetischen Reprogrammierung des Spermienmethyloms führt. Diese Änderungen werden auf die Nachkommen übertragen und führen dort zu einer erhöhten postnatalen Mortalität, sowie zu einer Beeinträchtigung der Gewichtsentwicklung. Als immunologische Konsequenz zeigen männliche Nachkommen postseptischer Väter eine gestörte systemische und pulmonale Immunreaktion und sind daher anfälliger für bakterielle und fungale Infektionen. Die Ergebnisse widerlegen zum einen die aktuelle Hypothese einer globalen Immunsuppression auf zellulärer Ebene und eröffnen zum anderen gleichzeitig neue Perspektiven im Bereich der epigenetischen, intergenerationalen Vererbung immunologischer Veränderungen durch eine Sepsis. Somit unterstreichen die Daten dieser Arbeit die Notwendigkeit weiterer Untersuchungen der postseptischen Immunreaktionen, um die molekularen Veränderungen besser zu verstehen und möglicherweise in ein therapeutisches Konzept für die klinische Anwendung umzuwandeln.
Multi-AXis Differential Optical Absorption Spectroscopy (MAX-DOAS) is a well-established remote sensing technique for the detection of atmospheric aerosol and trace gases. Ultra-violet and visible radiation spectra of skylight are analysed to obtain information on different atmospheric parameters. An appropriate set of spectra recorded under different viewing geometries ("Multi-Axis") allows to infer aerosol and trace gas vertical distributions as well as aerosol properties by applying numerical inversion methods. It is well known but not yet used in MAX-DOAS applications that, besides the spectra, the polarisation state of skylight provides additional information on the atmospheric conditions. The major aim of the presented work was to assess the potential of polarimetric MAX-DOAS observations. For this purpose, a novel polarization-sensitive MAX-DOAS instrument (PMAX-DOAS) and a corresponding inversion algorithm (RAPSODI) were developed, capable to record and process polarimetric information. Furthermore, RAPSODI is the first MAX-DOAS inversion algorithm allowing to retrieve aerosol microphysical properties.
Compared to conventional non-polarimetric MAX-DOAS approaches, the information on the atmospheric state contained in polarimetric observations is strongly enhanced: assuming typical viewing geometries, the degrees of freedom of signal increase by about 50% and 70% for aerosol vertical distributions and aerosol properties, respectively, and by approximately 10% for trace gas vertical profiles. For an ideal atmosphere, the studies on synthetic data predict an improvement in the results’ accuracy (root-mean-square differences to the true values) of about 60%, 40% and 10% for aerosol vertical columns, aerosol properties and trace gas vertical columns, respectively.
In this work, a framework for the computation of the time evolution of correlation functions is developed. For that purpose, techniques from the functional renormalisation group (fRG) are used, with the unique feature of a temporal regulator. This specific choice of regulator suppresses quantum fluctuations based on a time scale and yields causal properties for correlation functions. As a consequence, flow equations can always be integrated analytically, which in turn allows for the derivation of a one-loop exact functional relation for the inverse propagator. In addition to this formal result, the method is applied to the phi^3-theory, where the dynamics of the propagator is investigated. In this setup, the system is prepared far-from-equilibrium and the time evolution of the propagator is computed. Even in the simple truncation that is employed, there are already hints at a self-similar time evolution.
In a separate part, three-dimensional Yang-Mills theory is examined in equilibrium. Due to the simpler computation as opposed to non-equilibrium scenarios, it is possible to investigate this theory in view of different truncations. This study reveals that truncations have to be chosen carefully in order to achieve apparent convergence.
Carbon-ion beam radiotherapy has been developed as a highly effective modality for cancer treatment. Based on the precise dose distributions of carbon-ion treatments, a better sparing of healthy organs surrounding the tumor site compared to the standard photon radiotherapy are provided. However, dose distributions in car-bon-ion beam radiotherapy are more prone to uncertainties. Therefore, strategies to monitor the ion beam during a treatment delivery, ideally directly in the patient, are of great importance. In this thesis, a non-invasive methodology to measure the lateral pencil beam positions in the patient has been developed. It is based on detection and tracking of charged nuclear fragments leaving the treated patient as secondary radiation. The performance of the developed methodology was investigated in patient-like models mimicking clinical situations at the HIT facility in Heidelberg, Germany. Moreover, the developed methodology has been tested for the first time during a real patient treatment. Precision, accuracy and effectivity of the developed method were found to be clinically attractive compared to the maximal accepted uncertainties of 1 mm. Additionally, the beam movement was aiming towards online carbon-ion beam monitoring. This independent beam monitoring methodology is ready to be evaluated on a larger patient group in a clinical study.
Cell motility has a critical role in a range of biological processes including development, immunity and disease. Navigation through complex and ever-changing environments often relies on the activity of actin-rich protrusions at the leading edge, also referred to as lamellipodia. Lamellipodia are known to exhibit areas of continuously rearranging membrane curvature, and their dynamics determines motion persistence. One group of proteins interesting in the context of membrane curvature are BAR domain proteins. However, whether and how these curvature-sensitive proteins contribute to leading edge dynamics and function, remains poorly understood. Here, we use neutrophils as a vertebrate model system of a highly migratory cell type. By combining RNAseq with a localization screen we identify two BAR proteins that are relevant for cell surface organization during migration: SH3BP1 and Snx33.
First, using fluorescent imaging and Atomic Force Microscopy, we show that SH3BP1 responds to changes in membrane mechanics and, vice-versa, modulates membrane tension. Using microfluidics, we further demonstrate that SH3BP1 is important for cell navigation through complex environments. Namely, its knockout displays increased cell speed and decision making during directed cell migration.
Next, we used the above techniques complemented with machine learning-based segmentation for time-resolved TIRF microscopy to understand the role of Snx33. We show that motion persistence and directionality, in both freely moving and environmentally constrained cells, depends on Snx33 activity. Specifically, Snx33 has an inhibitory effect on the lamellipodia dynamics by regulating WAVE2-driven actin polymerization. Our work exposes a novel mechanism by which cells steer protrusions upon encountering obstacles that facilitates efficient migration. In summary, we discovered novel functions of the curvature-sensitive proteins SH3BP1 and Snx33 in regulating cell surface mechanics and efficiency of directed cell migration.
Despite extensive research and significant advances in the past decades, cancer is still the second leading cause of deaths worldwide. Within cancer research, a promising and growing field is the immuno-oncology, which takes advantage of a patient’s immune system to elicit a protective response against malignant cells. Personalized vaccination against neoantigens is an encouraging approach to target different types of cancer. Neoantigens result from point mutations in the cancer cell genome and transform originally non-immunogenic sequences into immunogenic epitopes that overcome central immune tolerance. Despite different vaccine designs, which are primarily based on dendritic cells, DNA, RNA or synthetic peptides, additional strategies are required to reach sufficient immune responses.
In this study, a novel approach was tested by displaying (neo-)antigens on adeno-associated virus-like particles (AAVLPs) to effectively prime CD8+ T cell responses. AAV was chosen as an antigen-presentation-scaffold owing to its excellent safety profile in humans and tolerance towards genetic engineering of the capsid, allowing presentation of 60 antigen copies per particle. The general vaccination strategy was tested in mice with AAVLPs displaying the ovalbumin-derived model antigen SIINFEKL. Initial experiments showed induction of long-lasting CD8+ T cell responses, sufficient to protect mice completely from B16F10-OVA tumor growth. Based on the SIINFEKL vaccine, the strategy was optimized by defining the most suitable injection routes, adjuvants and capsid insertion sites. Highest CD8+ T cell responses were achieved when the vaccine was I) injected s.c. into the hock, II) adjuvanted with Montanide ISA 51, III) injected at a high local concentration and IV) was composed of vector DNA-containing particles that V) display the antigen in the VR-IV loop of the capsid proteins.
While tested prime-boost strategies and coupling of anti-CD40 to the capsid had no benefit for the vaccination, co-display of the immune stimulatory peptide J-ICBL improved T cell responses significantly. Interestingly, the presence of B cells was disadvantageous for the induction of antigen-specific CD8+ T cells and tumor protection, while the presence of CD4+ T cells was essential. Accordingly, T helper epitopes were identified within the AAVLP capsid sequence. In addition to SIINFEKL-presenting AAVLPs, particles were designed to present a set of different B16F10-derived neoantigens. While a head-to-head comparison showed no effect of a peptide vaccine against B16F10 tumor growth, injection of neoantigen-displaying AAVLPs significantly reduced the tumor growth rate. Although the general strategy requires further refinement and mechanistic analyses, neoantigen-AAVLPs represent an alternative for current therapy approaches and could be a promising candidate for future clinical applications.
Type Ia supernovae (SNe Ia) are important for galactic chemical evolution (GCE) because they produce heavy elements. Sub-Chandrasekhar mass carbon-oxygen white dwarfs with helium shells are favored progenitors for SNe Ia. This thesis investigates the double detonation explosion scenario. A focus lies on an accurate calculation of the detonation propagation in the white dwarf shell and the assumption of core-shell mixing. Parameter studies were conducted to analyse whether variations found in observables of SNe Ia can be reproduced and to provide (metallicity-dependent) nucleosynthetic yields for subsequent radiative transfer calculations and GCE models. Three-dimensional simulations were carried out using the Arepo code. A previously neglected carbon detonation ignition mechanism was found showing that the helium detonation wave convergence is sufficient to ignite carbon in a core-shell transition region. The study shows that various luminosities coinciding with SNe Ia can be reproduced. Metallicity-dependent yields illustrate that a high stellar metallicity shifts the production to stable isotopes while supporting the manganese production. GCE models suggest that the inclusion of this explosion type allows to account for about 80% of the solar manganese abundance. The correlation of [Mn/Fe] with metallicity in the solar neighborhood is supported by the inclusion of metallicity-dependent SNe Ia yields.
Background & Aims: Under favorable microenvironment, nuclear receptor FXR controls apical ABC-transporter BSEP/ABCB11 for bile acid delivery. In acute-on-chronic liver failure (ACLF), most patients lack nuclear FXR in hepatocytes, but maintain apical BSEP expression. The current study investigated the mechanisms of how hepatocytes maintain BSEP expression at the absent of FXR.
Methods: We observed liver tissues from 3 controlled, 5 chronic HBV infected, and 18 ACLF patients. Among ACLF patients, 13 received liver transplantation and 5 recovered. BSEP and target transcription factors were examined by immunohistochemistry in the collected liver tissues. BSEP regulatory mechanisms were investigated in AML12 cell line, mouse primary hepatocytes, human primary hepatocytes and fxr knockout mice.
Results: Firstly, we used immunohistochemical staining (IHC) to examine BSEP expression in 26 liver tissues. IHC analysis showed intact BSEP apical expression in controlled, chronic HBV infected and recovered ACLF patients. In irreversible ACLF patients, 10 displayed BSEP expression in most hepatocytes while 3 did not have detectable BSEP expression in most areas of the specimens. Fifteen ACLF patients who maintained BSEP expression did not have detectable nuclear FXR expression. However, they expressed robust nuclear FOXA2 in hepatocytes. We further compared clinical parameters between ACLF patients possessing and lacking BSEP expression. The recovered patients showed remarkably improved serum total bilirubin, international normalized ratio and Model for End-stage Liver Disease score than those receiving liver transplantation (p<0.01 for all parameters). In addition, compared to the irreversible patients possessing BSEP, the patients who lost apical BSEP demonstrated higher serum total bilirubin concentration (p<0.05). These results suggest that the maintenance of BSEP in hepatocytes is important for the recovery of ACLF. In vitro, overexpression or knockdown FOXA2 induced or inhibited BSEP expression in hepatocytes. Furthermore, ectopic FOXA2 expression restored apical BSEP expression in hepatocytes of fxr knockout mice in vitro and in vivo. ChIP assay revealed that both FXR and FOXA2 initiated ABCB11/BSEP transcription through binding to the gene promoter. However, administration of FXR agonist reduced FOXA2 binding to the ABCB11/BSEP gene promoter, indicating a competitive effect between FOXA2 and FXR on binding to the ABCB11/bsep gene promoter. In normal hepatocytes, insulin impeded FOXA2 nuclear translocation while glucagon induced FOXA2 expression. Under inflammatory condition, inflammatory factors, e.g. LPS and TNFalpha, resulted in high levels of glucagon and inhibition of FXR expression. Further, TNFalpha induced hepatocyte insulin resistance and thus upregulated FOXA2-dependent BSEP expression. In addition, LPS exploited NFκB signaling and thus initiated foxa2 transcription through p65 binding to the promoter. Notably, high concentration of LPS led to FOXA2 nuclear exclusion through phosphorylating at Thr156 of FOXA2, which reduced apical BSEP expression in hepatocytes.
Conclusions: The findings of this study illustrate two regulatory mechanisms relevant for the maintenance of BSEP on bile canaliculi in physiological and pathological conditions. (1) In physiological condition, FXR regulates BSEP expression. (2) In inflammatory circumstance, FXR is significantly inhibited. Inflammation-dependent insulin resistance, high levels of glucagon and NFκB p65 signaling induce FOXA2 expression and activity to maintain BSEP expression, which is important for ACLF patients’ survival and recovery. (3) In sepsis condition, severe infection and inflammation, e.g. high levels of LPS, inhibit FOXA2 activation and thus lead to the loss of BSEP expression on bile canaliculi, which are associated with poor prognosis of ACLF patients. In conclusion, the maintenance of BSEP is essential for survival and recovery of ALCF patients.
Human Immunodeficiency Virus-1 (HIV-1) assembles and buds as non-infectious particles at the plasma membrane of host cells. During assembly, it forms an irregular hexameric ordered lattice consisting of the structural polyproteins Gag and GagProPol. To facilitate infectivity, the intrinsic protease (PR) of HIV-1 drives the maturation by processing these polyproteins at 5 and 9 cleavage sites (CS), respectively, after self-cleavage. This proteolytic part is tightly regulated temporally and sequentially, to ensure a correct morphological rearrangement by a specific release of subdomains inside the viral particle. Gag comprises the matrix (MA), capsid (CA), and nucleocapsid protein (NC) as well as two spacer peptides (SP1 and SP2) and the C-terminal p6-domain. After maturation, CA encapsulates a condensed complex of NC and the RNA genome copies as the conical core. Already subtle dynamical or structural changes can prevent a successful maturation and therefore impair viral infectivity. The concomitant start of maturation and assembly/budding processes so far prevented a precise time-course analysis of maturation in connection with structural and cofactor interactions, and the determination of pH during maturation. Previous proteolytic cleavage studies with in vitro translated Gag and viral particles already led to the categorization of the CSs regarding their processing rates: rapid (SP1-NC), intermediate (MA-CA, SP2-p6), and slow (CA-SP1, NC-SP2). However, these dynamics differ from the processing of synthetic CS peptides. That is why this work aimed to analyze the impact of Gag assembly, which is usually induced by the binding of nucleic acid, and other factors as specific mutations upon the dynamics of maturation. In previous studies, only the final products of maturation were analyzed in viral particles regarding morphology and processing results. Other time-course analyses excluded the verification of the Gag multimerization or the influence of nucleic acid as present in virus-producing cells. Thus, I wanted to compare the processing of Gag in an assembled structure or non-assembled state and additionally introduced specific cleavage site mutants and maturation altering compounds into my system. In order to tackle these open questions for Gag processing dynamics, an in vitro based processing approach for the analyses of proteolytic maturation was chosen, including non-assembled Gag and in vitro assembled ΔMACANCSP2, a truncated Gag variant. Therefore, I produced recombinant Gag with a C-terminal His-tag (Gag-His) in E. coli and optimized the protocol to yield high purity and no nucleic acid contamination, to avoid preliminary assembly. A given protocol to assemble ΔMACANCSP2 was optimized, which increased assembly efficiency and stability to endure the inconvenient conditions of the following processing experiment. Additionally, this newly created protocol could achieve assembly of ΔMACANCSP2 in the absence of any nucleic acid into curved filaments instead of spherical particles. As these filamentous structures are a novelty, further structural analysis of them could give more insight into the assembling properties of Gag in the future. Gag-His featured, independent of its intrinsic homodimerization, an altered order of processing in contrast to the assembled ΔMACANCSP2. The initial cleavage occurred at MA-CA and CA-SP1, followed by SP1-NC and SP2-p6, and at last, NC-SP2. In comparison, the processing of assembled ΔMACANCSP2 reproduced the same processing order shown in the literature, which was only marginally affected by the application of longer NA than 68 nt. The processing of assembled protein was finished up to six times faster than for the non-assembled, while the absence of or very shot (5 nucleotides) NA during the processing of assembled ΔMACANCSP2 caused a mixture of both results. While MA-CA and CA-SP1 are processed like assembled ΔMACANCSP2 with nucleic acid, SP1-NC and NC-SP2 are processed significantly slower than in the case of non-assembled Gag-His. These results suggest that the cleavage events of non-assembled Gag is dependent on the amino acid sequence of the CSs, and assembly causes for MA-CA and CA-SP1 a maturation restriction. Changing the pH of the processing procedure had a severe impact on the processing of CA-SP1 and NC-SP2 in an assembled or non-assembled protein. While the processing was fastest at pH 6.0, the optimum for PR activity, the processing was strongly reduced at pH 6.5 and even more at pH 7.0. The remaining three CSs were only marginally affected. Consequently, CA-SP1 and NC-SP2 might comprise pH-dependent structural domains or interactions, and a theoretical pH shift during maturation of viral particles could enable fast processing. The introduction of mutations known to inhibit proteolytic cleavage or a maturation inhibitor showed that the processing of each site, but CA-SP1, is independent of the cleavage of the other CSs. By inhibiting the processing at SP1-NC, the cleavage of CA-SP1 got delayed, which was observed in the presence and absence of nucleic acids. Interestingly, the inhibition of MA-CA cleavage led to the processing of a new cryptic CS, which was determined to be at the N-terminal region of CA. In summary of this work, assembly of Gag in the presence of nucleic acid accelerates maturation notably, whereas the single cleavage events are independent of each other and only temporally ordered. While assembly delays the processing at MA-CA and CA-SP1, the presence of nucleic acid is the actual key player to shorten the maturation, but it is not essential for Gag assembly.
In the last decades, molecular biology has transformed into a data-rich discipline. This trend is driven by developments in imaging and the continuous increase in available omics technologies which allow for high-throughput profiling of various types of molecules in a given biological system. Classical omics approaches profile the abundance of thousands of cellular biomolecules, e.g., RNAs or proteins. Recently developed assays, such as Thermal Proteome Profiling (TPP), however, can additionally inform on biophysical states of proteins. By choosing the right experimental design or through contextualization of TPP experiments they can reveal small molecule protein engagement, protein-protein interaction (PPI) dynamics or effects of post-translational modifications (PTM). However, while experimental de- signs, reproducibility, amenable organisms and throughput of the TPP assay are being advanced at a fast pace, computational methods for statistical analysis of obtained data are lagging behind.
This thesis proposes a suite of computational methods to provide tools for several of the aforementioned application areas of TPP. First, it describes a software package for analysis of TPP experiments in the context of PPIs and suggests a method for detection of differential PPIs across conditions. The application of this method to different TPP datasets revealed significantly changing PPIs during different phases of the human cell cycle and behavior of protein complexes in Escherichia coli within and across cellular compartments.
Second, this work addresses a specific experimental TPP setup called 2D-TPP in which thermal stability of proteins is measured as a function of temperature and concentration of a compound of interest to find proteome-wide interactions of the compound. This was done by implementation of a curve-based hypothesis test to analyze data obtained from such experiments with false discovery rate control. The method was benchmarked on simulated data and on several real datasets. Application of the software to 2D-TPP datasets profiling epigenetic drugs revealed hitherto unknown off-targets and downstream effects of these drugs.
Third, the same computational method was applied to a 2D-TPP dataset profiling ATP and GTP in a crude cell extract. The analysis of these datasets revealed functional roles of ATP in proteome regulation ranging from allosteric binding, over protein complex assembly and condensate formation. Last, a method for analysis of TPP experiments to profile the effect of PTMs is presented. While the application of this method led to the detection of phosphosites known to be involved in protein regulation, it also pointed out sites which appear to be involved in controlling the localization of proteins to membrane-less organelles. Taken together, this thesis introduces and showcases computational methods for different application areas of TPP. The presented methods are implemented as open source software packages to enable long-term availability and access to the broader community.
The morphology of plants can be highly adaptive due to the plasticity governed by postembryonic development. In plants, stem cell populations, called meristems are maintained through the entire life and enable lifelong development. Longitudinal growth is realized by the meristematic activity in the apices of the shoot and root, while lateral growth is enabled by the activity of the vascular cambium that is able to produce specialized tissues in a bifacial manner. Initially, the fascicular cambium is active solely in vascular bundles. At a later stage of development, the fascicular cambium extends and cells in the region between vascular bundles – the so-called interfascicular region – start to divide, cumulating in the post-embryonic establishment of the vascular cambium. Its formation is potentially based on de-differentiation of differentiated cells. However, how exactly this process is initiated is still poorly understood. Here, I used the process of vascular cambium formation, as a unique model to study cell fate change. To control the formation of vascular cambium, I used a genetic tool that allowed me to induce auxin biosynthesis in a cell type-specific manner. Strikingly, induction of auxin biosynthesis in fully differentiated starch sheath cells provoked a cell fate change and initiated the formation of interfascicular cambium. Based on detailed analysis of interfascicular cambium formation, nuclear auxin signaling is shown to be critical for this process. Furthermore, I investigated the role of cell wall during vascular cambium formation, as plant cells are immobilized by cell walls – a rigid type of extracellular matrix. The thereby fixed position of the cell can contain important information for cell fate determination. Interestingly, extensive cell wall remodeling takes place during cambium formation, as revealed by immunohistochemical quantification of extensin abundance and pectin modifications. Genome-wide transcriptional profiling upon auxin-induced interfascicular cambium formation further supports this result, as genes related to cell wall remodeling were disproportionately represented among differentially expressed genes. Finally, I demonstrated the importance of cell wall remodeling during interfascicular cambium formation by blocking auxin induced interfascicular cambium formation via inhibiting cell wall remodeling. The reported change in cell wall composition during vascular cambium formation may cause a change in the elasticity of the cell wall, which might in turn be a prerequisite for lateral growth. To quantitatively measure the elasticity of cell walls, I established Brillouin microscopy in our lab and investigated cell wall properties depending on their orientation and localization. I was able to show that Brillouin microscopy is a suitable method to further investigate cell wall mechanics and its impact in the field of plant science.
Trypanosoma parasites are the pathogenic agent causing human and animal African Trypanosomiasis. The parasites undergo a process called antigenic variation, which allows them to perpetually evade the host immune response. Trypanosomes are coated almost uniformly with a single protein, the Variant Surface Glycoprotein (VSG). This protein exists in over 1000 distinct alterations within the genetic repertoire of the parasite. These highly antigenic membrane proteins will trigger an extreme immune reaction, which are effective in clearing parasite. However, some cells in the pathogen population will switch to another VSG coat protein. This mechanism introduces a novel antigenic surface to the current Ig response, rendering it non-effective. The first two published protein structures lead to the assumption that all VSGs’ N-terminal architecture is highly conserved. However, these findings are deceptive. Recently published structures revealed that the N-terminus diverges far more than initially anticipated. Furthermore, it has been historically proposed that the VSG coat is not penetrable. High-density packing and the initial structural insight lead to the belief that only the upper portion of the N-terminal domain was likely to interact with the immune system. More recent reviews evaluated the data on this hypothesis and found that the evidence is lacking, suggesting that the coat is more accessible than previously claimed. This thesis aims to map binding epitopes on the VSG N-terminal domain, using high-resolution crystallographic data of VSG-nanobody complexes. Two epitopes could be verified, but these surprisingly did not occur on the upper surface of the VSG protein, but deeply buried. Three nanobodies were found to bind 50 Å below the top of protein at the three-helix bundle, and one other nanobody was found to bind the bottom lobe, 80 Å below the top surface. These findings suggest that, indeed, the antigenic surface coat of Trypanosoma parasites is not an inaccessible “shield”, but rather major parts of the N-terminal domain are exposed to the immune system. This data challenges the historical view on the parasite surface organisation and recasts the understanding of the host-pathogen interaction in this disease. Finally, in collaboration with the Engstler lab at the University Würzburg, we found that one of these nanobodies has a toxic effect on live parasites. Incubation with this nanobody induces high motility inhibition and eventually leads to death of the parasite. Electron micrographs revealed that the nanobody’s introduction results in the creation of excess membrane particles. These particles are likely the results of nanobody-induced membrane fission events. These events usually require a dedicated protein machinery paired with high energetic efforts, while the system described here in vivo is passively driven by protein-protein crowding on the dense VSG surface.
In humans, the perception of volatile substances is multimodal and requires both olfactory and the trigeminal system activation. While olfactory sensory neurons (OSNs) receptive fields are limited to the nasal cavity, trigeminal axons convey sensory information from the head and the neck. Trigeminal neurons are involved in the initiation to protective reflexes and respond to different stimuli modalities such as mechanical pressure, changes in temperature and chemicals, with intensities extending to the noxious range. In contrast, OSNs are mainly activated by odorant chemicals. In the nose, afferents originating from the ophthalmic and maxillary divisions of the trigeminal ganglion, involved in the detection of pungent substances, reside in parallel with olfactory sensory neurons. The proximity of these two systems and psychophysical evidence have led scientists to investigate the possibility of a cross-modality interaction. Most studies have focused on trigeminal modulation of olfactory signals, unravelling a suppressive effect mediated by calcitonine gene-related peptide (CGRP) released from trigeminal afferents. Recently, scientists began to explore the reverse interaction. However, the effect of olfactory stimuli on pungency perception, the possible site of interaction and the molecular pathways underlying it remain unclear. In this thesis, trigeminal innervation was systematically investigated in mice using imaging and electrophysiological techniques to map and characterize anterior ethmoidal nerve afferents within the nasal epithelium. Unexpectedly, during this study, nasal epithelium sections from transgenic reporter mouse line revealed the presence of a subpopulation olfactory sensory neurons expressing the voltage-gated sodium channel NaV 1.8, characteristic for sensory neurons present within dorsal root and trigeminal ganglia. The possibility of a cross-modal interaction between olfactory and trigeminal was interrogated in mice and human. The pure odorant phenylethyl alcohol mitigated irritant aversion to the TRPV1 agonist cyclohexanone and the TRPA1 agonist allyl isothiocyanate diluted in mice. However, these results could not be reproduced in human psychophysical tests. Therefore, the direct influence of OSN activation on trigeminal signalling was assessed using an optogenetic OMP-hChR2Venus mouse line expressing the light-sensitive channel rhodopsin 2 in mature OSNs. Concomitant OSN photo-activation did not affect action potentials signalling in individual trigeminal afferents within the nasal epithelium, suggesting that the mitigating effect observed at behavioural level in mice is unlikely to happen within the nose.
Ozon ist als starkes Oxidationsmittel ein wichtiges Spurengas in der Atmosphäre. In der arktischen Troposphäre liegt die Hintergrundkonzentration von Ozon bei etwa 30-50 nmol/mol. Das Ozon wird hauptsächlich durch photochemische Reaktionen von Stickoxiden und flüchtigen organischen Verbindungen gebildet oder aus niedrigeren Breiten in die Arktis transportiert. Im polaren Frühling werden regelmäßig troposphärische Ozonzerstörungsereignisse (Ozone Depletion Event, kurz ODE) beobachtet. Während eines ODE reduziert sich das Mischungsverhältnis von Ozon in der planetaren Grenzschicht innerhalb von Stunden bis Tagen von der Hintergrundkonzentration auf Werte von nahezu Null. Gleichzeitig werden ansteigende Konzentrationen von gasförmigen Halogenen, insbesondere Brom, beobachtet, welches aus Meersalz im Aerosol, Schnee oder Eis freigesetzt wird. Ein möglicher Emissionsmechanismus ist die autokatalytische "Bromexplosion". Da im Rahmen eines ODE das Oxidationspotential der Atmosphäre und die chemischen Prozesse von Ozon, organischen Gasen und Quecksilber stark verändert werden, besteht Interesse an der Untersuchung von ODEs.
Mit dem eindimensionalen, chemischen Transportmodell KINAL-T wird das Auftreten von oszillierenden ODEs untersucht. Nach dem Abbruch eines ODE ist das Mischungsverhältnis von Ozon auf nahezu Null gesunken, was zu einer Umwandlung von reaktivem Brom zu chemisch trägem Bromid führt. Ozon kann sich dann photochemisch oder durch Transportprozesse erneuern, was zu einer weiteren Bromexplosion und ODE führen kann. Um dies zu modellieren und in einer umfassenden Parameterstudie zu untersuchen, wird das Computermodell optimiert und unter anderem um komplexe Aerosolchemie erweitert. Es werden Oszillationsperioden von mindestens fünf Tagen für photochemisch erneuertes Ozon und von mindestens 30 Tagen für Ozonerneuerung durch vertikalen Transport aus der freien Troposphäre gefunden. Eine wichtige Voraussetzung für oszillierende ODEs ist nach den Ergebnissen dieser Arbeit eine ausreichend starke Inversionsschicht, die den Austausch der Luft zwischen der Grenzschicht und freien Troposphäre minimiert. In einer Parameterstudie wird die Abhängigkeit der Oszillationsperiode von der Stickoxidkonzentration, Stärke der Inversionsschicht, Lufttemperatur, Aerosoldichte und Sonneneinstrahlung untersucht und diskutiert.
Mit dem dreidimensionalen, regionalen Modell WRF-Chem werden Chemie und Transport in einem den Großteil der Arktis umfassenden Gebiet untersucht. Das Ziel ist eine möglichst genaue Vorhersage der Ozon- und Bromchemie im Frühjahr 2009, für das viele Messdaten zum Vergleich vorliegt. Hierzu wird ein bestehender Chemiemechanismus um Halogenchemie und Bromemissionsmechanismen erweitert, außerdem wird die Einspeisung von Daten zur Unterscheidung des Alters des Meereises ermöglicht. In einer Parameterstudie werden verschiedene Emissionsmechanismen getestet und mit GOME-2 Satellitendaten sowie Daten an zwei Messstationen verglichen. Die vom Modell gefundenen Strukturen sind mit den Messdaten konsistent. Eine Erhöhung der Emissionsstärke verbessert die Simulationsergebnisse, zudem verbessert die Annahmen einer Bromfreisetzung durch von Ozon oxidiertes Bromid die Simulation der Auslösung der Bromexplosion. Meterologische Relaxation ist zur korrekten Vorhersage der ODEs über die dreimonatige Simulationszeit nötig. Es stellte sich heraus, dass ohne den Halogenchemiemechanismus keine korrekte Vorhersage der arktischen Chemie im Frühling möglich ist.
Extraction of DNA from so-called difficult samples like feces and soil is problematic, because of the presence of compounds which are mutagenic and destructive against DNA and inhibitors that influence further processing of DNA. As fecal DNA contains DNA from various sources, like gut flora or intestinal mucosa, it has diagnostic relevance. Analysis of fecal DNA might therefore allow conclusions on the presence of (intestinal) diseases like tumors and infections at early stages in a quick and non-invasive manner. DNA extracted from soil, on the other hand, allows conclusions on the composition of the microflora and the purification and analysis of DNA from specific bacteria. Unfortunately, all methods for extraction of DNA from difficult samples currently available are neither quick nor automated nor easy to use. Thus, the development of an easy-to-use, automatable and portable system for extraction of DNA from difficult samples would be beneficial. At first, a suitable system was searched as base for further modifications. This system was then scaled down to microchip-size and several modifications to the original protocol were made to adapt the extraction system to difficult samples. A number of microchip designs were developed, built and tested with the new extraction method and the protocol was adapted to it. As examples for difficult samples soil and, in later experiments, human feces were used for further evaluations. In order to make extraction possible inside a chip without the use of a centrifuge, sedimentation was introduced to replace a centrifugation step in the extraction protocol. Finally, complete DNA extraction was performed inside a chip, from sample addition to final elution. As an advantage, the majority of parts are reusable, except for a small fraction of tubes. The chip itself might be reused as well. Bacteria DNA was successfully extracted from soil and from stool samples with the microsystem, even from spiked samples with very low bacteria count. However, DNA yield from stool was lower than from soil samples. Extraction and detection of human DNA from stool was not successful. For this, further modifications to the extraction protocol and / or the system itself are needed. The microsystem developed within this work is easy to use and the established protocol is quick to perform. It is significantly faster than current column-based methods and requires a minimal number of manual steps. The system offers options for automation, so that it might be possible to increase speed and simplicity even further. Additionally, a PCR step could be integrated in the chip as well as specific markers, so that the extraction system may be expanded to a detection system, e.g. for diseases (stool) or specific bacteria (soil).
The formation of multi-protein complexes is a key feature of the cellular proteome in all kingdoms of life. The biogenesis of protein complexes in vivo is still poorly understood, but recent methodological advances now make it possible to reveal the underlying mechanisms. One milestone method, termed Selective Ribosome Profiling (SeRP), allowed to demonstrate the omnipresence of co-translational assembly in bacteria and yeast (Shieh et al. 2015, Shiber et al. 2018). This method provides codon-resolved information about heterodimer formation between already completed and folded proteins and their nascent partner subunits (termed co-post assembly). The fact that assembly can occur during translation raised the question whether co-translational assembly could also involve interaction between two nascent proteins translated by two neighboring ribosomes (termed co-co assembly). So far, indirect evidence suggested co-co assembly of only a few protein complexes. However, direct evidence that two ribosome-nascent chain complexes interact via their nascent chains is still scarce and we lack any information about the prevalence of this proposed process.
This dissertation focused on the investigation of the hypothesized co-co assembly mode. In collaboration with Matilde Bertolini (PhD student in the Bukau lab), we first developed an unbiased, proteome-wide screen based on ribosome profiling (Disome Selective Profiling, DiSP), to reveal the prevalence of co-co assembly in human cells. By applying DiSP to HEK293-T and U2OS cells, we identified hundreds of high confidence co-co assembling nascent proteins. Our proteome-wide data suggest that up to 30% of all annotated homomer subunits employ co-co assembly, most frequently induced by the formation of N-terminal coiled coils (mostly partially exposed at assembly onset) or interactions of well-known globular dimerization domains (that are generally fully exposed at assembly onset). We further show that co-co assembly of two human homodimeric candidates can be recapitulated in bacteria, in the absence of any eukaryote specific machinery. This suggests that assembly is solely facilitated by the intrinsic propensities of the nascent proteins to form quaternary structures. In addition, we validate the existence of co-co assembly also for endogenous E. coli proteins by DiSP.
The main outcome of this dissertation is the demonstration of co-co assembly as a mechanism mainly employed for homomer formation. Our experimental data indicate the high prevalence of co-co assembly in human cells to ensure productive protein complex biogenesis in the crowded cytosol of cells. Initial findings suggest that co-co assembly is coordinated by a general transient slow-down of translation at the onset of assembly.
Facultatively intracellular pathogens adapt to and rewire host defenses to induce infection and promote survival and proliferation. Salmonella enterica serovar Typhimurium (STm) injects effector proteins via two Type 3 secretion systems into the host cytoplasm to usurp host cell machineries. Hence, investigating protein-protein interactions at the host-pathogen interface is essential for gaining a deeper insight into the interdependency between Salmonella and their host during infection. While more than 30 translocated effectors have been described, knowledge about their function during infection remains incomplete, and the number of proteins that are secreted during infection is likely underestimated.
In this work, l demonstrate the identification of interaction partners of known translocated effectors, maintaining the infection context and without relying on ectopic expression. The Affinity Purification Quantitative Mass Spectrometry (AP/QMS) workflow that I describe in this thesis bridges the gap between large-scale proteomics and high physiological relevance. In the process, I highlight, validate and characterize interactions occurring at endogenous levels of effector secretion. Many bacterial effector proteins displayed promiscuity and multifunctionality by targeting different host processes. Additionally, I assess effector convergence on distinct host processes, as well as effector cooperation and physical effector- effector interaction. These concepts are of groundbreaking importance to better understanding the reprograming of host pathways in different host backgrounds.
I identify cholesterol transport as a convergence point for multiple effector proteins and assess the impact of the involved effectors and targeted host proteins on cholesterol accumulation at the Salmonella-containing vacuole. In addition, I demonstrate that the Salmonella effector SteC is able to directly bind and phosphorylate formin-like proteins, thereby providing a missing link regarding the method by which Salmonella induces cytoskeletal rearrangements during infection. Furthermore, I describe an arrayed STm knockout screen in an infection context, relying on high-throughput microscopy and unbiased image feature extraction. This is used to showcase bacterial processes that are essential for infection and to predict a novel secreted effector protein, YebF, based on its feature fingerprint.
In conclusion, the work presented in this thesis provides the infection biology research community with a rich dataset of novel effector-target interactions, an adaptable and validated AP/QMS workflow, and a well-characterized strain collection for the identification of protein- protein interactions at the pathogen-host interface. Additionally, a database of unbiased phenotypic fingerprints obtained from high-throughput microscopy will be invaluable for the prediction of novel effector proteins and the dissection of host-pathogen interconnectivity. The multifaceted systems biology approach to Salmonella infection presented in this study will fuel hypothesis-driven research and provides a decisive step towards a holistic understanding of the host-pathogen interface.
Transcription is a multistep process that is tightly regulated by transcription factors (TFs). TFs typically comprise two subdomains - a DNA-binding domain (DBD) and an activation domain (AD). Properties of TF target site binding are attributed to the DBD. The AD is thought to determine interactions with the transcriptional machinery, the acquisition of co-activating chromatin modifications and, potentially, the formation of phase-separated nuclear compartments via multivalent interactions. However, it remains unclear if DBD and AD really act independently in determining crucial parameters that govern the induction of transcription. In particular, it is unknown if TF binding kinetics and binding site residence times regulate transcription independent of equilibrium binding parameters and whether phase separation caused by multivalent TF interactions is functionally relevant for activation. In this thesis, experimental and analytical approaches were developed and applied that provide mechanistic insights from the highly informative analysis of TF binding and transcription kinetics. Techniques were introduced to measure TF binding kinetics and to follow transcription using light dependent TF recruitment. These approaches were automated and software packages for the analysis of the resulting data were developed to test a large number of conditions in single cells with high time resolution. The wide applicability of light-induced transcription time courses was demonstrated by two proof-of-concept applications: the detection of transcriptional memory and the discrimination of stochastic models using heterogeneous single-cell trajectories. The framework was then applied to reveal a functional link between binding properties of the DBD, multivalent interactions of the AD and the dynamics of transcriptional (co-)activation. Specifically, the following conclusions could be reached: (1) Reduced TF residence time decreased transcription, even for identical binding site occupancy. (2) Multivalent interactions of the AD stabilized chromatin binding of weakly bound TFs and led to the recruitment of an indirectly bound fraction of molecules. (3) ADs with strong multivalent interactions activated faster and more strongly. (4) Phase-separation into macroscopic droplets did not enhance transcription and could in some conditions even have a suppressive effect. (5) Acetylation of histone at lysine residue 27 (H3K27ac) and the binding of BRD4, which interacts with H3K27ac, were induced by indirectly and transiently bound activators under conditions that were not sufficient to induce RNA production. (6) H3K27ac and BRD4 were not strictly necessary for transcription, but had an enhancing effect. Based on these findings the thesis provides an integrated view of TF activity, in which multiple, interdependent properties of DBD and AD increase transcriptional output. These include long TF residence time, high binding site occupancy, complex stabilization by multivalent interactions and interactions with co-activators, but not phase-separation into macroscopic compartments. These findings provide insights into the different TF features that govern their ability to activate transcription and for the design of synthetic TFs.
Cell polarity is a prerequisite for the formation of distinct cell shapes, which allow different cell types to fulfill their specialized functions. In plants, the family of Rho-Of-Plants (ROPs) controls cellular pathways required for the initiation symmetry breaking including cytoskeleton rearrangements and targeted vesicle transport. Because of their decisive role in these fundamental processes, the spatio-temporal regulation of ROP activation is a delicate task. Efficient ROP activation is mediated by plant-specific ROP guanine nucleotide exchange factors (RopGEFs), which share the central Plant-specific Rop Nucleotide Exchanger (PRONE) domain. While the PRONE domain of RopGEFs is conserved, the flanking N- and C-termini are variable in sequence and do not contain known functional domains. In this PhD thesis, selected RopGEFs were studied for their roles in the establishment of cell polarity during root hair development in the model plant Arabidopsis thaliana. I investigated the functions and regulation of RopGEFs, which were reported to polarize at the Root Hair Initiation Domain (RHID) early (RopGEF3, RopGEF14) or during the onset of polar growth (RopGEF4). In this thesis, I showed that the early polarizing RopGEF3 is crucial for early events including ROP2 recruitment and timing of growth initiation, while the late polarizing RopGEF4 is required for root hair elongation. Furthermore, the unrelated N-termini of early and late polarizing RopGEFs were characterized by exchange of N-termini in RopGEF3. I showed that the N-termini of early polarizing RopGEFs are functionally related in distinction to the N-terminus of RopGEF4. While N-termini of RopGEF3 and RopGEF14 promote RopGEF removal from the RHID, absence of these N-termini or presence of RopGEF4 N-terminus resulted in root hair phenotypes reminiscent of ROP overexpression as well as protein stabilization at the RHID. Cross-species and pairwise RopGEF N-termini sequence alignments combined with in silico prediction for phosphorylation sites in the RopGEF3 N-terminus revealed promising candidate amino acid residues possibly being involved in RopGEF3 regulation at the RHID. Taken together, in this PhD thesis evidence is provided for a putative task-sharing mechanism of early and late polarizing RopGEFs during root hair development as well as for the involvement of RopGEF N-termini in RopGEF protein abundance regulation at the RHID most likely involving regulatory phosphorylations.
Reaction-diffusion equations coupled with ordinary differential equations (ODEs) are used to model various biological, chemical and ecological processes. In case some diffusion coefficients tend to infinity, the reaction-diffusion-ODE system can be approximated by a reduced system. This system is called \emph{shadow limit} and is used to facilitate model analysis. A convergence result is well-known for time intervals which are finite compared to the large diffusion parameter. This research investigates the relation between a reaction-diffusion-ODE system endowed with zero flux boundary conditions and its shadow limit on long-time scales. Such long-time intervals scale with the diffusion coefficient and tend to infinity as diffusion tends to infinity. Solutions of both systems are compared with respect to the $L^\infty$ norm and errors are estimated in terms of the inverse of the large diffusion parameter. This work shows that an extension of uniform error estimates to large time intervals may fail without additional stability assumptions. Error estimates are derived by using a uniform stability condition for the evolution of the linearized subsystem of ODEs and of the linearized shadow system. The method is based on previous results for short-time intervals which use a cut-off technique applied to the system linearized at the shadow solution. The partial lack of diffusion implies low regularity in space of solutions to both systems. Hence, mild solutions are considered in this work. Moreover, two analytical ways of verifying the stability conditions are discussed in detail: dissipativity of evolution systems and linearized stability of stationary shadow solutions using a spectral analysis. The general framework applied in this thesis allows to study the uniform shadow limit approximation for reaction-diffusion systems and reaction-diffusion-ODE systems, under low regularity of the solutions and of the domain. The explicit error estimates provide information on the long-term dynamics of such models from results obtained for their shadow limit. Additionally, this detailed study shows that the shadow limit reduction exhibits characteristic time scales. Validity of the approximation on these time ranges can be verified under certain stability assumptions on the shadow system.
During the process of clathrin-mediated endocytosis (CME), an invagination of the plasma membrane is formed which finally gets pinched off to form an intracellular vesicle. CME is essential for cellular function as it is important for the uptake of nutrients, regulation of signaling, and membrane homeostasis. The machinery mediating CME consists of more than 50 different proteins which are conserved from yeast to mammals. Dynamic high-resolution studies of CME remain challenging due to the small scale (≈ 250 nm) and the fast sequence (≈ 20 s) of the process. This leaves the underlying mechanisms by which spatial rearrangements of endocytic components drive membrane invagination poorly understood.
In this work, we established a new approach to dynamically reconstruct the mobile phase of CME in the budding yeast Saccharomyces cerevisiae at the nanoscale from static super-resolution snapshots. The approach is based on dual-color single-molecule localization microscopy (SMLM) of hundreds of endocytic sites that have been fixed at random points along the regular timeline of endocytosis. We introduced a reference structure composed of two endocytic proteins to sort the sites temporally and align them spatially by fitting a geometric model. Imaging of this reference structure in the first channel alongside an endocytic (query) protein in the second channel allowed us to reconstruct the temporal rearrangements of the query protein.
To obtain large datasets with high quality, we increased the throughput of dual-color SMLM by testing different imaging buffers. Sealing of the sample holder turned out to be crucial to maintain good imaging conditions. Furthermore, we developed a computational pipeline to automatically segment and coarsely align endocytic sites from high-throughput datasets. The segmented sites were then fitted with a geometric model based on previous knowledge from super-resolution and electron microscopy studies. This fitting process helped us to retrieve parameters from the SMLM data which were used for spatial and temporal alignment. Importantly, the fitting is only performed on the reference channel, imposing no structural assumptions on the query protein.
We first validated that the reference structure can be used to temporally sort endocytic sites and then successfully demonstrated that our approach can faithfully recover the structural rearrangements of four proteins exemplary for different endocytic modules. Future progress will enable us to integrate multiple datasets into a single dynamic reconstruction of yeast CME with multiple query proteins.
During early kidney organogenesis, nephron progenitor cells move from the tip to the branch of the ureteral bud to form the so-called pretubular aggregate, the precursor structure of the later nephron. It is assumed that cell pattern formation during this critical phase of organogenesis is primarily controlled by chemotactic mechanisms and differential cell-cell adhesion. The spatial-temporal organization of this process is not yet fully understood. In recent studies, a nonlinear swarm-like pattern of cell movement has been observed.
In order to gain a better understanding of these processes, I elaborated a three-dimensional mathematical Cellular Potts model, and carried out, validated and applied corresponding model simulations. The model parameters were estimated from experimental data obtained in ex vivo kidney explant and dissociation-reaggregation organoid culture studies.
The simulations showed that an optimal enrichment and aggregation of nephron progenitor cells in the corner niche of the ureteral bud branch depends on three factors: the secretion of chemoattractant molecules by a) the epithelial cells of the ureteral bud and b) the nephron progenitor cells themselves, and c) by different adhesion energies between the different cell types. Furthermore, it was observed both experimentally and in the model simulation that nephron progenitor cells move at a higher speed in the corner region of the ureteral bud branches than in their region of origin at the tip of the bud from which they originate. The existence of different cell velocity domains along the ureteral bud was also evaluated with the self-organizing map technique.
In summary, I was able to confirm in the present work the suitability of the Cellular Potts model approach for simulating cell movements and pattern formation during early nephrogenesis. A further refinement of the model should allow the effects of developmental changes the cell phenotypes and the molecular interactions during organ development.
Determining conditions under which a given map is close to a homeomorphism has been an important problem in geometric topology. One of the major results related to the problem is the α-Approximation Theorem of Chapman and Ferry, which asserts that a small homotopy equivalence between manifolds is small homotopic to a homeomorphism. In this context, the smallness condition on a homotopy means that the size of the track covered by each point during the homotopy is small when measured by an open cover of the target space. In proving such a theorem, besides the original approach of ChapmanFerry which uses some results from topological surgery theory, there is another more geometric approach that is more suitable to establish a similar theorem for classes of spaces more general than manifolds. This second approach, due to Chapman himself, is to use controlled topological engulfing to prove a geometric result on approximate fibrations called the Sucking Principle. The α-Approximation Theorem then follows from an application of this principle together with the Cell-Like Approximation Theorem of Siebenmann. In this thesis, based on previous work of B. Hughes, we develop various tools that address the above approximation questions in a stratified setting of possibly singular spaces. In particular, we establish the Stratified Radial Engulfing Theorem, the Stratified Wrapping Up Theorem, the Stratified Handle Theorem, and the Stratified gamma-Sucking Theorem. As a consequence we obtain a Stratified Sucking Theorem with unstratified polyhedral target space.
Measurements of magnitude and composition of volcanic gas emissions allow insights in magmatic processes. Within the Network for Observation of Volcanic and Atmospheric Change(NOVAC) automatically scanning UV-spectrometers are monitoring gas emission at volcanoes. The emissions of BrO and SO2 can be retrieved from the recorded spectra by applying Differential Optical Absorption Spectroscopy(DOAS) and comparing the optical absorption of the volcanic plume to the background. Therefore, the background spectrum must not be affected by volcanic influence. Classically, the background spectrum is taken from the same scan but from an elevation angle which has been identified to be outside of the volcanic plume. However, experience shows those background spectra can still be contaminated by volcanic gases. Alternatively reference spectra can be derived from 1) a theoretical solar atlas spectrum or 2) a volcanic-gas-free reference spectrum recorded by the same instrument. 1) comes with a drawback of reduced precision, as the instrumental effects have to be modeled and added to the retrieval. For 2), the alternative reference spectrum should be recorded at similar conditions with respect to meteorology and radiation. We use the first option to check for contamination and the second to evaluate the spectra to maintain a goid fit quality. We present our approach and its results when applied on NOVAC data from Tungurahua and Nevado Del Ruiz.
Imaging of atmospheric trace gas distributions by optical remote sensing allows for a direct assessment of the dynamics of both physical and chemical processes. In particular, the fast evolution of trace gases emitted by point sources (e.g., volcanic plumes) can be studied comprehensively by imaging approaches. Presently applied imaging techniques still either lack in spatio-temporal resolution like, e.g., Imaging DOAS or in selectivity and sensitivity like sulphur dioxide (SO2) cameras. Throughout this thesis, a novel imaging approach based on Fabry-Perot interferometer correlation spectroscopy (FPI CS) is presented. The technique exploits the periodic transmission profile of a Fabry-Perot interferometer (FPI) and its correlation to the (approximately periodically varying) target trace gas spectral absorbance. The feasibility of the novel technique for SO2, bromine monoxide (BrO), and formaldehyde (HCHO) is examined in a model study. A prototype of a one-pixel FPI instrument for HCHO is characterised and tested in the laboratory yielding a good agreement between the modelled and measured sensitivity for HCHO. The sensitivity (weighted mean trace gas absorption cross section) of the one-pixel HCHO instrument is 2.28 × 10−20 cm2 molec−1. From the HCHO sensitivity, a BrO sensitivity of 6.21 × 10−18 cm2 molec−1 can be inferred due to the spectral similarity of the BrO and HCHO absorption cross section. Finally, an imaging FPI CS prototype is designed, built, and tested in field measurements. The SO2 detection limit of 3.8 × 1017 moleccm−2 s−1/2 is comparable to present SO2 cameras, however, the selectivity is drastically increased. FPI CS therefore shows a promising potential to allow for fast imaging measurements of most of the trace gases that can be measured by DOAS.
Die Datenaufbereitung für (umwelt-)wissenschaftliche Analysen stellt oftmals eine große Herausforderung dar, da Daten verschiedenster Quellen erst aufwändig umformatiert und präprozessiert werden müssen, um einen kohärenten Datensatz zu erhalten. Ziel der Forschungsumgebung von V-FOR-WaTer ist es, den Zugang zu Daten aus den terrestrischen Umweltwissenschaften zu vereinfachen, die Publikation von Daten zu unterstützen und die Datenaufbereitung sowie die Analyse von Daten mithilfe einer umfangreichen Auswahl von Werkzeugen zu erleichtern. Durch diesen einfachen Zugriff auf Daten und Werkzeuge, und deren Verknüpfung in ‚Workflows‘ für Wissenschaftler aus Universitäten und Landesämtern, wird die wissenschaftliche Arbeit beschleunigt, und die Reproduzierbarkeit von Analysen wird gefördert. Das Rückgrat des Prototyps der Forschungsumgebung bildet eine Datenbank mit einer detaillierten Metadatenbeschreibung, die auf die Anforderungen von Wasser- und terrestrischen Umweltdaten zugeschnitten ist. Die bisher integrierten Daten stammen aus Universitätsprojekten und von Landesämtern. Weiter wird an einer Verbindung zu den ‚GFZ Data Services‘, dem etablierten Repositorium für geowissenschaftliche Daten des Geoforschungszentrums Potsdam, gearbeitet. Dadurch wird zum einen die Publikation von Daten aus der Forschungsumgebung heraus vereinfacht und zum anderen der Zugriff auf externe Daten im Portal des GFZ ermöglicht. Der Grundlage, um mit den GFZ Data Services und anderen Systemen kompatibel zu sein, ist die Konformität unseres Metadatenschemas mit internationalen Standards (INSPIRE, ISO19115). Durch die Berücksichtigung der gängigen Standards kann das Portal - nach entsprechenden Anpassungen - auch von anderen Geo- und Umweltwissenschaftlichen Disziplinen genutzt werden. Das Design der Forschungsumgebung ist an typischen Arbeitsabläufen in den Umweltwissenschaften ausgerichtet. Über eine Karte und einen Filter können Daten einfach ausgewählt werden, während ein eigener Arbeitsbereich Werkzeuge für die Präprozessierung, Skalierung und häufige hydrologische Anwendungen bereithält. Darüber hinaus sind auch spezifischere Werkzeuge wie z.B. für die Geostatistik, und demnächst auch für Berechnungen zur Evapotranspiration verfügbar. Die Auswahl an Werkzeugen kann flexibel erweitert werden und wird letztendlich auch Werkzeuge enthalten, die von Nutzern entwickelt wurden, wodurch die aktuellen Forschungsthemen und ‑methoden der hydrologischen Gemeinschaft widergespiegelt werden. Die Werkzeuge sind als ‚Web Processing Services‘ (WPS) angebunden, die als ‚Workflows‘ verknüpft und gespeichert werden können. Dies ermöglicht auch komplexere Analysen und erhöht die Reproduzierbarkeit der Forschung.
We used knockout recombinant viruses to examine the functions of the M81 tegument proteins. It has recently been shown that functions of EBV proteins and of some EBV non-coding RNAs differ between the EBV M81 and B95.8 strains. EBV M81 strain has the unique feature to show a spontaneous lytic replication within B cells. In particular, this allows for an easy investigation without addition of chemical agents or artificial induction of the virus replication to the cells. The first part of our work underscored the importance of tegument proteins for an efficient virus production and for the infectivity of the produced viruses. We then found that the deletion of BGLF4 does not block production of virus and transmission across B cells but only reduces it on average three times. This suggests that the effects of the deletion are less pronounced than in 293 producer cells in which the absence of BGLF4 reduces the efficiency of transmission by a factor 30. Altogether, the effect of BGLF4 deletion affected lytic replication and propagation only mildly, suggesting that the multiple effects in the literature have little influence in the context of the whole virus. We could confirm viral targets of BGLF4 such as BMRF1 and the role of this kinase in late protein expression. However, our work has also evidenced the role of BGLF4 in B cell infection, a function that was not identified clearly before. Here the impact of BGLF4 on late gene expression through reduced binding and fusion is probably also crucial. Finally, although the BGLF4 deletion mutant virus could transform B cells normally, we found that BGLF4 surprisingly regulates the expression of latent proteins, in particular of LMP1. How a protein expressed only in lytically replicating cells can influence the expression of proteins in latently infected cells remains to be elucidated.
In this thesis, we present a novel mathematical model-based approach to optimize loading schemes of isometric resistance training (RT) sessions for different training goals. To this end, we develop a nonlinear ordinary differential equation model of the time course of maximum voluntary isometric (MVIC) force under external isometric loading. To validate the model, we set up multi-experiment parameter estimation problems using a comprehensive dataset from the literature. We solve these problems numerically via direct multiple shooting and the generalized Gauss-Newton method. Moreover, we use the proposed model to examine hypotheses about fatigue and recovery of MVIC force. Then, we mathematically formulate key performance indicators and optimality criteria for loading schemes of isometric RT sessions identified in sports science and incorporate these into multi-stage optimal control problems. We solve these problems numerically via direct multiple shooting and structure-exploiting sequential quadratic programming. We discuss the results from a numerical and sports scientific point of view. Based on the proposed model, we additionally formulate the estimation of critical torque as a nonlinear program. This allows us to reduce the experimental effort compared to conventional testing when estimating these quantities. Furthermore, we formulate multi-stage optimum experimental design problems to reduce the statistical uncertainty of the parameter estimates when calibrating the model. We solve these problems numerically via direct single shooting and sequential quadratic programming. We discuss the solutions from a numerical and physiological point of view. For our approach, a small amount of data obtained in a single testing session is sufficient. Our approach can be extended to more elaborate physiological models and other forms of resistance training once suitable models become available.
In this work the dynamics of molecular hydrogen (H 2 ) and molecular deuterium (D 2 ) are investigated in a series of kinematically complete experiments on an attosecond time scale using the RABBIT technique. To achieve this, a high-repetition rate attosecond beamline has been designed and constructed. A Mach-Zehnder interferometer konfiguration is used to perform pump-probe XUV+IR experiments with the help of a sub-8-fs 150-kHz fiber laser to produce XUV light using high harmonic generation. The beamline is combined with a state-of-the-art reaction microscope and coincident electron and ion detection is used to uncover the molecular fragmentation dynamics. The electron localization in the molecular frame of reference in photodissociation of H2 and D2 is demonstrated. A semi-classical simulation supports the observed phenomena. The localization of the electron can be manipulated by the number of absorbed photons in the system as well as the delay between the pump and probe pulses. Moreover, phases of the electronic wavepackets in the vicinity of resonant doubly-excited states are extracted, and isotope effects are presented.
Mammalian life starts with the formation of a zygote through fertilization of an oocyte by the sperm; subsequently the first mitotic divisions take place. Errors in these earliest divisions can easily propagate throughout preimplantation development and can lead to pregnancy loss prior to its clinical recognition. Albeit one would expect that these divisions should be particularly safeguarded due to their fundamental importance for reproduction, the first embryonic mitoses are much more error-prone than mitosis in somatic cells. Interestingly, this is a paradox observed in all mammalian species analyzed so far, including human. The essential structure mediating chromosome segregation is the microtubule-based mitotic spindle, which has been extensively studied in cell-free systems, somatic cells and embryonic model systems, such as Drosophila melanogaster and Caenorhabditis elegans. By contrast, in mammals, we understand surprisingly little about the mechanisms and regulation of spindle assembly during preimplantation development. In this thesis I therefore studied the assembly of the first mitotic spindle in two mammalian model systems, mouse and cow, to obtain insights into the mechanisms that may underlie early embryonic division errors. In both systems, I found that the first mitotic division is mediated by a pair of spindles that handle the parental genomes separately. The data indicate that this dual spindle assembly is predominantly driven by chromosomal microtubule nucleation and subsequent self-organization of the microtubules around the two spatially separated genomes. First, I used the mouse embryo—a powerful and well-established research model—in which the dual spindle assembles in the presence of many acentriolar cytoplasmic microtubule organizing centers (MTOCs) that were assumed to functionally replace centrosomes. I developed several imaging-based assays to investigate spindle assembly mechanisms in live and fixed mouse embryos and could show that, surprisingly, initial spindle microtubules in the zygote are formed by chromosomal and kinetochore-based microtubule nucleation. By contrast, only some of the many cytoplasmic MTOCs participate in spindle assembly and seem to be more important for stabilization of the spindle than for its initial assembly. Second, I studied zygotic spindle assembly in the bovine embryo, which is in general physiologically more similar to humans, and in particular, the cow zygote also inherits a centrosome from the sperm. However, it is much less established for cell biological research. Using custom-developed light sheet microscopy, I could image live bovine embryos with high spatial and unprecedented temporal resolution and thereby established this system as a valuable model to study cellular mechanisms of mammalian preimplantation development. I could show that dual spindle assembly is conserved in bovine zygotes, despite the presence of two centrosomes. Interestingly, I discovered that the centrosomes are not very active in spindle assembly, and it is thus very likely that the spindle assembly mechanism of chromosomal nucleation and microtubule self-organization, which I observed in the mouse model, is conserved. Overall, my work shows that dual spindle assembly can be found in both acentrosomal and centrosomal zygotes. It is thus likely that this mechanism is conserved across mammalian species, where the parental genomes are present in separate pronuclei in early mitosis. This is also the case in human embryos, and thus my work has significant biomedical implications. In addition, the data presented in this thesis further strengthen the hypothesis that likely also in human zygotes the parental genomes do not mix during the first mitosis, which would have substantial legal and ethical implications for the definition of the beginning of human life.
Gallbladder cancer (GBC) and cholangiocarcinoma (CCA) comprise the group of biliary tract cancers (BTC). They are a rather rarely occurring group of gastrointestinal tumors, however, with bad prognosis. Due to the lack of specific symptoms and late diagnosis, the only potentially curative treatment is surgical resection, but only few patients are eligible, while the only treatment option for unresectable patients lies in general chemotherapy with a platinum-derived agent and gemcitabine. However, this is usually not extending the life expectancy substantially. BTCs are understudied cancers, of which the pathological mechanisms behind tumor formation are mostly unknown, but it is thought that chronic inflammatory processes can contribute to carcinogenesis. In preceding studies to the here presented doctoral thesis, miRNA profiling of a large German cohort of 40 GBC and 8 healthy gallbladder samples was performed. This led to the identification of 24 human miRNAs, which are differentially expressed between healthy gallbladder epithelium and gallbladder cancer. These miRNAs could further be classified into pro-survival and anti survival associated miRNAs, with miR 145 5p being the most downregulated miRNA and miR 4502 representing a potentially oncogenic miRNA. Furthermore, it was shown that CCA cells overexpressing miR 145 5p, significantly upregulated the STAT1 signaling pathway. This led to a tumor suppressive phenotype, as assessed by decreased colony formation and cell viability in CCA cells. Within the scope of this study, two projects were pursued: firstly, confirming the upregulation of STAT1 signaling by miR 145 5p and sustained tumor suppressive STAT1 signaling by concomitant downregulation of the phosphatase PTPRF and secondly: proteomic profiling of FFPE (formalin fixed paraffin embedded) GBC and healthy gallbladder samples of the same cohort as mentioned above, were analyzed by in-depth quantitative proteomics. This resulted in the identification of differentially expressed proteins, leading to the identification of the tumor suppressor FHL1, which is also a target of the oncogenic miRNA miR 4502. Regarding the first part, STAT1 gene and protein expression, as well as STAT1-induced target genes were investigated in CCA, GBC and hepatocellular carcinoma (HCC) cells under the ectopic overexpression of miR 145 5p. This showed that miR 145 5p induced STAT1 upregulation is functional and predominantly occurs in CCA. This emphasizes the molecular distinction between these cancer entities. Additionally, it was shown that along the axis of miR 145 5p and STAT1 activation, the phosphatase PTPRF is downregulated. PTPRF has been predicted to be a miR 145 5p target, however, proving the direct binding and subsequent downregulation was not successful so far. Nevertheless, as STAT1 and PTPRF co-precipitate, it is likely for PTPRF to be able to dephosphorylate STAT1, thereby negatively regulating STAT1 signaling. This led to the conclusion that the tumor suppressive miR 145 5p can lead to the induction of STAT1 expression, along with sustained phosphorylation by concomitant downregulation of PTPRF. Thereby, STAT1 signaling was further enhanced, since the attenuation of tumor suppressive signaling by PTPRF was decreased. These findings suggest the use of the miR 145 5p/STAT1/PTPRF axis to develop a targeted therapy for CCA. For the second part of the studies, a new proteomic approach from FFPE-tissues of GBC and healthy gallbladder was established. Up to date, only few studies using this technology exist as it still challenging. However, the successful performance of quantitative proteomic profiling with a deep resolution of differentially expressed proteins of the GBC cohort is described here. This created the possibility to integrate the miRNA array data with the protein expression data. It was found that FHL1 is one of the topmost downregulated proteins in GBC. Furthermore, miR 4502 is an upregulated miRNA in the short-survival group and it was predicted by miRNA-binding algorithms to target FHL1. These predictions were confirmed by a luciferase assay, proving direct binding of miR 4502 to the 3’ untranslated region (3’UTR) of FHL1. In addition, miR 4502 is downregulating FHL1 in GBC cells after overexpression of mimic 4502 and miR 4502 inhibitor is upregulating FHL1 levels. FHL1 exhibited tumor suppressive properties by reducing colony formation, cell proliferation and cell viability in GBC cells. FHL1 contains protein-protein interaction domains and it has previously been shown to associate with transcription factors, such as the cofactor RBPJ, without being capable of binding DNA itself. In this study, the binding of FHL1 to RBPJ was confirmed and thus FHL1 is likely to replace the intracellular domain of NOTCH1 (N1ICD) in the N1ICD-RBPJ-complex, as N1ICD-mediated transcriptional activity is reduced after overexpression of FHL1. Concomitantly, NOTCH1 target genes are also reduced. As NOTCH1 signaling is frequently overactive in BTCs while FHL1 expression is decreased, this suggests an imbalance between FHL1 binding to RBPJ and N1ICD binding to RBPJ, thereby resulting in expression of oncogenic target genes. Reconstitution of FHL1 leads to tumor suppression, which could partially be attributed to attenuated NOTCH1 signaling. After co immunoprecipitation of FHL1 and subsequent mass-spectrometric analysis of the associated proteins, additional transcription factors have been found, such as GTF2I. Thus, this study shows that FHL1 is a tumor suppressor in GBC and indicates that the miR 4502/FHL1 axis with subsequently affected genes, can be an important new pathway in order to develop a targeted therapy for GBC. This may in the long term improve the outcome of GBC patients.
Antibiotic resistance (ABR) is currently one of the most significant global health challenges. In addition to the rapid development of resistance against new antibiotics, the transfer of existing ABR genes between bacteria leads to the growth of difficult-to-treat multidrug-resistant opportunistic pathogens, causing millions of infections and thousands of deaths every year worldwide. Mobile genetic elements (MGEs) provide a powerful mechanism to transfer ABR genes, because they can move across bacterial cells and species and carry ABR cargos within their sequence. However, their mechanisms of transfer are incompletely understood. Therefore, I investigated the molecular mechanism of a prominent but poorly characterized MGE, the vancomycin resistance carrying conjugative transposon (CTn) Tn1549 from Enterococcus spp. This element is responsible for propagating resistance to this last-resort antibiotic in a wide range of intestinal bacteria. I focused my work on the integrase (Int) and excisionase (Xis) proteins, which are responsible for performing all DNA cleavage and joining reactions during Tn1549 transposition.
In the first part, I reconstituted the complex of Int with a four-way Holliday Junction (HJ) DNA molecule in vitro and solved its crystal structure at 3.3 Å resolution. This is the first CTn integrase structure trapped with this reaction intermediate, showing that these enzymes assemble a stable tetramer to recombine two DNA substrates, in a similar way as the site- specific tyrosine recombinases. Comparison of both enzyme families shows that a cyclic exchange of the C-terminal protein segments promotes tetramerization in all cases. I further characterized the structure of the accessory Xis protein at 1.5 Å resolution. In the second part of my work, I validated the structure by performing HJ resolution experiments in vitro. I found that Int can resolve HJ intermediates both to products and to substrates, likely due to missing regulatory factors. This reaction leads to DNA products containing up to 3 nt long unpaired regions, reflecting Int’s ability to insert its cargo DNA at diverse genomic sequences. In the third part of my work, I show that novobiocin, an aminocoumarin antibiotic, can inhibit Int tetramerization and HJ resolution in vitro, highlighting the importance of the tetrameric state for the transposition reaction.
This work sheds light on an essential step of Tn1549 transposition and its regulation. Moreover, it highlights crucial similarities with site-specific recombinases, increasing our understanding of conjugative transposition. As a proof-of-principle, inhibition of Int activity by novobiocin may open doors to develop potent CTn inhibitors as a new strategy to limit ABR spreading among bacteria.
The prototype minute virus of mice (MVMp) and the H-1 virus belong to the family of Parvoviridae and can be used as oncolytic agents that infect, replicate in and kill human cancer cells. In preclinical trials the viruses were shown to be very successful to eradicate tumors in mice and rats. A phase I/IIa clinical trial with patients suffering from recurrent glioblastoma multiforme showed that parvovirus is safe to use in humans and that the therapy induces an infiltration of immune cells into the tumor tissue.
Many aspects of the parvoviral life cycle have been studied over the last decades. The entry, replication, transcription and packaging mechanisms of the virus are well understood. However, the process of the virally-induced lysis of the cell is still unknown. The viral non structural protein 1 (NS1) was suggested to have cytotoxic features but a direct lytic mechanism could not be shown. Recently, the porcine parvovirus (PPV) was found to express the short transmembrane protein SAT. Mészáros and coworkers showed that a knockout of SAT led to a reduced lytic capacity of the virus (Mészáros et al., 2017). In the current study we show that the SAT protein of MVMp is also important for the lysis of the cell. A knockout of SAT in the genome of MVMp reduced the lytic capacity of the virus and SAT-ko viruses were also found to be less infectious than the wild type virus. The sole expression of SAT induced lysis and it exceeded the lytic capacity of NS1 by multiple times. Mészáros and coworkers suspected SAT to induce an irreversible stress in the endoplasmic reticulum which eventually led to the death of the cell. However, a direct mechanism of this process could not be shown. We suspect SAT to function as a viroporin - a class of virally-encoded transmembrane proteins that oligomerise to form pores through membranes. We could show that SAT is transported to the plasma membrane where it oligomerises in multimers and makes the plasma membrane permeable to the small molecule Hygromycin B. A computer simulation of the protein confirmed that SAT oligomerises in symmetrical complexes. However, pore-like structures were not observed. Furthermore, an increase in the permeability of ions such as calcium and sodium, which is often seen for other viroporins, was not found.
In an attempt to increase the lytic capacity of MVMp we created the SUPER virus. The position of SAT was altered in the genome of SUPER in order to increase its translation. We could show that the translation of SAT was indeed increased for the SUPER virus which led to an accelerated lysis of the cells. However, the production of the SUPER virus was not very efficient. In order to increase the production of SUPER, we constructed shRNA constructs to knockdown SAT. Although the production of SAT was decreased, this approach did not increase the production of the virus. Nevertheless, the SUPER virus could be a promising candidate for the therapy with oncolytic parvoviruses. We suspect its increased lytic potential to release tumor antigens more efficiently compared to the lytic potential of the wild type virus. The released tumor antigens are suspected to stimulate cells of the immune system such as dendritic cells and cytotoxic T-cells in order to recognise and attack non-infected tumor cells.
High Grade Serous Ovarian Adenocarcinoma (HGSOC) is a highly aggressive disease with poor prognosis and the leading cause of gynecological tumor-related deaths. The poor prognosis is related to the fact that already during early stages tumor cells start to spread into the peritoneum. This metastatic spread and the colonization of organs located within the peritoneum are the biggest problems with regard to therapy of HGSOC. Tumors often consist of a functionally heterogeneous population of cancer cells with distinct features. Subsets of tumor cell populations are able to promote tumor progression, metastatic spread and colonization, as well as outgrowth of tumor cells at distant organs. Therefore, the identification and targeting of so-called tumor-initiating cells is crucial. Knowledge about the intrinsic features of tumor-initiating cells and targeting them may ultimately lead to tumor regression and improved patient survival. However, no conclusive evidence about markers for a tumor-initiating population has been provided so far and even less is understood regarding the molecular mechanisms driving tumor-initiating cancer cell populations in HGSOC.
My work shows that Stage Specific Embryonic Antigen 1 negative (SSEA1-) cells are enriched for tumor-initiating abilities in human HGSOC. Furthermore, SSEA1- cells can give rise to both SSEA1- and SSEA1+ cells whereas SSEA1+ cells only give rise to SSEA1+ cells demonstrating a hierarchical organization with SSEA1- cells being on top. Gene expression profiling demonstrated an enrichment of the transcription factor SAM-pointed ETS domaincontaining factor (SPDEF) in SSEA1- cells. Lentiviral knockdown of SPDEF impaired in vivo tumor growth and in vitro colony formation, whereas overexpression of SPDEF resulted in increased colony formation in vitro and tumor formation in vivo. Strikingly, also SSEA1+ cells acquired the capacity to initiate tumors in vivo and form colonies in vitro after SPDEF expression was re-introduced. I also show, that SPDEF negatively regulates the expression of the transcription factor Forkhead box protein A2 (FOXA2). FOXA2 overexpression resulted in decreased tumor-promoting capacity in an in vivo tumor formation assay. Based on these results, I propose that the transcriptional programs modulated by SPDEF, as well as those genes changed due to suppressed FOXA2 target gene transcription, lead to increased survival, clonogenicity and stemness of SSEA1- SPDEFhigh FOXA2low cells and may therefore promote ovarian cancer tumor initiation and metastatic spread.
In summary, the data I generated indicate that SSEA1- cells represent a cellular subpopulation with increased tumor-initiating ability in HGSOC. These cells express higher levels of SPDEF, which exerts its tumorigenic potential by suppressing FOXA2 expression. Developing SPDEF inhibitors might be promising to target the SSEA1- tumor-initiating population and might ultimately lead to tumor regression and improved patient survival.
Cerebral Malaria is a complex neurological condition that results from interaction between the host and the Plasmodium parasite through the different phases of parasite's life-cycle. This interaction ranges from infection to immune response triggered in the host system. Various strains of the Plasmodium parasites are found to have difference in the severity of disease after infection. However, the precise factors defining the infectivity of Plasmodium parasites and the resulting disease outcome have not been completely identified so far. In the thesis, the Plasmodium berghei mouse model for Malaria is used to characterize the infection dynamics of Plasmodium berghei ANKA (wild-type) and a mutant strain that lacks a Plasmodium antigen PbmaLS_05. The mutant infection leads to lower parasitemia in red blood cells and less severe disease outcome in contrast to mice infected with the wild-type strain. Moreover, the mice infected by injecting PbmaLS_05(-) KO-infected red blood cells show reduced immune response in contrast to infection with PbmaLS_05(-) KO-sporozoites. By developing mathematical models describing various mechanisms of the infection and fitting them to experimental data; I find factors that influence the difference in disease progression seen between the two strains. Most strikingly, the KO strain shows a decreased ability to infect immature red blood cells that are usually a preferred target of the parasite. This altered property of infection limits parasite burden and affects disease progression. In addition to this, a statistical analysis of immune activation and immune response data from the KO or WT infected mice was done, which resulted in selecting major indicators of cerebral Malaria. The analysis showed that the number of CD8+ T cells accumulated in the brain, the reduced proportion of CD8+ T cells to lymphocytes in the spleen, the increased presence of Malaria specific CD8+ IFN-+ T and the secondary activation of CD8+ T cells due to the antigens cross-presented by infected red blood cells sequestered in the brain are the prominent distinguishing factors between the ECM causing PbANKA and non-ECM causing PbmaLS_05 (-) infections. An exploratory analysis of the liver-stage of infection and immune response highlighted that PbmaLS_05 may not have an important role to play in triggered immune response during the liver-stage of Malaria. However, its absence may lead to a small decrease in number of productive infections during the liver-stage, which must be further investigated. The antigen PbmaLS_05 can potentially aid in discovering the factors that influence the activation of immune responses and that might contribute to vaccine development and efficient parasite control.
Singlet fission (SF) is a process where two triplet charge-carriers are generated from one photoexcited singlet state. This opens up the possibility to increase the efficiency limit for single-junction solar cells by one third from 33% to 44%. In this work, the long-lasting question of the effect of competing pathways on the efficiency of SF is addressed by time-resolved spectroscopy and a novel global fit approach. This is demonstrated on two examples. First, SF is established in a new class of molecules, the tetraazaperopyrenes. Here, substituent-dependent factors, namely excimer formation as well as vibronic and spin-orbit coupling, are identified to be decisive for SF efficiency. Subsequently, solutions of (hetero-) acenes are investigated, in which comparisons between ambient conditions and deaerated solutions highlight the importance of molecular oxygen for SF: A new, sequential mechanism including atmospheric oxygen as a catalyst is resolved, which allows for a step-wise doubling of triplet states even at low chromophore concentrations. In concentrated solutions, diffusion-controlled SF outcompetes other reaction pathways resulting in triplet yields close to 200%. The absence of any intermediate species emphasises the efficiency of this process.
We know precisely the position of the Sun in our Galaxy. Yet, like for most stars, we cannot tell where it was born. Stars undergo dynamical memory loss: their orbits evolve, because the Milky Way, like many galaxies, has non-axisymmetric structures (e.g. bar, spirals) that shuffle stellar orbits. My thesis quantifies the strength of that process to answer: How (much) do stars change orbit? Can we still infer their birth places, to constrain the formation of the Milky Way disk? I have combined data from the large stellar surveys APOGEE and Gaia, and developed a method to extract the information they contain on the Galactic disk evolution. I forward-modelled the formation of the stellar disk, the stars’ elemental abundances and their subsquent orbital diffusion, which then informs us about their birth radii through ’weak chemical tagging’. I have found that stars can change orbits by large amounts, and most of this evolution is cold (the orbits stay near- circular). Secular evolution determines how the Milky Way disk is structued. If the Milky Way is typical this explain what drives disk galaxies in general to their typical exponential disk density profiles.
The association of proteins into functional oligomeric complexes is crucial for nearly all cellular processes. Despite rapid progress in characterizing the structure of native assemblies, the underlying mechanisms that guide faithful complex formation in the crowded cellular environment are understood only superficially. To secure efficient complex biogenesis and limit the exposure of aggregation-prone intermediates, many proteins assemble co-translationally, via interaction of a fully synthetized and a nascent protein subunit (co-post assembly). Here, we explore the prevalence and the mechanistic principles of a putative co-translational assembly mechanism, which involves the direct interaction of nascent subunits emerging from proximal ribosomes (co-co assembly). To obtain direct evidence of this putative assembly mode, we apply a newly developed method based on Ribosome Profiling, named Disome Selective Profiling (DiSP), which allows to monitor the conversion of single ribosomes to nascent chain connected ribosome pairs across the proteome with high resolution. We use this approach to analyse co-co assembly in two human cell lines and demonstrate that it constitutes a general mechanism inside cells that is employed by hundreds of high confidence and thousands of low confidence candidates, comprising 11 to 32% of all complex subunits. Analysing the features of the co-co assembly proteome, we reveal that this mechanism guides formation of mostly homomeric complexes and typically relies on interaction of N-terminal nascent chain segments. We further identify five dimerization domains mediating the majority of co-co interactions, which are either partially or completely exposed at the onset of nascent chain dimerization, implying different folding and assembly mechanisms. The detectable fraction of each candidate’s nascent chains that co-co assemble is in median 40% and in some cases exceeds 90%, suggesting that this co-translational assembly path may be employed as the main route for complex formation. To gain deeper insights into the mechanistic basis of co-co assembly, we took a series of experimental approaches that distinguish between interactions of nascent chains emerging from the same or different polysomes (termed assembly in cis and in trans, respectively). These experiments could not support a model of assembly in trans. Conversely, we find indications supporting a cis assembly model for nuclear lamin C, one of our high confidence candidates. This mechanism provides a simple explanation for the remarkable specificity of lamin homodimer formation in vivo, where splice variants with largely overlapping sequences do not mix. We propose that assembly in cis more generally secures specific homomer formation of isoforms and structurally-related proteins which are highly prone to promiscuous interactions inside cells. In conclusion, this study provides a global annotation of nascent chain interactions across the human proteome and elucidates the basic principles of this widespread assembly pathway. Our findings raise a number of fundamental questions concerning the mechanisms ensuring high-fidelity protein biogenesis, including the implications of co-co assembly on polysome structure, the possible consequences of co-co assembly failure, the inter-dependence with co-translational folding and the synchronization and coordination with translation kinetics.
Chronic lymphocytic leukemia (CLL) is a lymphoid neoplasm characterized by an accumulation of mature B lymphocytes in blood and peripheral lymphoid organs which highly depend on a tumor-supportive microenvironment. Altered T-cell distribution and function have since long been observed in the CLL microenvironment, but the exact pathological role of the different T-cell subsets remains uncertain. In the present work, the spectrum of CLL-associated T-cell phenotypes were investigated by using leading-edge single-cell technologies. Mass cytometry analyses of lymph nodes (LN), peripheral blood and bone marrow of CLL patients together with reactive lymph nodes (rLNs) of donors without cancer identified the CLL LN as a distinct niche, where CD8+ effector memory T-cells with an exhausted phenotype accumulate. Single-cell transcriptome and TCR-clonality analyses of LN T-cells further revealed a clonal expansion restricted to effector memory CD8+ T-cells, and enabled the characterization of the specific cross-talk between CLL cells and T- cell subsets. Besides, the single-cell transcriptome of T-cells from the Eμ-TCL1 mouse model of CLL was examined and shown to be similar to that of T-cells from CLL patients. Since genome-wide association studies have identified that a single-nucleotide polymorphism affecting the T-cell master regulator EOMES is associated with CLL development, the role of this transcription factor in the disease was investigated. Epigenetic and single-cell RNA sequencing analyses revealed that EOMES is not expressed in CLL cells but in T-cells, and that its levels are highest in exhausted CD8+ T-cells. Interestingly, Eomes deficiency in CD8+ T-cells prevented their expansion and led to a decreased leukemia control in the TCL1 mouse model, providing a novel layer of evidence for an anti-tumor role of CD8+ T-cells in CLL. Furthermore, mass cytometry and single-cell RNA-sequencing analyses revealed an increase of T regulatory type 1 (TR1) CD4+ T-cells in CLL LNs compared to rLNs. Such accumulation was likewise confirmed in spleen of Eμ-TCL1 mice using flow cytometry. Strikingly, TR1 cells failed to expand from Eomes-deficient CD4+ T-cells adoptively transferred in leukemic mice, and consequently were less capable of controlling leukemia development. Moreover, TR1 cell-mediated CLL control required IL-10 receptor signaling, as Il10rb-/- CD4+ T- cells showed impaired anti-leukemia activity. Taken together, the data generated herein comprehensively and deeply characterize the composition and phenotype of the T-cell compartment of CLL patients in comparison to individuals without cancer, and significantly improve our understanding of the function of distinct T-cell subsets in CLL.
Injured neurons in the peripheral nervous system (PNS) can regenerate long-distances and re-establish function, while in the central nervous system (CNS) they do not. Anatomical and cellular signaling discrepancies set up different post-traumatic landscapes between the PNS and CNS, therefore residing neurons respond distinctively to axonal injury. A clear example is when the lesioned peripheral branches of dorsal root ganglia (DRG) regenerate spontaneously, while the lesioned central branches do not. Understanding why such differences occur is the underlying theme of the work presented here. The regenerative capacity of the lesioned PNS is known to be transcription-dependent. Recently, the histone acetyl-transferase p300/CBP-associated factor (PCAF) was discovered to enhance the axonal regenerative program of the DRG as well as prime growth of injured central DRG axons. Although PCAF was found to be an essential player in the transcriptional regulation of a handful of key regeneration-associated genes (RAGs), the precise mechanism of how PCAF enhances axonal regeneration remained elusive. We identified PCAF-dependent RAGs (PCAF-RAGs) through analysis of the DRG transcriptome (RNA-seq) from wildtype and PCAF knockout mice after a peripheral regenerative lesion. Transcription factor binding site analysis predicted potential co-regulators, Specificity protein 1 (Sp1) and myc-associated zinc finger protein (MAZ). Knockdown and overexpression experiments showed that Sp1 and MAZ can enhance neurite outgrowth in vitro, dependent upon the presence of PCAF. More importantly, pharmaceutical activation of PCAF allowed for enhanced neurite outgrowth of cultured DRG and systemic administration of the PCAF activator promoted increased regeneration of the PNS. Anatomically, the periphery subjects its neurons to mechanical forces that may influence its repair outcome. These mechanical cues are missing from the CNS. Therefore, we explored if and how sensory neurons can respond regeneratively to mechanical stimulation. An in-house built bioreactor applied cyclic mechanical tension to PNS- or CNS-located DRG-nerve explants. Neurite length analysis showed that stretch can induce outgrowth of medium to large diameter DRG, partially through the expression of activating transcription factor 3 (ATF3). Our work shows that either a regenerative injury or mechanical stimulation regulate specific outgrowth-related transcriptional machinery. Better understanding of such mechanisms will allow recapitulation of regenerative programs in non-regenerating neuronal populations, in hopes of promoting functional regeneration and recovery.
For the longest time, there was the widely held belief that drug uptake into a cell is mainly due to diffusion, channels and carriers. Only in the 1940s, there was the first drug-transporter interaction discovered. As more transporter-related diseases where discovered and transport proteins identified that had an important connection to cancer, like the breast cancer resistance protein, the research field gained more interest. Drug-Drug interactions were identified and an International Transporter Consortium was founded that had the task to identify relevant transport proteins and closely work together with the drug approval by the FDA. The majority of transport proteins either belong to the class of ABC transporters, which are primary active export transporters or to the class of solute carrier (SLC) transporters, which are secondary active uptake transporters. OATP2B1 is an organic anion transporting polypeptide and is part of the group of SLC transporters. It is seen as an emerging transport protein and has gained attention due to many drug-fruit juice interactions. As the simultaneous intake of fruit juices and drugs are likely to happen in everyday life and OATP2B1 is a human transporter expressed mostly in liver and intestine, it is important to understand more about its uptake mechanism and possible inhibition.
Until now, the emerging field of transporters are examined by either radioactive or fluorescence-based assays. Radioactive assays render a rather unpopular method with many obstacles like cost, safety-issues, labelling and no possibility of high-throughput. Fluorescence-based assays are widespread and have the positive property that they can be automated and used in HTS. The negative aspects here are also labelling and the false negatives and positives prediction that comes through autofluorescence and quenching effects. Our goal therefore was to develop an alternative cell based assay based on a different technology: MALDI MS. MALDI MS brings the advantage that it is a label-free technique and it also is HTS-compatible, like it was already shown in the past. Cell-based MALDI MS assays have gained recognition in the last years in consequence of their speed, robustness and ease in setup and are suitable for the investigation of transport mechanisms due to the abundance of additional information gathered by this technique.
For the MALDI MS method development, the use of E3S as a substrate provided the best results. The optimal matrix composition for the detection of E3S in the cells had to be found. 2.5 mg/mL Ph-CCA-NH2 in 70 % ACN were identified as the best composition and were further used for transport characterisation with the confirmation of time-dependence and concentration-dependence of E3S uptake. The optimal assay conditions (2 min, 10 µM E3S) were used to screen a set of 294 compounds consisting of the top 300 marketed drugs and a set of compounds that are known to interact with OATP2B1. The used compounds were tested in their ability to inhibit the uptake of E3S into the cells. There were 76 compounds found with an inhibition of more than 50 %, which were then further analysed by pIC50 determination. 67 of those compounds could be verified as hits, leading also to 14 very potent inhibitors with a pIC50 over 6. With an average CV % under 10 for 6 biological replicates, the method confirms reproducibility and reliability of the data.
As a reference assay to the aspired MALDI MS assay, also a fluorescence-based assay was developed to examine the uptake of DBF through OATP2B1. This assay was also used to screen the 294 compound set and 67 compounds with an inhibition ≥50 % were identified in the course of the experiments. 57 could be verified as a hit. There was a calculation being done to examine the clinical relevance of those transporters showing a clinical relevance of more than 60 % in the intestine reinforcing the meaning of transporter studies. By the comparison of the two techniques, it was found that only 47 inhibitors overlapped leading to compounds that were not found with one of both methods. This can eventually be explained by the use of different substrates and the multiple binding sites of OATP2B1, but still has to be addressed further. The comparison of the data with the previously published Karlgren et al. data set showed an overlap of 83 % and therefore shows the applicability of the MALDI MS method. To conclude, there were two assay systems developed that are suitable to examine the emerging transport protein OATP2B1. The importance of this transport protein has been shown through the amount of identified (clinically relevant) inhibitors. While the developed fluorescence-based method acts as a good reference method, the newly developed MALDI MS method represents a completely new way to analyse substrate and inhibitor of transporters. With its ease and speed in handling and most notably the label-free approach, the MALDI MS method is an indispensable tool for transporter characterisation and DDI analysis.
Soil water flow is a key process in Earth's hydrological cycle and an essential part of many ecosystem services. Soils are porous media and exhibit a heterogeneous, multi-scale architecture. Their non-linear material properties have a significant influence on the soil water dynamics, which poses difficulties for numerical models. These material properties cannot be measured directly, but data assimilation methods can estimate them by combining information from measurements of soil hydraulic states and from numerical models. The validity of the estimation results can be strongly affected by model errors. This dissertation (i) presents a versatile software package for modeling soil water flow and analyzes the accuracy and efficiency of its numerical discretization schemes, and (ii) employs this software in synthetic data assimilation tasks to investigate the effects of unrepresented dynamics, topography, and small-scale heterogeneity on estimated material properties and forecasts conducted with them. The results reveal that favoring low-order numerical methods over more accurate ones can be justified for use cases in soil hydrology. Moreover, the findings indicate that one-dimensional models with estimated effective material properties can reasonably replicate the dynamics of heterogeneous, two-dimensional domains with complicated topography, if boundary conditions are represented correctly.
We describe mirror symmetry as an equivalence of D-modules. On the A-side we give an introduction to Gromov-Witten invariants, quantum cohomology and the Dubrovin connection. In particular we compute the small quantum cohomology for Del Pezzo surfaces in general and the Dubrovin connection for X_4 explicitly. On the B-side a mirror D-module is constructed as some Fourier-Laplace transformed Gauß-Manin system. We consider its Brieskorn lattice and explicitly compute it for the toric variety X^o_4. Furthermore we derive a solution to Birkhoff’s problem by determining concretely a good basis in the sense of M. Saito. Consequently we prove a mirror theorem for X_4.
Chronic lymphocytic leukemia (CLL) is an indolent B-cell malignancy with a very heterogeneous clinical course. Even though many aspects of the biology of CLL have been thoroughly described, the underlying molecular cause for this heterogeneity has still not been completely understood. To fill this gap, this thesis presents a comprehensive analysis of cancer cell-intrinsic and extrinsic factors which modify drug response phenotypes and patient outcome in a cohort of 81 primary CLL patient samples.
Some cancer cell-intrinsic factors, like the genome or transcriptome of CLL, have been comprehensively explored. However, proteomic profiling of a large CLL patient cohort and its integration with other molecular layers is currently lacking. Therefore, this study performed a thorough characterisation of multiple CLL cell-intrinsic factors, including the proteome, the transcriptome and the genome. These were additionally linked to ex-vivo drug response profiles (43 drugs). This revealed associations between the different layers and functional consequences for drug response and clinical outcome. nsupervised clustering of protein levels uncovered a previously unappreciated poor prognosis CLL subgroup, which was independent of established risk factors and characterised by a distinct protein and drug response profile. The existence of this subgroup could be validated in an external cohort. This comprehensive multi-omics analysis represents the first proteogenomic study of a large CLL patient cohort.
CLL cells additionally depend on cell-extrinsic signals provided by the microenvironment, such as the bone marrow niche. Such signals can modify and reduce the activity of selected drugs. However, a systematic analysis of how the bone marrow microenvironment influences drug response and resistance is lacking, because appropriate bone marrow model systems for high-throughput drug screening do currently not exist. To this end, a high-throughput co-culture drug-sensitivity testing platform was established. During the careful evaluation of different stroma cells as CLL cell support for the system, an unexpected phenomenon was discovered. Some bone marrow stroma cells had the ability to phagocytose apoptotic cells in large amounts. Phagocytosis decreased the total amount of cells and, thus, artificially increased the percentage of alive cells. This has implications for co-culture studies in general, as phagocytosis can cause a systematic bias and the misinterpretation of results if left unconsidered. Consequently, nonphagocytic stroma cells were chosen for the final screening platform.
Using this optimised system, responses to 43 different drugs were measured. A linear model was employed to distinguish between the effect of stroma cells on spontaneous and on druginduced apoptosis of CLL cells. In accordance with the literature, stroma cells protected CLL cells from spontaneous apoptosis ex-vivo. Interestingly, effect sizes varied between patients and especially samples with unmutated immunoglobulin heavy chain variable region and high degrees of spontaneous apoptosis profited from co-culturing. Moreover, the influence of stroma cells on drug responses was systematically assessed. While some drugs, like chemotherapeutics, were less active in co-cultures, other drugs had unchanged activity or were even more effcient in the context of stroma cells. Especially Janus kinase inhibitors could overcome the protective effect by stroma cells and kill CLL cells despite the presence of stroma. The systematic analysis of the impact of the bone marrow niche on drug response can help to understand and overcome microenvironment-induced resistances.
In conclusions, this thesis provides a systematic overview of how leukemia cell-intrinsic layers of CLL and the microenvironment determine drug response and patient outcome.
Interferon beta (IFNβ) triggers the JAK-STAT signaling cascade to induce IFN-stimulated genes (ISGs), which is a hallmark of innate immune response against viral infections. The transcription factors STAT1 and STAT2 become activated and, together with IRF9, assemble into the ISGF3 complex. This complex translocates to the nucleus and activates ISGs by binding to its DNA recognition motif. Most cell types have the potential to activate ISGs upon IFNs signaling but embryonic stem cells (ESC) have a very different response compared to differentiated cells. However, the exact molecular mechanisms that drive this cell type specific interferon signaling are poorly characterized.
In this thesis, the cell type specific IFNβ response was compared between mouse ESCs and differentiated cells like mouse embryonic fibroblasts (MEFs) that carry the same genome. I tested the hypothesis that the cell type specific differences in IFNβ response originate from distinct epigenetic states by applying a genome-wide multiomics approach: (i) A differential gene expression analysis by RNA-seq of IFNβ stimulation defined a total of 513 ISGs and allowed it to identify cell type specific ISG signatures. The bulk sequencing analysis was complemented with single cell RNA-seq to resolve heterogeneity of gene expression response. (ii) By TF chromatin immunoprecipitation followed by sequencing (ChIP-seq) the STAT1 and STAT2 binding sites were mapped across cell types. (iii) Active chromatin regions were detected with the assay for transposase accessible chromatin with high-throughput sequencing (ATAC-seq). Single cell ATAC-seq was used to identify coregulated enhancers and promoters. (iv) ChIP-seq of histone acetylation (ac) and mono- and tri-methylation (me1, me3) marks at histone lysine residues for H3K4me1, H3K4me3, H3K9ac, H3K27ac, H3K9me3 and H3K27me3 was conducted.
The analysis of this comprehensive data set yielded cell type specific patterns of ISGs, ISGF3 binding and chromatin features. The overall stronger IFNβ response in MEFs could be rationalized by factors from the JAK/STAT signaling cascade being constitutively more strongly expressed. In addition, 33 ISGs in ESCs and 305 ISGs in MEFs were found to be cell type specific and thus candidates for epigenetic regulation. To characterize the underlying mechanism, the genomic location, chromatin context and target genes of ISGF3 were characterized. While 92 ISGF3 sites were shared between ESCs and MEFs, 116 and 184 sites were specific for one cell type and found at promoters and putative enhancers. Based on a co-regulation analysis of single cell ATAC-seq data, many of these enhancers could be linked to specific ISGs. Furthermore, the analysis revealed that a pre-existing enrichment of H3K4me1 and open chromatin loci at ISGF3 sites was positively correlated with ISGF3 binding while H3K27me3 showed the opposite effect.
In summary, this thesis characterizes the contribution of epigenetic gene regulation mechanisms to the cell type specific IFNβ response and rationalizes how chromatin features direct cell type specific ISGF3 binding. The insight gained opens up new possibilities for targeted interference with interferon response in anti-viral drug development by accounting for the contribution of chromatin to this process.
Accelerated mixed-signal neuromorphic hardware refers to electronic systems that emulate electrophysiological aspects of biological nervous systems in analog voltages and currents in an accelerated manner. While the functional spectrum of these systems already includes many observed neuronal capabilities, such as learning or classification, some areas remain largely unexplored. In particular, this concerns cybernetic scenarios in which nervous systems engage in closed interaction with their bodies and environments. Since the control of behavior and movement in animals is both the purpose and the cause of the development of nervous systems, such processes are, however, of essential importance in nature. Besides the design of neuromorphic circuit- and system components, the main focus of this work is therefore the construction and analysis of accelerated neuromorphic agents that are integrated into cybernetic chains of action. These agents are, on the one hand, an accelerated mechanical robot, on the other hand, an accelerated virtual insect. In both cases, the sensory organs and actuators of their artificial bodies are derived from the neurophysiology of the biological prototypes and are reproduced as faithfully as possible. In addition, each of the two biomimetic organisms is subjected to evolutionary optimization, which illustrates the advantages of accelerated neuromorphic nervous systems through significant time savings.
The human induced pluripotent stem cells (h-iPSCs) are valuable and promising tools for regenerative medicine and disease modelling because of their capacity to differentiate into multiple types of cells. For the application of female h-iPSCs, an important open question is whether they possess abnormal X chromosome inactivation (XCI) levels which might result in the alteration of gene expression and further downstream consequences. This thesis investigates a population-level set of 273 female h-iPSCs from the Human Induced Pluripotent Stem Cell Initiative (HipSci) and shows a clear line-to-line variety in XCI levels, with four lines (1%) showing complete XCI loss. XCI level is associated with the expression of 2,086 genes (q-value < 0.1), 85% of which are on autosomes. XCI level is inherited in cells differentiated from h-iPSCs. Therefore, the variance of XCI might have an impact on downstream phenotypes, such as immune response. To allow researchers to quality control their h-iPSCs and to maximize the utility of existing h-iPSC banks, methylation-based and expression-based XCI metrics are proposed. These XCI metrics show a clear association between each other and can be used as covariates in further analysis. To explore potential causal factors of XCI loss, variance component analyses are carried out with multiple potential sources, including donor information and technical or biological explanatory variables. These analyses reveal that culture time explains little of the XCI variation, that the expression of XIST is one of the most important explanatory factors, while still not a perfect marker, and that there is a significant donor effect. To identify potential genetic determinants of XCI level, a genome-wide association study (GWAS) and a linear analysis with a subset of expression-related genetic variants are carried out. With cross-check of two XCI metrics, a variant region, as well as a single variant rs3790598, which is associated with the putative RNA helicase MOV10, are found as promising genetic sources of XCI variation.
Particle therapy exploits the highly localized depth dose profile of protons and light ions to deliver a high dose to the target while largely sparing surrounding healthy tissue. The steep dose gradient at the end of the ions range, known as the Bragg peak, however, also makes particle therapy sensitive to range uncertainties. In current clinical practice, a major cause of range uncertainties resides in the conversion of the treatment planning x-ray CT to the patient specific relative stopping power (RSP) map that is crucial for accurate treatment planning. By measuring the energy loss of particles after traversing the patient, particle imaging enables a more direct reconstruction of the RSP. In this thesis, different aspects towards the clinical implementation of particle imaging are investigated. First, a theoretical description of the point-spread function for different particle radiography algorithms is developed in order to explain observed limitations. A novel filtering technique to remove nuclear interaction events in particle imaging is proposed and high quality experimental helium ion CTs are demonstrated. First results from an experimental comparison between particle and x-ray CT modalities for RSP prediction in animal tissue samples are presented. Furthermore, a novel technique for intra-treatment helium ion imaging based on a mixed helium/carbon beam is explored with that relative range changes in the millimeter regime were observable. Finally, novel particle imaging detector designs are investigated. The thesis highlights the potential of helium ion imaging for pre- and intra-treatment image guidance in particle therapy.
This thesis deals with non-parametric hypothesis testing for ill-posed inverse problems, where optimality is measured in a non-asymptotic minimax sense. Loosely speaking, we observe only an approximation of a transformed version of the quantity of interest. Statistical inference, which usually requires an inversion of the transformation, is thus an inverse problem. Particularly challenging are ill-posed inverse problems, where the inverse transformation is not stable.
The thesis is divided into two parts, which investigate different ill-posed inverse models: the inverse Gaussian sequence space model with partially unknown operator and a circular convolution model. In both models we derive minimax separation radii of testing, which characterise how much an object has to differ from the null hypothesis to be detectable by a statistical test. We propose two types of testing procedures, an indirect and a direct one. The indirect test is based on a projection-type estimation of the distance to the null and we prove its minimax optimality under mild assumptions. The direct test is instead based on estimating the energy in the image space and thus avoids an inversion of the operator. We highlight the situations in which also the direct test performs optimally. As usual in non-parametric statistics, the performance of our tests depends on the optimal choice of a dimension parameter, which relies on prior knowledge of the underlying structure of the model. We derive adaptive testing strategies by applying a classical Bonferroni aggregation to both the direct and the indirect testing procedures and analyse their performance. Compared with the non-adaptive tests their radii face a deterioration by a log-factor, which we show to be an unavoidable cost to pay for adaptation. Since our minimax optimal testing procedures are based on estimators of a quadratic functional, we further explore the connection between the two problems – quadratic functional estimation and minimax testing – in the circular convolution model. We show how results from one framework can be exploited in the other. Lastly, we consider minimax testing under privacy constraints, where the observations are deliberately transformed before being released to the statistician in order to protect the privacy of an individual.
Antibodies play an increasingly important role in modern medicine, with monoclonal antibody sales expected to reach nearly USD 200 billion by 2024. High-throughput antibody screening is possible with phage display technologies, however, these are based on antibody binding, which does not necessarily correlate with the phenotypic effects of antibodies on target cells. Traditional functional antibody screening can also be performed with hybridoma cell technology, but as individual hybridoma cells have to be grown up into colonies and tested, this process is both time-consuming and expensive, and only a mere few thousand clones can be obtained and screened.
Droplet microfluidics has been utilised for the screening of individual antibody-secreting cells, as the small droplet volumes enable the accumulation of antibodies to a functional concentration within hours. Droplet microfluidics is also the basis of emulsion-based single-cell transcriptomic assays, where the co-encapsulation of barcoded entities with cells enables the labelling of all mRNA from an individual cell with the same barcode, permitting high-throughput single-cell transcriptomic analyses.
In this thesis, we aimed to evaluate the feasibility of an antibody screening technology that would combine these two applications of droplet microfluidics, and to set up the individual components necessary for such a technology. This technology would enable the study of the effects of single antibody-secreting cells on the transcriptomes of single target cells of interest, in order to identify antibodies of interest.
This requires the co-encapsulation of two different cell types. As this process is governed by Poisson distribution, most droplets cannot reach the desired cell occupancy. To overcome this, we have optimised a picoinjection workflow to selectively inject lysis buffer into droplets with desired cell occupancies, in order to maximise the utility of the sequencing results in our eventual screens.
In addition, we have identified appropriate model systems in which induced transcriptomic changes can be identified at a single-cell level, which can thus be used for further proof of concept and optimisation experiments. We have also evaluated the feasibility of detecting perturbations from the transcriptomic data of a small number of cells treated with drugs, as the identification of antibody hits in our future screens would require the identification of single perturbed transcriptomes in a background of untreated transcriptomes.
Furthermore, as our antibody screening technology would require a means of antibody sequencing, we have developed a Drop-seq-compatible antibody sequencing methodology that enables the identification and elucidation of heavy and light chain antibody variable region sequences. We apply this methodology to sequence mixtures of four different hybridoma cell lines mixed at different ratios and to sequence a diverse population of antibody-secreting cells, for which we have no prior knowledge of the heavy and light chain variable regions present.
These individual components pave the way for the development of a microfluidic antibody screening pipeline that employs single-cell transcriptomics. By studying gene expression changes as a proxy for the global phenotypes of target cells, the effects of different antibodies on various different targets can be simultaneously monitored, permitting highly multiplexed screening campaigns.
Functional specialization of singles cells but also of whole tissues requires the division of labor and leads to the establishment of unequal distribution and polar organization. As a consequence, a diverse spectrum of cells with polar shapes has arisen in all kingdoms of life and conceptual similarities between these cells indicate the presence of a general unifying machinery. Even though we have a good understanding of the process of polar growth itself, we still know very little about the initial steps that lead to cellular asymmetry and polarity establishment. The root hair system of Arabidopsis thaliana is an excellent model to study all aspects of polarity and especially its initiation. One of the first proteins to localize at the root hair initiation domain (RHID) in Arabidopsis is the small GTPase RHO-OF PLANTS 2 (ROP2), which is a central regulator of the root hair growth machinery. ROP2 is a molecular switch that shuffles between an active (GTP-bound) and an inactive (GDP-bound) state. It is obvious that the spatio-temporal control of ROP2 positioning and its activation is of critical importance for proper root hair development, however to date it remains unclear how ROP2 is recruited to the RHID, how ROP2 polarization is timed and how its polar accumulation is maintained. In this thesis I could show that the poly-basic region close to the C-terminus of ROP2 is necessary, but not sufficient for its accumulation at the RHID. I could further demonstrate that polar accumulation of ROP2 depends on its N-terminus, which we have reported to be involved in the interaction with its putative activator and landmark protein GEF3. I found that the ability to shuffle between its active and inactive state is critical for ROP2 polarization and have presented evidence for an activation-dependent immobilization of ROP2 that involves the interaction with GEF3, activation of GDP-ROP2 and differential protein mobility. Using single-molecule localization microscopy I was able to show that ROP2, GEF3 and other proteins of the root hair growth machinery localize into nanoclusters at the PM. To fully characterize the proteome of these nanoclusters, I established biotin-ligase based proximity labelling in Arabidopsis root hairs. This will allow us to elucidate whether the structuring of the PM (at the RHID) into nanoclusters plays a role in the establishment, maintenance and plasticity of cellular polarity.
The amplitude and shape of the density fluctuation power spectrum today are determined by the initial conditions set after inflation, the properties of dark matter particles and the growth of structures due to the gravitational interaction in an expanding spacetime. Since cosmic structure formation is highly non-linear, the impact of the properties of dark matter on today’s structure or results from N-body simulations like the observed universal halo density profiles, are hard to understand with conventional analytical methods. While these approaches break down at small scales when particle streams cross, Kinetic Field Theory (KFT) operates with a generating functional in classical N-particle phase space, circumventing those problems. In this work, we present novel asymptotic methods that apply to rapidly oscillating integrals with two large parameters. Applying these methods to KFT, we derive the asymptotic limit of the power spectrum on small scales in the Zel’dovich approximation. The power spectrum universally develops a k^−3 tail, independent of the steepness of the initial spectrum, suggesting that scale-invariant structures form below a characteristic length scale already early in cosmic history. Finally, we derive the asymptotics of the factors of the factorized generating functional to guide their numerical implementation. These factors are indispensable for the numerical evaluation of perturbation theory and density correlation functions of high order within the framework of KFT.
This work presents several contributions on the topic of learning representations of function spaces, as well as on learning the dynamics of glioma growth as a particular instance thereof. We begin with two preparatory efforts, showing how expert knowledge can be leveraged efficiently in an interactive segmentation context, and presenting a proof of concept for inferring non-deterministic glioma growth patterns purely from data. The remainder of our work builds upon the framework of Neural Processes. We show how these models represent function spaces and discover that they can implicitly decompose the space into different frequency components, not unlike a Fourier transform. In this context we derive an upper bound on the maximum signal frequency Neural Processes can represent and show how to control the learned representations to only contain certain frequencies. We continue with an improvement of a more recent addition to the Neural Process family called ConvCNP, which we combine with a Gaussian Process to make it non-deterministic and to improve generalization. Finally, we show how to perform segmentation in the Neural Process framework by extending a typical segmentation architecture with spatio-temporal attention. The resulting model can interpolate complex spatial variations of segmentations over time and, applied to glioma growth, it is able to represent multiple temporally consistent growth trajectories, exhibiting realistic and diverse spatial growth patterns.
Intense high-energy particle beams are used in fundamental sciences, material sciences, relativistic laboratory astrophysics, and in the industry. Traditionally, dense collimated multi-GeV photon and electron-positron beams are generated via bremsstrahlung and Bethe-Heitler (BH) electron-positron pair creation, respectively. Recent research has focused on strong-field QED processes for greatly enhancing the flux and intensity of the generated beams. To determine the relative role of collisional and strong-field QED processes, we implemented bremsstrahlung and BH pair production processes in the particle-in-cell code Smilei. Using simulations, we show that a high-current ultrarelativistic electron beam interacting with a submicrometer-thick conducting foils can undergo strong self-focusing accompanied by efficient emission of gamma-ray photons. We study the effect of varying electron beam shape, radius and length on the final radiated energy. We show that the self-generated fields can be strong enough that emission occurs in the strong-field QED regime, where a single emitted photon can carry away a significant fraction of the emitting electron energy. We demonstrate that, after beam collision with multiple foils, femtosecond collimated electron and photon beams with particle number density exceeding that of a solid are obtained. This study is timely as it enables laserless strong-field QED investigations with a single high-current electron beam, particularly relevant for the upcoming FACET II facility.
Synchronization is everywhere in nature. It is an emergent property arising in systems of interacting oscillatory entities, regardless if these entities are physical (e.g. in an electrical circuit) or biological (e.g. a group of fireflies). In vertebrate embryos, synchronization of intracellular signaling oscillations regulates the precise and periodic formation of somites, the precursors of vertebrae. In this system, oscillations are coordinated between neighbors via intercellular coupling, and such coordination results in a phase shift between oscillations, giving the impression of a spatiotemporal wave pattern travelling through the presomitic mesoderm (PSM) from posterior to anterior. Where this wave arrests at the anterior PSM, a new somite forms. The timing of such an event is mediated by the segmentation clock, an elaborate molecular signaling network between Notch, Wnt, and FGF signaling.
While there are numerous studies focusing on the molecular details underlying such spatiotemporal regulation from the bottom-up, research delving into (1) the nature and mechanism of its synchronization and (2) its impact on tissue patterning during embryogenesis remains limited. To address this, we thus instead focus on a principles-based, top-down, approach. Accordingly, we recently developed a microfluidics-based experimental platform allowing entrainment of the signaling oscillations in the PSM to periodic pulses of signaling modulators, leveraging fundamental entrainment principles that have also been studied in other complex physical and biological oscillatory systems (e.g. the circadian clock).
In this current research, we use such experimental platform to map Arnold tongues, to systematically control both the period and the phase of oscillations in the PSM, and to precisely modulate the segmentation clock. We report how the systems-level entrainment behavior of oscillations in an embryonic tissue follows dynamical systems theory, despite its complexity. Furthermore, we uncover- and elaborate on a peculiar behavior in our system (i.e. emergence of a period gradient even upon tissue-level entrainment), providing insight into the nature of the underlying oscillatory network in the PSM. This finding has enabled us to generate testable hypotheses about the importance of the period gradient for the processing of spatiotemporal cues and proper tissue patterning. Moreover, experiments with intact embryonic tissue have allowed us to link modulation of the segmentation clock and its consequences on patterning of the PSM. We specifically record observations in apparent contradiction with traditional interpretations of a well-known model of periodic patterning during somitogenesis (i.e. the classical clock and wavefront model). Our observations instead support the proposition that the oscillatory dynamics encode both the timing and spacing of somite formation.
More generally, we here highlight the power of our experimental approach to precisely control the period and phase of a complex spatially-extended system of coupled and phase-shifted oscillations in an embryonic tissue, which had not been possible before using genetics and pharmacological intervention. We hope this research provides further experimental evidence of the universality of fundamental entrainment principles, and offers an alternative top-down approach to the study of synchronization of biological oscillations in embryonic development.
The human neocortex is greatly expanded and exhibits a highly organized and extensively folded (gyrencephalic) structure. Model systems gave a fundamental understanding about how the cortex is generated although the applied models often involve species with a smooth (lissencephalic) brain surface, such as mice. Thus, key cellular events that impact human-specific brain expansion and our understanding of how disease-linked mutations disrupt human cortical development remains elusive. Lissencephaly is a malformation of cortical development which is characterized by a smooth brain and a disorganized cortex. Heterozygous deletions or mutations in the LIS1 gene, encoding a microtubule-associated protein in humans, were identified to cause lissencephaly with diverse clinical phenotypic variations ranging from mild pachygyria (broad gyri) to severe agyria (no gyri) resulting in epilepsy and intellectual disabilities. While the clinical severity generally correlates with the degree of agyria, the location and type of mutation in the LIS1 gene does not. From LIS1 mouse models we know that LIS1 regulates the microtubule motor cytoplasmic dynein and by that dynein-dependent processes such as neuronal migration, nucleokinesis, interkinetic nuclear migration and mitotic spindle orientation. Even though the observed LIS1-deficiency-associated phenotypes appeared drastically milder in murine systems compared to humans these studies suggest that LIS1 gene dosage is relevant for the phenotypic severities. However, why a specific mutation within the LIS1 gene as identified in LIS1-lissencephalic patients (LIS1-patients) leads to different disease severities and whether human-specific processes during cortical development are differentially affected by the specific mutations could, due to a lack of adequate model systems, so far not been investigated. Here, I explore the ability to recapitulate different disease severities of LIS1-lissencephaly using LIS1-patient-specific iPS cells and thereof derived forebrain-type cerebral organoids. To do so, I selected from a LIS1-patient cohort comprising 63 cases 7 patients who cover the whole spectrum of gyrification alterations of LIS1-lissencephaly ranging from Dobyns grade 5 (mild) to 1 (severe). Each patient harbors a different molecular characterized heterozygous mutation in the LIS1 gene. To analyze the consequences of each LIS1 mutation on human brain development a 3D cell culture forebrain-organoid protocol was developed. Following reprogramming of patient-derived somatic cells and basic characterization (2 clones each) the iPS cells were applied to the organoid protocol. Organoids reproduced, in correlation with the patient’s severity, alterations in organoid cytoarchitecture and premature neurogenesis. To assess the direct consequences of the patient-specific mutations on LIS1 microtubule stabilizing function I investigated the stability of the cytoskeleton of apical (a) RG cells within the cortical ventricular-like zone (VZ) structures and found a progressive collapse of tubulin strand stability with increasing patient disease severity leading to a disruption of cellular organization. These phenotypic alterations could in part be reversed by stabilizing the microtubule array using the FDA-approved drug EpothiloneD. In addition, organoids from individuals with severe but not mild disease showed a non-random aRG cell division switch from proliferative to neurogenic division. As an underlying molecular cause, WNT-signaling alterations were identified, most prominently in severe conditions. To test to what extend perturbed WNT-signaling contributes to the observed patient-specific alterations, organoids were exposed to the GSK3ß inhibitor CHIR99021 leading to a significant rescue of non-random aRG cell division switch in severe organoids and to enlarged VZ diameters as well as reduced neurogenesis in all patient derived organoids. The here demonstrated research underlines the capability of cerebral organoids to sensitively model individual disease severities, a so far not addressed major challenge of the system. My data show that different patient-specific mutations in the LIS1 gene have divergent direct impact on microtubule stability, which directly and/or indirectly lead to perturbed human corticogenesis providing the missing link between the patient-specific LIS1 mutation and the clinical severity grade. Future applications analyzing individual diseases have the potential to advance personalize medicine and improve the understanding of individual pathology for personalized therapy.
Hepatocellular carcinoma (HCC) is a complex disease with a poor prognosis which has increased the survival rates on account to the improvement in patient stratification and the introduction of new targeted therapies. However, there is still an urgent need for early diagnostic markers and personalized treatments in order to enhance the survival and reduce HCC´s recurrence. BAMBI, a transmembrane glycoprotein that regulates several biological activities through TGF-β signalling inhibition, was shown to have increased expression levels in colorectal, gastric and ovarian cancers where it correlated with metastasis, invasion and poor prognosis. Although in HCC BAMBI was reported to be upregulated, no molecular and functional studies were done so far to unravel its participation in hepatocarcinogenesis. Our meta-analysis in publicly available HCC data cohorts confirmed BAMBI overexpression in 78% of HCC patients (n=803) being upregulated and also present in cirrhotic samples and the tumour stroma. Further, BAMBI expression was also confirmed in MDR2-KO and DEN mice. Parallel to these results, the immunohistochemical (IHC) staining of a human HCC tissue microarray revealed the upregulation in 76% of patients with positive staining for BAMBI in the surrounding tissues. In HCC cell lines, BAMBI expression appeared only in the cells that present early TGF-β signature (Hep3B, HepG2 and HUH7), which corresponds to an epithelial phenotype and less aggressiveness. BAMBI knockdown in Hep3B cells produced a strong TGF-β-mediated apoptosis and reduced cell proliferation. In less differentiated HLE cells with low intrinsic BAMBI expression, BAMBI overexpression enhanced proliferation, migration and invasion in vitro. Immunoblot assays additionally show BAMBI expression dependent modulation of ERK1/2, NFκB, Wnt/β-catenin, AKT and JNK/p38 MAPK pathways. In conclusion, we report that bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) is upregulated in livers of HCC patients and differently expressed in HCC cell models. High BAMBI expression is blocking TGFβ-mediated apoptosis and increases proliferation in epithelial HCC cells, and reduces proliferation, migration and invasion with impact on Wnt/β-catenin, ERK1/2, AKT, NFκB and JNK/p38 MAPK pathways in mesenchymal HCC. The activation of these functions depends on the TGF-β signature stage and cell context. We postulate BAMBI as a potential target in personalized therapies for human hepatocellular carcinoma.
Fear and aggression are evolutionary conserved emotional responses that can be evoked by different stimuli. One of these stimuli is exposure to a threatening conspecific that depending on the context and history of previous encounters can elicit either defence and avoidance or approach and aggression. The ventrolateral division of the ventromedial hypothalamus (VMHvl) has been recently identified as a structure involved in both behaviours. Neural activity in the ventromedial hypothalamus has been shown to be necessary for defensive and aggressive behavioural responses to conspecific threats. In male mice, inhibition of neural activity in VMHvl reduces avoidance behaviour following exposure to an aggressive male, as well as attack behaviour following exposure to a subordinate male. However, whether the same or different neurons in VMHvl are responsible for defence and aggression toward social threat, how experience affects these responses and the identity of defence neurons in VMHvl remains unknown. Here we performed serial cFos labelling experiments and found that defence and aggression recruited partially overlapping populations in VMHvl. Using in vivo calcium endoscopy of VMHvl neuron activity during social defence and aggression we found that strong calcium responses were elicited upon exposure to the social stimulus and these were further modulated as the animal exhibited defensive or aggressive behaviours. Notably, specific neuronal calcium responses were identified that were correlated with defensive behaviours, some of these neurons were reacted to more than one behaviour, showing complex patterns of activity during aggression and defence. Moreover, calcium recordings over several days of either defence or aggression revealed a change in the ensemble activity between defence and aggression and this effect was dependent on the previous experience of an animal. At the same time we performed a series of functional manipulation experiments blocking or activating neuronal activity in different cell types of the VMHvl. We found separate populations of VMHvl Esr1+ and Nos1+ neurons that were able to modulate defensive responses to social threat. Together, these results demonstrate that the VMHvl encodes and controls both specific and overlapping features of defensive and aggressive behavioural responses to social threat.
The sinoatrial node (SAN) is the natural pacemaker of the heart and initiates the rhythmic contractions of this organ. Its unique genetic profile is mediated by a network of transcriptional regulators. Among them is the homeodomain transcription factor SHOX2, which plays a major role in maintaining the phenotypic border between the SAN and the surrounding tissue. Mutations in this gene have been associated with early-onset and familial forms of Atrial Fibrillation (AF). AF is the most common cardiac rhythm disorder, affecting 1-2% of the general population. In the clinical context, it often co-exists with malfunctions of the sinus node (sinus node dysfunction, SND), however, it is unknown if both diseases interact, perpetuate, or initiate each other. In the first part of this project, a candidate gene study was combined with functional analyses to identify a causal relationship between novel SHOX2 gene variants and the development of AF and SND. Screening 98 SND patients and 450 individuals with AF led to the identification of four heterozygous variants in SHOX2 (p.P33R in the SND cohort and p.G77D, p.L129=, p.L130F, p.A293= in the AF cohort). We selected mutations based on their in silico predicted pathogenic potential and overexpressed them in embryonic zebrafish hearts. A dominant-negative effect leading to bradycardia and pericardial edema was detected for p.G77D, while no effect was revealed for the p.P33R and p.A293= variants. A significantly impaired transactivation activity for both missense variants p.P33R and p.G77D was demonstrated by in vitro reporter assays. Moreover, upon overexpression of the p.P33R mutant in zebrafish hearts, a reduced Bmp4 target gene expression was revealed. This study demonstrated for the first time a genetic link between SND and AF involving SHOX2. Patient-specific human induced pluripotent stem cells (iPSCs) harboring putative disease-causing variants offer unprecedented opportunities for the investigation of cardiovascular diseases. We generated and characterized iPSCs from patients with previously identified heterozygous SHOX2 mutations (SHOX2 c.849C>A and SHOX2 c.*28T>C). To establish an isogenic control, we developed a novel strategy for the scarless correction of heterozygous mutations. Patient-derived iPSCs were gene-edited with the CRISPR/Cas system and subdivided into small cell pools (sib-selection). We quantified wildtype and mutant alleles via digital PCR and next generation sequencing to detect shifts in the wildtype/mutant allele ratio that indicated the presence of gene-corrected cells. Using this method, we managed to enrich our target cells 8-10-fold before generating a monoclonal cell population via single-cell cloning. The recharacterization of the new lines confirmed a preserved pluripotency and a normal karyotype. Future electrophysiological and molecular analysis will give further insights into the contribution of SHOX2 to onset and progression of AF.
Intensity-modulated scanned particle therapy in combination with the characteristic depth dose deposition of carbon ions entail a higher sensitivity to physical changes of the patient geometry as compared to photons. As a result, carbon ions may stop at different spatial locations than predicted during treatment planning. But also the patient's response to radiation is uncertain thereby further compromising the quality of the radiation treatment plan. The unknown level of uncertainty in the carbon ion dose requires a patient specific uncertainty analysis and uncertainty mitigation. For this reason the thesis at hand presents a novel method to assess and quantify carbon ion treatment plan uncertainties considering physical uncertainties, biological uncertainties as well as fractionation effects. Second, the manuscript demonstrated how uncertainties were in a subsequent probabilistic optimization mitigated. The proposed methodology was applied to multiple clinical scenarios and its advantageous impact on the carbon ion treatment plan robustness was demonstrated.
Unlike protons, carbon ion treatment planning needs to account for the increased nonlinear cell killing of carbon ions in a mixed radiation field which increases the treatment planning complexity. With respect to uncertainties, not only the location of dose deposition is uncertain for carbon ions but also their effectiveness which consequently introduces biological uncertainties to treatment planning.
Different to scenario based approaches, this work presents exact and approximated nonlinear closed-form calculations of the expectation value and covariance of the RBE weighted dose accounting for setup-, range- and biological-uncertainties in fractionated carbon ion therapy. The developed analytical pipeline allows propagating linearly correlated Gaussian input uncertainties through the carbon ion pencil beam dose calculation algorithm to obtain uncertainties in dose.
With I and J being the number of voxels and pencil beams, respectively, low-rank tensor approximations were derived for the expectation value and standard deviation reducing the computational complexity from O(I x J^2) to O(I x J) and from O(I x J^4) to O(I x J^2) with minimal loss in accuracy. The consideration of biological errors introduces a new uncertainty structure in the analytical pipeline without increasing the computational complexity. The calculation of expected dose and variance influence information via APM allows performing a subsequent probabilistic optimization.
A proof of concept and several aspects such as accuracy, fractionation and the impact of different probability densities to model input uncertainties were studied in detail on a one-dimensional phantom case. Further, basic three-dimensional dose calculation and optimization functionalities were implemented in the open-source treatment planning system matRad. A subsequent validation against a clinical reference system revealed excellent agreement for elementary pencil beams and patient cases as indicated by γ-pass rates above 99.67%. Theoretical APM derivations were implemented on top and were then applied to clinical carbon ion patient cases. The expectation value and standard deviation of the RBE weighted dose were compared to estimated analogs stemming from 5000 random samples. The γ-pass rate exceeded 94.95% in all patient cases thereby proving the validity of the proposed analytical pipeline. A subsequent probabilistic optimization avoided underdosage of the target volume, reduced the integral dose and resulted in carbon ion treatment plans with a minimized standard deviation of RBE weighted dose. Thus the developed Analytical Probabilistic Model facilitates a flexible, effective and accurate probabilistic description of the radiation treatment plan and generalizes to probabilistic optimization.
In conclusion, the manuscript presents an analytical method to quantify and minimize the uncertainty in the delivery of carbon ion treatment plans. As a result, treatment plans became more robust against the involved uncertainties as demonstrated for a number of clinical scenarios.
The nature of dark matter is one of the most exciting questions of fundamental physics. In terms of a particle physics model the mass spectrum for possible dark matter candidates is huge. Extensive experimental programs seek to unveil the microscopic physics governing dark matter. While indirect and direct detection as well as collider searches have excluded a large class of dark matter models at the weak scale, the region below the GeV scale is relatively unexplored by these experiments. Therefore, it is particularly interesting to study the phenomenology of sub-GeV dark matter. One of the leading constraints in the MeV to GeV range is expected to be set by indirect detection. However, a theoretical description of dark matter annihilation processes into Standard Model quarks is missing yet. In a Monte-Carlo based implementation in Herwig, we provide, for the first time, a modeling of these annihilations for dark matter models with vector mediators. This allows for a comprehensive study of sub-GeV dark matter annihilations in indirect detection searches. In the sub-MeV mass range, low energy experiments and astrophysical as well as cosmological ob- servations can set constraints on dark matter couplings to the Standard Model. If the dark matter candidate does not couple to the Standard Model Higgs, standard collider searches are not able to probe dark matter masses at the order of a few eV. We sytematically study a wealth of constraints on scalar and pseudoscalar dark matter candidates over a large range of dark matter masses. In addition, we introduce a novel search strategy at the LHC that extends the reach of complementary searches for light dark matter candidates.
At the center of black holes, the theory of general relativity breaks down. The resolution of such singularities could require a theory of quantum gravity which describes the fundamental nature of space-time at shortest distances. In this thesis, we explore the tensor model approach to quantum gravity and inspect its relation to other theories of quantum gravity, such as, e.g., asymptotic safety, through a universal continuum limit. Even though at microscopic distances, general relativity breaks down, at large distances this theory is highly successful. We will inspect how one of the predictions of general relativity, gravitational waves, can help us to learn more about new physics beyond the Standard Model.
The interplay of multiple RNA binding domains (RBD) in a single RNA binding protein (RBP) to achieve RNA target specificity is far from being understood. In this thesis, a multidisciplinary study of Upstream of N-Ras (Unr) is presented. Unr is an RBP that has been predicted to contain five single-stranded RNA binding cold shock-domains (CSD). The thesis aims to unravel how Unr binds RNA targets specifically in a cellular context. Several NMR and crystal structures of multidomain constructs were determined. As a result, four non canonical CSDs in addition to the five previously known canonical CSDs were discovered. These non-canonical CSDs play a scaffolding role between the canonical domains, but do not bind RNA independently. Using NMR relaxation and small angle X-ray scattering, it could be shown that the linker between most of the canonical and non-canonical domains is rigid, leading to a restricted flexibility of the full-length protein. Different in vitro and cellular mutational studies, including a reporter gene assay and a RIP seq experiment showed that a disruption of the fixed domain arrangement has influence on RNA recognition and the protein function. Additionally, a crystal structure of a multidomain construct of Unr bound to poly-A RNA provides further information on the complexity of the multidomain RNA binding mechanism of Unr. Several non-canonical binding residues, some even in the non-canonical CSDs, contribute to cooperative RNA binding, suggesting that RNA binding of the full-length protein is likely to be of even higher complexity and plasticity. Further insights into Unr-RNA binding are provided by a full-length protein model, that describes a restricted flexibility of the protein, which might play an essential role within target specificity. To expand the studies towards Unr-ribonucleotide complexes a quantitative mass spectrometry analysis was conducted, that defined several Unr interactors. The protein-protein interaction with the top hit of this result, namely pAbP, was further characterized, which paves the way towards future structure analysis of a larger translation repressor complex, as it assembles on the 3’ UTR of the msl2 mRNA.
Neuroblastoma (NB) is the most common extracranial solid tumor in infants, arising from the developing sympathetic nervous system. A subgroup of approximately 6-8% of NB patients harbors an alteration in the anaplastic lymphoma kinase ALK gene, making this receptor tyrosine kinase an important therapeutic target. Small-molecule inhibitors targeting ALK are already in clinical use as monotherapies, but an optimal integration into the standard care has yet to be established. This study aims at characterizing the inhibitory properties of the third generation ALK inhibitor lorlatinib (LOR) on proliferation of tumor cells and its potential in a combinatorial therapy approach.
In this study, treatment with LOR was investigated in a selection of ALK amplified, F1174L and R1275Q mutated, as well as wild-type NB cell lines. We found that the sensitivity to LOR correlated with expression levels of activated ALK protein. ALK addicted NB-1 cells, which expressed the highest ALK protein levels, were the most sensitive amongst all tested cell lines and were further used as model system to study ALK inhibition in vitro. In these cells, LOR treatment reduced cell viability and proliferation by inhibiting the Ras-MAPK and PI3K-AKT signaling pathways, leading to a predominant G1 cell cycle arrest. Cell death was induced only at concentrations above a clinically achievable level. Despite a high initial sensitivity to LOR, cells could overcome this arrest and gain resistance after long-term treatment. Thus, LOR may not be appropriate for sustained monotherapy. Therefore, we tested whether the treatment of NB-1 cells could benefit from combination therapy with the two first-line chemotherapeutics cisplatin (CDDP) and vincristine (VCR), both in a classical two-dimensional in vitro setting and also in multicellular tumor spheroids. Indeed, combination therapy could significantly reduce cell viability in NB-1 cells. In contrast, NB cell lines that harbor an ALK alteration but express lower amounts of active ALK protein were less sensitive or even resistant to LOR and could not benefit from combination therapies. Furthermore, basal expression of ALK signature genes did not reflect the sensitivity of the different cell lines to LOR. Therefore, RNAseq data that are frequently acquired for NB patients may not be adequate to predict therapy response.
Taken together, this study shows that not only the genomic ALK status is relevant for therapeutic success, suggesting that protein levels should be examined before the treatment with ALK inhibitors. Furthermore, this study indicates that a novel combinatorial approach of LOR with CDDP or VCR is feasible for ALK addicted NBs.
The microenvironment is essential to the development of blood cells. Hematopoietic progenitors receive stimuli from neighbouring cells in the form of secreted cytokines or direct contacts via adhesion molecules. These signals regulate the proliferation and differentiation of the developing cells. In this work, we study interactions between haematopoetic progenitors and their microenvironment from different angles and in different biological systems. We first develop a method to identify candidate ligands-receptors interactions based on transcriptomic data, and using this method, we recover known interactions and predict new candidate interactions between hematopoietic stem and progenitor cells and their niche in the bone marrow. Our analysis notably emphasizes the unique role of mesenchymal stem cells and endothelial cells and further reveals that a niche cell population expressing a certain ligand often expresses its antagonist. This finding suggests a dynamic regulation of HSPCs by the niche. We then undertake to characterize quantitatively cell proliferation, a feature which can be regulated by the microenvironment in vivo. We develop a method to determine the length of each cell cycle phase as well as the relative size of the quiescent fraction. We show that our method, which combines a short pulse-chase experiment with EdU, a thymidin analogue, and mathematical modeling, can accurately determine the cell cycle length of a cancer cell line (Tet21N). We apply our method to double positive thymocytes and establish that most cells from this population are quiescent but that a small fraction of these cells is highly proliferative with a mean cell cycle length of 9h, half the time of the Tet21N. In a final part, we study T cell development in the thymus. We study how the murine thymus, an organ which normally depends on influx from bone marrow progenitor can become self sufficient when transplanted in a mouse which does not produce competent bone marrow progenitors. We show that in this context where resident thymocytes do not have to compete for Il-7with incoming 3 progenitors, a specific population, the double negative 3 (DN3), can self-renew despite receiving the preTCR signal which normally drives their differentiation. We further show that the longer dwelling time of these DN3 progenitors permits their differentiation into γ δ T cells which is normally precluded by the preTCR signal. We show how the limited resources available in the thymus and the flow of incoming progenitor shape the cellular response to the preTCR signal and thereby directly influence the fate of the DN3.
The next generation of Gamma-ray observatory - Cherenkov Telescope Array (CTA) - aims at improving the detection capabilities at a broad energy range of the gamma-ray spectrum by a factor of 10 in sensitivity. The FlashCam camera is one of the camera types mounted on the medium-sized telescopes (MST) of CTA, responsible for the observation of the core energy range between 150 GeV and 5 TeV. The first part of this thesis assumed the task of verifying the trigger system of FlashCam. Studies of the trigger efficiency and night-sky background light trigger response were performed, whilst also improving the Monte-Carlo description of the detector. The second part was dedicated to the research of the fluorescence light detection capabilities of the full 25 FlashCam-MST sub-array using simulations. A trigger logic was developed, which allows the detection of air showers with primary energies higher than 1 PeV through their fluorescence emission. The effective area of this detection method was determined and the angular resolution using a shower axis reconstruction calculated. The combination of these individual studies allowed the estimation of the sensitivity on point-like gamma-ray sources emitting in the energy range above 1 PeV.
Das grundlegende Ziel dieser Dissertation war es, Gelenkknorpel von jungen und alten porkinen Kniegelenken vor und nach einer tribologischen Belastung zu vergleichen. Hierfür wurde auf Kniegelenke aus dem örtlichen Schlachthof zurückgegriffen, der von 16 jungen (6 Monate) und 15 alten (5 Jahre) Schweinen stammte. Die folgenden Fragestellungen wurden hierbei untersucht: 1. Konnte an dem Gelenkknorpel Osteoarthrose nachgewiesen werden? Falls Osteoarthrose vorhanden gewesen sein sollte, wie stark war diese ausgeprägt? 2. Gab es einen statistisch belastbaren Unterschied zwischen dem Knorpelbelag der alten und jungen Schweine? 3. Konnte ein Unterschied im biomechanischen Verhalten nach einer tribologischen Belastung zwischen den jungen und alten Knorpelproben nachgewiesen werden? 4. Welchen Effekt auf der morphologischen Ebene hinterließ die tribologische Belastung an den Knorpelproben? Gab es einen Unterschied zwischen den jungen und alten Tieren im Hinblick auf diesen Effekt? Zur Beantwortung von 1. und 2. wurde unter Zuhilfenahme von radiologischen (Kellgren&Lawrence- Score), makroskopischen (ICRS-Score) und mikroskopischen (Little-Score) Bewertungskriterien der Knorpel beider Gruppen untersucht und verglichen. Hierbei konnte nachgewiesen werden, dass alte Kniegelenke osteoarthrotischen Veränderungen unterlagen und sich in allen drei Bewertungskriterien signifikant von jungen Kniegelenken unterscheiden. Im Anschluss (3.) wurde der Knorpel mit Hilfe eines tribologischen Prüfsystems - basierend auf dem Pin-on-Plate-Prinzip - getestet. Als Pin diente ein osteochondraler Zylinder aus der Femurkondyle, der sich oszillierend auf der korrespondierenden, quadratischen Plate aus dem Tibiaplateau bewegte. Mithilfe eines Höhenmessers wurde hierbei die kontinuierliche Abnahme der Knorpelhöhe der Proben während der 1108 Test-Zyklen (2,03 Stunden) andauernden Belastung gemessen. Hier konnte ebenfalls ein statistisch belastbarer Unterschied nachgewiesen werden. Im Mittel verloren junge Knorpelproben nach der tribologischen Belastung 0,86 mm und alte 0,50 mm an Knorpelhöhe. Eine negative Korrelation zwischen den Arthrose-Scores und der Höhenabnahme des Knorpels zeigte, dass je stärker der Knorpel von Osteoarthrose befallen war, er umso weniger an Höhe während der tribologischen Testung verlor. Für 4. wurde mit denselben makro- und mikroskopischen Bewertungskriterien der Effekt der Belastung auf die jeweilige Gruppe evaluiert und beide Gruppen miteinander verglichen. Der makro- sowie mikroskopisch erkennbare Effekt führte in beiden Gruppen zu einem signifikanten Anstieg des Scores im Vergleich zur Situation vor der Belastung. Auf der tibialen (Plate-) Seite war eine ausgeprägte Riefenbildung erkennbar, welche in der jungen Gruppe stärker zur Geltung kam. Ein eigener, adaptierter Score, bei dem der Fokus der mikroskopischen Veränderungen ausschließlich auf den durch die Reibeversuche verursachten Effekte auf der Oberfläche des Knorpels und nicht auf degenerativen Prozessen lag, konnte in der Situation nach Belastung kein Unterschied zwischen den beiden Gruppen feststellen. Die Ergebnisse dieser Dissertation lassen die Interpretation zu, dass arthrotischer Knorpel resistenter gegenüber mechanischen Beanspruchungen ist, als junger. Diskutiert werden sollte, inwieweit dem tribologischen Prüfsystem mit seiner Pin-on-Plate Konfiguration ein eventueller Einfluss auf die Ergebnisse zugeschrieben werden muss. Die vielversprechenden Ergebnisse dieser Dissertation sollten in weiterführenden Projekten verfolgt werden, da sie wichtige Charakterisierungen der Biomechanik des Knorpels liefern können.
Extensive efforts in characterizing the biological architecture of schizophrenia have moved psychiatric research closer towards clinical application. As our understanding of psychiatric illness is slowly shifting towards a conceptualization as dimensional constructs that cut across traditional diagnostic boundaries, opportunities for personalized medicine applications that are afforded by the application of advanced data science methods on the increasingly available, large-scale and multimodal data repositories are starting to be more broadly recognized. A particularly intriguing phenomenon is the discrepancy between the high heritability of schizophrenia and the difficulty in identifying predictive genetic signatures, for which polygenic risk scores of common variants that explain approximately 18% of illness-associated variance remain the gold standard. A substantial body of research points towards two lines of investigation that may lead to a significant advance, resolve at least in part the ‘missing heritability’ phenomenon, and potentially provide the basis for more predictive, personalized clinical tools.
First, it is paramount to better understand the impact of environmental factors on illness risk and elucidate the biology underlying their impact on altered brain function in schizophrenia. This thesis aims to close a major gap in our understanding of the multivariate, epigenetic landscape associated with schizophrenia, its interaction with polygenic risk and its association with DLPFC-HC connectivity, a well-established and robust neural intermediate phenotype of schizophrenia. As a basis for this, we have developed a novel biologically-informed machine learning framework by incorporating systems-level biological domain knowledge, i.e., gene ontological pathways, entitled ‘BioMM’ using genome-wide DNA methylation data obtained from whole blood samples. An epigenetic poly-methylation score termed ‘PMS’ was estimated at the individual level using BioMM, trained and validated using a total of 2230 whole-blood samples and 244 post-mortem brain samples. The pathways contributing most to this PMS were strongly associated with synaptic, neural and immune system-related functions. The identified PMS could be successfully validated in two independent cohorts, demonstrating the robust generalizability of the identified model. Furthermore, the PMS could significantly differentiate patients with schizophrenia from healthy controls when predicted in DLPFC post-mortem brain samples, suggesting that the epigenetic landscape of schizophrenia is to a certain extent shared between the central and peripheral tissues. Importantly, the peripheral PMS was associated with an intermediate neuroimaging phenotype (i.e., DLPFC-HC functional connectivity) in two independent imaging samples under the working memory paradigm. However, we did not find sufficient evidence for a combined genetic and epigenetic effect on brain function by integrating PRS derived from GWAS data, which suggested that DLPFC-HC coupling was predominantly impacted by environmental risk components, rather than polygenic risk of common variants. The epigenetic signature was further not associated with GWAS-derived risk scores implying the observed epigenetic effect did likely not depend on the underlying genetics, and this was further substantiated by investigation of data from unaffected first-degree relatives of patients with SCZ, BD, MDD and autism. In summary, the characterization of PMS through the systems-level integration of multimodal data elucidates the multivariate impact of epigenetic effects on schizophrenia-relevant brain function and its interdependence with genetic illness risk.
Second, the limited predictive value of polygenic risk scores and the difficulty in identifying associations with heritable neural differences found in schizophrenia may be due to the possibility that the manifestation of the functional consequences of genetic risk is modulated by spatio-temporal as well as sex-specific effects. To address this, this thesis identifies sex-differences in the spatio-temporal expression trajectories during human development of genes that showed significant prefrontal co-expression with schizophrenia risk genes during the fetal phase and adolescence, consistent with a core developmental hypothesis of schizophrenia. More specifically, it was found that during these two time-periods, prefrontal expression was significantly more variable in males compared to females, a finding that could be validated in an independent data source and that was specific for schizophrenia compared to other psychiatric as well as somatic illnesses. Similar to the epigenetic differences described above, the genes underlying the risk-associated gene expression differences were significantly linked to synaptic function. Notably, individual genes with male-specific variability increases were distinct between the fetal phase and adolescence, potentially suggesting different risk associated mechanisms that converge on the shared synaptic involvement of these genes. These results provide substantial support to the hypothesis that the functional consequences of genetic risk show spatiotemporal specificity. Importantly, the temporal specificity was linked to the fetal phase and adolescence, time-periods that are thought to be of predominant importance for the brain-functional consequences of environmental risk exposure. Therefore, the presented results provide the basis for future studies exploring the polygenic risk architecture and its interaction with environmental effects in a multivariate and spatiotemporally stratified manner.
In summary, the work presented in this thesis describes multivariate, multimodal approaches to characterize the (epi-)genetic basis of schizophrenia, explores its association with a well-established neural intermediate phenotype of the illness and investigates the spatio-temporal specificity of schizophrenia-relevant gene expression effects. This work expands our knowledge of the complex biology underlying schizophrenia and provides the basis for the future development of more predictive biological algorithms that may aid in advancing personalized medicine in psychiatry.
The genetic information of all life is encoded within DNA molecules that are translated into functional entities, so-called proteins. They are responsible for operating and controlling a vast array of molecular mechanisms in any biological system and ubiquitous in (patho)physiology as a result. Besides, proteins are the primary target of drugs and can have a central role as biomarkers for diagnostic, prognostic, or predictive purposes. Here, many regulatory mechanisms and spatiotemporal influences prevent an accurate prediction of a proteins’ abundance and its associated functionality based on the genome information alone. Nowadays, it has become possible to measure and quantify thousands of proteins simultaneously, however, involving comprehensive sample preparation procedures. Currently, no universally standardized method enables a routine application of proteome profiling in a clinical environment. In this thesis, an automated workflow for the efficient processing of the most common and quantity-limited specimens is described. In order to demonstrate the usefulness of the end-to- end pipeline, which was termed autoSP3, it was applied to the proteome profiling of histologically defined and WHO recognized growth patterns of pulmonary adenocarcinoma (ADC) that currently have a limited clinical implication. Secondly, we investigated the proteome composition of a molecularly well-defined cohort of Ependymoma (EPN) pediatric brain tumors. Despite the availability of substantial NGS data and their ability to differentiate nine distinct subgroups, the majority of tumors remained without a functional insight. Here, the proteome profiling could provide a missing link and emphasize several subgroup-specific protein targets. In summary, this thesis describes the optimization of SP3 and its automation into a robust and cost-efficient pipeline for quantity-limited sample preparation and biological insight into the proteome composition of ADC growth patterns and EPN tumor subgroups.
The aim of this work is the analysis of the vacuolar pH homeostasis in Arabidopsis thaliana root cells by means of computational modeling. The pH is an important parameter for a range of cellular processes such as the control of enzyme activity and the maintenance of osmotic pressure acting through the establishment of a proton motive force across the vacuolar membrane that in turn is used in the homeostasis of other ions on both sides of the membrane. Although many processes are known to be important for the establishment and maintenance of an acidic vacuolar lumen, recent experimental results have shown that our current understanding of those processes is not complete. To study the vacuolar pH homeostasis in an integrative manner, this work focuses on three different aspects. In the first part, an overview over computational systems biology approaches in Arabidopsis thaliana is given to demonstrate the state of the art and put the rest of the work in a broader context. The second part then focuses on transmembrane transport reactions and the importance of the correct scaling of the kinetic rate laws of those reactions in mathematical models employing sets of ordinary differential equations, which is of importance for any multi-compartment model such as the one presented in part three of this thesis. In the third part, a mathematical modeling approach is subsequently used to explain experimental data concerning the vacuolar pH homeostasis. To do so, three hypotheses of the mechanisms contributing to vacuolar acidification are developed: An as of yet unknown direct proton import, protons released by protein degradation and the reversal of a proton-calcium antiporter. Each of those hypotheses is implemented in an ordinary differential equations model and tested for feasibility against the experimental data.
Aberrations in signaling networks have long been studied for their contribution to tissue degeneration and cancer. Chemical modulation of such pathways provides an excellent opportunity to fine-tune their activity, owing to the rapid and dose-dependent effects of small molecules. The results of the presented thesis demonstrate the application of small molecules in: (i) studying one of the key pathways in cancer and development–TGFb/Smad signaling; and (ii)modulation of cellular fate towards stemness via targeting self-renewal- and pluripotency-associated signaling molecules. In the first chapter, I describe the use of newly-synthesized molecules derived from the indirubin family of natural compounds in the regulation of R-Smad signaling, with a focus on TGFb-related Smad2/3. Using a large screen encompassing cell lines of various cancerous- and non-cancerous tissue origins, I show that indirubin derivatives (IRDs) collectively, and the IRD E738 analogue specifically, interfere with TGFb/Smad signaling via a profound reduction in steady-state R-Smad levels. My analyses illustrate that this effect is not only due to reduced R-Smad gene transcription by E738, but also a result of protein degradation, demonstrating that the IRD modulates the TGFb/Smad pathway through different mechanisms. Further investigation by transient over-expression of wild-type-, truncated-, and phosphorylation-defective mutant forms of Smad2 and Smad3 reveals the significance of the "linker domain", in particular its phosphorylation status, in the small molecule's regulation of basal Smad stability. Given the role of Smad linker phosphorylation–referred to as "phospho-Smad signaling"–in the malignant switch of TGFb in some tumor types, the potential connection between E738's regulation of steady-state Smad2/3 stability and the phospho-Smad pathway is explored. Using patient-derived cholangiocarcinoma (CCA) cell lines, I show that IRD E738 inhibits oncogenic phospho-Smad isoforms (linker region sites), while maintaining those associated with a cytostatic phenotype (C-terminally phosphorylated sites). This effect reflects the molecule's inhibitory activity on kinases (e.g., GSK3 and CDKs) involved in the phosphorylation of Smad2/3 linker region. The results of the second chapter outline a combined chemical and genetic approach for enhancing the generation and maintenance of human iPSCs. Using a cell-based high-throughput screen, a series of OCT4-inducing compounds (O4Is) are identified, including imidazopyridine analogues, with the lead compound termed O4I3, as well as 4-(tert-Butyl)-N-(2,3-dimethylphenyl) thiazol-2-amine, herein O4I4. The small molecules either alone or in combination are shown to activate pluripotency-associated signaling, and to increase the reprogramming efficiency of human fibroblasts into iPSCs when combined with ectopic expression of the master pluripotency transcription factors: OCT4, SOX2, KLF4, and MYC (known as "OSKM"). Transcriptomic analyses (DNA microarray/RNA-sequencing) and ATAC-sequencing revealed previously unrecognized, targetable molecular events in the path towards pluripotency. Indeed, O4I3 lifts an epigenetic barrier of reprogramming through inhibiting the histone demethylase enzyme, KDM5A, thus allowing the enrichment of H3K4Me3 at the OCT4 promoter. Additionally, applying a combination of O4I3 and O4I4 to OSKM-based reprogramming reveals the involvement of bone morphogenetic protein (BMP)/Smad signaling upstream of high mobility group A1 (HMGA1) in O4I3/4-mediated induction of endogenous OCT4, which in turn initiates the reprogramming process. Altogether, the results of both chapters demonstrate the potential use of new classes of small molecules in targeting signaling networks associated with cancer and somatic cell reprogramming.
Injury to the adult spinal cord damages ascending and descending spinal fiber tracts thereby disrupting proper information transmission between the brain, spinal cord and periphery of the body. Restoring neural connectivity beyond the site of injury is the essential prerequisite for functional recovery to occur. Without intervention, central nervous system (CNS) axons fail to regenerate, resulting in tremendous impairment of sensorimotor function as well as autonomic dysfunction and, consequently, a significant reduction of the patients’ quality of life. Hence, a massive effort has been spent to develop effective repair strategies for spinal cord injury (SCI) including cell transplantation and biomaterial implantation. However, functional axonal growth past the lesion site remains insufficient due to inappropriate implant integration, detri-mental fibroglial scarring and failure of spinal axons to grow beyond the site of injury. Recently, astrocytes were identified as essential key players for neuroregeneration due to their neuro-protective and supportive functions after CNS injury. Further, immature astrocytes not only fulfil scaffolding functions during development, but might also adapt to the harsh lesion envi-ronment without adopting detrimental phenotypes. Thus, astrocyte are prime candidates to provide structural as well as trophic support for growing axons in combination with biomaterial implants at SCI lesion sites. In the present study, novel alginate-based hydrogel implants with a defined channel micro-structure were combined with cellular grafts of immature astrocytes derived either from the cortex or the spinal cord of neonatal Fischer-344 rats to: (1) provide a physical guidance structure for regrowing axons at the site of injury; and (2) establish a permissive cellular growth substrate within and beyond the hydrogel implant supporting axonal crossing of the lesion cavity of a cervical unilateral hemisection of the spinal cord in adult rats. First, alginate-based hydrogel implants were modified with polypetides to improve their bio-compatibility and cell viability in vitro and in vivo. Afterwards, immature astrocytes from neona-tal rat cortex were cultivated and enriched in vitro. Seeding of alginate-based hydrogel im-plants with immature cortex-derived astrocytes improved axonal regrowth compared to non-seeded hydrogel implants following SCI. The grafted astrocytes interacted with the host as-trocytic network and aligned into tissue bridges structurally guiding axons across the host-graft interface. To elucidate whether astrocytes with a spinal cord identity would elicit superior pro-regenerative effects after SCI, immature astrocytes were isolated from the spinal cord of neonatal rats and compared with cortex-derived astrocytes. Phenotypic characterization re-vealed minor molecular and morphological differences between both astrocyte populations in vitro and in vivo. Particularly, cortex-derived astrocytes were found to have a more mature phenotype compared to spinal cord-derived astrocytes in vitro, however, both cell populations adopted a differentiated morphology and expressed functional molecular astrocytic markers in vivo after transplantation into the intact spinal cord. After SCI, seeded hydrogel implants to-gether with additional caudal grafts of either immature astrocyte population further enhanced axonal growth through the implantation site and promoted revascularization. The grafted cells connected with the host spinal parenchyma facilitating tissue bridging between implant and host. Finally, seeded hydrogel implants in combination with rostral and caudal immature astro-cyte grafts were shown to additionally increase axonal growth through the hydrogel implants after SCI by 70% compared to the previous transplantation paradigms. Thus, the combination of biomaterial implantation with cell transplantation superiorly promotes axonal growth through sites of acute SCI compared to treatment paradigms based only upon biomaterial implants. Moreover, additional grafts of immature astrocytes into the surrounding host tissue improve host-graft interactions by formation of a continuous cellular substrate spanning the SCI lesion site. Nonetheless, axonal re-entry into the distal host spinal cord may require additional trophic attraction.
Inflammation or infections have a great impact on an organism. In order to protect the hematopoietic system from exhaustion during pathogenic insult, heterogeneity in metabolic activity, gene expression patterns, differentiation capacity, and responsiveness to cytokines such as interferons (IFN) have been revealed. However, how this diversity in the system is generated and maintained remains poorly understood. In this thesis, I explored three aspects of IFN signaling heterogeneity in the hematopoietic system. First, I investigated the origin of IFN signaling heterogeneity during hematopoietic development (1.). Here, I was able to show that differences in baseline IFN signaling are already present at the onset of definitive hematopoiesis at embryonal day (E) 10.5 of development. In addition, I identified the placenta as an embryonic niche that provides definitive hematopoietic stem cells (HSCs) with stable high basal IFN signaling. Second, I investigated the function of IFN signaling heterogeneity both during embryonic development and in different cell populations of the adult system (2.). I uncovered a potential role for higher baseline IFN signaling in mediating protection against infections and pathogen invasion in hematopoietic cells during development. Furthermore, in the adult my data indicate that T cells with different levels of baseline IFN signaling display different activation efficiencies and expression of immune checkpoint molecules following in vitro stimulation. Thirdly, I investigated the mechanisms of IFN signaling heterogeneity (3.). Analysis of DNA methylation patterns of cells with different levels of basal IFN signaling revealed possible epigenetic differences both during development as well as in the adult hematopoietic system. Understanding the origin, function, and mechanism of IFN signaling heterogeneity during hematopoietic development and in the adult hematopoietic system will allow targeting of specific aspects of the pathway to improve response to infections, inhibit development of infectious diseases and possible hematological malignancies, improve existing treatments or develop new treatment approaches. In addition to the investigation of IFN signaling heterogeneity in the hematopoietic system, a fourth aim of my PhD thesis concentrated on the role of the extracellular matrix (ECM) in the stress-induced activation of HSCs (4.). Previous studies from our group (Uckelmann et al., 2016) could show that the ECM component Matrilin-4 (Matn4) plays an indispensable direct role in the activation of HSCs upon inflammatory stress. I investigated several other ECM components, but only found minor differences in the hematopoietic compartment of mice lacking these components during homeostasis and under inflammatory stress. However, the ECM is a complex and dynamic network of constantly interacting components and novel approaches will be necessary to understand the two-way communication between HSCs and the ECM under inflammatory stress.
In this thesis numerical methods for the solution process of the Newman-type lithium ion battery (LIB) model with a distribution of spherical particles with different radii are derived. Since the Newman-type LIB model is a multiscale model, where the scales cannot be completely separated, it has a pseudo twodimensional structure. To be solved it needs the solution of a so called cell problem. Furthermore, it consists of a homogenized and a microscopic part that have to be solved coupled. The solution process of the cell problem is done with the CutCell method to improve reconstruction properties of the microstructure. Therefore, the condition number of a Laplace problem with the CutCell method is investigated and it is shown that a simple preconditioner can improve the condition problems due to the CutCell method. Furthermore, a goal oriented a posteriori error estimator based on the dual weighted residual (DWR) method is derived for the CutCell method. For the LIB model the condition number is investigated and a simple preconditioner is suggested. Based on the DWR method a goal oriented error estimator is derived and tested for the pseudo twodimensional problem. The model behavior of this extension is investigated, especially the occurrence of the different deintercalation properties of the particles with different radii. Furthermore, a simplified model for small charge/discharge rates for an anode is derived and compared to the full model. In the end, the sensitivity of full Newman-type model with respect to two parameters, which are hard to determine experimentally, is investigated.
In recent years, hydrogels developed to promising tools for biomedical and industrial applications. For biomedical approaches hydrogels, possess the capacity to immobilize and release cells, they offer the desired 3D environment to induce cell specific behaviour or serves as a drug delivery system. Moreover, they can be used for tissue engineering approaches by mimicking the ECM. In this thesis, a novel hybrid double cross-linked hydrogel is presented and designed based on the bottom-up approach of synthetic biology. It consists of simultaneously formed chemical and physical cross-links and made out of two components: (1) thiol functionalized HA (74 kDa) (HA-DTPH) and (2) ionic crosslinker (Cl+). HA-DTPH provides the chemical cross-link by forming disulphide bonds and the ionic cross-linker forms physical cross-links, such as hydrogen bonds and salt bridges. Three different ionic cross-linker were used: (1) deacetylated disaccharide unit of HA (dHA+) (2) charged glucosamine (GluA+) and (3) ammonium chloride (NH4+). These ionic cross-linker were chosen due to their biocompatibility and ability to form physical cross-links, such as hydrogen bonds and salt bridges. The increasing capacity to form hydrogen bonds from NH4+ to dHA+ enabled us to study the influence of the physical cross-link on the hydrogel properties. I could show that the disulphide bond formation was enhanced, by adding an ionic cross-linker and led to the formation of stable hydrogels. Under the same reaction conditions, HA-DTPH without an ionic cross-linker, needed further oxidation with hydrogen peroxide to result in a stable hydrogel (HA-DTPH-Ox.). By varying the degree of thiolation on HA and additionally by varying the type and concentrations of the used ionic cross-linker, the mechanical stiffness, swelling properties and response to external stimuli were tuneable. Varying the degree of modification and used ionic cross-linker enables a specific adjustment of the hydrogels specifically the hydrogel suitable for cell studies with mechanical range of 0.1 Pa to 8 kPa. Furthermore, swelling ratios of HA-DTPH-Cl+ hydrogels are highly influenced by the ionic strength and pH. Remarkably HA- DTPH- dHA+ hydrogels upon incubation in a solution of pH 7 showed a feedback loop swelling behaviour. At the swollen state of the hydrogel, the ionic cross-linker dHA+, leaked out of the hydrogel network, acidified the solution, which resulted in shrinking of the hydrogel. Biological properties like enzymatic degradability showed that the half-live of HA-DTPH-Cl+ hydrogels are increasing with increasing capacity of the ionic cross-linker to form hydrogen bonds. Moreover, due to the absence of any toxic agent during the hydrogel formation the hydrogel system was used for live cell applications such as cell encapsulation or cell adhesion studies. To conclude, a hybrid double cross-linked hydrogel system could be presented, mimicking the ECM, in a minimal model and a critical influence of physical cross-links is observed from results obtained by characterizing the physical and biochemical properties by investigating the gels’ swelling capability, response to environmental changes and sensitivity to hyaluronidases. Depending on the desired biomedical application, these hydrogel systems can be tuned in regards to their stiffness, swelling behavior and degradability enabling applications in 3D tissue engineering, drug delivery and regenerative medicine.
A functioning nervous systems results from complex developmental processes. One requirement is that individual neurons need to form sufficient synaptic connections with adequate partners. Here, molecular signaling and neural activity control morphological development of axons and dendrites and synaptogenesis in order to establish and maintain stable networks. However, mechanisms maintaining stable postembryonic circuits are not well understood and the long-term effects of embryonic neural activity on neuronal morphology and connectivity are unkown. This thesis investigates trans-synaptic, anterograde Jelly-Belly-Anaplastic lymphoma kinase signaling in postembryonic circuit development and elucidates the establishment of synaptic patterns by embryonic neural activity in the motor circuit of Drosophila larva. I demonstrate that Alk activity inhibits the formation of postsynaptic specializations on motoneurons during postembryonic circuit growth by analyzing single cell connectivity. I employ a new Bxb1 integrase-based technique for targeted mutations to show that presynaptic release site number of an upstream interneuron is unchanged but Jeb-Alk seems to elicit a negative feedback that limits the formation of presynaptic filopodia. These Jeb-Alk devoid circuits with altered synaptic patterns produce epilepsy-like seizure behavior. Additionally, in vivo time lapse imaging of dendrites reveals that dendritic growth and postsynaptic synaptogenesis are regulated independently and presynaptic filopodia likely promote dendritic elaboration. During embryogenesis, neural activity adjusts the establishment of synaptic patterns in motoneurons. In a picrotoxin-induced epilepsy-like model, dendritic growth is unaffected, but synaptic input is increased. The number of release sites of an upstream interneuron is again unaffected. In summary, I identified cellular and molecular mechanisms required for the establishment and maintenance of synaptic patterns for reliable circuit function. With novel genetic and imaging techniques, I show embryonic neural activity is pivotal for the formation of functionally stable synaptic patterns, and establish Jeb-Alk signaling as a negative regulator of circuit expansion maintaining embryonically established connectivity. These developmental mechanisms highlight that balancing pre- and postsynaptic growth and synaptogenesis is central to stable network function.
Coral reefs are the most biodiverse ecosystems on Earth. Their productivity is powered by the symbiotic association between corals and unicellular photosynthetic dinoflagellates of the family Symbiodiniaceae. These symbionts reside inside the corals’ cells in specialized organelles, the symbiosomes, from where they transfer energy-rich compounds to the corals to support their nutrition. Interestingly, symbiosis is re-established every generation, as most corals produce symbiont-free offspring, which acquire symbionts from the surrounding sea water by phagocytosis. Despite the importance of symbiosis establishment for the coral life cycle, it is unclear how corals identify compatible symbionts and which mechanisms allow the symbionts to persist in their intracellular niche. For example, it is unknown how symbionts escape the host immune response and how symbiont-derived nutrients are integrated into the host cell metabolism. Using the sea anemone Aiptasia as a model, I characterized the underlying processes of symbiosis establishment. Aiptasia engages in symbiosis with similar species of Symbiodiniaceae as corals, and can be induced to produce larvae under laboratory conditions, providing access to symbiont-free larvae to study symbiosis establishment year-round. In a first step, I compared the uptake of compatible symbionts to that of free-living Symbiodiniaceae and inert beads in Aiptasia larvae. I uncovered that selection of symbionts occurs already prior to their phagocytosis and that while phagocytosis was size-selective, all tested particles were phagocytosed. This implies that additional mechanisms for the selection of compatible symbionts occur once potential symbionts have been phagocytosed. To assess the molecular mechanisms underlying symbiosis establishment in more detail, I developed a cell-specific method to compare gene expression in symbiotic and non-symbiotic cells. This revealed a major down regulation of various catabolic processes, including autophagy, as a response to symbiont uptake. Specifically, I found evidence that the shutdown of autophagy is regulated by a conserved gene-regulatory network under the control of the master regulator of cell growth and proliferation, mTOR (mechanistic target of rapamycin). mTOR is activated by symbiont-derived nutrients and symbiosis resulted in increased lipid storage and cell proliferation. Thus, I propose a model where symbiont-derived nutrients activate mTOR signaling, which acts as a mechanism of symbiont selection. Beneficial symbionts activate mTOR, resulting in their maintenance and allowing them to proliferate in the host tissue, while failure to activate mTOR would result in their autophagy. Currently, coral reefs are declining world-wide at unprecedented rates, mostly caused by global warming disrupting the symbiotic interaction leading to loss of symbionts from the host, a process known as ‘coral bleaching’. This thesis lays the foundation for future work studying the molecular mechanisms underlying symbiont selection and symbiosis establishment in cnidarian-dinoflagellate symbiosis. Understanding how corals select and stably integrate symbionts may help to predict how corals adapt to changing environments, a prerequisite to combat the loss of these important ecosystems.
Cell migration of Plasmodium parasites, the causative agents of malaria, is powered by an actomyosin motor. This substrate-dependent type of movement termed gliding motility is important at different stages throughout the complex life cycle of these parasites and is required to traverse tissues and invade host cells. The motor complex is located beneath the plasma membrane and consists of anchored myosin A proteins that exert forces on actin filaments which can be transmitted to a substrate by transmembrane proteins. How the surrounding environment impacts parasite motility is not fully understood. Furthermore, it is unclear how the actomyosin motor can be modulated to allow for stage-specific regulation of parasite motility. In the first part of this study, I show how substrate elasticity, confinement and pore size affect Plasmodium motility using polyacrylamide hydrogels. I found that the parasites were not capable to move persistently on flat soft substrates, while the migratory capacity increased with substrate stiffness. In confined environments on the other hand parasites moved robustly even if the surrounding matrix was extremely soft. Plasmodium sporozoites, the stages transmitted into the dermis by a mosquito, could squeeze through the tiny pores of polyacrylamide hydrogels. This 3D hydrogel assay can be used as an in vitro model to test drugs or antibodies against the parasite after transmission. The second part of this thesis deals with the role of myosin A for Plasmodium gliding motility. Using a reverse genetic approach, I could demonstrate the importance of amino acids within the unusual N-terminal extension of myosin A for maximum speed of sporozoites in vitro. I found that phosphorylation of one of these residues is required for efficient salivary gland invasion of sporozoites in vivo. These results show for the first time, how modulation of motor properties by post-translational modification of myosin A could regulate parasite motility to allow for successful transmission from mosquito vector to mammalian host.
Die Anzahl der interventionellen Eingriffe steigt und die Unterstützung durch robotische oder digitale Assistenzsysteme nimmt zu. Zeitgleich steigt der Informationsgehalt der diagnostischen Daten, die vor der Intervention über den Patienten erhoben werden. Der Transfer der präinterventionellen Daten in den Interventionsraum kann für Interventionalist und Assistenzsystem entscheidende Vorteile in Planung und Durchführung bedeuten. Derzeit bedeutet die interventionelle Bildgebung entweder zweidimensionale Projektionen ohne Tiefeninformation oder dreidimensionale Aufnahmen, die unter hohem Strahlungsaufwand gewonnen werden. Die interventionelle 2D-3D-Registrierung schließt diesen Informationsbruch durch die räumliche Registrierung der drei-dimensionalen präinterventionellen Daten durch eine oder mehrere Projektionen. In dieser Arbeit wird ein flexibles Bildregistrierungsframework vorgestellt, dass dreidimensionale Daten mit zweidimensionalen Aufnahmen registriert. Dazu wurde die mathematische Beschreibung der interdimensionalen Registrierung analysiert, die Komponenten identifiziert und deren Einfluss auf die Registrierungsperformanz evaluiert. Als Ergebnis der Laufzeitanalyse ist in das Framework ein hardware-beschleunigter Projektionsalgorithmus mit minimalen Datentransfer integriert. Für spezifische Anwendungen können sowohl die Komponenten, Transformationstyp, metrische Funktion und Optimierungsalgorithmus, gewählt werden, als auch die Anzahl der Objekte, die mit den Referenzaufnahmen registriert werden. Für die Instantiierung und Initialisierung des Frameworks werden weniger als eine Millisekunde benötigt, so dass für neue Aufnahmen das Framework rekonfiguriert werden kann. Es ist vollständig in die weitverbreitete open-source-Software Insight Segmentation and Registration Toolkit für die Bildverarbeitung integrierbar und funktioniert mit verschiedenen Kombinationen von Metriken und Optimierungsfunktionen. Stellvertretend wurde die erfolgreiche 2D-3D-Registrierung mit der Mutual-Information-Metrik und der Powell-Optimierungsfunktion gezeigt.
Since the 1980s, the human immunodeficiency virus 1 (HIV-1) has been acknowledged as the trigger for AIDS, the acquired immunodeficiency syndrome. Every year, worldwide approximately 700,000 people die from late effects of HIV-1 infection and AIDS (UNAIDS, 2018). Thus, continuous research is important to better understand the interaction of the virus with the human host and to develop a cure. Host cell proteins that promote or fight infection are referred to as co- and restriction factors, respectively. Innate immunity restriction factors are, for example, TRIM5 or tetherin and some of them are induced by interferons (IFNs). Cyclophilin A (CypA), a small protein that influences the folding and thus the function of several cellular proteins, is a co-factor for HIV-1 infection. According to current knowledge, CypA shields cellular HIV-1 capsid cores form restriction factors after viral cell entry and thus ensures the safe transport of the virus genome into the cell nucleus. There, the HIV-1 genome can integrate into the host genome. In addition to CypA, the protein family of cyclophilins (Cyps) contains at least 16 other proteins in humans, all of which have similar cyclophilin domain structures, but have diverse cellular functions. While the role of CypA during HIV-1 infection is reasonably well characterized, almost no information is available for the other Cyps. This work investigated the influence of Cyps on early HIV-1 infection events in connection with the antiviral effects of type I IFNs. In general, some type I IFN-stimulated cells exert a significantly reduced HIV-1 infection. Interestingly, this early block to infection is amplified in CypA deficient cells. This indicates a role of CypA in the immune defense against HIV-1. Furthermore, an increase in infection after treatment with a cyclophilin inhibitor, Cyclosporin A (CsA) was observed. This can also be observed in the absence of CypA, the supposedly main target of CsA inhibition. This suggested the presence of CsA-sensitive factors that affect HIV-1 infection in type I IFN treated cells. Since both, CsA and type I IFNs have been proposed and tested as possible therapy strategies, however with little success, this observation warrants further investigation to reveal the underlying mechanisms, which could lead towards an adapted therapeutic strategy. The most obvious candidate targets are other members of the cyclophilin family. Therefore, CypB, CypC, CypD, CypE and CypH deficient THP-1 cells were generated using CRISPR/Cas9, and the effect of type I IFN treatment and CsA stimulation on HIV-1 infection was examined. While knockout of CypB, CypC and CypD modulated infection but showed no effect in response to IFN or CsA, CypE and CypH knockout cells showed a significantly increased sensitivity of HIV-1 infection to type I IFN-induced blocks. Due to these different phenotypes, the interplay of several Cyps was examined by generating double knockout cell lines. Depletion of CypB alone had no effect on HIV-1 infection, but a significantly increased sensitivity to type I IFN-induced post entry blocks was observed in the absence of both, CypA and CypB. The same could be observed for the double knockout of CypA and CypE. This suggests that Cyp functions on early HIV-1 infection events are complex and that some functions may depend on other members of this protein family. Furthermore, the results of this study show that CypA is not the only member of this family that has a function during early HIV-1 infection. The results from this study suggest that knockout of single cyclophilin genes was insufficient to render THP-1 cells insensitive to the CsA-induced increase in HIV-1 infection in type I IFN-induced cells, i.e. the phenotype could not be explained by one the candidates tested. To conduct a more unbiased approach, a mass spectrometry screen based on thermal protein stability was carried out covering the entire cellular proteome. In addition to known CsA targets, several new factors could be identified for which protein stability was sometimes dramatically altered upon CsA treatment of cells, indicating possible functional sensitivity to CsA. Some of these were IFN-induced proteins, such as members of the Retinoic Acid inducible gene I (RIG-I) signaling pathway. RIG-I recognizes viral RNA and induces an antiviral signaling cascade within the cell, which among other things leads to type I IFN production. Since this signaling pathway has been already associated with HIV-1 infection in the literature, some members of this signaling pathway were examined in more detail regarding HIV-1 infection and sensitivity to type I IFN and CsA. Knockout of RIG-I, MDA5, MAVS, TRADD or IRF3 in CypA deficient THP-1 cells showed increased sensitivity to type I IFN-induced early infection blocks. In addition, an increased infection in IRF3 knockout cells was observed, which indicates a function of IRF3 in the restriction of HIV-1. It was also seen that the CsA-induced effects in CypA knockout cells were no longer observable when either RIG-I, MDA5, MAVS or IRF3 were knocked out on top. While a mechanism of action of CsA on the RIG-I signaling pathway could unfortunately not be identified due to time limitations, the generated cell lines in this study are excellent tools for future studies that will aim to reveal mechanistic insights. The complex interplay between HIV-1 co-factors and type I IFN-induced cellular restriction factors in early infection events may yet again underline how perfectly well HIV-1 has adapted to exploit cellular pathways.
Airways muco-obstruction and irreversible neutrophil-driven inflammation cause bronchiectasis in lung diseases such as cystic fibrosis (CF) and chronic obstructive pulmonary diseases (COPD). To enter the airway lumen, neutrophils secrete their proteases, namely cathepsin G (CG), neutrophil elastase (NE), proteinase 3 (PR3) and neutrophil serine protease 4 (NSP4). The released neutrophil serine proteases (NSPs) contribute directly and indirectly to the innate immunity. Released NSPs’ action is usually counteracted by endogenous antiproteases. However, the delicate balance between these two components is broken in chronic inflammation. Strikingly, NSPs greedily associate to the surface of the secreting neutrophil, to the myriad of extracellular vesicles filling the airway fluid and to the tangled DNA webs made of neutrophil extracellular traps (NETs). When fastened to such structures, NSPs seem to be less accessible to antiproteases and their persisting activity damages the connective tissue. As a result, more proinflammatory stimuli are released and the outcome is a vicious cycle leading to non-resolving airway neutrophilia. In order to expand our palette of fluorescent tools and to propose an alternative drug target and inflammatory biomarker, we developed of a new series of Förster resonance energy transfer (FRET)-based reporters, which revealed high cathepsin G activity in CF and COPD airways. Also, we were inspired by the demand of novel advanced diagnostic technologies to examine sputum samples in a hospital environment. Therefore, we established a new assay based on the combination of spatially localized FRET probes and flow cytometry. This combination was shown to be a valuable diagnostic technique applicable in a basic and translational biomedical context. The simplicity and throughput of the new method opened the doors to two novel biomedically relevant projects. First, to identify new inflammatory markers, we investigated the discriminants and common traits of inflammation in CF and COPD airways. We carried out a comprehensive characterization of sputum samples via analysis of protease activities, cytokines and antiprotease levels. We found that COPD airways appear to be characterized by less severe inflammation featuring elevated but not uttermost marker levels, compared to CF airways. As a key marker, high membrane-bound protease activity was the most significant indicator for COPD, suggesting this trait as a highly relevant early-inflammation biomarker. Second, we wondered if in addition to the neutrophil surface, CF- and COPD-derived exosomes carry active NE and how to measure such activity at a single nanoparticle level. Therefore, we adapted our cytometric assay to monitor protease activity on human sputum particles as small as 100 nm in diameter. We showed that CF exosomes acquired NE at their surface in the inflamed airways and exported it to surrounding cells. Finally, we synthesized small-molecule probes designed to attach to DNA with the help of a DNA minor groove binder (Hoechst). The respective reporters were able to detect and quantify NE and CG activity on NETs, making them valuable tools to study the eclectic effect these enzymes have when embedded in DNA webs. Our reporters revealed that DNA-bound NE retained its catalytical activity. When applied to 5 μm mouse lung slices, the probe allowed to both distinguish single cell nuclei and to quantify cell-specific NE activity within the section. In conclusion, the activity of enzymes like CG and NE can now be studied with unprecedented spatial resolution. Furthermore, this work brings a flow cytometric assay into biomedical research which, in combination with an expanding palette of FRET-based tools, bears the potential to allow for rapid and detailed diagnosis and treatment evaluation for lung disease patients, ideally at the early stage of the disease.
Commissural neurons have their cell body on one side of the body and project their axon across the midline to the other side of the body. The midline is a structure that is specific to Bilaterian animals and is formed when the blastopore closes. Whether the blastopore was closed in Urbilateria, the last common ancestor of Bilaterians, is an open question. Since commissural neurons require the blastopore to close, finding that homologous neurons are commissural across Bilateria would suggest that the blastopore was closed in Urbilateria. On the other hand, finding that commissural neurons have acquired the ability to cross the midline convergently would suggest that the blastopore closed independently in the Protosome and Deuterostome lineages. Transcription factors play a pivotal role in specifying neuronal cell fate. The analysis and comparison of transcription factor molecular signatures across Bilaterian animals thus represents a powerful approach to put forth and validate hypotheses about the homology of commissural neurons. One putatively-conserved commissural cell type is made up of ascending neurons that express the homeobox transcription factor even- skipped (eve/evx) in insects and vertebrates. To advance the molecular characterisation and comparison of the commissural neurons in Bilateria, I have studied the commissural neurons of the ventral nerve cord of the marine annelid Platynereis dumerilii. Several characteristics of this animal make it particularly well suited for evolutionary studies, including its ancient mode of development and life style. It is also a model for which powerful tools have been developed to study its morphology and gene expression, such as a serial block face scanning-electron microscopy dataset of the full 6-days-post-fertilization (dpf) larva and gene expression atlases at several developmental stages. Platynereis’ commissural neurons were identified following both a biased approach searching for molecular signatures known from insects or vertebrates, and an unbiased approach taking the axonal traces of an entire segmental complement of commissural neurons as a starting point. I thus determined that in the second segment of the 6 dpf larva, fewer than 15% of the neurons are commissural. Analysis of their specific transcription factor-expression pattern allowed for the classification of seven cell types, characterised by the expression of i) Phox2, Hox1, and Brn3, ii) Phox2, Hox1, and Isl, iii) Dbx1, Pax6, and Irx6, iv) Eve and Lhx1/5, v) Nk6, PitxB, and Maf, vi) Nk6, PitxB, and Tal, and vii) Nk6, PitxB, and Irx6. There are commissural neurons in the vertebrate neural tube with a similar transcription factor-expression pattern and developmental origin to the cell types i, ii and iv, consistent with these being conserved in Bilateria. This would support the hypothesis that the midline existed in Urbilateria. More comparative research will be needed to settle this question.
High-content microscopy led to many advances in biology and medicine. This fast emerging technology is transforming cell biology into a big data driven science. Computer vision methods are used to automate the analysis of microscopy image data. In recent years, deep learning became popular and had major success in computer vision. Most of the available methods are developed to process natural images. Compared to natural images, microscopy images pose domain specific challenges such as small training datasets, clustered objects, and class imbalance.
In this thesis, new deep learning methods for object detection and cell segmentation in microscopy images are introduced. For particle detection in fluorescence microscopy images, a deep learning method based on a domain-adapted Deconvolution Network is presented. In addition, a method for mitotic cell detection in heterogeneous histopathology images is proposed, which combines a deep residual network with Hough voting. The method is used for grading of whole-slide histology images of breast carcinoma. Moreover, a method for both particle detection and cell detection based on object centroids is introduced, which is trainable end-to-end. It comprises a novel Centroid Proposal Network, a layer for ensembling detection hypotheses over image scales and anchors, an anchor regularization scheme which favours prior anchors over regressed locations, and an improved algorithm for Non-Maximum Suppression. Furthermore, a novel loss function based on Normalized Mutual Information is proposed which can cope with strong class imbalance and is derived within a Bayesian framework.
For cell segmentation, a deep neural network with increased receptive field to capture rich semantic information is introduced. Moreover, a deep neural network which combines both paradigms of multi-scale feature aggregation of Convolutional Neural Networks and iterative refinement of Recurrent Neural Networks is proposed. To increase the robustness of the training and improve segmentation, a novel focal loss function is presented.
In addition, a framework for black-box hyperparameter optimization for biomedical image analysis pipelines is proposed. The framework has a modular architecture that separates hyperparameter sampling and hyperparameter optimization. A visualization of the loss function based on infimum projections is suggested to obtain further insights into the optimization problem. Also, a transfer learning approach is presented, which uses only one color channel for pre-training and performs fine-tuning on more color channels. Furthermore, an approach for unsupervised domain adaptation for histopathological slides is presented.
Finally, Galaxy Image Analysis is presented, a platform for web-based microscopy image analysis. Galaxy Image Analysis workflows for cell segmentation in cell cultures, particle detection in mice brain tissue, and MALDI/H&E image registration have been developed.
The proposed methods were applied to challenging synthetic as well as real microscopy image data from various microscopy modalities. It turned out that the proposed methods yield state-of-the-art or improved results. The methods were benchmarked in international image analysis challenges and used in various cooperation projects with biomedical researchers.
Glomerulosclerosis is a hallmark of diabetes (D) and hypertension (HBP) induced kidney failure. Here, we studied the combined effect of D-HBP on the kidney using expansion microscopy for nanoscale imaging of podocyte foot processes (FP). Method: In 6 weeks old male double transgenic rats (dTGRNeph-hAT1R;Cyp1a1mRen2dF1=dTGR) and TGRCyp1a1mRen2d (WT) we induced diabetes by streptozotocin (60mg/kg i.p.) for 8 weeks, and Hypertension by indole-3-carbinol (IC3) (0.0125% in chow), alone or in combination (D-HBP). After perfusion with 2%PFA, kidneys were ~4.5 times expanded using acrylamide/sodium-acrylate gel embedding, SDS/75°C+90°C denaturation, podocin immunohistochemistry and imaging by confocal microscopy after expansion in H2O. We measured FP width as the distance between two podocin signals and normalized for the expansion factor. Results: D-HBP rats developed massive albuminuria, and shown a drop in GFR which correlated with histological alterations in glomeruli, including mesangial expansion, podocyte loss and adhesion of the glomerular tuft to the Bowman’s capsule. HBP rats showed very mild histological injury. D rats did not differ from controls. AT1R overexpression in podocyte aggravated the glomerular damage, drop in renal function in HBP and D-HBP but did not affect controls. Glomerular size increased similarly in both HBP and D-HBP and did not differ between dTGR and WT. In dTGR- and WT-controls (CTRL), podocyte FP architecture was regular with long thin processes (200±20 nm width). In HBP, FPs were shortened and widened, but regularly organized. In D-HBP, FP structure within one glomerulus varied from regular with shortened and widened FP to irregular structures to regions with the total loss of FP (average FP width 400±20 nm). Conclusions: In contrast to D and HBP, combined D-HBP induced rapid progression of glomerulosclerosis and podocyte damage. Podocyte’s AT1Rs aggravate damage index. To able to visualize those alterations, we established a method, by modifying ExM, that enables the nanoscale evaluation of podocyte FPs and slit membrane in 3-D by using traditional immunohistochemistry and confocal microscopy.
Multimodal image registration benefits the diagnosis, treatment planning and the performance of image-guided procedures in the liver, since it enables the fusion of complementary information provided by pre- and intrainterventional data about tumor localization and access. Although there exist various registration methods, approaches which are specifically optimized for the registration of multimodal abdominal scans are only scarcely available. The work presented in this thesis aims to tackle this problem by focusing on the development, optimization and evaluation of registration methods specifically for the registration of multimodal liver scans. The contributions to the research field of medical image registration include the development of a registration evaluation methodology that enables the comparison and optimization of linear and non-linear registration algorithms using a point-based accuracy measure. This methodology has been used to benchmark standard registration methods as well as novel approaches that were developed within the frame of this thesis. The results of the methodology showed that the employed similarity measure used during the registration has a major impact on the registration accuracy of the method. Due to this influence, two alternative similarity metrics bearing the potential to be used on multimodal image data are proposed and evaluated. The first metric relies on the use of gradient information in form of Histograms of Oriented Gradients (HOG) whereas the second metric employs a siamese neural network to learn a similarity measure directly on the image data. The evaluation showed, that both metrics could compete with state of the art similarity measures in terms of registration accuracy. The HOG-metric offers the advantage that it does not require ground truth data to learn a similarity estimation, but instead it is applicable to various data sets with the sole requirement of distinct gradients. However, the Siamese metric is characterized by a higher robustness for large rotations than the HOG-metric. To train such a network, registered ground truth data is required which may be critical for multimodal image data. Yet, the results show that it is possible to apply models trained on registered synthetic data on real patient data. The last part of this thesis focuses on methods to learn an entire registration process using neural networks, thereby offering the advantage to replace the traditional, time-consuming iterative registration procedure. Within the frame of this thesis, the so-called VoxelMorph network which was originally proposed for monomodal, non-linear registration learning is extended for affine and multimodal registration learning tasks. This extension includes the consideration of an image mask during metric evaluation as well as loss functions for multimodal data, such as the pretrained Siamese metric and a loss relying on the comparison of deformation fields. Based on the developed registration evaluation methodology, the performance of the original network as well as the extended variants are evaluated for monomodal and multimodal registration tasks using multiple data sets. With the extended network variants, it is possible to learn an entire multimodal registration process for the correction of large image displacements. As for the Siamese metric, the results imply a general transferability of models trained with synthetic data to registration tasks including real patient data. Due to the lack of multimodal ground truth data, this transfer represents an important step towards making Deep Learning based registration procedures clinically usable.
Recent breakthroughs in sequencing technologies allowed researchers to generate extensive amounts of data characterizing cellular regulation at many levels. Consequently, this boosted our understanding of gene regulatory networks responsible for different biological processes and highlighted the overall importance of transcription factors (TFs). TFs are dynamic mediators that react to both intra- and extracellular changes in order to ultimately transmit signals and execute genetically inherited gene regulatory programs in a time- and location-specific manner. However, it is still challenging to quantify in vivo TF specific binding occupancy and dynamics due to the high complexity of the regulatory part of the genome. Modern technologies measuring chromatin changes (e.g., chromatin accessibility, DNA methylation, histone modifications) can now generate testable hypotheses about the effects of TF binding on gene regulation. In this thesis, I mainly describe the novel computational tool diffTF, a multiomics data integration tool for globally assessing differential TF activity and classifying TFs into transcriptional activators and repressors (by integrating chromatin accessibility and gene expression data). We applied it to a recently published ATAC-seq dataset from a cohort of chronic lymphocytic leukemia (CLL) patients and identified dozens of differential active TFs representing two different CLL subtypes that are inherently linked to tumour progression. In addition, we integrated gene expression data from corresponding RNA-seq and were able to globally predict an activating or repressive role for 40% of the expressed TFs. We validated the approach on an independent CLL dataset and showed that the majority of TFs does not change their mode of action upon genetic or environmental perturbations. Finally, we extensively tested and benchmarked diffTF to validate its technical robustness. We also applied diffTF to a multiomics dataset from the mouse hematopoietic differentiation system and targeted potential TFs that are disturbed upon epigenetic dysregulation driven by a Tet methylcytosine dioxygenase 2 (TET2) knockout in acute myeloid leukemia (AML). TET2 plays an essential role in the cellular DNA methylation balance and is known to be frequently mutated in leukemia. We used the first high-quality TET2 binding map to identify TF families that can facilitate TET2 binding in the genome. In summary, we developed a novel hypothesis-generation computational tool that can, in a data-driven way, identify key regulators of cellular biological processes based on chromatin and expression data.
Diffusion Tensor Imaging (DTI) is a method widely used in research and clinic, especially for imaging and connectivity analysis of the white brain matter. Despite the many possibilities offered by DTI, this method suffers from an inherently low signal-to-noise ratio (SNR), since both the long echo time and the diffusion gradients weaken the signal. The SNR is particularly low at high spatial resolution, e.g. in the DTI of nerves. A low SNR leads to systematic and statistical errors in parameters calculated from the DTI, e.g. fractional anisotropy (FA). A low SNR can be partially compensated by increasing the number of diffusion directions or using methods for a posteriori noise correction. The most robust method for anatomical structures with unknown orientation is to distribute the diffusion gradients evenly in space. However, if the preferred direction of the anatomical structure is known in advance, it may be advantageous to limit the diffusion gradients to a cone centered on the axis of the structure. The aim of this work was to develop a DTI method with high accuracy and reliability for application in peripheral nerves. Two methods to reduce image noise were investigated: (1) A newly developed scheme of diffusion gradient vectors (DGV), where the vectors are restricted to a cone with an aperture angle Theta around the axis of the nerve and (2) different methods for a posteriori noise correction. For this purpose, Monte Carlo simulations were performed based on realistic values for diffusivity, FA and noise obtained from clinical investigations and studies. Furthermore, the methods were tested in a specially designed phantom simulating diffusion in peripheral nerves (FA = 0.65). These investigations were performed on a 3 Tesla whole-body magnetic resonance (MR) scanner. To determine the accuracy and reliability of the DTI using the appropriate measurement or correction procedures, systematic deviations of FA from baseline and the statistical error of FA were measured. The newly developed DGV scheme with limited space coverage was compared with gradient schemes with uniform space coverage (Jones, Downhill Simplex Method (DSM), gradient scheme of the manufacturer) based on their condition number (CN). The study showed that with the newly developed DGV scheme FA can be measured with high accuracy when the angle Theta is at least 45° or 60°. The minimum Theta depends on the number of gradient directions and on FA. Basically, the higher the FA value and the greater the number of gradients, the better the accuracy of the DGV scheme. For N = 30, the DGV allowed an exact determination of FA for the entire FA range (0.4 - 0.8) investigated in this study, if Theta ≥45° was. It could be shown that when using the new DGV scheme, a slight inclination of the investigated structure (≤30°) does not affect the accuracy of FA. CN of the developed DGV-scheme was higher than CN of the Jones-scheme and the DSM-scheme for N = 6; for N≥10 CN of the new DSM-scheme was lower than that of the Jones-scheme. However, it is also not to be expected that a method that concentrates the gradient vectors on a limited segment of space is as insensitive to interference as schemes with uniform gradient distribution. Nevertheless, the CN of the new DGV method was in the same order of magnitude as that of the other methods. A comparison of the different a posteriori correction methods showed that the power image method is the most effective and robust method and compensates for both the systematic and statistical errors of FA. The efficiency of the power image method is independent of the number of diffusion gradients used. In addition, the method works reliably - regardless of the method used for the coil combination (square sum versus adaptive combination). In contrast, both correction factor methods used in this study were less efficient in terms of noise correction; furthermore, the correction efficiency depended on the coil combination method. In conclusion, a combination of the newly developed DGV scheme with the power image method for a posteriori correction allows DTI of peripheral nerves with high SNR, high accuracy and reliability of the calculated parameters (e.g. FA) without the need for additional acquisition time. So far, however, these newly developed and tested methods have not yet been applied in studies or clinical trials.
CD8+ T cells play a crucial role in fighting infectious diseases and cancers, but it is frequently observed that CD8+ T cells are functionally compromised or “exhausted” in chronic infections and solid tumors. Exhausted T cells are characterized by the expression of immune checkpoint molecules, reduction in effector cytokine secretion, and alterations in cellular metabolism, such as loss of mitochondrial membrane potential (ΔΨm). Immune checkpoint blockade-based immunotherapies revive exhausted T cells, enhance effector cytokine production, and have achieved clinical success in several types of cancers. It is unknown whether and how ΔΨm regulates T cell exhaustion in chronic infection and cancer. Mitochondrial complexes accept electrons from tricarboxylic acid (TCA) cycle-derived electron carriers, transfer electrons, and pump protons to establish a membrane potential. The malate aspartate shuttle (MAS) produces and transports electron carriers into the mitochondrial matrix and donates electrons to the electron transport chain (ETC). We, therefore, hypothesize that MAS may regulate ΔΨm and antagonize T cell exhaustion. To test this hypothesis, we have generated a mouse model with T cell-specific deficiency of glutamic oxaloacetic transaminase 1 (Got1, encoding the MAS enzyme GOT1). We chose GOT1 because it is a key enzyme of the MAS, and we observed that GOT1 expression was induced by persistent antigenic stimulation in mouse CD8+ T cells and upregulated in CD8+ tumor-infiltrating lymphocytes (TILs) from human colon cancer. T cell-specific ablation of Got1 impaired tumor-specific effector CD8+ T cell accumulation, effector cytokine production and potential to sustain long term immune responses in a mouse melanoma model. Using the lymphocytic choriomeningitis virus (LCMV) Armstrong acute infection and the LCMV clone 13 chronic infection model, we found that Got1 deficiency affected the ongoing antiviral effector responses, but not the memory T cell formation after viral clearance, suggesting that CD8+ responses required GOT1 in the presence of persistent, but not transient antigenic stimulation. Further analysis revealed that Got1 deficiency alone did not affect CD8+ T cell metabolism or ΔΨm when cells were cultured in complete medium. Instead, Got1 deficiency became only catastrophic for CD8+ T cells when extracellular nutrients were restricted, and antigenic stimulation was persistent. Got1 deficiency reduced the NAD+/NADH ratio, increased reactive oxygen species (ROS) production and decreased ΔΨm. This study reveals, GOT1 selectively supports effector CD8+ T cells that are nutrient restricted and chronically stimulated. Summarized, the presented work revealed that persistent stimulation with tumor and viral antigens induced GOT1 expression on CD8+ T cells. GOT1 was shown to desensitize CD8+ T cells to environmental nutrient limitation and persistent antigenic stimulation-induced loss of ΔΨm and T cell exhaustion. These results present an example of how immune cells alter their metabolic pathways to adapt to nutrient and antigen availability.
In this thesis, mid-infrared (MIR) pulses with arbitrary temporal and spectral shape are generated via a difference-frequency process for application in a non-linear Raman microscope. Solely by shaping the sub 10 fs driving pulses, the broadband spectra of the MIR pulses are switched to narrowband and tuneable ones. In MIR transmission spectroscopy, these narrowband MIR spectra allow for investigating molecular vibrations from 1250 to 3250 cm-1 with spectral resolutions below 20 cm-1. Furthermore, MIR transmission microspectroscopy is combined with coherent-anti-Stokes Raman scattering (CARS) to provide a direct comparison of spectra and images obtained in one spot of the sample. Sum-frequency (SF) microspectroscopy is an additional technique, which complements the toolbox of this non-linear Raman microscope with the potential to investigate non-centrosymmetric systems. The flexibility of the pulse shaper allows for implementing two different SF-methods. Whereas the heterodyne multiplex method acquires the whole SF spectrum by imprinting only three different phase functions, the homodyne MIR-scanning method generates a high SF intensity directly linked to one vibrational mode. In all applications, the phase of MIR pulses must be well-known. This phase is determined in the focal plane of the microscope over more than 1000 cm-1 via two methods based on the dispersion-scan.
This thesis describes the development of a high-resolution soft X-ray detector based on metallic magnetic calorimeters (MMCs). MMCs are cryogenic, energy dispersive particle detectors which consist of a particle absorber that is thermally coupled to a paramagnetic temperature sensor. The latter is placed in a weak magnetic field, hence exhibiting a temperature dependent magnetization M(T). Upon X-ray photon absorption, the rise of detector temperature causes a change of sensor magnetization, which is usually read out with a current-sensing dc-SQUID via a superconducting flux transformer. Here, an imperfect transformer matching, as well as a transformer intrinsic energy coupling losses, limit the achievable energy resolution. To challenge this limit, a novel integrated detector was developed, in which the temperature sensor is integrated into a custom-designed dc-SQUID to maximize signal coupling. A major challenge of this configuration is the Joule heating of the SQUID, since heating effects prevent cooling of the detector and thus limit its performance. For this reason, the developed 32 pixel detector makes use of a newly developed thermalization scheme for the SQUID’s shunt resistors, resulting in operation temperatures below 20 mK for the detector. With this kind of detector, a baseline energy resolution of dE = 1.3 eV, and dE = 1.8 eV at 5.9 keV was achieved.
Evolution and adaptation through natural selection are cornerstone concepts of Biological sciences. The recent advances in the fields of Microbiology and Molecular Biology allowed scientists to introduce evolution in controlled laboratory settings. Adaptive laboratory evolution (ALE) has been successfully applied to better understand the effect of natural selection on individuals, as well as to obtain cells with improved phenotypic characteristics. In the majority of the reported cases, the characteristics that are targeted for improvement are related to biotechnological processes, aiming to create improved microbial strains for industrial applications. However, ALE is limited to growth-associated traits, such as substrate utilization and increased tolerance of compounds that inhibit growth. The aim of this PhD thesis was to develop novel methodologies that could overcome the major bottleneck of ALE to enable the improvement of non-growth associated traits for non-genetically modified organism (GMO) biotechnological applications. In the first approach, small synthetic obligatory mutualistic communities were established. The design of a metabolic cross-feeding relationship between the species in the community couples the production of a target metabolite to the survival and proliferation of the community. Increased concentration of the target metabolite in the environment results in improved community fitness, despite of any potential production cost. Communities consisting of natural vitamin secreting lactic acid bacteria and engineered Saccharomyces cerevisiae were successfully evolved for the improved production of two different B group vitamins (riboflavin and folate). The isolated evolved overproducing bacterial strains can be used for the production of food with increased nutritional value. The second approach described in this PhD thesis is a novel algorithm that uses genome-scale metabolic model simulations to identify the environmental conditions that will create selection pressure for the pathways involved in the production of a target compound. The computed chemical composition will be used as the environment (evolution niche) for ALE with straightforward growth selection. The resulting adapted metabolic network is expected to manifest the enhanced compound production when cells are switched back to their natural environment. As a proof-of-concept, we successfully applied this approach for the increased production of aroma compounds originating from the branched-chain or the aromatic amino acid pathways in wine yeast strains. Together, the results of this thesis demonstrate that the developed methods can increase the precision of laboratory evolution and allow the selective production of fitness-costly metabolites. The phenotypic characteristics of both prokaryotes and eukaryotes could be improved, and the obtained strains hold potential for biotechnological applications, especially when the use of genetically engineered strains is restricted. Apart from the potential biotechnological applications, the designed laboratory evolution strategies can also be exploited to shed light on open questions about the physiology, the ecology and the social life of microbial species and communities.
It has long been assumed that the world's oceans are homogeneous in δ234U, even on a sub-‰ scale, however this has not been comprehensively investigated outside of the North Pacific using modern high-precision MC-ICP-MS. In this study, the δ234U of water samples from across the Mediterranean Sea, Amazon Estuary, and North Atlantic is presented to show that the oceanic uranium system is much more variable and dynamic than previously believed and variable on a ‰-scale. Mediterranean water masses are elevated in δ234U compared to the Atlantic by ~1-2‰, allowing for the estimation of the δ234U of riverine and groundwater inputs to the basin. Analysis within the Amazon Estuary shows that there are significant geographical differences in the non-conservative behavior of U, and that the Amazon has little to no effect on the δ234U of the nearby Atlantic. The upper Atlantic is on average 1‰ lower in δ234U than the deep Atlantic, indicating that oceanic δ234U is not in steady-state but rather decreasing. Results point towards the offset seen in the upper Atlantic possibly being the result of inputs from the Indian Ocean (which has yet to be investigated at such high-precision), indicating that such oceanic δ234U variability may be a widespread phenomena.
Scalar fields play an important role in cosmology. They can be responsible for cosmic inflation in the very early universe, as well as are among well-motivated dark matter (DM) candidates. The aim of this dissertation is to contribute to a better understanding of the dynamics of such fields in the nonperturbative regime.
Motivated by cosmological scenarios, we consider coherent oscillations of a scalar field in potentials that are summed from periodic and monomial terms, which have recently attracted much attention in the context of axion-like particles (ALP). We investigate the resonant amplification of quantum fluctuations, as well as the subsequent nonlinear dynamics after the fragmentation of the field.
Our studies are extended to the nonthermal production of ALP DM. It is found that the process of fragmentation imprints strong overdensities of DM on small scales, as well as can produce a stochastic gravitational wave background, potentially within reach of future detectors.
Finally, we investigate the role of experiments with ultracold atoms for the quantum simulation of nonperturbative dynamics and describe how, by means of a modulation of the interatomic interaction strength, such Bose gases can go through the characteristic stages of the dynamics of relativistic systems in the early universe.
Due to their ability to record climate change over large periods of time, stalagmites remain a primary focus of paleoclimatology. Utilizing high-precision age determination methods such as U-series dating, it is possible to measure climate-induced variations of the geochemical composition of speleothems over the time of their growth periods. This dissertation focuses on the investigation of radiocarbon (14C) concentration in several stalagmites. To this end, a new setup for the chemical preparation of carbonate samples for 14C measurements was planned and successfully put into operation. The incorporation of 14C into stalagmites is dependent on various climate-related processes in the soil and in the karst host rock above the cave. Using stable isotope ratios and trace element concentrations, this was investigated in two case studies with high-resolution 14C measurements. Reduced 14C concentration was observed in a stalagmite originating from Moomi Cave on Socotra Island which is indicative of aged soil organic matter influencing the stalagmite formation. It was demonstrated that a combination of higher soil humidity and denser vegetation towards the end of the last glacial period caused higher stalagmite 14C concentration. In a second study on a stalagmite from Sofular Cave in Turkey, the 14C signature allowed for the observation of various aspects of soil carbon dynamics from the last glacial period through into the Holocene. Moreover, the record suggests an increase of atmospheric 14C concentration coincident with the geomagnetic minimum approximately 41000 years ago. This may contribute to the ongoing effort to improve radiocarbon calibration datasets, on which the 14C dating method is based. Lastly, a summary of various stalagmite studies conducted at the Institute of Environmental Physics and a review of the relevant processes of stalagmite 14C incorporation is presented.
Every living organism, including viruses, has to undergo reproduction in order to successfully establish a niche on this planet. One of the vital steps in the reproduction process is replication of the genetic material and its subsequent segregation between the two daughter cells. Errors in these processes may be fatal to the organism causing it to disappear from this planet.
Eukaryotic cells have evolved an energy-consuming dedicated machinery to properly segregate the replicated genetic material between the two daughter cells. Unlike eukaryotes, prokaryotes adopt different mechanisms for chromosome segregation, which are in some cases still poorly understood. Moreover, in prokaryotes, chromosome replication and segregation happen simultaneously. While a dedicated chromosome segregation machinery has been identified in Caulobacter crescentus, such machinery has not been identified in many other prokaryotes. There is an ongoing debate in the prokaryotic field whether mere entropic forces of repulsion between the duplicated chromosomes alone can achieve full and precise chromosome segregation, or whether additional machineries are needed.
A previous study from our group has shown that Escherichia coli MinD binds to DNA in a non-sequence specific manner. Based on computational, in vitro and in vivo analyses, it was hypothesized that such binding would be used by E. coli cells to properly segregate their chromosomes. Residues on MinD that, either directly or indirectly, affect DNA binding have been identified; however, the direct MinD-DNA binding interface is so far unknown.
E. coli MinD, together with MinC and MinE, constitutes the so-called Min system, which has been extensively studied for its role in mid-cell determination. MinC actively counteracts FtsZ polymerization. It would do this anywhere in the cell, if it were not for MinD and MinE, which regulate MinC localization so that it is minimal at mid-cell, where the FtsZ ring can be assembled. The way in which MinD and MinE keep MinC away from mid-cell is very dynamic, and consists in pole-to pole oscillations that never cease. These oscillations are self-organized and occur also in the absence of MinC, as far as MinD, MinE and the membrane are present. Since MinC forms a complex with MinD, it effectively gets carried along in the oscillations, spending on average more time at the poles and being at low concentration at the centre. Without MinC, the Min oscillations do not exert any activity towards FtsZ. Thus, the three Min proteins ought to work together to achieve their goal of mid-cell determination. It is plausible that a concerted action of all Min proteins is required also for proper chromosome segregation, since we discovered that MinC strongly enhances the DNA-binding activity of MinD, while MinE terminates the binding by releasing MinC and the DNA off MinD (1).
In my thesis, I aimed to identify the residues of MinC that either directly or indirectly aid MinD in DNA binding. To this end, potential MinC residues that could bind to the DNA were first computationally predicted and then mutated to experimentally test the consequences of such mutations on the DNA binding activity of MinC and MinD. Using electrophoretic mobility shift assay (EMSA) experiments, I found that glycine at position 10 and lysine at position 66 on MinC are involved in DNA binding since the MinCG10D and MinCK66A mutants showed strongly reduced binding. By performing circular dichroism experiments, I could exclude that the impairment of MinCG10D-MinD in DNA binding is due to changes in the secondary structure of the mutant protein, suggesting that the DNA is repelled by the negative charge of the aspartic acid. Most importantly, I discovered that not only the length, but also the amino acid sequence of the unstructured linker region of MinC, which connects the N- and the C-terminal domains of the protein, play a vital role in the DNA-binding activity of MinCD. Since MinC is also involved in inhibiting FtsZ polymerization, by performing cell viability spot assays I found that the linker should consist of at least two amino acids in order to efficiently inhibit FtsZ polymerization. EMSA assays with various synthetic constructs I made to test the necessity of different elements (MinC N- and C-terminal domain, linker region, MinD) for DNA binding revealed that the linker region of MinC is necessary for DNA binding. Further experiments are needed to understand if MinC N- and C-terminal domains and MinD are needed solely to place the linker in the proper orientation for it to bind to the DNA or if they contribute directly to the binding with specific residues. Interestingly, microscopy experiments performed using a synthetic construct made of the N-terminal MinC domain, the linker, a bZIP dimerizing domain and mRuby showed that this construct co-localizes with the E. coli nucleoid. Introduction of the G10D mutation on the synthetic construct did not alter its in vivo association with the nucleoid, suggesting that G10 is not used to directly bind to the DNA.
Beyond studying the DNA binding activity of MinC and MinD, during my Ph.D. I analyzed the mechanism by which MinE is impaired when eYFP is C-terminally fused to it. By combining in vivo and in vitro assays, I show that eYFP makes the fusion protein prone to aggregation, and reduces the accessibility of MinE MTS as well as of arginine at position 21, needed to activate the ATPase activity of MinD.
Finally, as to study biological processes it is often necessary to co-transform two plasmids in the cells of interest, I wanted to devise a method to reduce the requirement from two to one antibiotic to maintain two plasmids. To this aim, I employed split inteins to reconstitute full-length, functional enzymes conferring resistance towards antibiotics, which are expressed as two dysfunctional halves each on one plasmid. This method, which we called SiMPl, allows maintaining two plasmids in bacteria and mammalian cells using a single antibiotic chosen between kanamycin, chloramphenicol, ampicillin, hygromycin and puromycin.
Unveiling the mystery of dark matter is one of the most sought-after goals of modern physics. Working towards this aim, this thesis explores new links between the possibility that dark matter is made up of light scalar particles and other open questions in particle physics and cosmology. In the first part of the thesis, scenarios in which the dark matter contains a particle-antiparticle asymmetry are considered. As an example, the production of such an asymmetry in a model of scalar dark matter is studied. Furthermore, a setup is presented where baryogenesis and asymmetric dark matter have a common origin linked to the dynamics of neutral B mesons. In the second part, the emphasis is put on axions. After assuring the cosmological stability of general axion-like particles, a non-canonical kinetic term is introduced, which leads to an enhanced production of this kind of dark matter. Finally, the collider phenomenology of axions and their coupling to electroweak gauge bosons is scrutinized. The third and final part is devoted to the production of light bosons during inflation. A non-minimal coupling to gravity leads to rich inflationary dynamics and precise predictions regarding the properties of the sub-galactic distribution of dark matter.
Protein O-mannosylation is an essential, conserved and abundant post-translational modification in the eukaryotic secretory pathway. Protein O-mannosylation is initiated at the endoplasmic reticulum (ER) by the covalent addition of single mannoses to serine and threonine residues of target polypeptides. This reaction is catalyzed by members of the evolutionarily conserved protein mannosyltransferase family (PMT). In the model organism Saccaromyces Cerevisiae, among its different biological roles, protein O-mannosylation has been described to target both unfolded and irreversibly misfolded proteins. The recently named unfolded protein O-mannosylation (UPOM) pathway is hypothesized to function as a fail-safe mechanism to prevent ineffective folding attempts for polypeptides that did not achieve the native conformation within certain time window. However, although UPOM has been described to target multiple misfolded model proteins, the mechanism by which the PMT machinery discriminates misfolded polypeptides is unknown. In fact, the question of whether UPOM is a physiologically relevant mechanism contributing to maintain homeostasis in the ER remains unanswered. In this current work, it is shown: I) The relevance of the Pmt1-Pmt2 complex as necessary to maintain ER homeostasis being integrated in the unfolded protein response (UPR). II) Aiming to unravel the UPOM machinery, the genes PGI1 (phosphoglucose isomerase) and BFR1 (Brefeldin A resistance) were found as necessary for efficient UPOM. On one hand, the characterization of PGI1 highlighted a direct link between O-mannosylation, cytosolic sugar metabolism, and ER stress. On the other hand, the RNA binding protein (RBP) Bfr1 was found to modulate the translational state of PMT mRNAs among many other ORFs, mainly coding for proteins localizing to the secretory pathway, suggesting a role as a local translational control factor. III) Aiming to address the physiological relevance of UPOM in the ER protein quality control system, the impact of O-mannosylation on the stability of a subset of PMT target proteins was investigated. Both, protein stabilization and de-stabilization effects were presented. The prevalence of either effect was largely dependent on the nature of the O mannosylation substrate. In line with this, Pmt1 is shown to be necessary for the degradation of the beta-1,6 synthase Kre6 when mislocalized in the vacuole.
During post-embryonic development, plants rely on the integrity of phloem within their root systems. The phloem is part of the vasculature and transports energy metabolites from leaves into mitotically active regions such as the root apical meristem (RAM). Loss of function of genes regulating phloem development can result in severe changes in root growth and plant body architecture. The redundantly active genes SUPPRESSOR OF MAX2 1-LIKE3 (SMXL3), SMXL4 and SMXL5 are central regulators of early phloem formation. However, molecular mechanisms underlying SMXL3/4/5 gene activities during early phloem development are mostly unknown. The functional relevance of SMXL3/4/5 protein domains including the EAR motif is also unclear. The aim of my dissertation was to characterise the mode of action of SMXL3/4/5 during early events of phloem development in detail using Arabidopsis thaliana roots as model organ to investigate spatiotemporal tissue formation.
First, I investigated at which developmental steps SMXL3/4/5 genes are required to promote phloem development in the RAM, how they interact genetically with positive regulators (OPS, BRX) and how their function is affected by negative regulators (CLE26, CLE45). I found that SMXL4/5 function is required to initiate and promote the activities other of of genes regulating phloem development (OPS, BRX, BAM3, CVP2 and APL), and that SMXL4/5 protein functions are possibly required to attenuate CLE-mediated suppression of phloem differentiation. Furthermore, I examined whether the highly conserved EAR motif of SMXL5 is functionally relevant to promote early phloem development. Here, I tested whether protein accumulation was altered for EAR motif-mutated SMXL5 proteins (SMXL5mEAR) in planta, and if phloem formation could be restored in smxl4;smxl5 double mutants complemented with SMXL5mEAR proteins. My data suggest that SMXL5 protein function is independent from the EAR motif indicating that SMXL5 proteins do not act as canonical EAR repressors in the context of phloem development. Last, I aimed at identifying new genes that are functionally related to SMXL3/4/5 during early phloem development. Therefore, I performed an ethyl methanesulfonate (EMS)-based mutagenesis of smxl4;smxl5 double mutants to screen for genetic suppressors that alleviate the phloem defects characteristic for smxl4;smxl5 mutants. I found that mutagenesis of yet unknown suppressor genes in the smxl4;smxl5 background could indeed restore phloem development. Further analysis including genome mapping is required to identify candidate genes that result in the suppression of the smxl4;smxl5 mutant phenotype.
In conclusion, I postulate that SMXL3, SMXL4 and SMXL5 genes are required to establish the post-embryonic phloem lineage and regulate the phloem-specific developmental program in the RAM. Together, a complex, tightly balanced network of molecular players depending on SMXL3/4/5 activities ensures the formation of phloem within the root system.
TRIM25 is an E3 ligase of the tripartite motif protein family, that is best known for its function in innate immunity, where it activates the pattern recognition receptor RIG-I. More recently, it was identified as a putative RNA binding protein, though lacking domains with known RNA-binding potential. In this thesis, I present evidence that RNA binding is mediated by the coiled-coil (CC) and PRY/SPRY domain with possible contributions of the disordered linker connecting the domains. Using NMR spectroscopy and mutational analysis, I could map the RNA binding site on these domains. Small-angle X-ray scattering indicates that RNA-binding stabilizes an inherent, but weak interaction between these domains leading to a more rigid domain architecture possibly explaining the increase in ubiquitination activity in the presence of RNA observed by us and others. In line with that, mutants affecting RNA binding or the weak CC:PRY/SPRY interaction also reduced ubiquitination of the RIG-I caspase-activation and recruitment domains (CARDs). RNA binding in addition promotes phase-separation and association with RIG-I, as our results indicate that there is no direct protein-protein interaction between TRIM25 and RIG-I. This reconciles seemingly controversial results in recent studies and contributes to further unravel the mechanism behind the immune response activation upon viral infection.
Hepatitis C virus (HCV) is a blood-borne, enveloped, single-stranded, (+)-oriented RNA virus that mainly infects hepatocytes. Most infections progress into chronicity and eventually lead to severe liver disease. Although effective treatments have been developed, access to diagnosis and treatment is low, particularly in non-developed countries. Thus, eradication of the disease is unlikely without a prophylactic vaccine. Research, therefore, has to continue despite the high cure rates of today’s HCV regimens. We use mathematical modeling to study HCV replication and its intricate connection with the infected host cell. A model that is able to simulate intracellular HCV RNA replication suggested a host factor species (HF), representing a protein (complex) or a host process, to be critically involved in HCV replication. Gene expression profiling revealed several candidates potentially representing this HF. We validated those candidates in two variants of the human hepatoma cell line Huh7 and could confirm that five of them indeed played a role for HCV replication, namely CRAMP1, LBHD1, CRYM, THAP7, and NR0B2. The latter three are nuclear receptors or transcriptional (co )repressors, suggesting they could influence HCV replication indirectly, e.g. through glucose, lipid, or cholesterol metabolism. Follow-up studies will help to understand the implication of those factors in HCV replication and reveal important insights into the metabolic pathways regulating HCV replication. Model analyses also revealed the most sensitive steps in HCV RNA replication that could potentially be targeted by specific intervention. The standard of care for chronic HCV infection has been interferon alpha (IFN α) therapy that elicited a very broad but rather unspecific antiviral response of the host cell and came along with severe side effects. IFN-α activates signaling cascades that lead to the expression of hundreds of interferon stimulated genes that exert antiviral action. Despite its decades-long use, the exact mechanism of the suppression of HCV replication by IFN α treatment remains elusive. We thus combined experimental data with an intracellular model for HCV replication and revealed the steps in the viral replication cycle that are most probably affected by IFN α treatment. The obtained findings were well in line with in vitro data and confirmed the validity of our intracellular model to make such analyses. Recently, direct-acting antivirals (DAAs) have replaced IFN-α-containing regimens as the standard of care for chronic HCV infection. Those DAAs possess much less side effects, can be taken orally, and give extraordinarily high cure rates. Mainly three classes exist: inhibitors of the viral protease, the viral polymerase, and a viral multifunctional phosphoprotein. The latter class constitutes highly potent inhibitors of the HCV NS5A protein, exerting effects in the low picomolar range. However, due to the many roles of NS5A in the HCV life cycle, the exact mechanism of action of those DAAs remains unclear. For the other two classes, the mode of action is distinct and well defined. We, thus, used one representative member of each of these classes to validate the capacity of our model to implement drug effects and predict HCV replication correctly. Model predictions upon a priori fixing of the affected parameters in the model qualitatively resembled HCV replication dynamics under the respective drug treatment. This allowed us to apply our model to HCV replication data under treatment with an NS5A inhibitor in order to gain insights into its mode of action. The model revealed that the translation rate of HCV RNA as well as RNA synthesis steps in the HCV replication compartment are most probably affected by the drug. These findings were reasonable and supported by known roles of NS5A in the HCV life cycle. However, our model was limited to intracellular HCV replication and did not account for steps like particle assembly or infection of target cells. Therefore, we extended our intracellular model to cover the full viral life cycle. Our new full life cycle model could simulate viral (+)- and (-)-strand RNA, viral titers as well as spread of the infection, and was able to correctly predict HCV replication under drug treatment. Our new model will be helpful in further elucidating the mode of action of NS5A inhibitors and IFN α and in deciphering the role of host factors that determine permissiveness for HCV. Hence, this study provides a novel, extended mathematical model of the full HCV life cycle with the proven capacity of simulating and analyzing HCV replication even under pharmacological intervention. It can serve as an invaluable tool to study further molecular details of HCV replication and to devise and test novel therapeutic approaches.
The topic of this work is the synthesis and properties of silanes with amino substituents and their corresponding silylium ions. In the first part of this thesis, the first aminosilylium ion was synthesized by hydride abstraction of (Me2N)3SiH. The dictionic ((Me2N)3Si+)2 could be fully characterized in solution and in the solid state. The compound can be described as the dimeric form of the silaguanidinium. Different factors that influence the dimerization process could be determined by suitable theoretical methods. In comparison to silylium ions with aliphatic substituents, the aminosilylium ion shows higher stability and compatibility with common organic solvents. Nevertheless, the dication is able to perform electrophilic, aromatic Friedel-Crafts silylation reactions with electron-rich, aromatic compounds or to initiate hydrodefluorination reactions. In the presence of acetonitrile, an insertion of the nitrile in one of the Si-N-bonds was achieved and the structure was characterized in the solid state. The synthesis of more sterically demanding, monomeric aminosilylium ions and the challenges around this are described in detail. In the second part of this thesis, the influence of aromatic triamido (NNN3−) and amidodiphenolato (ONO3−) substituents of the resulting silanes was investigated. The enforced geometrical strain resulted in a higher levels of Lewis acidity and thus dimerization of the silicon compounds. By installing substituents with different sterical and electronical properties around silicon, it was possible to influence the dimerization process on purpose. The species could be grouped in monomeric, structural-reversible and dimeric compounds. The high Lewis acidity resulting from the pre-organisation of the substituent was evident from the deviation from tetrahedral geometry and the planarization of the compounds in the solid state structures. Different factors which contribute to the dimerization process were determined by theoretical methods and their influence was explained in detail. For effective dimerization, the dispersion interactions of the substituents, low deformation energy at silicon, electrostatic attraction between silicon and nitrogen and a special σ-bond resonance phenomenon were identified as the main factors. The obtained results suggest unique stabilising effects and can be applied for more general conclusions about aggregation of amphiphilic species and Lewis acidity at silicon. The reactivity of the compounds towards different substrates was investigated with help of a model compound. In the third part, a silylium ylidene-like valence isomer will be synthesised in due course. With help of the redox-active substituent, a silylium ylidene compound was obtained via charge redistribution. The combination of the Lewis basic lone pair and the highly electrophilic silicon cation resulted in extraordinary reactivity. Although it was not possible to isolate the silylium ylidene compound, different adducts of the silylium ion with donor molecules could be obtained and its exceptionally high Lewis acidity could be proven by the Gutmann-Beckett method. By investigating secondary reaction products, the silylium ion showed a strong fluoride ion affinity in presence of SbF6− and a particular reactivity towards the weakly coordinating anion B(C6F5)4−.
Cervical cancer is the fourth most common cancer in women worldwide. It is estimated that more than one million women are currently suffering from cervical cancer, and there are 570,000 new cases in 2018. The majority of cases (>80%) occur in less developed region. There are three HPV prophylactic vaccines in the market currently. They are designed to induce L1-specific antibodies blocking the infection of epithelial cells. The therapeutic efficacy was neither observed for Cervarix nor for Gardasil. Therefore, preventive vaccines cannot benefit individuals with already existing viral infections. As a result, a high prevalence of cervical cancer still threatens human life worldwide. Until now, there are no anti-HPV drugs available, an effective strategy should be the therapeutic vaccination to eliminate HPV-transformed cells by the activated immune system. The objective of my PhD project is to combine prophylactic and therapeutic value in one vaccine. The combined vaccine would ideally resolve productive infections and HPV-related diseases benefitting both uninfected and already infected individuals. Thioredoxin (Trx) was applied as a scaffold to develop HPV prophylactic vaccine Trx-L2 and Trx-8mer-OVX313 in our lab previously. In my project, we firstly verified that Trx was also able to induce CD8+ cytotoxic T cell responses. Then, we designed our prophylactic and therapeutic combined vaccines based on Trx-L2 and Trx-8mer-OVX313. HPV16 E749-57 was chosen as CTL epitope since it is considered a tumor specific antigen as well as an oncoprotein being expressed throughout the whole HPV life cycle. We developed the combined vaccines PADRE-Trx-L2-flank E7 (monomeric) and Trx-8mer-flank E7-OVX313 (heptameric). The E7-specific T cell responses were compared via IFN-gamma ELISpot between these two vaccines and the data indicates that heptamerization leads to a stronger T cell response. We therefore continued investigating the B cell responses induced by the heptameric antigen. From pseudovirion-based neutralization assay (PBNA), we saw that presence of CD8 T cell epitopes on the antigen does not interfere with the induction of neutralizing antibodies. In view of the in vitro promising results of Trx-8mer-flank E7-OVX313, we were encouraged to evaluate the therapeutic potential of Trx-8mer-flank E7-OVX313 in vivo. C57BL/6N mice were administrated with two doses vaccination after TC-1 cells challenge, and a potent antitumor activity was observed. These results demonstrate that antigen Trx-8mer-flank E7OVX313 is a promising and cost-efficient candidate with both prophylactic and therapeutic effectiveness.
In this thesis the reconstruction of climatic conditions and paleotemperatures by the analysis of oxygen (δ18O) and hydrogen (δ2H) isotopes of fluid inclusions in speleothems from two different climatic zones (mid-latitudes and tropics) was performed. An extraction and processing system for fluid inclusion analysis with laser spectroscopy was developed and characterized with respect to the memory or amount effect. I could exclude disturbance effects and achieve a precision of 0.5‰ for δ18O and 1.5‰ for δ2H in replicated measurements with water volumes >0.2μl. The analysis of stable water isotopes allows the determination of paleotemperatures using the classic carbonate thermometer as well as the application of the δ2H/T - relationship with a precision down to ±0.45°C. For the western tropical Atlantic I identified a cooling during the Heinrich stadials (2 and 3) of ~3°C applying the classic carbonate oxygen isotope thermometer. Beyond direct temperature determination, the analysis of fluid inclusions provides information about karst water availability during stalagmite formation. For instance, the δ18O and δ2H values of two speleothems from Germany and Puerto Rico, show slopes of +2.3±1.1 and +3.7±0.2, respectively, that are significantly lower compared to meteoric water lines (slope of ~8). This clear evaporation signal in the related fluid inclusions probably indicates evaporation effects (cave or epikarst), which are enhanced during drier climate conditions.
In dieser Dissertation wird der Einfluss elektronenreicher Guanidin-Liganden auf O‒O und B‒B Bindungsaktivierungsprozesse vorgestellt. Zu Beginn wurde die Aktivierung von molekularem Sauerstoff an Kupfer(I)-Komplexen mit den Liganden 2,6-Bis(tetramethylguanidino-methyl)pyridin (L1) und 2,6-Bis(tetramethyl-guanidino-methyl-ethyl)pyridin (L2) untersucht. Für den dinuklearen Komplex [(L1Cu)2]2+ wurde eine durch Sauerstoff initiierte aliphatische Ligandhydroxylierung beobachtet und mit der Reaktivität des Enzyms Peptidylglycin-α-hydroxylierende Monooxygenase (PHM) verglichen. Experimentelle und theoretische Untersuchungen an dem Modellkomplex lassen einen kooperativen Effekt zwischen den beiden Kupferatomen als Schlüsselschritt für den Hydroxylierungsprozess vermuten. In dem mononuklearen Komplex [L2Cu]+ wurden die zuvor hydroxylierten Stellen des Liganden L1 durch zusätzliche Methylgruppen blockiert. Die Oxygenierung führte dadurch zur Bildung eines Kupfer(II)-Superoxid-Komplexes, welcher mittels in situ UV/Vis- und Raman-Spektroskopie charakterisiert werden konnte. Weiterhin wurden Kupfer(I)-Komplexe mit dem Liganden 1,2-Bis(tetramethylguanidino)-benzol (L3) und dem redoxaktiven Liganden 1,2,4,5-Tetrakis(tetramethylguanidino)benzol (L4) für oxidative C‒C Homo- und Kreuzkupplungsreaktionen von Phenolen mit dem grünen Oxidationsmittel Sauerstoff eingesetzt. Komplexe mit dem elektronenreichen Liganden L4 zeigten hierbei eine signifikant höhere Aktivität und Chemoselektivität der Kreuzkupplungsreaktion. Auf der Grundlage von zahlreichen Testreaktionen wurde ein Reaktions¬mechanismus postuliert. Weiterhin wurden die Synthesen der neuen redoxaktiven Liganden Bis(diisopropylguanidino)dimethylbenzodioxol (L5) und 1,2-Bis(tetramethyl-guanidino)-4,5-bis(dimethylamino)benzol (L6) und ihrer entsprechenden Kupfer(I)-Komplexen entwickelt und erste Untersuchungen einer Sauerstoffaktivierung durchgeführt. Abschließend wurden die Liganden L3, L4 und L6 zur Aktivierung der B‒B Bindung in kationischen Diboranen eingesetzt. Mit den Bisguanidinen L3 und L6 gelang erstmals die Synthese von unsymmetrisch substituierten dikationischen Diboranen mit sp2-sp2-hybridi-sierten Boratomen. Die Verbindungen werden durch eine intramolekulare Umlagerung der initial gebildeten symmetrisch substituierten dikationischen Diborane erhalten. Diese beispiellose, nukleophil katalysierte Isomerisierung wurde im Detail untersucht. Aus der Temperaturabhängigkeit der Reaktionsgeschwindigkeit konnten die Aktivierungsparameter der Reaktion bestimmt werden. Die unterschiedliche Fluorid-Ionen-Affinität der beiden Boratome sowie die Bindungssituation dieser einzigartigen, unsymmetrischen dikationischen Diborane wurde mit Hilfe von computerchemischen Methoden untersucht.
The neglected tropical diseases leishmaniasis, Chagas disease and African trypanosomiasis, are inflicted by different trypanosomatid parasites and continue to spread. The limited number of available treatment options suffer from side effects and resistance issues, creating a need for novel anti-parasitic medicines. A target pathway of interest for developing anti-trypanosomatidic agents is the folate and biopterin metabolism. Trypanosomatids are auxotrophs for these metabolites and depend on their reductive activation by dihydrofolate reductase (DHFR) and pteridine reductase 1 (PTR1). However, inhibition of the anti-cancer and anti-bacterial target DHFR failed in trypanosomatids, since PTR1 provides a metabolic bypass of the DHFR activity. Thus, targeting of more than a single protein is required to interfere with the trypanosomatidic folate and biopterin pathway function. PTR1 is unique to the parasites, whereas DHFR has a human homolog representing an important off-target for compound development. Comparative studies of the sequences, structural data and physicochemical properties of the binding pockets were carried out for PTR1 and DHFR. The computational mapping revealed similarities between the different trypanosomatidic targets and important differences to human off-targets, which were translated into guidelines for the optimization of specific inhibitors of the parasitic target enzymes. Comparative modeling of ten further Leishmania major folate and biopterin pathway proteins expanded the comparison to a near-complete folate pathway pocketome and three biopterin-binding enzyme pockets. From this analysis, further potential off-targets for PTR1-specific inhibitors and additional side targets, for example the methylene tetrahydrofolate reductase or the folylpolyglutamate synthase, were suggested. Structure-based design and optimization were then carried out based on the target mapping. Building on previously developed thiadiazole-based Leishmania major PTR1 inhibitors, computational docking supported the determination of a structure-activity relationship (SAR) and the design of more effective thiadiazole-based and benzothiazole-based Trypanosoma brucei PTR1 inhibitors. Docking approaches further revealed the SAR for flavonoid inhibitors of different PTR1 variants and allowed for the proposal of core-hopping strategies. Novel pteridine-based inhibitors permitted the combined selective targeting of PTR1, with picomolar binding affinity, and parasite DHFR. Their design and SAR evaluation was informed by the computational docking predictions and additional efforts to improve the in vitro on-parasite effect on the basis of computationally predicted physicochemical compound descriptors supported the development of compounds with low micromolar in vitro activity against T. brucei bloodstream forms. Computational docking-derived SARs and their use in the design of improved inhibitors were thus successfully coupled with comparative mapping of protein binding pockets and computation-based optimization routines beyond the target level. This computational framework is applicable to the future development of anti-trypanosomatidic agents with different chemical scaffolds.
The binding process of quarks in the early time evolution in heavy-ion collisions in the presence of strongly interacting, highly occupied fields is an inherent non-equilibrium phenomenon. Understanding whether such a binding occurs, its time scale and its mechanism is important for predictions of the production of hadrons. Based on the works by Berges et al. [1–3] in the nonequilibrium as well as by Laine et al. [4] in the classical thermal equilibrium, we present a method for the evolution of heavy quark fields in the background of highly occupied gauge fields whose dynamics are obtained from a classical statistical lattice simulation as it is valid within the Colour Glass Condensate framework. We reproduce known results out of as well as in the classical thermal equilibrium. Employing NRQCD for the heavy quark degrees of freedom, we obtain heavy-quarkonium spectra from an in the light fermions quenched Minkowskian lattice simulation. For that we employ a leapfrog-algorithm in the gauge field dynamics, which serves as input for the integration of the heavy-quark propagators which are evolved with the Crank-Nicholson method. We find that the back-coupling of the heavy quarks to the gauge fields is essential for the binding process to occur which is in contrast to the intuition from Euclidean lattice simulations. We present the static potential evaluated in a simulation that was corrected by this back-coupling via the Gauß law. We find in contrast to Laine et al. that this potential obtains a real part which shows clear hints on Debye-screening.
Many metabolic pathways in bacteria are modulated by metabolite-sensing riboswitches, which regulate gene expression at the level of transcription elongation or translation initiation. Riboswitches represent promising targets to modulate expression of genes and operons relevant for the biotechnological production of commercially relevant compounds. In Firmicutes, approximately 70% of all putative and validated riboswitches (are predicted to) act exclusively at the transcriptional level using a termination-antitermination mechanism.
In a first attempt to interfere with purine-sensing riboswitches and deregulate purine metabolism in Bacillus subtilis, a set of synthetic small RNAs (sRNAs) targeting the purine-sensing aptamers were designed to impair ligand binding using rational design combined with in silico evolution. However, the designed sRNAs did not show any activity in vivo on the riboswitch controlling purine biosynthesis (pur operon riboswitch). The effect of the antisense RNA (asRNA) perfectly complementary to the aptamer of the pur operon riboswitch was also tested; The asRNA did not affect negatively expression of a riboswitch-regulated lacZ gene, yet similarly to the partially complementary sRNAs, the asRNA did not impair the downregulation exerted by the riboswitch in the presence of ligand. Finally, expression of the small RNAs in B. subtiliswas quantified, and the kinetic limitations for their hybridization with the aptamer and their competition with the ligand are discussed.
A second metabolic engineering strategy based on editing the genome of B. subtilis with regard to transcriptional riboswitches was investigated. Removal of the riboswitches that control purine biosynthesis and riboflavin biosynthesis in B. subtilis led to auxotrophic strains. As an alternative, a rational approach was developed for engineering transcriptional riboswitches independently from the availability of their 3D structures. This approach consists in the identification and deletion of a key nucleotide sequence exclusively involved in transcription termination without affecting formation of other secondary and tertiary structures potentially involved in other roles. To demonstrate the efficacy of the approach, it was applied to derepress the purine and the riboflavin biosynthetic pathways in B. subtilis. Following the proof of concept using specialized reporter strains, the approach was implemented into a B. subtilis wild-type strain employing CRISPR-Cas genome editing. The CRISPRCas9 system displayed an efficiency of 61% in editing the genome, and the resulting purine and riboflavin production strains were characterized at the level of gene expression, metabolite synthesis, and growth. With a substantial enhancement observed at each level, the strategy established here represents a powerful tool for deregulating pathways modulated by transcriptional riboswitches. Finally, applying this strategy to derepress the purine pathway of an industrial riboflavin overproducing strain, with impaired growth, led to an increase in biomass by 53% and resulted in an enhanced total production of riboflavin in the culture.
In an aging world, neurodegenerative diseases, such as Alzheimer’s Diseases start to appear more and more in society. Unfortunately, only drugs ameliorating the symptoms, but no preventive or curative medications are available. One underlying reason is that only animal models exist that do not fully reflect the human pathophysiology, leading to the difficulty of translating findings into humans. Therefore, it is of great importance to access an authentic in vitro cell culture system to study disease. Such a cell culture system would also have the potential to be used in later stages of drug discovery and drug development. Here, a quick, robust and chemically-defined xeno-free differentiation protocol was developed to obtain a human induced pluripotent stem cell (hiPSC)-derived cortical neuron cell culture system. Several factors influencing variability of differentiation were addressed and identified, leading to faster generation of cells, greater robustness and wide applicability among different hiPSC lines. Further, it was shown that the derived culture system is suitable for disease modeling in Alzheimer’s Disease as the Aβ-pathology could be recapitulated in the cells, already at a very early time point of differentiation. The established differentiation protocol is a promising tool in disease modeling of Alzheimer’s Disease and other tauopathies, without the need of animal-derived cell culture supplements and reagents.
Despite the recent success of deep learning, the mammalian brain is still unrivaled when it comes to interpreting complex, high-dimensional data streams like visual, auditory and somatosensory stimuli. However, the underlying computational principles allowing the brain to deal with unreliable, high-dimensional and often incomplete data while having a power consumption on the order of a few watt are still mostly unknown. In this work, we investigate how specific functionalities emerge from simple structures observed in the mammalian cortex, and how these might be utilized in non-von Neumann devices like “neuromorphic hardware”. Firstly, we show that an ensemble of deterministic, spiking neural networks can be shaped by a simple, local learning rule to perform sampling-based Bayesian inference. This suggests a coding scheme where spikes (or “action potentials”) represent samples of a posterior distribution, constrained by sensory input, without the need for any source of stochasticity. Secondly, we introduce a top-down framework where neuronal and synaptic dynamics are derived using a least action principle and gradient-based minimization. Combined, neurosynaptic dynamics approximate real-time error backpropagation, mappable to mechanistic components of cortical networks, whose dynamics can again be described within the proposed framework. The presented models narrow the gap between well-defined, functional algorithms and their biophysical implementation, improving our understanding of the computational principles the brain might employ. Furthermore, such models are naturally translated to hardware mimicking the vastly parallel neural structure of the brain, promising a strongly accelerated and energy-efficient implementation of powerful learning and inference algorithms, which we demonstrate for the physical model system “BrainScaleS–1”.
Metal microfibers have a wide range of industrial applications, e.g. as filters, fiber-reinforced composites, electrodes, catalysts, sensors, or magnetic shielding materials. In this project, we modified melt-spinning device and its experimental parameters to fabricate metal microfibers. It was shown for the first time that metal microfibers down to 5 μm could be fabricated using a melt spinning device. The size and circularity of formed fibers could be controlled by experimental parameters.e.g. slit distance to the wheel, chamber pressure, slit size, wheel speed. The mechanism of fiber formation relies on two main steps; i)thin film formation on the rotating wheel ii)spontaneous breaking of the film to smaller widths, dewetting the wheel. It was shown that this process is reproducible and could be used for different classes of materials. e.g. intermetallic alloys, conventional alloys, metal elements and amorphous alloys. The modification of the melt spinning device leads to higher quenching rates up to 108!C/s. The high quenching rate made it possible to make fully amorphous stainless steel fibers for the very first time. Heat-treatment of amorphous stainless steel leads to dual-phase microstructure (nanocrystals embedded together with a glassy phase) which was responsible for its ultra-high hardness value, 14GPa. This value is 7 times higher than the original stainless steel hardness. Thus, the technique opens new possibilities for working with conventional and amorphous alloys e.g. mechanically improved conventional alloy microfibers/ribbons, introducing new alloy microstructures.
Magnetic resonance imaging (MRI) provides a versatile tool to determine a variety of fluid mechanical properties, but similarly the quantification is biased by the acquisition itself and the technique has limitations among which are limited spatial resolution and low signal-to-noise-ratio.
To overcome such issues, phase contrast MRI methods have been investigated at 7 T. At this field strength variations of the transmit field pose a substantial problem. Additionally, so-called displacement artifacts become important, since they scale with spatial resolution. Transmit field variations are addressed in this work by multi-spoke RF pulses, which are not straightforwardly applicable to velocity quantification. They require a detailed investigation of displacement artifacts that arise due to differences in the encoding time points of velocity and space. This work investigates three different encoding schemes, for conventional excitation as well as for multi-spoke excitation, which yield different displacement artifacts. Their impact on derived haemodynamic parameters, such as wall shear stress, which had been unknown so far, are investigated. Moreover, spoke pulses are further fine-tuned by using asymmetric pulse shapes. Besides the correct determination of the velocity at 7 T, the precise quantification of acceleration is another important factor, which is solved in this work by developing an acceleration-encoded sequence free of artifacts. Furthermore, another confounding factor in MRI-based velocitmetry, the intravoxel velocity distributions, affect the velocity encoding process. This effect has been investigated and, based on measured velocity spectra, noise-optimized velocity encoding sensitivity (VENC) values have been proposed. Finally, the potential of precise MR-based velocity measurements is demonstrated for a well-known fluid dynamic test case (flow over periodic hills) with a Reynolds number of 60,000. For this case, the Reynolds stress tensor has been quantified.
In conclusion, the presented techniques improve the precision at which fluid mechanical properties can be quantified by means of MRI.
Silk fibers are outstandingly tough biomaterials, a result of the controlled self-assembly of their protein building blocks, spidroins. The combination of extensibility and high tensile strength relies on the microscopic composition within the fiber: small and strong beta-sheet crystals formed mainly by poly-alanine repeats enclosed into a flexible amorphous matrix of glycine-rich repeats. The internal molecular structure of silk proteins makes them sensitive to an elongational flow, which is a crucial factor for spider silk fiber spinning. However, the mechanism of flow-induced silk self-assembly, as well as the relevant dynamics of single silk proteins under flow remain largely unknown. In the present work, a bottom-up approach was used to study the dynamics and self-assembly of spider silk proteins under uniform flow conditions. We used non-equilibrium molecular dynamics (MD) simulations to study these processes at two scales, an atomistic model with explicit water, and a coarse-grained model with hydrodynamics incorporated by multi-particle collision dynamics. To be able to analyze the role of the flow on spider silk molecules atomistically, a prior implementation and systematic study of uniform flow MD simulations were carried out based on the GROMACS MD software. Subjecting a tethered single silk peptide to uniform flow leads to a coiled-to-stretch transition involving a multitude of intermediates states, the process of which depends on the mean flow velocity. The flow-induced structural changes of single spidroins exhibit a prominent tendency of alanine residues to be in beta-sheet conformation. All-atom simulations of the assembly process at low flow regimes revealed that the interchain contacts happen primarily in the poly-alanine repeats, which is a suitable condition for crystal formation and fibrillation. We also found beta-sheets formation at low flow regimes, confirming that flow promotes crystal formation. We complemented these findings to the more coarse-grained hydrodynamic simulations at aminoacid resolution, treating the silk proteins as semi-flexible block copolymers. We observed that the spidroins aggregate faster when they are less extended by monitoring oligomer formation in time. At medium peptide extensions (around 60-70%), the spidroin alignment increases, while their assembly slows down because of the reduced fluctuations orthogonal to the flow direction. The microscopic understanding of the spidroin dynamics provided in this work is likely relevant for other flow-dependent proteins.
The transport of charge and energy are two essential processes in optoelectronic devices. In this thesis, using quantum chemical methods, molecular properties, as well as charge and energy transfer performance are studied in novel N-heteropolycycles. N-heteropolycycles are formally N-doped heterocyclic nanographene segments. The position and number of the nitrogen substitution, as well as further modification, can fine-tune their molecular properties such as energy levels, diradical characters, and charge and energy transfer rates. For the investigation of energy transfer, particular interest lies in singlet fission (SF), which has the potential to dramatically increase solar cell efficiency by converting one singlet exciton to two free triplet excitons or a correlated triplet pair. In chapter 3, quantum chemical methods based on DFT and constrained DFT are applied to rationalize how SF is affected by systematic chemical modifications introduced into phenazinothiadiazoles (PTD). The results indicate that unlike unsubstituted tetracene, PTDs fulfill the energetic requirement of SF (E(S_1)≥2×E(T_1)), and the effective coupling can be up to 75.8 meV. Hence, PTDs are promising candidates for SF. In chapter 4, a single-reference DFT-based protocol is proposed to simulate the absorption spectra of excited states involved in SF. The resulting spectra show good agreement with the experiment. This could be helpful for the identification of various species in SF and the understanding of SF dynamics. On the other hand, N-heteroacenes are known as electron-poor counterparts of the acenes, and they are electron transport (n-type) materials. Since the charge transport moiety in bulk films of azaacenes is thought to be the radical anion, in chapter 5, the energetics, electronic structures, and spectroscopic properties of negatively charged N-heteroacenes are investigated. It is found that the anions of the azapentacenes and their derivatives are stable with respect to electron loss and disproportionation into the dianion and the neutral compound. This motivates a further look into their electron transport properties. The results of electron transfer integrals and charge mobilities are demonstrated in chapter 6. Excellent performance of electron transport has been proved for halogenated 6,13-Diethynyl-5,7,12,14-tetraazapentacenes, especially for the bromine and iodine derivatives.
Das Ziel dieser Arbeit ist die Untersuchung und Entwicklung numerischer Methoden zur Bewegungserzeugung von humanoiden Robotern basierend auf nichtlinearer modell-prädiktiver Regelung. Ausgehend von der Modellierung der Humanoiden als komplexe Mehrkörpermodelle, die sowohl durch unilaterale Kontaktbedingungen beschränkt als auch durch die Formulierung unteraktuiert sind, wird die Bewegungserzeugung als Optimalsteuerungsproblem formuliert.
In dieser Arbeit werden numerische Erweiterungen basierend auf den Prinzipien der Automatischen Differentiation für rekursive Algorithmen, die eine effiziente Auswertung der dynamischen Größen der oben genannten Mehrkörperformulierung erlauben, hergeleitet, sodass sowohl die nominellen Größen als auch deren ersten Ableitungen effizient ausgewertet werden können. Basierend auf diesen Ideen werden Erweiterungen für die Auswertung der Kontaktdynamik und der Berechnung des Kontaktimpulses vorgeschlagen.
Die Echtzeitfähigkeit der Berechnung von Regelantworten hängt stark von der Komplexität der für die Bewegungerzeugung gewählten Mehrkörperformulierung und der zur Verfügung stehenden Rechenleistung ab. Um einen optimalen Trade-Off zu ermöglichen, untersucht diese Arbeit einerseits die mögliche Reduktion der Mehrkörperdynamik und andererseits werden maßgeschneiderte numerische Methoden entwickelt, um die Echtzeitfähigkeit der Regelung zu realisieren.
Im Rahmen dieser Arbeit werden hierfür zwei reduzierte Modelle hergeleitet: eine nichtlineare Erweiterung des linearen inversen Pendelmodells sowie eine reduzierte Modellvariante basierend auf der centroidalen Mehrkörperdynamik. Ferner wird ein Regelaufbau zur GanzkörperBewegungserzeugung vorgestellt, deren Hauptbestandteil jeweils aus einem speziell diskretisierten Problem der nichtlinearen modell-prädiktiven Regelung sowie einer maßgeschneiderter Optimierungsmethode besteht. Die Echtzeitfähigkeit des Ansatzes wird durch Experimente mit den Robotern HRP-2 und HeiCub verifiziert.
Diese Arbeit schlägt eine Methode der nichtlinear modell-prädiktiven Regelung vor, die trotz der Komplexität der vollen Mehrkörperformulierung eine Berechnung der Regelungsantwort in Echtzeit ermöglicht. Dies wird durch die geschickte Kombination von linearer und nichtlinearer modell-prädiktiver Regelung auf der aktuellen beziehungsweise der letzten Linearisierung des Problems in einer parallelen Regelstrategie realisiert. Experimente mit dem humanoiden Roboter Leo zeigen, dass, im Vergleich zur nominellen Strategie, erst durch den Einsatz dieser Methode eine Bewegungserzeugung auf dem Roboter möglich ist.
Neben Methoden der modell-basierten Optimalsteuerung werden auch modell-freie Methoden des verstärkenden Lernens (Reinforcement Learning) für die Bewegungserzeugung untersucht, mit dem Fokus auf den schwierig zu modellierenden Modellunsicherheiten der Roboter. Im Rahmen dieser Arbeit werden eine allgemeine vergleichende Studie sowie Leistungskennzahlen entwickelt, die es erlauben, modell-basierte und -freie Methoden quantitativ bezüglich ihres Lösungsverhaltens zu vergleichen. Die Anwendung der Studie auf ein akademisches Beispiel zeigt Unterschiede und Kompromisse sowie Break-Even-Punkte zwischen den Problemformulierungen. Diese Arbeit schlägt basierend auf dieser Grundlage zwei mögliche Kombinationen vor, deren Eigenschaften bewiesen und in Simulation untersucht werden. Außerdem wird die besser abschneidende Variante auf dem humanoiden Roboter Leo implementiert und mit einem nominellen modell-basierten Regler verglichen.
Bis heute ist die Anzahl an Patienten aus der Gruppe der angeborenen Glykosylierungsdefekte (CDG) gering, wobei zu vermuten ist, dass dieser Stoffwechselbereich hochgradig unterdiagnostiziert ist. CDG zählen daher noch immer zu den seltenen Stoffwechselerkrankungen. Die Entdeckung und Charakterisierung neuer Glykosylierungsdefekte sollten daher einen noch höheren Stellenwert im Bereich der pädiatrischen Stoffwechselstörungen einnehmen. Mit meiner Arbeit konnte erstmals eine Verbindung zwischen Glykosylierungsdefekten und dem Larsen Syndrom hergestellt werden. Hierfür wurden Serumproben und Hautfibroblasten der Patientin molekularbiologisch und biochemisch charakterisiert. Das multisystemische Krankheitsbild unserer Patientin äußerte sich bereits zum Zeitpunkt der Geburt durch Kleinwuchs, skelettale Dysplasien und multiple Gelenkluxationen. Die übrigen Symptome, wie Dysmorphien, Hyperlordose der Wirbelsäule, Gelenkanomalien und der Verlust des Hör und Sehsinns, folgten im Laufe der ersten Lebensjahre. Aufgrund ihres multisystemischen Phänotyps wurde die CDG Diagnostik eingeleitet, wobei ein kombinierter Defekt der N und O Glykosylierung im Serum festgestellt und darauffolgend auch in den Fibroblasten der Patientin bestätigt wurde. Durch „Whole Exome Sequencing“ konnte die homozygote Variante c.865G>T (p.Glu289*) im GDNF Transduzierbaren Zink Finger Protein 1 (GZF1) nachgewiesen werden, die zum Larsen-Syndrom führt. Weiterführende Untersuchungen ergaben eine aus der Mutation resultierende Verminderung der mRNA und Proteinexpression sowie das Fehlen von GZF1 in den Zellkernen der Patientenfibroblasten. In Komplementationsstudien nach viraler Infektion mit der Wildtyp-GZF1-cDNA konnte eine Erhöhung der GZF1-Proteinexpression und dessen wiederkehrende Lokalisierung im Zellkern erreicht werden. Weiterhin ergab das eingebrachte GZF1 eine teilweise Re-Glykosylierung und erhöhte Expression der beiden Glykomarkerproteine GP130 und LAMP2. Der Glykosylierungsdefekt der Patientin ist aber vermutlich noch in dem durch die homozygote Variante c.1174G>A (p.Ala392Thr) betroffenen Vacuolar-Sorting Protein 1 Homolog (VPS45)-Gen begründet. Transkript- und Proteinexpressionsstudien von VPS45 und von drei Untereinheiten des mit VPS45 in Verbindung stehenden Conserved Oligomeric Golgi (COG)-Komplexes ergaben dementsprechend deutliche Auffälligkeiten, wie sie bereits von den COG-CDG-Defekten her bekannt sind. Ein hierdurch verursachtes Missorting von Glykosyltransferasen im Golgi ist vermutlich neben GZF1 mitverantwortlich für den bei der Patientin beobachtete schweren Glykosylierungsdefekt.
Image labeling is a fundamental problem in the area of low-level image analysis. In this work, we present novel approaches to maximum a posteriori (MAP) inference and model parameter learning for image labeling, respectively. Both approaches are formulated in a smooth geometric setting, whose respective solution space is a simple Riemannian manifold. Optimization consists of multiplicative updates that geometrically integrate the resulting Riemannian gradient flow.
Our novel approach to MAP inference is based on discrete graphical models. By utilizing local Wasserstein distances for coupling assignment measures across edges of the underlying graph, we smoothly approximate a given discrete objective function and restrict it to the assignment manifold. A corresponding update scheme combines geometric integration of the resulting gradient flow, and rounding to integral solutions that represent valid labelings. This formulation constitutes an inner relaxation of the discrete labeling problem, i.e. throughout this process local marginalization constraints known from the established linear programming relaxation are satisfied.
Furthermore, we study the inverse problem of model parameter learning using the linear assignment flow and training data with ground truth. This is accomplished by a Riemannian gradient flow on the manifold of parameters that determine the regularization properties of the assignment flow. This smooth formulation enables us to tackle the model parameter learning problem from the perspective of parameter estimation of dynamical systems. By using symplectic partitioned Runge--Kutta methods for numerical integration, we show that deriving the sensitivity conditions of the parameter learning problem and its discretization commute. A favorable property of our approach is that learning is based on exact inference.
Mechanical and chemical pattern formation in the development of biological tissue is a fundamental and fascinating process of self-complexation and self-organization. Yet, the understanding of the underlying mechanisms and their mathematical description still lacks in many interesting cases such as embryogenesis. In this thesis, we combine recent experimental and theoretical insights and numerically investigate the capacity of mechano-chemical processes to spontaneously generate patterns in biological tissue.
Firstly, we develop and numerically analyze a prototypical system of partial differential equations (PDEs) leading to mechanochemical pattern formation in evolving tissues. Based on recent experimental data, we propose a novel coupling by tensor invariants describing stretch, stress or strain of tissue mechanics on the production of signaling molecules (morphogens). In turn, morphogen leads to piecewise-defined active deformations of individual biological cells. The presented approach is flexible and applied to two prominent examples of evolving tissue: We show how these simple interaction rules (“feedback loops”) lead to spontaneous, robust mechanochemical patterns in the applications to embryogenesis and to symmetry breaking in the sweet water polyp Hydra. Our results reveal that the full 3D model geometry is essential to obtain realistic results such as gastrulation events. Also, we highlight predictive numerical experiments that assess the sensitivity of biological tissue with regard to mechanical stimuli, namely to micropipette aspiration. These numerical experiments allow for a cross-validation with experimental observations. Besides, we apply our modeling approach to growing tips in colonial hydroids and investigate the role of rotational and shearing active deformations by comparison to experimental data.
Secondly, we develop an efficient, numerical method to reliably solve these strongly coupled, prototypical systems of PDEs that model mechanochemical long-term problems. We employ state-of-the-art finite element methods, parallel geometric multigrid solvers and present a simple, local mesh refinement strategy to obtain an efficient solution approach. Parallel solvers are essential to deal with the huge problem size in 3D and were modified to keep track of biological cells. Further, we propose a stabilization of the structural equation to deal with the strongly coupled system of equations and the challenges of the different timescales of growth (days) and nonlinear elasticity (seconds). Also, this addresses the instabilities which result form the description of homogeneous Neumann values on the entire boundary that is necessary since the locations of patterns is a priori unknown.
In many parts of physics, chemistry, biology, or material science, excited electronic states, accessible via the interaction of atoms or molecules with electromagnetic radiation, play an essential role. Experimental spectra, however, generally provide only indirect information on molecular structure and dynamics. Thus, a theoretical description of excitation energies and transition strengths is crucial for a comprehensive understanding of light-induced processes. In this dissertation, the theory, implementation, and application of several Hermitian methods to calculate the properties mentioned above are described. If excitation energies are obtained by diagonalization of a non-Hermitian secular matrix, both left and right eigenvectors need to be calculated to obtain spectral intensities and other properties. In this case, the eigenvectors are not orthogonal to each other, and the energy may become complex. Hermiticity is thus a very desirable property since none of the aforementioned problems occurs. Thus, several approaches based on the algebraic-diagrammatic construction (ADC) scheme, as well as the related unitary coupled-cluster (UCC) method, are presented. Within these methods, one-electron properties such as dipole moments are available via the so-called intermediate state representation (ISR) approach, which corresponds to an expectation value of the respective one-electron operator with the wave function. The ISR formalism is also used to derive explicit working equations for the second-order ADC scheme, which is based on a ground state described by Møller–Plesset (MP) perturbation theory. This implies that ADC inherits all weaknesses from the underlying MP model. For the ADC(2) scheme, merely the first-order MP wave function is required, which contains only doubly-excited determinants for a Hartree–Fock reference. Due to the form of the first-order doubles amplitudes, several cancellations occur in the singles block of the ADC(2) matrix. In order to remedy the breakdown of MP2, the first-order doubles amplitudes from MP are replaced by the ones obtained from a coupled-cluster (CC) calculation, which are formally correct through infinite order. The resulting schemes, referred to as CC-ADC(2), are applied to several sets of small to medium-sized molecular systems, where generally minor improvements in excitation energies compared to the standard ADC(2) scheme can be observed. For the ozone molecule, which is known to be a difficult test case for quantum-chemical methods, the experimental first excitation energy is 1.6 eV; standard ADC(2) is far off with 2.14 eV, and CCD-ADC(2) yields 1.59 eV. Excited-state potential energy curves along the dissociation of the nitrogen molecule calculated with ADC(2) break down at around 2 Å due to the failure of MP2. The CCD-ADC(2) curves remain reasonable up to about 3.5 Å. The CC-ADC(2) methods are successively extended to the calculation of static dipole polarizabilities. It is shown that the correlation amplitudes play a more important role in the modified transition moments than in the ADC secular matrix itself, and consistent improvement is obtained for static polarizabilities with the CC-ADC schemes compared to standard ADC, particularly for aromatic systems like benzene or pyridine, which had proven difficult cases for standard ADC. Specifically, the CC-ADC(2) schemes yield significantly better results than the ADC(3/2) scheme, at a computational cost amounting to only 1% of the latter. The ISR derivation can also be carried out with a CC wave function correct through first order instead of the MP one. However, having converged CCD amplitudes instead of the first-order MP ones, the aforementioned cancellations in the second-order singles block do not occur. Hence, the final matrix elements differ between CCD-ADC(2) and this scheme referred to as CCD-ISR(2). As the expansion of the UCC similarity-transformed Hamiltonian does not truncate naturally, it needs to be truncated manually, usually by using arguments from MP perturbation theory. The UCC2 doubles amplitudes correspond to those from LCCD, but the secular matrix elements depend on the treatment of the similarity-transformed Hamiltonian is treated. By employing the Baker–Campbell–Hausdorff expansion, the second-order singles block is equivalent to CCD-ISR(2), but by employing the Bernoulli expansion, the matrix elements are equivalent to CCD-ADC(2), with differences only in the correlation amplitudes. In a strict perturbation-theoretical framework, all methods turn out to be identical. All different Hermitian second-order methods have been implemented and tested on a set of small molecules, where it turned out that the differences in excitation energies between the methods are small whenever the systems are well described by means of perturbation theory. The Bernoulli UCC scheme is further extended to third order, where excitation energies and oscillator strengths on medium-sized organic molecules as well as ground- and excited-state dipole moments are reported for the first time. While vertical excitation energies of the UCC3 scheme are similar to those obtained with ADC(3), significant improvements can be observed for the dipole moments in the ground and excited states. Furthermore, this UCC scheme is applied to the electron propagator, and ionization potentials of the IP-UCC2 and IP-UCC3 schemes of selected amino acids are reported for the first time. Apart from expectation values, molecular properties can be calculated as derivatives of the energy with respect to a perturbation connected to the observable. The two approaches are only equivalent if the Hellmann–Feynman theorem is fulfilled. By using explicit working equations, the relationship between the two approaches is investigated with a focus on orbital relaxation for all standard quantum-chemical methods, in particular MP and ADC. It is shown that for MP2 the expectation value is very close to the orbital-relaxed property. In contrast, for ADC(1) the expectation value includes no orbital relaxation and for ADC(2) only a small fraction. With ADC(3) eigenvectors, on the other hand, the ISR gets closer to the relaxed values, but only for singly-excited states. Numerical investigations underline all the theoretical predictions.
Fluid flow is governed by primary and secondary porosity of rocks but also by their permeability. Often the values of primary porosities and permeabilities are not sufficient to allow fluids to flow from potential geothermal or hydrocarbon reservoirs. To ensure an efficient productivity, fractured reservoirs come into focus as they might provide an economically viable fluid flow. Subsurface fractured reservoirs are difficult to investigate, outcrop analogues like the one investigated help in a better understanding. The studied outcrop represents a Lower Triassic braided river succession within an arid alluvial plain, affected by the main fault of the western Rhine Graben (southwestern Germany). The research thesis was carried out with the help of terrestrial laser scanning (TLS) to generate a digital outcrop model (DOM), used to digitize data and serve as basis for the subsequent modeling in two steps. These are (i) the volumetric modeling of the investigated fault zone within the Triassic Lower Buntsandstein, and (ii) subsequent modeling of the discrete fracture network (DFN). Volumetric modeling comprises three main points: (i) the application of a fault zone facies concept, (ii) stair-stepped fault gridding, and (iii) splitting the fault zone into two geobodies, well established in structural terminology, the damage zone ‘DZ’ and the fault core ‘FC’. For the subsequent DFN calculations a thorough fracture data parametrization was carried out providing six defined fracture sets, the fracture shape, the log-normal aperture distribution, the log-normal length distribution, the P32 intensity, and fracture truncation percentages at bed boundaries (DZ only). DFN upscaling was then conducted with the “Oda” and “Oda Corrected” methods for the fracture permeability calculations. The resulting volumetric model comprises 13 fault zone facies types. Their distribution within the DZ follows the encountered beds’ morphology. Within the FC three facies distribution cases were modeled. Seven different DFN configurations were calculated, consisting of 162 fracture sets in total. Fracture permeability amounts between 190 and 720 D within the DZ and 14,130 to 55,189 D within the FC, while the fracture porosity shows values of about 0.4 % for the DZ and 2.38 % for the FC. The study shows that volumetric fault zone modeling requires a simultaneous fault facies analysis and grid construction. Because stair-stepped fault grids facilitate a high complexity but lack cell size flexibility, a thoroughly considered choice of the cell size, dependent on the smallest geological objects present, is crucial. Characterization and processing of fracture aperture constitutes the most important part of the parametrization, as different methods can lead to distinct differences in the modeled final fracture permeabilities, spanning multiple orders of magnitude, even for exactly the same values of mechanical aperture. Inclusion of fracture connectivity lowers the resultant horizontal fracture permeability by 26 to 38 %, while truncation of fractures on bed boundaries can overestimate permeability values. Although the FC shows a significantly higher fracture permeability than the DZ it is affected by extreme fracture permeability cutoffs due to the fault cores’ specific architecture, resulting in a conduitbarrier system. Fracture porosities are more insensitive to parameter changes, because of its dependence on the mechanical aperture only. The presented multi-approach thesis highlights the challenges, limitations, and great possibilities of fault zone models, to help in a better understanding of the impact fault zones might have on geothermal and hydrocarbon reservoirs, and thereby support exploration.
Recent advances in the fields of genome editing, whole-genome sequencing, single-cell RNA sequencing, and in situ spatial transcriptomics have enabled the cost-efficient production of high-throughput big data. However, the lack of dedicated bioinformatics algorithms to analyze such data has been a big hurdle. In this thesis, several novel bioinformatics tools applicable to each field are presented. First, a series of web-based tools for genome editing are presented: Cpf1-Database, Cas-Analyzer, web-based Digenome-seq software, BE-Designer/Analyzer. These tools have been developed to guide researchers to easily use genome editing systems, using Cas9 or Cpf1, by providing an easily accessible web-based interface. Second, the development of two bioinformatics pipelines are described: a small variant calling pipeline to process tumor genome sequencing data without a matched control, and a pipeline to pre-process single-cell RNA sequencing data. Third, a novel segmentation-free algorithm to call cell-types from in situ transcriptomics data, namely Spot-based Spatial cell-type Analysis by Multidimensional mRNA density estimation (SSAM) is presented. Recent advances of in situ spatial transcriptomics techniques, such as multiplexed fluorescence in situ hybridization or in situ/intact tissue sequencing have enabled the discovery of spatial heterogeneity of cell types at the tissue level. However, cell type calling methods are often limited by cell segmentation algorithms due to various imaging problems. SSAM circumvents these problems by estimating spatial gene expressions as a density estimation of the mRNA in a spatial context and identifying de novo cell-types and their spatial organization without the need to segment cells. Optionally, SSAM can be guided by external sources of cell-type information, integrating them in a spatial context. In this thesis, SSAM is demonstrated with three different mouse brain tissues imaged by different imaging techniques: the somatosensory cortex (SSp) imaged by osmFISH; the hypothalamic preoptic region (POA) by MERFISH; and the visual cortex (VISp) by multiplexed smFISH. SSAM can produce similar results compared to segmentation-based methods and outperforms them when cell segmentation is the limiting factor. In summary, the bioinformatics tools presented in this thesis overcome major obstacles that would normally hinder effective analysis: the web-based tools for genome editing have a wide user base due to their easy-to-use web-based interfaces; omics data analysis pipeline that enables fast analysis of omics data utilizing a compute cluster and facilitate hypothesis generation when lacking control tissue; and SSAM that enables the analysis of in situ spatial transcriptomics data without being limited by cell segmentation. All of the tools and pipelines described in this thesis are open-sourced and freely accessible for non-profit, research-purpose use.
Interactions of atoms or molecules with electromagnetic radiation or free electrons can induce a variety of transformations. Apart from elastic scattering processes, in which the quantum states of the involved particles are preserved, inelastic scattering may occur. The distribution of product states depends on the kind of the interacting particles and the energy transferred in the scattering process.
Among the possible transformations are electronic excitation, photoionization and the formation of electronic resonances, i.e., metastable electronic states which undergo subse quent decay by emission of an electron. The latter states can evolve in electronic excitation processes or as a result of electron attachment. In this dissertation, the implementation and application of quantum chemical propagator methods for the description of the above-mentioned processes are presented.
More specifically, a number of perturbation theoretical methods based on the algebraic diagrammatic construction (ADC) schemes for the electron propagator and the polarization propagator are considered. In the framework of these methods, one-electron properties are available via the intermediate state representation (ISR) approach, which enables the computation of the explicit form of the respective wave functions. The third-order static self-energy Σ(3) appearing in the third-order ADC(3) equations can thereby be replaced by an improved fourth-order quantity resulting from the so-called Σ(4+)-procedure, and this option has been explored in the context of ADC for ionization potentials (IP-ADC), electron affinities (EA-ADC) and, for the first time, excitation energies (PP-ADC).
In the first part of this dissertation, photoionization processes are considered, whose theoretical treatment is possible using IP-ADC(3). In the course of this work, the existing implementation of IP-ADC(3) in the Q-Chem quantum chemical program package has been extended by the possibility to compute photoelectron intensities, and therefore, to simulate photoelectron spectra. Other newly implemented features enable the interpretation of ionization transitions by means of visualization of Dyson orbitals and one-particle density matrix-based quantities as, e.g., detachment and attachment densities, which are available via the second-order ISR(2) approach.
The accuracy of the IP-ADC(3)/ISR(2) methodology with respect to ionization potentials and one-particle properties of electron-detached states has been evaluated in a subsequent benchmark study. Therein, the results obtained for 44 electronic states of small molecules are compared to high-level configuration interaction results. For this set of transitions, ionization potentials exhibit a mean absolute error of |∆| ≈ 0.2 eV. For dipole moments, a relative error of |∆| = 19 % is found. In a second IP-ADC(3) study, the applicability of the newly implemented density matrix-based analyses for the interpretation of photoelectron spectra is demonstrated using the example of the galvinoxyl free radical.
In the second part of this dissertation, electronic resonances are addressed. Due to the unbound nature of the involved electronic states, their theoretical treatment is challenging. Different theoretical approaches for their description within the framework of standard quantum chemical methods have been devised, two of which are considered in this work.
First, the efficient implementation of the Fano-Stieltjes-ADC method in the Q-Chem program is presented. For the first time, the third-order PP-ADC(3) scheme as well as various unrestricted PP-ADC schemes have been combined with the Fano-Stieltjes formalism. The applicability of the implementation for the description of resonances in medium-sized organic molecules is demonstrated in a study of a Feshbach resonance in the naphthalene molecule.
As a second option for the theoretical treatment of electronic resonances, the combination of the subspace-projected complex absorbing potential (CAP) method with PP- ADC(3) and EA-ADC(3) is considered. Results obtained using the novel CAP-EA-ADC and CAP-PP-ADC methods as implemented in the Q-Chem quantum chemical program package show an excellent agreement with theoretical best estimates and experimental data in studies of π* shape resonances in unsaturated molecules. Among the studied resonance states are the ²Πg resonance of the dinitrogen anion as well as the lowest π* resonances of the anions of the non-conjugated organic dienes norbornadiene and 1,4-cyclohexadiene. CAP-EA-ADC(3) calculations are in line with previous findings and show that a strong through-bond interaction mechanism reverses the natural ordering of the π* molecular orbitals in 1,4-cyclohexadiene.
The hematopoietic system is a highly versatile regenerative tissue, in which hematopoietic stem cells drive the life-long production of multiple mature blood cell types. During hematopoietic differentiation, the regulation of genome-wide epigenetic patterns of histone modification or DNA methylation marks is an essential process orchestrating cell identities, lineage decisions and developmental cell fates. In acute myeloid leukemia, mutations frequently affect direct and indirect epigenetic regulators and modifiers such as isocitrate dehydrogenase 1 (IDH1) or DNA methyltransferase 3 alpha (DNMT3A), and result in disturbed epigenetic landscapes and differentiation patterns. Here, IDH1 mutations promote oncogenic transformation through the de novo production of the metabolite D2-hydroxyglutarate, which induces a genome and epigenome instability by inhibiting multiple histone and DNA demethylases. Yet, molecular details of how IDH1 mutations alter characteristics of individual hematopoietic cell types remain poorly understood. In the course of this thesis, combinatorial mouse models carrying specific Idh1-R132H and DNMT3A-R882H mutations, which frequently co-occur in acute myeloid leukemia patients, were extensively characterized. By integrating phenotypic readouts in combination with latest advances in high-throughput single-cell RNA-sequencing approaches, cooperativity and impact of these mutations on individual cell types of the hematopoietic system were delineated and gene regulatory networks which are altered upon the expression of an Idh1-R132H or a DNMT3A-R882H mutation were identified. At a phenotypic level, neither an Idh1-R132H mutation alone nor in combination with a DNMT3A-R882H mutation resulted in the development of myeloid malignancies, suggesting a restricted oncogenic potential of these mutations and additional intrinsic or extrinsic factors to be required for further malignant transformation. However, Idh1-R132H mutated hematopoietic stem cells displayed increased engraftment and reconstitution potential during serial transplantations and featured aberrant expression of genes associated with DNA damage and DNA repair. Furthermore, both Idh1-R132H single-mutant and Idh1-R132H DNMT3A-R882H double-mutant mice displayed aberrant differentiation patterns predominantly affecting the myelo-monocytic lineage, culminating in a favored monocytic cell fate and increased monocyte and monocyte progenitor counts in the bone marrow. By employing a multi-layered single-cell transcriptome analysis of nearly all cell types within the hematopoietic compartment, differentiation trajectories from hematopoietic stem cells towards mature differentiated cells were reconstructed and underlying molecular defects characterized. Pseudotime-inferred myeloid lineage trajectories revealed an aberrant lineage specification in particular for Idh1-R132H DNMT3A-R882H double-mutated myeloid progenitor cells, resembling a differentiation arrest at the stage of common myeloid progenitors and an ineffective hematopoietic differentiation as seen in myelodysplastic syndromes. At the molecular level, this aberrant population was characterized by an altered metabolic signature and elevated Myc signaling, which is involved in the regulation of terminal myeloid differentiation. Importantly, we could correlate this transcriptome-defined population to a surface marker-defined population, allowing the prospective isolation of these cells for further investigation. Independent of a DNMT3A-R882H mutation, the expression of an Idh1-R132H mutation resulted in the deregulation of several key regulatory factors which either orchestrate monocyte and macrophage development or their activation upon inflammatory stimuli. In line with this, monocyte progenitor cells displayed elevated interferon signaling levels, suggesting that a proinflammatory environment is a common characteristic of an Idh1-R132H mutated hematopoietic compartment and could contribute to leukemic transformation upon additional events. In summary, the experimental framework presented in this thesis enhanced our understanding of how IDH1-R132H mutations alone or in combination with a DNMT3A-R882H mutation in patients synergistically drive leukemia initiation and progression. The identified molecular characteristics will be of benefit in designing treatment strategies for patients carrying IDH1-R132H and DNMT3A-R882H mutations and can be used as a resource when studying these mutations in the context of altered physiological conditions and upon additional extrinsic stimuli.
Durch die Messung der 23Na-Konzentration ist eine Beobachtung von physiologischen Prozessen in vivo möglich, da Natrium an essentiellen Zellprozessen maßgeblich beteiligt ist. Mit der 23Na-Magnetresonanztomographie kann die 23Na-Konzentration im Gewebe nicht-invasiv quantifiziert werden. Obwohl bereits Studien zur 23Na-Quantifizierung existieren, werden Variationen im Messaufbau und in der Versuchsdurchführung noch nicht vollständig durch Korrekturmethoden ausgeglichen. Dadurch sind bisherige quantitative Studien nur schwer miteinander vergleichbar und können systematische Abweichungen in der Quantifizierungsgenauigkeit aufweisen. Ziel dieser Arbeit war die Entwicklung und Analyse von Korrekturmethoden, um eine akkurate Quantifizierung der Gewebe-Natriumkonzentration im Kopf und Abdomen zu ermöglichen.
Bei Kopfaufnahmen wurden unter Verwendung der, in der Literatur vorgeschlagenen, Birdcage-Spule gute Signalstärken erreicht. Im Gegensatz dazu konnte bei der abdominellen Anwendung eine optimale Spulenkonfiguration mit größeren Kanaldurchmessern (200 mm) als erwartet gefunden werden, wodurch beispielsweise das Signal-zu-Rausch Verhältnis im Zentrum der Aufnahme von 48 auf 112 gesteigert wurde. Bestehende Korrekturmethoden des Sendefeldes erwiesen sich als ausreichend. Änderungen in der Quantifizierung durch ein inhomogenes Sendefeld waren für typische Geweberegionen nicht signifikant. Bei der Auswertung von Geweberegionen in der Spulenperipherie traten jedoch im ungünstigsten Fall Abweichungen von bis zu 35,7% auf. Bei der Korrektur des Empfangsfeldes war die bestehende Stand-der-Technik Methode nicht ausreichend. Eine neue Methode (Modell-Methode) wurde entwickelt, welche die Quantifizierungsgenauigkeit um 52,3% steigerte und eine 6,7% höhere Genauigkeit als der Stand-der-Technik erzielte. Gleichzeitig benötigt die Modell-Methode keine zusätzliche Messzeit und ist unabhängig vom Messaufbau. Eine Untersuchung der Auswirkung von klinisch genutztem Kontrastmittel auf die 23Na-Quantifizierung ergab keinen signifikanten Einfluss bei der Nutzung einer Quantifizierungssequenz. In jeweils einer Schlaganfall- und Prostatastudie wurden die Korrekturmethoden evaluiert und führten zu gemessenen quantifizierten 23Na-Konzentrationen in Übereinstimmung mit Literaturwerten.
Für klinische Studien ergibt sich, dass zur Messzeitreduktion bei den meisten Anwendungen die Korrektur des Sendefeldes im Kopf und Abdomen sowie die Korrektur des Empfangsfeldes im Kopf vernachlässigt werden kann. Im Abdomen ist die Korrektur des Empfangsfeldes essentiell. Statt der bisherigen Stand-der-Technik Methode sollte dazu die neu entwickelte Modell-Methode verwendet werden. Die 23Na-Messung zur Quantifizierung kann flexibel ohne Beachtung einer Kontrastmittelgabe im klinischen Protokoll platziert werden. Eine flexible Platzierung im klinischen Protokoll ermöglicht es beispielsweise Protokollpausen auszunutzen. Eine Verwendung dieser ermittelten Erkenntnisse in einer klinischen Studie ermöglicht eine akkurate Quantifizierung der Gewebe-Natriumkonzentration und führt zu einem Biomarker in der klinischen Diagnostik und Therapieplanung bei Pathologien wie beispielsweise dem Schlaganfall oder der Tumorerkrankung.
Schlafstörungen treten häufig in Zusammenhang mit chronischen Schmerzerkrankungen auf. Sowohl die Prävalenz von Schlafstörungen, als auch die Prävalenz von Schmerzerkrankungen liegen bei Frauen deutlich höher als bei Männern. Die Dissertation „Humanexperimentelle Untersuchungen zu geschlechtsabhängigen Effekten von Schlafentzug auf Nozizeption und Ängstlichkeit und ihre Korrelation mit (neuro)endokrinen Parametern" befasst sich mit den Auswirkungen von Schlafentzug auf das Schmerzempfinden und die möglichen dahinterstehenden Pathomechanismen. Dazu wurden zusätzlich die Auswirkungen von Schlafentzug auf das schmerzmodulierende System, auf psychopathologische Eigenschaften und auf hormonelle Parameter untersucht. Um geschlechtsspezifische Unterschiede darstellen zu können, wurde das Schmerzempfinden in einem cross-over Design sowohl bei 10 männlichen, als auch bei 10 weiblichen, gesunden Probanden (23,7 ± 2,2 Jahre) unter den Schlafkonditionen „eine Nacht Schlafentzug“ (SE) und „habitueller Schlaf“ (HS) untersucht. Dazu verwendeten wir das standardisierte Protokoll zur Quantitativen Sensorischen Testung und den Cold Pressor Test. Eine Nacht SE führt unabhängig vom Geschlecht zur Hyperalgesie (CPT, HPT, MDT, P<0.05), jedoch nicht zu Spontanschmerz. Im Vergleich zeigt sich bei Frauen eine stärkere Hitzeschmerzempfindlichkeit, während Männer eine höhere Empfindlichkeit für spitze Schmerzreize entwickeln (Schlaf * Geschlecht < 0.05). Weiterhin konnte ein signifikanter geschlechtsspezifischer Unterschied in Bezug auf die zeitliche Summation spitzer Schmerzreize und die konditionierte Schmerzmodulation beobachtet werden. Psychopathologisch führt SE zu erhöhter Ängstlichkeit (P<0.05), einer Reduktion des positiven Affekts (P<0.01) und zu verminderter Vigilanz (P<0.05). Auf hormoneller Ebene bewirkt SE unabhängig vom Geschlecht eine Reduktion von Testosteron (P<0.01). Bei Frauen führt SE zu einem Anstieg von Oxytocin (P<0.05). In Zusammenschau bestätigt unsere Studie mit gesunden Probanden, dass eine Nacht Schlafentzug zu Hyperalgesie führen kann (Kundermann et al., 2004a; Lautenbacher et al., 2006; Moldofsky and Scarisbrick, 1976). Dabei wurden geschlechtsspezifische Unterschiede entdeckt, die das Verständnis über die Pathomechanismen bei der Entwicklung von Hyperalgesie nach Schlafentzug erweitern. Die Studie hat klinische Bedeutung in Bezug auf mögliche geschlechtsspezifische therapeutische Ansätze bei Schmerzerkrankungen und Schlafstörungen.
The etiology of breast cancer (BC) involves both non-genetic and genetic factors. Environmental and lifestyle factors such as age, use of menopausal hormone therapy, smoking, and body mass index have been associated with the risk of developing BC. Genetic susceptibility determined mainly by family history and ethnic background have an important role in the risk of developing this disease (Dossus and Benusiglio, 2015). In recent years, novel variants robustly associated with BC risk have been identified in large-scale genetic association studies in women of European and Asian origin. However, few studies directed towards the identification of BC susceptibility variants have been conducted among Latin American and Hispanic populations.
This thesis examined the contributions of genetic ancestry, established risk factors, and newly identified susceptibility variants to BC risk in Colombia. A total of 2,045 participants from the Colombian Breast Cancer Case-Control study were included in this analysis: 1,022 BC patients, and 1,023 healthy controls. BC patients were unselected for family history and age at BC diagnosis. European, Native American, and African ancestry proportion were quantified in each woman based on 30 ancestry informative markers, aiming to obtain the relationship between ancestry and BC risk. Seventy-eight previously identified common BC susceptibility variants were genotyped and associations of these variants with BC risk in the Colombian population were determined. To assess the interactions between the variants and ancestry proportions logistic regression models were applied. Native American proportions were lower in Colombian BC patients than in unaffected controls (P-value=5.2x10-16). This difference translated into an unadjusted decreased BC risk of 2.6% per each 1% increase in the Native American proportion (95% CI: 2.0-3.2). Associations with BC risk in Colombian women were obtained for thirteen variants, which in comparison with European women have partially different risk effects and allele frequencies. The risk effects of rs941764 (CCDC88C) and rs3803662 (TOX3) was controlled for ancestry proportions. One variant was associated with estrogen receptor negative (ER-), seven with estrogen receptor positive (ER+), and three with ER+ and ER- disease. The variance in BC liability due to susceptibility variants in European and Colombian women was estimated. Out of 13 variants associated with BC risk in Colombia, four explained a larger attributable heritability in Europe than in Colombia and nine revealed larger attributable heritability in Colombia than in Europe.
Area under the Receiver Operating Characteristic curves (AUCs) with their corresponding 95% CIs were estimated for established risk factors, genetic ancestry, and common BC susceptibility variants based on risk estimates from the literature and own Colombian data. The discriminative ability to separate Colombian cases and controls of family history of BC in first-degree female relatives (AUC=0.58) and the combination of all 13 associated risk variants (AUC=0.57) were similar to the discriminative ability of Native American proportions (AUC=0.61).
The findings demonstrate that individual ancestry proportions predict BC risk in Colombia as accurately as established BC risk factors. Combining Native American proportions, established risk factors, and newly identified genetic susceptibility variants could translate in promising clinical strategies on BC prevention in Latin American and Hispanic women.
Magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) can be used to investigate the quadrupolar nuclei 23Na and 35Cl, each with a nuclear spin of 3/2. The Na+ cations and Cl- anions are involved in cellular functions and can undergo quadrupolar interactions with oppositely charged macromolecules. These interactions give rise to triple-quantum (TQ) signals. Compromised physiological functions change the macromolecular composition and ion content, which can be investigated with TQ MRI/MRS. The goal was to develop a sequence to acquire single-quantum (SQ) and TQ images and to map relaxation parameters in vivo within one measurement. First, a density-adapted radial MRI technique (DA-R) was implemented at a 9.4 T scanner. Phantom images demonstrated superior image quality and measurement time efficiency. High-resolution 23Na and 35Cl images allowed for distinction of anatomical features in rat. Second, TQ spectroscopy with time-proportional phase incrementation (TQ-TPPI) was used to acquire data in cells and in rat head. The results revealed interesting discrepancies in 23Na and 35Cl TQ signals, uncovering differences in the quadrupolar interactions of Na+ and Cl- on a molecular level. Finally, TQ-TPPI was combined with DA-R to create TQ and SQ TPPI imaging (TASTI). The sequence was sucsessfully applied to rat head. For the first time, localized ratios between TQ and SQ signal were mapped in different head regions. Furthermore, it enabled the distinction between TQ signal fractions in the intra- and extracellular space. With its ability to analyze local changes in ion content, relaxation times and TQ signal, the TASTI sequence has the potential to become the one tool to combine all major approaches to address 23Na NMR.
Malaria is still a disease of global health significance. Its causative agent, Plasmodium, has a complex lifecycle alternating between the female Anopheles mosquito and the vertebrate host. The blood stage of the infection is thought to cause the clinical symptoms of the disease and is also targeted by the humoral immune response of the host. Natural acquired immunity to malaria is largely mediated by immunoglobulins and is reflected both by a lower prevalence of infection with age and lower rates of disease. Previous studies have generally focussed on understanding the protective role of naturally acquired functional antibodies targeting the mature infected erythrocyte (mIE) and highly immunogenic merozoite. However, the ring-infected erythrocyte (rIE), has hardly been explored to date in the context of naturally acquired protective immunity. As it is the stage of the infection that is predominantly found in the peripheral circulation, its immunological significance is uncertain. This thesis was aimed at contributing to this knowledge gap by investigating the role and significance of rIEs in protective immunity. Firstly, to investigate the immunogenicity of rIEs, I tested whether they were recognised by malaria-immune antibodies using immunofluorescence and flow cytometry. I confirmed that malaria-immune antibodies do bind to rIEs using both laboratory-adapted (lab) and field isolates. This binding was strongly correlated with that directed against mIEs and merozoites, both of which have been shown to be important targets of protective immunity. Malaria-immune antibody binding to rIEs had no direct inhibitory effect on parasite survival and maturation. In separate assays, I tested whether rIEs were retained in the spleen independently of antibody using a microsphiltration assay. As has been previously reported, rIEs were retained in the spleen and I demonstrated for the first time that the retention rate was comparable between lab and field parasite isolates of the same age, suggesting that this may be a mechanism of parasite clearance that contributes to the low parasite densities observed in malaria endemic areas. Secondly, to investigate the likely physiological relevance of antibody binding to rIEs during P. falciparum infection, I tested whether the in vitro opsonic phagocytosis of rIEs predicted the outcome of infection in a controlled human malaria infection (CHMI) study. I established the flow cytometry based opsonic phagocytosis assay (OPA) of rIEs in our laboratory. I showed for the first time, to the best of my knowledge, that opsonic phagocytosis of rIEs was significantly associated with the outcome of infection (lower parasite densities and the need for treatment) in the CHMI study. Interestingly, phagocytosis of uninfected erythrocytes (uEs) in the ring culture was higher in untreated as compared to the treated individuals, suggesting a potential contribution to anaemia, as others have reported. In addition, I developed competition OPAs and used stage-specific spent media, to demonstrate that merozoite specific antibodies mediated the observed opsonic phagocytosis of the ring culture cells and uEs incubated in spent media respectively. Finally, to investigate the specific parasite antigens on the surface of ring culture cells and uEs incubated in spent media, I employed surface trypsinization, followed by mass spectrometry of supernatants. This is the first surface proteomics study to be conducted on rIEs and in this single study I was able to identify parasite proteins on the surface of rIEs and uEs that had previously been identified singly, in a piecemeal fashion (EBA140, EBA175, RAP1, RAP2, RhopH1, RhopH2, RhopH3 and MCP1), and ENO which I showed for the first time. These proteins have been speculated to be transferred by the merozoite to the surface of the newly invaded erythrocyte during invasion and have also been shown to be correlates of protective immunity, with the exception of MCP1, whose role in parasite clearance and protective immunity has not yet been explored. This thesis shows for the first time that antibody mediated clearance of rIEs in vitro, is correlated with the outcome of P. falciparum infection in a CHMI study and was mediated by anti-merozoite antibodies. This study also demonstrates that there are shared parasite targets between merozoites and rIEs. I conclude that rIEs are not immunologically inert and are targeted by antibodies leading to parasite clearance through mechanisms such as phagocytosis. This activity was significantly associated with lower parasite densities and resistance against clinical symptoms in a human malaria challenge study suggesting that it contributes to protective immunity. The enhanced phagocytosis of uEs in protected individuals suggest a potential detrimental impact of anaemia that requires further investigation.
This study investigates the topic of rainwater harvesting on the Karak Plateau located in rural Jor-dan. The term rainwater harvesting describes various methods and structures employed for the collection, storage and use of rainwater and resulting (on-site) surface runoff. Within the scope of traditional water management, over millennia, many of these techniques were developed, refined and applied in Jordan, as well as in other, especially semiarid, regions of the world. This tradition is rooted in the natural water shortage of the plateau and frequent absence of other reliable sources of fresh water. Today, population growth, climate change and local effects of globalization and global change are leading to growing water shortages in the MENA region (Middle East and North Africa) and many other parts of the world. In the search for sustainable solutions for this problem, traditional as well as new strategies of rainwater harvesting have recently been receiving increasing interest. The present study contributes to an enhanced understanding of the applicability and the potential of some of the most widely-used, traditional rainwater harvesting methods, especially the use of cisterns. The mapped structures were examined taking into account the settlement history and the respective circumstances of the natural and human environment. Possible determining factors concerning site preferences and resulting patterns in the spatial distribution of rainwater harvesting sites have been detected. The diachronic comparative analysis revealed changes in human-environment-interactions, particularly with regard to the significance and management of local water resources under natural shortage. The collected data enabled the detailed estimation of the rainwater harvesting potential and the suggestion of possible ways to improve and expand current rainwater harvesting schemes and efforts. The integration and possible role of rainwater harvesting strategies were discussed with regard to modern, sustainable water management and supply. Additionally, the applicability of modern geoinformation techniques was evaluated. Remote sensing techniques and methods of image analysis, particularly with regard to the interpretation of satellite images of very high resolution, were examined especially. The combination of ground truth and other information from fieldwork and remote sensing-based data and techniques has proven most suitable and efficient. The mostly remote sensing-based mapping of rainwater harvesting structures and the establishment of a comprehensive database formed the basis for all subsequent analysis and possible further, sustainable planning steps. The semiautomatic analysis of the satellite imagery provided detailed information on land use/land cover and building rooftops and thus decisively contributed to the improvement of the (input) data basis. All in all, the collected data enabled a significantly enhanced, quantitative estimation of the rainwater harvesting potential of the study area. Many of the gained findings and insights can be transferred onto other dry areas and regions with similar environmental or socio-economic conditions.
The tumor suppressor p53 primarily functions as a transcription factor responding to a myriad of cellular stresses. It is a pivotal and pleiotropic regulator in the stress-induced cellular response networks. Diverse activities of p53 are important not only in DNA repair, induction of cell cycle arrest and apoptosis, but also in senescence, autophagy and metabolism. In cells infected with human papillomaviruses (HPVs), the viral oncoproteins E6 and E7 target the tumor suppressors p53 and pRb, respectively, for degradation and inactivation. HPV E6 and E7 synergistically act to promote uncontrolled cell divisions and inhibit apoptosis. Persistent infections with high-risk HPVs are closely linked to cervical cancer as well as other malignancies in the anogenital and oropharyngeal region. Our previous lab results found that HPV E6/E7 oncogenes are repressed under hypoxia, a condition that is frequently detected in solid tumors. Unlike the reactivation of pRb, p53 protein levels did not increase in E6/E7-repressed hypoxic HPV16-positive cancer cells, but even decreased further. The present study aimed to delineate the dynamics of p53 under hypoxic conditions as well as the mechanisms underlying this regulation and elucidate the role of p53 regulation for downstream responses and cellular outcomes/fates in hypoxic HPV16-positive cancer cells. It was revealed that despite a continuous repression of E6/E7 oncogenes, p53 did not immediately recover, but instead showed a biphasic regulation (rapid and strong depletion, then marked recovery). The initial hypoxic reduction of p53 was predominantly mediated via a lysosome-dependent mechanism. The biphasic regulation of p53 appears to serve as a survival and protective strategy of hypoxic HPV16-positive cancer cells under stress conditions. The modulation on p53 downstream target genes that coincides with p53 protein dynamics may contribute to enhance cellular adaptation to hypoxia. p53 target genes associated with terminal fates such as cell death (apoptosis) and permanent cell cycle arrest (senescence) are inactivated through p53 depletion by hypoxia, protecting cells from committing to an irreversible fate. After prolonged hypoxia, the restored p53 might be required by HPV16-positive cancer cells to maintain cellular homeostasis and select cells resistant to cell death by induction of apoptotic genes. Hypoxia-associated initial reduction of p53 facilitates the induction of autophagy, which is critical for the evasion of senescence by hypoxic HPV16-positive cancer cells. Collectively, these findings reveal a new regulation pattern of p53 by hypoxia and provide new insights into the role of p53 regulation in downstream responses and cellular adaptation to hypoxia in HPV16-positive cancer cells. This study further has implications for the development of new treatment strategies.
Magnetic resonance (MR)-guided radiotherapy involves complex irradiation- and imaging devices (MR-Linacs), as well as complex treatment procedures. To assure accurate patient treatments, both have to be tested for proper functioning. Here, new methods to simultane-ously measure the isocenter alignment accuracy and the geometric image distortions of clin-ical MR-Linacs were developed. A new phantom was designed, which includes a polymer dosimetry gel-container to visualize the beams from a star shot and to identify the radiation isocenter position in MR as well as a regular grid used to visualize the MR image distortions in 3D. It was found that the gel can be evaluated immediately after irradiation with a geo-metric accuracy comparable to that of radiochromic films. The method was applied on a clinical 0.35 T MR-Linac and the isocenter alignment accuracy in 3D was found to be (0.8 ± 0.9) mm. The spatial image distortions after machine-specific correction were (0.60 ± 0.28) mm and 99.82% of the 1330 evaluated control points within a 140 mm sphere had devi-ations below 1.5 mm. Finally, a 3D printing materials and printing technique, compatible with the polymer dosimetry gel, was identified for future designs of anthropomorphic phantoms to be used in end-to-end tests in MR-guided radiotherapy.
Cased-based Reasoning (CBR) ist ein Verfahren des maschinellen Lernens, das aufgrund des Wissens vergangener ähnlicher Fälle und ihren Lösungen Prognosen und Hilfestellungen zu einem neuen Fall liefert. Es besteht aus vier eigenständigen Phasen, die einen Kreislauf bilden. Der neue Fall und seine gewonnene Lösung werden im nächsten Durchlauf Teil dieses Kreislaufes und führen zu einer Erweiterung des bisherigen Wissens. Die Leistung des CBR hängt zum einen von der Größe und Informationsdichte der Datenbank ab und zum anderen maßgeblich von der korrekten Rangfolge der ähnlichen Fälle in der Retrieve-Phase, da alle folgenden Phasen von deren Ergebnissen betroffen sind. Mit den rasant wachsenden Datenbanken der heutigen Zeit steigert sich allerdings aus den verschiedensten Gründen auch die Menge an fehlenden Daten innerhalb dieser Datenbanken. Diese Tatsache hat zur Folge, dass das gesamte CBR auf solchen Datenbanken destabilisiert wird und besonders die Rangfolge der Retrieve-Phase darunter leidet, weil unvollständige Fälle weniger verlässlich gewertet werden können als vollständige Fälle. Überraschenderweise existieren für diese Problematik bisher kaum Arbeiten, welche den Einfluss der fehlenden Daten auf die Retrieve-Phase des CBR untersuchen und eine zuverlässige Lösung basierend auf modernen Verfahren bieten. Insbesondere fehlt es an einer umfassenden Lösung, welche unterschiedliche Arten von Variablen betroffen von unterschiedlichen Typen von fehlenden Daten verarbeiten kann. Als Antwort auf diese Fragestellung wird in dieser Arbeit das Multiple Retrieval Case-based Reasoning (MRCBR) vorgestellt und evaluiert. MRCBR ist ein Framework für CBR auf unvollständigen Datenbanken, das eine möglichste korrekte Rangfolge der ähnlichen Fälle mit Hilfe von modernen Methoden des maschinellen Lernens und der Statistik gewährleistet. Es bezieht die Verteilung der vollständigen Daten und die mögliche Verteilung der unvollständigen Daten in seine Berechnungen mit ein, indem es die Vorteile der Multiple Imputation und CBR in einem Verfahren effizient vereint. Das Verfahren wurde als Erweiterung des klinischen Entscheidungsunterstützungssystems für das Tumorboard (CBR-TDS) entworfen, damit dieses auf der unvollständigen Datenbank der Klinik für Strahlentherapie und Radioonkologie (MOSAIQ) fehlerfrei und vertrauenswürdig arbeiten kann. In diesem Hinblick wurde es optimiert und getestet. Des Weiteren ist es jedoch ein allgemeines Verfahren, das nicht auf diesen Anwendungsbereich allein beschränkt ist und für jedes CBR System angepasst werden kann. Es erlaubt die Verarbeitung der gängigen Arten von Variablen in medizinischen Datenbanken, numerische und kategoriale Variable, und aller Typen von fehlenden Daten. Die Methodik des MRCBR wurde mit acht konkurrierenden Methoden des letzten Standes der Technik verglichen, welche in der Lage sind CBR im Kontext von fehlenden Daten auszuführen. Die Ergebnisse auf der wahren vollständigen Datenbank bildeten die Referenz für die Einstufung der Ergebnisse der unterschiedlichen Verfahren mit Hilfe zweier verschiedener Fehlermaße. In vier repräsentativen Experimenten bestehend aus mehreren eigenständigen Versuchen wurden verschiedene Umgebungen und Bedingungen der fehlenden Daten realistisch simuliert und untersucht. Auch der Einfluss der Größe der Datenbank und andere Parameter des CBR wurden in Betracht gezogen. Für eine korrekte statistische Auswertung entsteht das Ergebnis jeder Methode in jedem Versuch aus der Mittelung von 200 einzelnen Ergebnissen. MRCBR hat in so gut wie allen Versuchen die bestehenden Methoden übertroffen und zeigte verlässliche stabile Ergebnisse in fast jedem der Experimente. Besonders in großen Datenbanken und einer großen Anzahl von unvollständigen Variablen konnte es seinen Abstand zu den anderen Methoden noch vergrößern. Die Analyse des Verhaltens der Verfahren zeigte, dass es keine Möglichkeit gibt fehlende Daten zu ignorieren ohne die Leistung des CBR drastisch zu reduzieren.
Accurate models of turbulent dispersion are required for simulating the near-field concentration distribution of pollutants. However, high-resolution measurements of turbulent dispersion in the atmospheric boundary layer are sparse. This thesis describes the three artificial release experiments in the summers of 2017-2019 within the comtessa (Camera Observation and Modelling of 4D Tracer Dispersion in the Atmosphere)project and presents derived results for the turbulent dispersion of tracer puffs. Instantaneous puffs of sulfur dioxide (SO2) were released from a tower on a military site in Norway. Column-integrated SO2 concentrations were observed with SO2 cameras from up to six viewing directions while the atmospheric flow was characterised by eddy covariance measurements at different altitudes along the release tower. A novel simplified tomographic approach was applied to reconstruct the dispersion of tracer puffs separated into their centre of mass trajectories and their dispersion around the centre of mass. Using ensembles of puff releases, the meandering, relative and absolute dispersion as well as the Lagrangian velocity autocorrelations were measured. The ratio of Lagrangian and Eulerian time scales was estimated to a lower bound of TL/TE = 0.33 * 1/i where i is the turbulence intensity; agreeing with previous studies.
Verbindungen des Phosphors spielen eine Schlüsselrolle als Flammschutzmittel FSM)für Polymere und Coatings, da sie aufgrund der chemischen Vielseitigkeit von Phosphor, aus Gründen der Nachhaltigkeit und nicht zuletzt einer hohen Effektivität auch bei niedrigen Beladungen zunehmend halogenierte FSM substituieren. Das Ziel dieser Dissertation war die Entwicklung neuer phosphorbasierter FSM für Epoxidharze, die über mindestens zwei Phosphoratome mit idealerweise unterschiedlichen Substitutionsmustern verfügen, sodass wichtige Struktur-Eigenschaftsbeziehungen hergestellt werden können. Dazu wurden vor allem Derivate des 9,10-Dihydro-10-oxa-phosphaphenanthren-10-oxids (DOPO), einem wichtigen gasphasenaktiven FSM, dargestellt. Das erste Synthesekonzept beinhaltete zunächst die Darstellung N-phosphorylierter Iminophosphorane mittels Staudinger-Reaktion durch die Umsetzung von Phosphoryla- ziden mit verschiedenen trivalenten Phosphorverbindungen (Phosphine, Phosphonite und Phosphite). Die thermischen Stabilitäten betreffende Untersuchungen bestätigten, dass phosphin-basierte N-phosphorylierte Iminophosphorane wie z.B. DOPO-N=PPh3 thermisch stabiler sind als deren phosphitstämmige Analoga wie z.B. DOPO-N=P(OPh)3. Diese zunehmende thermische Stabilität spiegelt sich in den entsprechenden Winkeln und Abständen der N-phosphorylierten Iminophosphorane wider. Das zweite Synthesekonzept hatte die Erweiterung der Staudinger-Reaktion von DOPO-N3 mit pentavalenten Phosphorverbindungen des Typs RR‘(O)P-H zum Ziel. Mit Hilfe der Röntgenkristallstrukturanalyse wurden den Produkten Imidodiphosphorstrukturen (R2(O)P-NH-P(O)R2) zugewiesen. In einem dritten Synthesekonzept wurden ausgehend vom Vinylphosphonsäuredimethylester (VPADME) und unterschiedlichen P-H-Verbindungen zunächst mittels Phospha-Michael-Addition Phosphonsäureester dargestellt. Diese wurden zu den entsprechenden freien Phosphonsäuren hydrolysiert und anschließend in die korrespondie- renden Melaminsalze überführt. Zur Untersuchung der FSM-Wirkung wurden ausgewählte Verbindungen dieser Stoffklassen in unterschiedliche Epoxidharze eingearbeitet - einem Diglycidylether von Bisphenol A (DGEBA) und einem glycidierten Phenolnovolak (DEN 438) und jeweils mit Dicyandiamid (D) als Härter sowie Fenuron (F) als Beschleuniger ausgehärtet. Dabei wurde die Reaktivität gegenüber der Oxirangruppe eingehend untersucht. Es wurde gezeigt, dass sich alle VPADME-stämmigen ethylenverbrückten Bisphosphorverbindungen, die Iminophosphorane DOPO-N=P(OMe)3 und DOPO-N=P(OPh)3 sowie die Imidodiphosphorverbindung DOPO-NH-DOPO reaktiv in die Epoxidharzmatrix einarbeiten lassen. Alle flammgeschützten Epoxidharze wurden auf ihre Materialeigenschaften und Brennbarkeit untersucht und unter Berücksichtigung der Substitutionsmuster an den Phosphoratomen bewertet. Zusätzlich wurden die Flammschutzmechanismen ausgewählter FSM in DGEBA/D/F und DEN 438/D/F mittels Cone-Kalorimetrie untersucht. Die Flammschutztests ergaben, dass die DOPO-stämmigen N-phosphorylierten Iminophosphorane vor allem in dem Harzsystem DEN 438/D/F einen guten Flammschutz bewirken. Allen voran bewirkte DOPO-N=PPh3 aufgrund seiner primären Gasphasenaktivität in diesem Harzsystem den besten Flammschutz, der sämtliche Referenzverbindungen übertraf. In DGEBA/D/F zeigte der ethylenverbrückte Bisphosphonsäurester (EBBPE) aufgrund seiner primären Aktivität in der kondensierten Phase sehr gute Flammschutzeigenschaften, die denen gängiger Referenzverbindungen entsprechen. Mit dieser Arbeit wurde gezeigt, dass sich phosphororganische Verbindungen, die über mindestens zwei Phosphoratome mit unterschiedlichen Substitutionsmustern verfügen, in einer Eintopfreaktion in guten Ausbeuten herstellbar sind und sehr gute Brandschutzergebnisse in unterschiedlichen Epoxidharzen erzielen.
The ability to cope with large perturbations is essential to avoid falling for humans as well as for humanoid robots. Every day millions of people are affected by injuries due to falling. This is a huge problem not only for the individuum but also for the society as it costs the health care systems billions of euros. Also in the field of humanoid robots fall avoidance is very important as it protects robots against breakage. In this thesis, the problem of fall avoidance is addressed using a combination of optimization, human-modeling and recorded push recovery motions. The aim is to identify the principles that lead to human-like push recovery motions. The human is modeled by rigid segments combined by joints leading to an underactuated multi-body representation. These models are included in multiple stage optimal control problems to reconstruct and sythesize human push recovery motions considering the dynamics of a human over the whole time horizon. Due to the high nonlinearity, the optimization problem is solved based on a direct multiple shooting method. To analyze the human push recovery motions, dynamically-consistent motions for the model that closely track experimental data are produced. The joint angles and joint torques for the human model controlled by joint torque derivatives are compared for perturbed and unperturbed motions from two subjects. The results verify the assumption that the heavier the perturbation is and the higher it is applied at the upper body, the larger are the resulting joint torques. We show that including optimally chosen spring-damper elements in the joints can reduce the active joint torques significantly. We further exploit our motion reconstruction approach to determine the states that are most affected during a perturbation. Relevant parameters such as the orientation and position of the head and body, joint angles and torques of the perturbed motions are analyzed for deviations to the unperturbed motions at the point in time when the push occurs. Identifying the point in time when the model states of the perturbed motions differ from the unperturbed motions, the reaction times are determined. To better understand human push recovery motions, we also investigate in a motion sythesis approach. This approach enables a control hypothesis, in the form of a specific objective function, to be formed. The minimization of effort combined with a periodicity formulation results in human-like motions and the influence of the push strength is analyzed. Formulating the objective function as a weighted linear combination of possible optimality criteria provides the possibility to analyze different optimality criteria and their resulting motion. The difficulty is, that for a given motion, it is not known, which criteria lead to that specific motion. In this thesis, the results for different basal objective functions are analyzed. These studies prepare to determine the optimal weights of the criteria by including the presented motion generation formulation in an inverse optimal control problem. Having analyzed general weights that lead to a good approximation of the human recovery motions, the resulting objective function can be used to generate push recovery motions also for humanoid robots or assistive devices such as exoskeletons. To show another application in the improvement of technical assistive devices, we include two combined human exoskeleton models of different weights in our calculations. This allows us to analyze the joint torques for these models including the exoskeletons and compare the results to a human model. As the resulting joint torques are quite large, we also formulate combined human exoskeleton models with passive spring-damper elements that act in parallel to the active torques. This compliant formulation leads to a significant reduction of the active joint torque needed for the recovery motion. The reduction of the active joint torques allows the reduction of energy needed for the recovery motion or can enable the recovery from stronger perturbations.
Considering a family of statistical, linear, ill-posed inverse problems, we propose their study from two perspectives, the Bayesian and frequentist paradigms. Under the Bayesian paradigm, we investigate two different asymptotic analyses for Gaus- sian sieve priors and their hierarchical counterpart. The first analysis is with respect to an iteration procedure, where the posterior distribution is used as a prior to compute a new posterior distribution while using the same likelihood and data. We are interested in the limit of the sequence of distributions generated this way, if it exists. The second analysis, more traditionally, investigates the behaviour of the posterior distri- bution as the amount of data increases. Assuming the existence of a true parameter, one is then interested in showing that the posterior distribution contracts around the truth at an optimal rate. We illustrate all those results by their application to the inverse Gaussian sequence space model. Finally we exhibit that the posterior mean of the hierarchical Gaussian sieve prior is both a shrinkage and an aggregation estimator, with interesting optimality properties. Motivated by the last findings about posterior mean of hierarchical Gaussian sieves, we propose to investigate the quadratic risk of aggregation estimators, which shape mimics the one of the above-mentioned posterior means. We introduce a strategy, relying on the decomposition of the risk, which allows to obtain optimal convergence rates in the cases of known and unknown operator, for dependent as well as absolutely regular data. We demonstrate the use of this method on the inverse Gaussian sequence space model as well as the circular density deconvolution and obtained optimality results under mild hypotheses.
This dissertation deals with the efficient numerical solution of switched optimal control problems whose dynamics may coincidentally be affected by both explicit and implicit switches. A framework is being developed for this purpose, in which both problem classes are uniformly converted into a mixed–integer optimal control problem with combinatorial constraints. Recent research results relate this problem class to a continuous optimal control problem with vanishing constraints, which in turn represents a considerable subclass of an optimal control problem with equilibrium constraints. In this thesis, this connection forms the foundation for a numerical treatment. We employ numerical algorithms that are based on a direct collocation approach and require, in particular, a highly accurate determination of the switching structure of the original problem. Due to the fact that the switching structure is a priori unknown in general, our approach aims to identify it successively. During this process, a sequence of nonlinear programs, which are derived by applying discretization schemes to optimal control problems, is solved approximatively. After each iteration, the discretization grid is updated according to the currently estimated switching structure. Besides a precise determination of the switching structure, it is of central importance to estimate the global error that occurs when optimal control problems are solved numerically. Again, we focus on certain direct collocation discretization schemes and analyze error contributions of individual discretization intervals. For this purpose, we exploit a relationship between discrete adjoints and the Lagrange multipliers associated with those nonlinear programs that arise from the collocation transcription process. This relationship can be derived with the help of a functional analytic framework and by interrelating collocation methods and Petrov–Galerkin finite element methods. In analogy to the dual-weighted residual methodology for Galerkin methods, which is well–known in the partial differential equation community, we then derive goal–oriented global error estimators. Based on those error estimators, we present mesh refinement strategies that allow for an equilibration and an efficient reduction of the global error. In doing so we note that the grid adaption processes with respect to both switching structure detection and global error reduction get along with each other. This allows us to distill an iterative solution framework. Usually, individual state and control components have the same polynomial degree if they originate from a collocation discretization scheme. Due to the special role which some control components have in the proposed solution framework it is desirable to allow varying polynomial degrees. This results in implementation problems, which can be solved by means of clever structure exploitation techniques and a suitable permutation of variables and equations. The resulting algorithm was developed in parallel to this work and implemented in a software package. The presented methods are implemented and evaluated on the basis of several benchmark problems. Furthermore, their applicability and efficiency is demonstrated. With regard to a future embedding of the described methods in an online optimal control context and the associated real-time requirements, an extension of the well–known multi–level iteration schemes is proposed. This approach is based on the trapezoidal rule and, compared to a full evaluation of the involved Jacobians, it significantly reduces the computational costs in case of sparse data matrices.
Aufgrund ihrer physikalischen Hauptwirkung ist die HIT-Anlage ein Medizinprodukt, das aus einer Industrieanlage (Beschleuniger, HOAI-Leistung der GSI), einem Medizinprodukt (PT-System, Siemens AG) und einer Maschine (Gantry, MT Mechatronics) zusammengesetzt ist. Da sie vom Universitätsklinikum Heidelberg herstellt, betrieben und angewendet wird, und keine Abgabe an Dritte (Inverkehrbringen) erfolgt, gilt die HIT-Anlage als Eigenherstellung gemäß § 3 Nr. 21 MPG und muss keine CE-Kennzeichnung tragen. Gemäß § 12 Abs. 1 MPG ist die Inbetriebnahme einer Eigenherstellung nur zulässig, wenn deren Hersteller die auf sie anwendbaren Grundlegenden Anforderungen des Anhangs I der Richtlinie 93/42/EWG über Medizinprodukte erfüllt und das für sie vorgesehene Konformitätsbewertungsverfahren gemäß § 7 Abs. 9 MPV durchgeführt hat. Die erstgenannte und somit zentrale Grundlegende Anforderung ist der Nachweis eines positiven Nutzen-/Risiko-Verhältnisses für den Patienten sowie die Gewährleistung der Sicherheit von Patienten, Anwendern und ggf. Dritten bei zweckbestimmungsgemäßer Anwendung des Medizinprodukts, was eine Risikoanalyse, bewertung und beherrschung während seines gesamten Lebenszyklus und somit ein auf objektiven Nachweisen basierendes Risikomanagement impliziert. Artikel 5 Abs. 1 der o.g. Richtlinie enthält ebenso wie § 8 Abs. 1 MPG einen Verweis auf harmonisierte Normen und die mit ihrer Anwendung einhergehende Konformitätsvermutung. Die harmonisierte Norm für das Risikomanagement von Medizinprodukten ist die EN ISO 14971. Im Rahmen meiner Tätigkeit als Risikomanagement-Beauftragte war es meine Aufgabe, die DIN EN ISO 14971 auf die HIT-Anlage anzuwenden, als Dreh- und Angelpunkt der aufgrund der raum- und versionsweisen Übergabe des PT-Systems zwischen 06/2006 und 10/2012 durchgeführten vier Konformitätsbewertungsverfahren und zur Gewährleistung der Nachhaltigkeit des dadurch erzielten Sicherheitsniveaus für Patienten, Anwender und Dritte während der erwarteten Lebensdauer von 25 Jahren. Durch Entwicklung eines Risikomanagementplans und eines sog. RA Gesamtdokuments wurde zunächst der formale Rahmen für die Planung und Erstellung von Risikoanalysen geschaffen. Aufgrund der Größe und Komplexität der HIT-Anlage wurden die Gebäude Infrastruktur und der Beschleuniger hierfür in 9 Gewerke unterteilt, für Zubehör, Arbeitsabläufe und MPG-technische Aspekte der Gantry wurden weitere Risikoanalysen aufgesetzt. Auf Grundlage von Funktionsanalysen wurden diese 12 Risikoanalysen von mit Experten besetzten RA-Teams durchgeführt, wobei systematisch gelistet wurde, welche Ursachen zu welchen Fehlern und welche Fehler zu welchen Gefährdungen führen können, und wie die daraus resultierenden Risiken – Auswirkungen und Wahrscheinlichkeiten – bewertet werden (Risiken vor Maßnahmen). Falls aufgrund der im o.g. Risikomanagementplan festgelegten Akzeptanzkriterien eine Risikominderung erforderlich war, wurden eine oder mehrere Maßnahmen zur Risikobeherrschung spezifiziert, die geeignet waren, das jeweilige Risiko auf ein akzeptables Maß zu mindern. Anschließend wurden die Restrisiken unter der Prämisse bewertet, dass diese Maßnahmen implementiert und wirksam sind (Risiken nach Maßnahmen). Nach vorläufigem Abschluss einer Risikoanalyse wurden die Maßnahmen thematisch zusammengefasst in sog Maßnahmenblätter exportiert, wobei jede Maßnahme einem Maßnahmen- und einem Prüfverantwortlichen zugeordnet wurde (4-Augen-Prinzip), deren Aufgabe es war, die Implementierung der Anforderung(en) durch Bereitstellung eines oder mehrerer objektiver Nachweise zu bestätigen. Danach wurde jede Maßnahme von einem Audit-Team verifiziert, das sie in ein Nachaudit verwies, falls die Anforderung(en) nicht vollumfänglich erfüllt war(en). Am Ende jedes Konformitätsbewertungsverfahrens wurden die Ergebnisse aller Audits des ihm zugeordneten Maßnahmenpakets RA-weise zusammengefasst und den RA-Teams zur Überprüfung vorgelegt, um evtl. Änderungen zu bestätigen, die o.g. Restrisiken zu überprüfen und ggf. zu korrigieren oder durch weitere Maßnahmen zu mindern. Ein übergeordnetes Kern-Team entschied abschließend über die Akzeptanz evtl. Restrisiken oberhalb der Akzeptanzgrenze und die Akzeptanz des Gesamt-Restrisikos. Die Ergebnisse des Risikomanagement-Prozesses wurden in einem Risikomanagementbericht aufgezeichnet. Aus den o.g. von 42 Personen in 3959,85 Stunden erstellten Risikoanalysen wurden 1387 Maßnahmen abgeleitet, die zu 681 Kombi-Maßnahmen zusammengefasst und von 45 Maßnahmen- und Prüfverantwortlichen implementiert wurden. Im Rahmen der vier Konformitätsbewertungsverfahren wurden in 80 Audits in 734,15 Personenstunden aufgrund von Nachaudits und Wiederholungen (bei raum- und/oder versionsspezifischen Anforderungen) insgesamt 1187 Kombi-Maßnahmen verifiziert. Durch aus der Gap Analyse anlässlich eines Normenwechsels (EN ISO 14971:2009 → EN ISO 14971:2012) abgeleitete Aktivitäten konnte die Anzahl der Restrisiken oberhalb der Akzeptanzgrenze von initial 25 bzw. (ohne Mehrfachnennungen) 16 auf 15 bzw. (ohne Mehrfachnennungen) 9 gesenkt werden, wobei es sich z.T. um nicht Ionenstrahl-Therapiespezifische Restrisiken handelt, wie die Bestrahlung von Patienten mit aktiven (z.B. Herzschrittmacher) und nicht-aktiven (z.B. Endoprothesen) Implantaten. Das Gesamt-Restrisiko wurde jedoch stets als akzeptabel bewertet. Um die mit der Anwendung der HIT-Anlage verbundenen Risiken für Patienten, Anwender und Dritte dauerhaft zu beherrschen muss der Risikomanagement-Prozess während ihres gesamten Lebenszyklus aufrechterhalten werden. Nur durch regelmäßige Überprüfung der Wirksamkeit des Maßnahmenbestands, strukturierte Aufarbeitung potentiell sicherheitsrelevanter Informationen und bedarfsorientierte Anpassung der Risikoanalyse(n) bei geplanten Änderungen kann der Regelkreis des Risikomanagements geschlossen und die Sicherheit, Eignung und Leistung der HIT-Anlage auch langfristig gewährleistet werden. Ein Teil des o.g. Maßnahmenvolumens ist auf Wiederholung ausgelegt und wird i.d.R. jährlich und/oder bedarfsorientiert durchlaufen. Die Sammlung und Überprüfung von Informationen aus der Herstellung nachgelagerten Phasen erfolgt sowohl produkt- als auch prozessbezogen: Ersteres geschieht regelmäßig im Rahmen der Marktbeobachtung, bei der HIT-interne und öffentlich zugängliche Informationen auf evtl. Handlungsbedarf bewertet werden. Letzteres findet bedarfsorientiert mittels eines Critical Incident Reporting Systems statt, mit dem Meldungen über kritische Ereignisse bei der Vorbereitung und Durchführung der Ionenstrahl-Therapie aufgearbeitet werden. Unwesentliche Änderungen der HIT-Anlage werden über ein Änderungsverfahren abgewickelt, in dem u.a. eine Klärung der risikotechnischen Implikationen – Neudurchlauf von Maßnahmen und/oder Aktualisierung der Risikoanalyse(n) – enthalten ist. Bei wesentlichen Änderungen ist jeweils ein Konformitätsbewertungsverfahren erforderlich. Darüber hinaus wird die Entwicklung der für die HIT-Anlage relevanten regulatorischen Anforderungen mittels eines Rechtskatasters verfolgt, um darauf ggf. fristgerecht reagieren zu können. Der Vorstand des Universitätsklinikums erhält Jahresberichte, in denen das aktuelle Risikoprofil der HIT-Anlage und die geplanten risikorelevanten Aktivitäten dargestellt werden. Das für die HIT-Anlage etablierte Risikomanagement gemäß DIN EN ISO 14971 erfüllt alle normativen Anforderungen und wird seit Aufnahme des Patientenbetriebs am 15. November 2009 vom gesamten an der Ionenstrahl-Therapie beteiligten Team erfolgreich gelebt. Bisher wurden mehr als 5000 Patienten bestrahlt, wobei die Anzahl der applizierten Fraktionen ca. 14000 pro Jahr beträgt. Verglichen damit ist die Anzahl von 19 gemeldeten CIRS-Fällen in einem Zeitraum von 6 Jahren äußerst gering, zumal es sich bei keinem dieser Ereignisse um ein meldepflichtiges Vorkommnis gemäß § 2 Nr. 1 MPSV handelte. Die Risikomanagementakte umfasst derzeit (u.a. aus Gründen der Transparenz und Nachvollziehbarkeit) mehr als 35000 Anforderungs- und Nachweisdokumente, davon ca. 500 DIN A3 Seiten Risikoanalysen, und wächst aufgrund o.g. Aktivitäten zur Nachhaltigkeit stetig weiter. Unabhängig von geplanten administrativen Vereinfachungen ist das Risikomanagement der HIT-Anlage auch für künftige Weiterentwicklungen der Technologie und ihrer Verwendung gut aufgestellt.
MDSC play a major role in immunosuppression and tumor progression in melanoma. Their recruitment to the tumor is mediated by chemokine receptors and their ligands, in particular by chemokine receptor CCR5. The aims of this thesis were to study the mechanisms of CCR5 upregulation on murine MDSC in melanoma and of the previously observed stronger immunosuppressive phenotype of CCR5+ MDSC as compared to their CCR5- counterparts. IL-6, GM-CSF and IFN-g upregulated Ccr5 mRNA expression in murine myeloid cells, whereas the CCR5 ligands, tumor-derived extracellular vesicles, toll-like receptor ligands and IL-1b failed to affect Ccr5 expression. IL-6 and GM-CSF were able to induce CCR5 surface expression during MDSC in vitro differentiation for four days by a STAT3 dependent mechanism. Importantly, we found four putative STAT3 binding sites in the murine Ccr5 promoters. In addition, the STAT3 inhibitor Stattic abrogated Ccr5 upregulation induced by IL-6 and GM-CSF. In the RET transgenic mouse model of malignant melanoma, IL-6 levels in the tumors correlated with the frequency of tumor-infiltrating CCR5+ MDSC. CCR5+ MDSC showed increased phosphorylated STAT3 levels. In addition to the upregulation of CCR5, IL-6 was responsible for stimulation of Arginase (Arg)1 activity and ROS production upon MDSC in vitro differentiation, inducing increased capacity of MDSC to suppress CD8+ T cell proliferation in a co-culture assay. The upregulation of Arg1 by IL-6 was also STAT3 dependent. In contrast to IL-6, CCR5 ligands failed to induce increased immunosuppressive capacity of MDSC. Altogether, IL-6 upregulated CCR5 and immunosuppressive capacity of MDSC in vitro in parallel, which could explain the elevated expression of immunosuppressive factors Arg1 and ROS on CCR5+ MDSC and their increased ability to suppress CD8+ T cell proliferation. However, we found only a slight increase in tumor-infiltrating MDSC and no increase in CCR5 expression or immunosuppressive factors detectable upon s.c. injection of IL-6 overexpressing Ret cells into wild type mice. In the same model, the tumor growth and mouse survival remained unaltered. Finally, we blocked IL-6 in vivo in RET transgenic melanoma-bearing mice by an anti-IL-6 antibody to decrease CCR5+ MDSC recruitment to the tumor and to inhibit IL-6-induced increase in MDSC immunosuppressive capacity, thereby neutralizing the immunosuppression in the tumor and preventing melanoma progression. Unexpectedly, the anti-IL-6 therapy resulted in accelerated tumor progression and earlier death of mice, which was most likely due to the negative effect of anti-IL-6 on T cell activation which was reflected by decreased CD4+ conventional T cells in the tumor. Altogether, we found IL-6 to upregulate CCR5 and immunosuppressive capacity of MDSC in vitro making this cytokine an interesting target for immunotherapy. However, further research should be performed to understand the delicate balance of IL-6 signaling in melanoma immunity in vivo and the challenges of IL-6 blocking immunotherapy for melanoma treatment.
Persistent infection with high-risk types of human papillomavirus (HPV) can cause several malignancies, in particular oropharyngeal and anogenital cancers. HPV16 has been identified as the most prevalent high-risk type, being related to 60% of cervical cancers, 75% of oropharyngeal cancers, 71% of anal cancers and the majority of precancerous lesions. As standard of care treatment is invasive, and harbors risks and side effects, there is a need for new approaches. For rationally designing a therapeutic vaccine against HPV-induced malignancies, it is essential to identify suitable target epitopes, which are presented on the surface of an HPV-transformed cell and induce immune responses that eventually mediate target cell death. The HPV16 oncoproteins E6 and E7 represent ideal targets for immunotherapy as they mediate the transforming potential of the virus and are constitutively expressed in all malignant cells. In order to define HPV16 target epitopes, in this thesis several algorithms were used to predict potential HPV16 E6- and E7-derived binders of human leukocyte antigen (HLA) class I in silico. Predicted peptides were synthesized and HLA binding capacity was validated in competition-based cellular binding assays. To ensure broad population coverage, predictions and validations were performed for seven frequent HLA alleles: A*01:01, A*02:01, A*03:01, A*11:01, A*24:02, B*07:02 and B*15:01. Including peptides derived from HPV16 E6/E7 variants containing amino acid changes, 271 peptides were experimentally assessed and 69 binders were identified. Combined with previous results, the total HPV16 E6/E7 dataset comprised 779 peptide-HLA measurements. The HPV16 E6/E7 dataset was used to evaluate the performance of employed predictors. No single algorithm was outperforming other methods, but different predictors were found to be best for different settings, depending on investigated HLA type and peptide length. As applying commonly used decision threshold yielded only low sensitivity, criteria for optimal decision thresholds were defined and optimal thresholds were calculated for individual predictors, HLA-types and peptide lengths. Comparing threshold-dependent performance of predictors showed that using criteria-based thresholds allowed more sensitive prediction of HLA-binding peptides without a strong negative influence on prediction accuracy. To identify T cell epitopes among the HPV16 E6- and E7-derived HLA ligands, their capacity to induce immune responses was investigated. To this end, peripheral blood mononuclear cells of healthy donors were HLA-typed and stimulated with respective peptides to generate epitope-specific T cell lines. By assessing interferon-γ-secretion of these T cells, 31 immunogenic peptides were identified. Further characterizing the functionality of epitopes in cytotoxicity assays, five of ten immunogenic HLA-A*02:01-peptides mediated specific killing of HPV16+ target cells by CD8+ T cells. In conclusion, several immunogenic HPV16 E6-and E7-derived epitopes were identified, which are the basis for rational design of a therapeutic HPV vaccine. Additionally, this thesis provides an evaluation of peptide–HLA class-I binding prediction method and recommendations to increase prediction sensitivity to extend the number of potential epitopes as targets for immunotherapy.
This thesis is concerned with the acquisition, modeling, and augmentation of material reflectance to simulate high-fidelity synthetic data for computer vision tasks. The topic is covered in three chapters: I commence with exploring the upper limits of reflectance acquisition. I analyze state-of-the-art BTF reflectance field renderings and show that they can be applied to optical flow performance analysis with closely matching performance to real-world images. Next, I present two methods for fitting efficient BRDF reflectance models to measured BTF data. Both methods combined retain all relevant reflectance information as well as the surface normal details on a pixel level. I further show that the resulting synthesized images are suited for optical flow performance analysis, with a virtually identical performance for all material types. Finally, I present a novel method for augmenting real-world datasets with physically plausible precipitation effects, including ground surface wetting, water droplets on the windshield, and water spray and mists. This is achieved by projecting the realworld image data onto a reconstructed virtual scene, manipulating the scene and the surface reflectance, and performing unbiased light transport simulation of the precipitation effects.
Epidemiological data indicate a potential relation between the consumption of milk and dairy products and the incidence of breast cancer. Particularly, the consumption of milk and dairy products originating from specific cattle species seems to play a significant role in the development of breast cancer. Our group has isolated a number of circular single-stranded DNAs (Bovine Milk and Meat Factors, BMMFs) from bovine sera of healthy cattle and cow milk, as well as from brain and serum samples of patients suffering from Multiple Sclerosis (MS). This project aims at the identification of such episomal DNA agents in commercially available dairy products and milk samples, resulting in the re-isolation of three of the previously identified agents in milk, yoghurt and crème fraiche and the isolation of two novel DNA agents from milk. Additionally, a similar molecule has been identified in a tissue sample adjacent to breast cancer tissue following a High Throughput Sequencing protocol that has been developed as part of this thesis. In addition, our efforts focus on the search for a permissive system for the identified BMMFs. Replication competence of the MS brain isolate MSBI1.176 was shown in two Hodgkin’s Lymphoma cell lines, the L-1236 and L-428, establishing a long-term persistence in human cells. Co-transfection experiments with more than one agent did not show regulatory effects. Additionally, a potential role of N-Glycolylneuraminic acid (Neu5Gc) as receptor for BMMF infection has been suggested, especially since it has already been reported that it acts as receptor for several viruses. However, the experiments performed in this study only showed a slight increase of replication after external addition of Neu5Gc, but they did not serve conclusive results regarding infectivity of BMMFs. Finally, analysis of sera from breast cancer and Hodgkin’s disease patients showed elevated anti-MSBI1.176 Rep immune response in breast cancer patients compared to healthy donors indicating that infection with BMMF related agents could result in neutralizing antibody formation. This study provides information about potential risk factors of bovine origin contributing to the pathogenesis of breast cancer and Hodgkin’s Lymphoma, which could allow for novel therapeutic strategies for fighting these diseases.
Dose accumulation (DA), the computation of the total delivered 3D dose distribution Da of a fractionated radiotherapy treatment using daily patient imaging, is getting established in clinical practice. Although the aim is to estimate the achieved biological outcome of the treatment, the principles of biological effect estimation are currently not followed consistently in the process. In this thesis, the biological effect accumulation approach and total biological dose (bEQD) were derived as a biologically consistent DA workflow. Clinical relevance of bEQD and its dependence on individual workflow aspects were investigated in data from three patient cohorts. It was found that Da systematically underestimates the obtained biological effect, which can be avoided by the use of bEQD. Results showed that this is strongest for late-responding organs at risk (OAR) with low �=� values in dose gradient regions around the target that are prone to organ motion. bEQD to Da deviations occurred locally, in so-called hotspots, showing individual cases of high difference magnitude but only small statistical impact. Hotspots of bEQD - Da deviation around 4 Gy in bladder and rectum were found in patients treated for prostate carcinoma. Hypofractionation increased these deviations strongly up to 8 Gy and also showed clinically relevant deviations in dose-volume analysis. Dose-response correlation in standard fractionation showed only little impact on the DA approaches. Workflow uncertainties are dominated by those from deformable image registration, which are in the same range as the difference between bEQD and Da. bEQD should be considered in the application of treatment adaptation, especially to avoid damage to OARs in individual cases.
Metastatic and recurrent malignant thymomas (TH) and thymic carcinomas (TC) are generally refractory to current empiric radio chemotherapies, thus warranting the search for novel second-line therapeutic strategies. The multi-target tyrosine kinase inhibitor sunitinib has been shown to induce partial remissions and to prolong overall survival of patients with aggressive TH and TC. However, although sunitinib has been proposed as an alternative treatment option in refractory disease, treatment response is only partial, and not all patients benefit equally. For a more accurate response prediction, new biomarkers are needed. In this study, we used phospho receptor tyrosine kinase (RTK) and MAPK arrays as well as a multiplex tyrosine phosphorylation assay containing 144 kinase substrates to characterize malignant TH and TC and to generate a sunitinib response index (SRI) using sunitinib resistance-induced cell lines. The RTK and MAPK arrays stratified the patients into two main dominant phosphor patterns: Pattern 1 was characterized by a strong activation of EGFR while pattern 2 was typified by a strong activation of TYRO3/Dtk. This activation of TYRO3/Dtk correlated with recurrence and metastatic spread of malignant TH. p38 andRSK1 were cluster-specifically activated in the MAPKs but showed no specific disease correlation. The SRI was functionally validated in several cell lines, and the application of the SRI to native malignant TH and TC samples identified a potentially sunitinib-responsive and a potentially sunitinib-resistant group. Among the predicted upstream kinases, TYRO3/Dtk belonged to the top candidates responsible for sunitinib response. TYRO3/Dtk and the highly activated IGF-1R in responsive patients were also functionally validated. Specific siRNA knockdowns in sunitinib-resistant cell lines and overexpression confirmed the functional relevance of TYRO3/Dtk and IGF1-R for sunitinib resistance. We present the SRI as a new approach to the resistance prediction of sunitinib in malignant TH and TC and propose that the activity level of TYRO3/Dtk and IGF-1R could serve as markers to aid in treatment decisions. The results of this in vitro investigation need validation of the SRI in prospective clinical trials.
Imaging atmospheric Cherenkov telescopes are used for the detection of highest-energy γ-rays. This thesis focuses on two experiments equipped with such telescopes: The operating High Energy Stereoscopic System (H.E.S.S.) and the future Cherenkov Telescope Array (CTA). Four of the five H.E.S.S. cameras saw a major electronics upgrade a few years ago enabling improved readout and analysis techniques mainly at the highest energies. The Compact High-Energy Camera (CHEC) is a design for the Small-Sized Telescopes of CTA focusing on the detection of γ-rays with energies exceeding 1 TeV. The first part of the thesis is dedicated to the characterisation of two CHEC prototype cameras developed successively: CHEC-M and CHEC-S. I present results of laboratory and on-telescope measurements for both cameras. In the case of CHEC-S, I focus on those parameters that had been shown to be performance-limiting in CHEC-M and which were therefore addressed in the design iteration for CHEC-S. The second part is devoted to the upgraded H.E.S.S. cameras. I present results of Monte-Carlo simulation studies, analysis developments, and performance measurements using full-waveform readout. In the former case I demonstrate a general consistency between simulations and measurements, in the latter case I show that the use of full-sampled waveform readout improves the performance, especially at the highest energies. In the last part, I present a new analysis of the Galactic γ-ray source HESS J1646–458 which is associated with Westerlund 1, the most massive stellar cluster in our Galaxy.
Influenza A virus (IAV) is a pleomorphic, enveloped virus known for its yearly epidemics and occasional, but fatal pandemics. The outer surface glycoprotein hemagglutinin (HA) together with the matrix protein 1 (M1) are the most abundant protein components of assembled virions. HA, located at the outside of virions, is involved in cell receptor recognition, membrane fusion and is the most relevant protein for antibody binding. Therefore the structure of isolated HA has been extensively characterised by X-ray crystallography. However, it remains unclear to which extent the structure of isolated HA corresponds to the in situ HA structure on the surface of IAV. M1 determines the morphology of the virus by forming a matrix layer underneath the viral membrane. A high resolution structure of full length M1 is missing and the lack of information about the in situ arrangement of the M1 matrix layer currently limits our understanding of how M1 functions. Here, I set out to determine the structures of HA and M1 directly from IAV particles using high resolution cryo-electron tomography (cryoET) and subtomogram averaging. I found that virus purification can affect the integrity of the virus HA glycoprotein layer and the morphology of virus particles. I therefore adapted a workflow which allows studying the structure of viral proteins directly from viruses in the vicinity of virus-producing cells. Biosafety regulations required inactivation of IAV samples by chemical fixation prior to cryoEM imaging. To assess effects of fixation, I complemented structural studies of HA from pathogenic, fixed IAV particles with studies of HA from non-infectious, unfixed virus-like particles (VLPs). These studies revealed that fixation captures HA in an open conformation while HA structures determined from unfixed samples perfectly match the closed conformation observed in the trimeric crystal structure. In concordance with recent work by others, this observation suggests that fixation captures HA in a an open, otherwise transient conformation, which is part of a constant opening and closing motion known as breathing motion. To characterise the in situ structure and arrangement of M1, I established a subtomogram averaging workflow to cope with the challenges presented by the small size of M1. I successfully obtained two independent structures of M1 directly from viruses and VLPs. Comparisons of my structures to existing high resolution models of the N-terminal domain (NTD) of M1 revealed that M1 monomers arrange as parallel strands, with a helical propensity and directly underneath the membrane. For the first time, my data allow to describe the M1-membrane interface as well as relevant M1-M1 interfaces within the matrix layer. Finally, I have gained first structural insights into the M1 C-terminal domain (CTD). I further combined the obtained structural information for M1 with a theoretical model of the mechanics of M1 polymerization and membrane deformation during virus assembly. The obtained results suggest that linear polymerization of M1 into multiple parallel strands efficiently provides energy to drive assembly of new virus particles. The results presented in this thesis improve our understanding of the arrangement and structure of the two influenza proteins HA and M1 in situ which has implications for current models of HA-mediated membrane fusion, virus architecture and virus assembly.
Diffusion is the major short-range transport mechanism in living cells. Within individual compartments of a eukaryotic cell, such as the nucleus, mitochondria or the cytosol, biological macromolecules find their targets mostly by thermally driven random motion. For instance, specific access of DNA-binding proteins to their target sequences in the genome occurs through a sequence of three-dimensional diffusion, DNA-binding and one-dimensional search events on the DNA. The DNA/chromatin network in the cell nucleus thus has two effects on protein diffusion: obstruction due to crowding and accelerated association to specific sequences through guided diffusion along the DNA chain. The problem of target finding of proteins in the cell nucleus is only one example of diffusion-controlled reactions in a dense polymer network. Outside the direct relevance for molecular and cellular biology, the study of diffusing particles in viscoelastic media has important applications in many fields of physics. By recording fast image series of two-dimensional sections of live cells, we monitor these diffusion processes in real time and gain better understanding of the underlying physics. The method used is light sheet fluorescence microscopy followed by auto (-cross) correlation analysis. We particularly studied the random motion of chromatin and its interconnection with nucleoplasmic A-type lamins. Utilizing this method, we find that 1. Nucleoplasmic lamin As and chromatin show significant co-mobility, indicating that their motions are interconnected in the nucleus. 2. The random motion of histones H2A within the chromatin network is subdiffusive, i.e. the effective diffusion coefficient decreases for slow timescales. Knocking out lamin A changes the diffusion back to normal. Thus, lamin A influences the dynamics of the entire chromatin network. 3. A-type lamins affect the spatial organisation of chromatin inside the cellular interior. We have also attempted to develop a modelling framework that describes chromatin dynamics within the cell nucleus in the presence and absence of nucleoplasmic A-type lamins. Our conclusion is that lamin A plays a central role in determining the viscoelasticity of the chromatin network and helping to maintain local ordering of interphase chromosomes. These findings enabled us to derive a qualitative description of diffusion based on the viscoelasticity of the cellular environment.
Scratch assays are standard in-vitro experimental procedures for studying cell migration. In these experiments, a scratch is made on a cell monolayer. By imaging the recolonisation process of the scratched region, we are able to quantify cell migration rates. This experimental technique is commonly used in the pharmaceutical industry to identify new compounds that target cell migration, and to evaluate the efficacy of potential drugs that inhibit cancer invasion.
Given the key role this method plays in assessing the potential of new compounds for clinical use, it is important to develop robust quantification frameworks that accurately describe the movement of the front of migrating cells. We develop a migration quantification method that fits experimental data more closely than existing methods, provides a more accurate statistical classification of the migration rate between different assays and is able to cope with experimental data of lower quality than the classic quantification methods can handle. The robustness of our new method is validated using both in-vitro and in-silico data.
Developing robust quantification methods allows the validation of mathematical models that can be used to test hypotheses about the physical and biological mechanisms that govern cell migration. Typically scratch assays are modelled by continuum reaction-diffusion equations depicting cell migration by diffusion and carrying capacity-limited proliferation by a logistic source term. An age-structured population model is presented that aims to explain the two phases of proliferation in scratch assays previously observed experimentally: where an initial phase is observed where proliferation is not logistic, followed by a second phase where proliferation appears to be logistic. The cell population is modelled by a McKendrick-von Foerster partial differential equation. The conditions under which the model captures this two-phase behaviour are presented.
Finally, an important aspect of modelling biological systems is the development of efficient algorithms. The scratch assay is a classical example of a system in which there is low cell number in some regions of the spatial domain and high cell number in others. When the cell number is sufficiently high, mean-field models, like partial differential equations, can capture the relevant dynamics. However, when the cell number is low, such models are not appropriate and stochastic representations must be employed. Hybrid algorithms allow multiple modelling frameworks for the same species in different parts of the spatial domain. Typically hybrid algorithms consider heuristic methods based on the cell density for determining which compartments will be updated deterministically or stochastically. We introduce a hybrid algorithm that couples the mesoscopic description of a reaction-diffusion system with its mean-field analogue. We consider a natural indicator of when the mean-field approximation is valid: the system variance. We estimate the system variance using the intrinsic noise approximation and use this estimate to determine the regions in which the system is updated stochastically or deterministically over time. We apply the hybrid algorithm to the stochastic Fisher-Kolmogorov-Petrovsky-Piscounov model, a typical model of scratch assays. We analyse systematically how good is the approximation to the stochastic process and compare its performance to another hybrid algorithm.
Detecting repeating firing motifs of neuron groups (so-called neuronal assemblies) and cell segmentation in calcium imaging, a microscopy technique enabling the observation of neuronal activity, are two fundamental and challenging tasks in neurophysiological data analysis. In this thesis, three novel approaches are presented, which use machine learning to tackle both problems from different perspectives. First, SCC is presented for the detection of motifs in neuronal spike matrices, which are gained from calcium imaging data by cell segmentation. SCC uses sparse convolutional coding and outperforms established motif detection methods by leveraging sparsity constraints specifically designed for this data type combined with a method to avoid false-positive detections. Second, LeMoNADe is the first method ever to detect spatio-temporal motifs directly in calcium imaging videos, eliminating the cumbersome extraction of individual cells. It is a variational autoencoder framework tailored for the extraction of neuronal assemblies from videos and matches the performance of state-of-the-art detection methods requiring cell extraction. Although LeMoNADe enables the detection of neuronal assemblies without previous cell extraction, this step is still essential for a wide range of downstream analyses. Therefore, the third method, DISCo, combines a deep learning model with an instance segmentation algorithm to address this task from a new perspective and thereby outperforms similarly trained existing models.
Postembryonic stem cell niches are present throughout the vertebrate clade to facilitate development, homeostasis, regeneration and growth. While teleosts and amphibians display sustained stem cell activity in most organs after embryogenesis, higher vertebrates retain stem cell activity only in specific tissues. Despite these differences, similar challenges are imposed on all vertebrate organisms: new tissue has to be generated to expand or replace existent one while simultaneously ensuring integrity and functionality of the organ. Tight control of stem and progenitor cell proliferation is necessary to avoid aberrant growth such as in cancer. In the retina of the teleost medaka (Oryzias latipes), retinal stem (RSC) and progenitor (RPC) cells are located in the ciliary marginal zone (CMZ) and mediate postembryonic growth and neurogenesis. Since function and shape of the eye are intimately linked, the activity of RSCs and RPCs is tightly coordinated to establish proper cell type composition and number. In this thesis I addressed intrinsic and extrinsic regulation mechanisms of the RSC niche. I hypothesised that retinal growth underlies intrinsically active growth factor signaling, and that immune cells safeguard the RSC niche in homeostasis and injury. To analyse intrinsic regulation of RSC proliferation, I assessed the function of insulin-like growth factor (Igf) signaling in the CMZ using gain- and loss- of-function approaches. I found that Igf1 receptor over-activation increased cell cycle speed, RPC number and consequently retinal size, while simultaneously preserving the stereotypical retinal architecture. Strikingly, RSCs were not susceptible to mitogenic stimuli, indicating that RPC amplification is the determinant of retinal size and composition. To understand the extrinsic regulation of the RSC niche, I examined the interplay of immune cells and RSCs. I found that Ccl25b-positive RSCs are phagocytosed by Ccr9a-positive immune cells located in the CMZ. Ccl25b mutation abrogates reactivation of immune cells upon RSC injury, implicating Ccl25b–Ccr9a signaling in the immune–stem cell interaction during homeostatic surveillance and injury response. In summary, my results propose that accurate postembryonic growth and tissue integrity depend on both cell intrinsic and extrinsic mechanisms of growth control in the RSC niche of medaka.
Various modeling and simulations have been done to study the organization of chromosomes under different circumstance and for different purposes. In this thesis, we would first study the influence of bending rigidity and spatial confinement on the organization of the chromatin. More concretely, the effect of heterogeneity and the definition of contacts will be addressed. We find that the definition of a contact does not change the asymptotic behavior of the contact probability. The heterogeneity of bending rigidity is shown to render the chain more flexible by comparing the persistent length and the contact probability of homogeneous and heterogeneous chains. In addition, we simulate semiflexible chains in rectangular confinements with different aspect ratios. An oscillation in the contact probability and the orientational correlation function is found due to the spiraling of polymer when the box size is small enough.
The entanglement of chains is another important aspect when studying chromatins. The processes of disentanglement of two flexible chains are studied using the Monte Carlo simulation. Specifically, several measurements such as the inter-contact of chains, dynamic structure factor are analyzed in the process. When only the excluded volume interaction exists in the system, the average time required for segregation is barely influenced by the initial configurations of the two chains according to our results. However, the intertwinement of chains indeed could impede the segregation at a small time scale. The number of contacts inside a self-avoiding chain is also analyzed. It is found that the total number $N_c$ grows linearly with the length of a free chain, while in cubic confinement it grows quadratically. The distribution function of contacts number between two halves $N_c(AB)$ shows a power-law decay behavior and then an exponential decay for a free chain. In confinement, the function has a maximum. As the chain becomes longer, the percentage of inter-half contacts among the total contacts has a power-law decay behavior with an exponent close to -1, which supports that the number of contacts between two halves is finite even when the chain is infinitely long.
Finally, we studied the fractality and the topology in the self-avoiding walks. Specifically, we calculate the fractal dimension and growth rates of the Betti numbers of the system. These growth rates can be viewed as a topological signature for different systems. The intra-contacts of the self-avoiding walk is a subset of the original walk, and we find that this subset may have a slight multifractal property. In addition, the topological exponents are also different from the self-avoiding walk. Further, each contact gives rise to the formation of a loop. To elucidate how these loops influence the structure of the self-avoiding walk, we delete the loops in a similar way to the loop-erased random walk, thus producing a new walk: loop-deleted self-avoiding walk (LDSAW). The critical exponent of LDSAW is approximated by studying the scaling behavior of mean end-to-end distance, and the dependence of the mean length of LDSAW on the length of the original self-avoiding walk. Afterward, the fractal dimension and growth rates of Betti numbers of this LDSAW are calculated. The same calculations are also performed on the projection and random subsets of self-avoiding walks.
The regulation of biological processes relies on a complex nucleotide code embedded in our DNA. Its decoding and interpretation is the main task of Transcription Factors (TFs), which altogether enable the recognition and modulation of gene expression. Whenever factors bind to DNA, a set of additional criteria and conditions need to be satisfied, such as TF concentration, DNA openness, and cooperativity with other binding factors. Such combinations of DNA-bound TFs, as well as their structural and functional cooperativity, allow a more fine-grained control of gene expression due to subtle changes in specificity in both DNA recognition and functional outcomes. This thesis explores the prediction of structural TF cooperativity and its biological consequences. Additionally, examples of functional cooperativity are presented and discussed in the context of neuronal activity and reprogramming. Altogether, this dissertation provides an extensive set of insights to better understand the complex interplay between TFs cooperativity and phenotypes.
Breast cancer is the leading cause of death in women worldwide and these deaths are mostly attributed to metastasis and tumour recurrence following initially successful therapy. Metastasis refers to the development of invasive disease, wherein malignant cells dissociate from primary tumours, infiltrating other organs and tissues to give rise to secondary outgrowths. Previously, metastasis was thought to be initiated in advanced tumours, but breast cancer cellsh with metastatic potential have now been shown to disseminate very early from the primary site via largely unknown mechanisms. These early interactions of tumour cells with their cellular micro-environment and normal neighbours also results in early tumour cell heterogeneity and must therefore be elucidated such that we can prevent metastatic spread in the patient situation and better treat the resulting heterogenous tumours. However, studying tumour initiation is not possible in patients because it happens on a cellular level not detectable by current technology. Tumour recurrence is another major cause of breast cancer related death and is believed to be caused by residual disease cells that survive initial therapy. These are a reservoir of refractory cells that can lay dormant for many years (sometimes decades) before resulting in relapse tumours. They are also difficult to obtain from human patients, since they are very few and cannot be detected easily, and thus their molecular mechanisms have not been fully explored. In addition to the unavailability of human tissue, mouse models of breast cancer also fall short in helping us study early cancer initiation, because they allow oncogenic expression in all cells of the tissue instead of initiating cancer like in the human situation|one neoplastic transformed cell proliferating unchecked in a normal epithelium. To address this issue, we used primary organoids from an inducible mouse model of breast cancer and lentivirally transduced single cells within these organoids to express oncogenes. We further optimized parameters for long term imaging using light sheet microscopy and developed big data analysis pipelines that lead us to discern that single transformed cells had a lower chance at establishing tumorigenic foci, when compared to clusters of cells. Thus, we postulate a proximity-controlled signalling that is imperative to tumour initiation within epithelial tissues using the first ever in vitro stochastic breast tumorigenesis model system. This new stochastic tumorigenesis system can be further used to identify the molecular interactions in the early breast cancer cells. Our group has already revealed distinct characteristics, such as dysregulated lipid metabolism, of the residual disease correlate obtained from an inducible mouse model. As survival mechanisms invoked by residual cells remain largely unknown, we analysed the dynamic transcriptome of regressing tumours at important timepoints during the establishment of residual disease. Key molecular players upregulated during regression {like c-Jun and BCL6 { were identified and the inflammatory arm of the Nf-kB cascade was found to be dysregulated among others. Further validation of these molecular targets as potentially synthetic lethal interactors remains to be performed so that they can be used to limit the residual disease reservoir and eventually tumour recurrence.
Bacteria control import and export of compounds, adhesion to surfaces, cell-to-cell interactions and interactions with host via proteins they feature on their cell surface. Recent evidence has emerged that members of a category of proteins anchored to bacterial membranes through their lipidated tails, called lipoproteins, can be exposed at the cell surface of Gram-negative bacteria. Escherichia coli is thought to carry around 100 lipoproteins, but what fraction of them is surface exposed and whether this is a general phenomenon remains unknown. In my PhD project, I systematically investigated whether lipoproteins are surface exposed in E. coli. In this work, I developed a Surface Proteome Quantification (SPQ) method, which systematically and quantitatively assesses protein cell-surface exposure at a proteome-wide level, using surface biotin labelling combined with quantitative mass spectrometry. Thereby I identified at least 23 outer membrane (OM) lipoproteins to be significantly surface exposed (out of 62 quantified OM lipoproteins) to similar levels as the bona-fide surface exposed OM β-barrel proteins. Then, 5 surface identified lipoproteins, 3 OM lipoproteins and 2 inner membrane lipoproteins, were verified for their surface exposure using independent biochemical methods. Furthermore, using the SPQ method, I identified a strong dependency of the lipoprotein cell-surface exposure on the BAM (β-barrel assembly machinery) and explored the role of the different components of the BAM complex. In parallel, I generated the first proteome-wide quantitative examination of the E. coli membrane proteome after physical separation of the inner- and outer-membrane, revealing an additional complexity and some mis-annotations in protein localization within the bacterial cell envelope. Finally, I investigated the physiological consequences on the Rcs stress response system through a mislocalized model surface exposed lipoprotein, RcsF. Overall, the complexity and importance of protein localization in the bacterial cell envelope, with the focus on lipoproteins, is investigated and discussed in this thesis.
Blood pressure is one of the vital signs and its regulation is crucial for survival. Several mechanisms contribute to maintain it in a physiological range: renin-angiotensin-aldosterone system, the autonomous nervous system and specialized baroreceptors neurons. In this study, we demonstrate the existence of a new population of sensory neurons marked by TrkC and TH that innervate blood vessels and are important in the control of blood pressure, blood flow and heart rate. Using an inducible Cre line driven from the TrkC locus, we show that TrkC is expressed in 30% of DRG neurons and that a fourth of these neurons are TH+ and project to blood vessels. Activation of TrkC+ TH+ neurons leads to high blood pressure, decreased blood flow and increased heart rate variability. Loss of function experiments revealed that TrkC+ TH+ sensory neurons are crucial for life. Ablation of TrkC+ neurons results in low blood pressure, alteration of blood flow and increased heart rate variability. All these cardiovascular alterations lead ablate mice to death within 48 hours. We also demonstrate that TrkC+ neurons do not act directly on blood vessels, but they exert their functions through a circuit with the sympathetic nervous system. We thus identified a new population of sensory neurons involved in the regulation of blood pressure, blood flow and heart rate and we hope that this can lead to the development of new therapeutic strategies in the near future.
RNA Polymerase III (Pol III) produces small, non-coding RNAs with fundamental functions in the eukaryotic cell, including translation, splicing and protein sorting. While structures of unbound and transcribing Pol III have been solved and provided valuable mechanistic insights into Pol III transcription, snapshots of molecular interactions that underlie Pol III activation and repression are lacking. In this thesis I address these questions with structural studies of the Sarrachomyces cerevisiae Pol III transcription apparatus. I present high-resolution cryo-EM reconstructions of Pol III bound to its principal transcription initiation factor TFIIIB that were used to build atomic models. The complex was observed in different functional states, including two early intermediates in which the DNA duplex is closed, an open DNA complex, and an initially transcribing complex with RNA in the active site. The structures reveal an extremely tight, multivalent interaction between TFIIIB and promoter DNA, and explain how TFIIIB recruits Pol III. Together, TFIIIB and Pol III subunit C37 activate the intrinsic transcription factor-like function of the Pol III-specific heterotrimer to initiate the melting of double-stranded DNA, in a mechanism similar to that of the Pol II system. I further present a high resolution structure of Pol III bound to the negative regulator Maf1, that explains how Maf1 achieves transcription repression by preventing interaction with TFIIIB. Maf1 occupies a position on Pol III that overlaps with the binding site of promoter DNA and TFIIIB. Furthermore, by mimicking the shape and electrostatic charge of a double-stranded DNA backbone, Maf1 further sequesters a mobile domain of Pol III subunit C34, which seals off the active site cleft and makes it inaccessible to bind DNA. Lastly, I describe a recombinant expression system for the six-subunit, 520 kDa transcription factor TFIIIC and subcomplexes thereof. Negative stain electron microscopy of a complex between the tA module of TFIIIC and TFIIIB provide the first molecular insights into how TFIIIC recruits TFIIIB and positions it upstream of the transcription start site. Biochemical experiments further show that the tA module is displaced after or concomitant with Pol III recruitment, establishing it as an assembly factor rather than a bona fide transcription factor.
miRNAs cooperate and fine tune gene expression on the post-transcriptional level and can therefore be seen as an additional regulatory layer during mouse embryonic stem cell (mESC) self-renewal and differentiation. However, the biological activity of conserved miRNAs during those processes is poorly understood, as most studies cannot uncouple miRNA activity from miRNA expression levels. Therefore, my PhD project focused on studying single cell miRNA dynamics during mESC differentiation towards the three major germ layers and measured miRNA affinities to a miRNA reporter in vivo. I established stable mESC lines expressing fluorescent reporters for the 162 miRNAs conserved in vertebrates and could show that the temporal miRNA activity profile is tightly regulated throughout mESC differentiation. Interestingly, miRNAs exhibit activity changes at early, mid and late stages of stem cell differentiation. Moreover, miRNAs seem to regulate differentiation in a cooperative manner on a global level rather than being germ layer specific. However, this does not exclude single miRNA clusters from being potentially germ layer specific as shown for the highly conserved miR-302 cluster. Strikingly, based on principle component analysis, miRNA activity diverged between germ layer fates already 48 hours after onset of differentiation. In addition, this PhD project experimentally determined miRNA affinities for 119 conserved miRNAs by integrating measurements of miRNA activity and expression levels. I could show that miRNA affinities span several orders of magnitude and are negatively correlated to miRNA expression levels, which suggests that weakly expressed miRNAs can be as potent as highly expressed ones. Knowing the affinity and expression levels in a given cell type, enabled me to rank miRNAs according to their effective potency. This will potentially help to determine which genes are targets of a given miRNA. In summary, this thesis project provides a comprehensive picture of changes in miRNA activity upon mESC differentiation in addition to experimentally determined miRNA affinities. Future gain and loss-of-function experiments of interesting miRNAs in fluorescently labeled fate marker cell lines will potentially reveal miRNAs indispensable for stem cell differentiation.
The drug-receptor binding kinetics are defined by the rate at which a given drug associates with and dissociates from its binding site on its macromolecular receptor. The lead optimization stage of drug discovery programs usually emphasizes optimizing the affinity (as described by the equilibrium dissociation constant, Kd) of a drug which depends on the strength of its binding to a specific target. Since affinity is optimized under equilibrium conditions, it does not always ensures higher potency in vivo. There has been a growing consensus that, in addition to Kd, kinetic parameters (kon and koff ) should be optimized to improve the chances of a good clinical outcome. However, current understanding of the physicochemical features that contribute to differences in binding kinetics is limited. Experimental methods that are used to determine kinetic parameters for drug binding and unbinding are often time consuming and labor-intensive. Therefore, robust, high-throughput in silico methods are needed to predict binding kinetic parameters and to explore the mechanistic determinants of drug-protein binding. As the experimental data on drug-binding kinetics is continuously growing and the number of crystallographic structures of ligand-receptor complexes is also increasing, methods to compute three dimensional (3D) Quantitative-Structure-Kinetics relationships (QSKRs) offer great potential for predicting kinetic rate constants for new compounds. COMparative BINding Energy(COMBINE) analysis is one example of such approach that was developed to derive target-specific scoring functions based on molecular mechanics calculations. It has been used extensively to predict properties such as binding affinity, target selectivity, and substrate specificity. In this thesis, I made the first application of COMBINE analysis to derive Quantitative Structure-Kinetics Relationships (QSKRs) for the dissociation rates. I obtained models for koff of inhibitors of HIV-1 protease and heat shock protein 90 (HSP90) with very good predictive power and identified the key ligand-receptor interactions that contribute to the variance in binding kinetics.
With technological and methodological advances, the use of all-atom unbiased Molecular Dynamics (MD) simulations can allow sampling upto the millisecond timescale and investigation of the kinetic profile of drug binding and unbinding to a receptor. However, the residence times of drug-receptor complexes are usually longer than the timescales that are feasible to simulate using conventional molecular dynamics techniques. Enhanced sampling methods can allow faster sampling of protein and ligand dynamics, thereby resulting in application of MD techniques to study longer timescale processes. I have evaluated the application of Tau-Random Acceleration Molecular Dynamics (Tau-RAMD), an enhanced sampling method based on MD, to compute the relative residence times of a series of compounds binding to Haspin kinase. A good correlation (R2 = 0.86) was observed between the computed residence times and the experimental residence times of these compounds. I also performed interaction energy calculations, both at the quantum chemical level and at the molecular mechanics level, to explain the experimental observation that the residence times of kinase inhibitors can be prolonged by introducing halogen-aromatic pi interactions between halogen atoms of inhibitors and aromatic residues at the binding site of kinases. I determined different energetic contributions to this highly polar and directional halogen-bonding interaction by partitioning the total interaction energy calculated at the quantum-chemical level into its constituent energy components. It was observed that the major contribution to this interaction energy comes from the correlation energy which describes second-order intermolecular dispersion interactions and the correlation corrections to the Hartree-Fock energy.
In addition, a protocol to determine diffusional kon rates of low molecular weight compounds from Brownian Dynamics (BD) simulations of protein-ligand association was established using SDA 7 software. The widely studied test case of benzamidine binding to trypsin was used to evaluate a set of parameters and a robust set of optimal parameters was determined that should be generally applicable for computing the diffusional association rate constants of a wide range of protein-ligand binding pairs. I validated this protocol on inhibitors of several targets with varying complexity such as Human Coagulation Factor Xa, Haspin kinase and N1 Neuraminidase, and the computed diffusional association rate constants were compared with the experiments. I contributed to the development of a toolbox of computational methods: KBbox (http://kbbox.h-its.org/toolbox/), which provides information about various computational methods to study molecular binding kinetics, and different computational tools that employ them. It was developed to guide researchers on the use of the different computational and simulation approaches available to compute the kinetic parameters of drug-protein binding.
The consistent definition of a quantum gravity theory has to overcome several obstacles. Here we take important steps in the development of three approaches to quantum gravity. By utilising matter fields as mediators from ultraviolet to infrared energies, we study a coupling between asymptotically-safe quantum gravity and the hypercharge. The resulting symmetry enhancement allows for a possible ultraviolet completion of the joined system, predicting the infrared value of the hypercharge within estimated systematic errors, thereby increasing the predictive power of the model. Additionally, previous studies suggest that K ̈ahler-Dirac fermions on Euclidean dynamical triangulations do not spontaneously break chiral symmetry. Here we develop computational tools accounting for the back reaction of fermions on the lattice. If extended studies support the evidence that chiral symmetry remains intact, then the model passes an important observational viability test. Lastly, we provide procedures allowing for the extraction of geometrical and topological properties from a causal set. Specifically, we build a spatial distance function which can be used to construct dimensional estimators for the Hausdorff and spectral dimension. In agreement with other quantum-gravity approaches, the latter exhibits a form of dimensional reduction at high energies on account of the inherent non-localness of causal sets.
In this thesis, we investigate optimization problems with partial differential equation (PDE) constraints. In particular we are concerned with the efficient numerical solution of optimum experimental design (OED) problems for parameter estimation (PE) with PDE models, among them sampling design problems. We consider two dimensional (2D) stationary diffusion advection reaction PDE models, including the challenging case of an advection dominated PDE. For the simulation of the PDE boundary value problem, we utilize discontinuous Galerkin finite element methods and adaptive spatial grid refinement. We solve the optimization problems with derivative-based algorithms. For the optimization algorithms to converge fast and to converge to the ”true“ optimum, we need to provide accurate sensitivities. It is a challenge to evaluate the sensitivities, which correspond to the approximate solution of the primal PDE model and are in this sense consistent. In this thesis we develop efficient and accurate methods for sensitivity generation. We transfer the principle of internal numerical differentiation (IND) from ordinary differential equations (ODE)s to PDEs. That means, we incorporate the sensitivity generation in the solution process. The standard upwind discontinuous Galerkin method is not differentiable. Therefore, we propose a differentiable discontinuous Galerkin method and give a rigorous convergence analysis of it. We develop methods for structure exploitation of the primal and tangential discretization schemes to efficiently generate the sensitivities with automatic differentiation (AD). Furthermore, we establish methods for frozen adaptivity to generate consistent sensitivities. We are especially concerned with frozen spatial grid refinement and the adaptive step number of the linear solver. We implement the developed methods in the software SeafaND-Optimizer, short for structure exploiting and frozen adaptivity numerical differentiation optimizer. It is a software for efficient and accurate simulation, PE and OED with PDE models. We perform numerical case studies for PE and OED problems with advection dominated 2D diffusion advection PDE models. With the structure exploiting techniques developed in this thesis, the example problems are solved with efficient memory usage. Due to the frozen adaptivity methods, we computed efficiently the consistent sensitivities. We test the PE algorithm with different noise levels. We perform a case study with different diffusion coefficients for sequential OED. Finally, we investigate, whether the developed methods are stable under mesh refinements.
With little to no progress in the treatment of pancreatic cancer over the past decades, novel approaches to cover the high unmet medical need are long overdue. However, the long lasting notion of PDAC being a ‘non-immunogenic’ neoplasm lacking uniform infiltration of effector lym- phocytes and holding an immunosuppressive microenvironment has so far precluded the use of adoptive T cell therapy in this malignancy. Furthermore, the overall low mutational burden of pancreatic cancer led to the assumption of decreased frequencies in immunogenic neoepi- topes targeted by T cells. Nevertheless, the recent identification of frequently occurring effector T cells recognizing their autologous tumor in pancreatic cancer indicates a potential use of PDAC derived TILs against the malignancy. In addition, exome analyses of larger patient co- horts indicated that formation of immunogenic, mutation derived epitopes in PDAC might be more frequent than previously expected. Nevertheless, to date functional analyses identifying and characterizing the epitopes recognized by pancreatic cancer TIL are yet to be carried out. Thus, we set out to develop screening approaches to reliably identify mutation derived epitopes within pancreatic cancer patients. This development was approached with two complementary strategies, first by looking into the antigens recognized by PDAC TILs and second by directly identifying the epitopes presented on the surface of the PDAC derived tumor cells. We addressed the antigen recognition by developing a novel expression based screening sys- tem. Since PDAC is frequently infiltrated by CD4+ and CD8+ T cells (both of which populations potentially mount anti tumor immune responses), combined with a low number of tumor muta- tions, we needed an approach able to screen for both, MHC-I and MHC-II restricted antigens. Therefore, we designed a system based on shuttling antigens into both, the major histocom- patibility complex (MHC)-I and -II antigen presentation pathways, in order to define the reac- tivity of PDAC TILs in a completely unbiased fashion (i.e. not depending on MHC restriction and/or epitope prediction algorithms). Furthermore, we validated the use of a targeted mass spectrometry based approach to directly identify epitopes presented on the surface of PDAC derived cells lines. In addition, the application of both approaches in a small patient cohort was used to draw initial conclusions on the feasibility of both approaches for an extension to larger patient cohorts. Taken together, the presented study describes the development and validation of screening methodologies capable of identifying antigens in the context of low mutational burden malig- nancies such as pancreatic cancer. These methodologies lay the foundation for the functional proof of concept studies of antigen reactive T cells infiltrating pancreatic cancer, opening new ways for the application of adoptive T cell transfer in this fatal disease.
Sind Sie neugierig? Klingt nicht so nett, oder? Dabei ist Neugierde eigentlich ganz natürlich. Menschen sind von Geburt an neugierig. Und das ist gut so. Denn durch Fragen und Ausprobieren lernen wir, wie man das Leben meistert. Wie kann man Kinder so fördern, dass sie neugierig bleiben und ihren Forschergeist entdecken? Das vermittelt das Forum „Forsch mit!“, nächste Woche in der Forscherstation Heidelberg. Campus Reporter Nils Birschmann hat dazu ein paar besonders neugierige Fragen gestellt.
Der Beitrag erschien in der Sendereihe "Campus-Report" – einer Beitragsreihe, in der über aktuelle Themen aus Forschung und Wissenschaft der Universitäten Heidelberg, Mannheim, Karlsruhe und Freiburg berichtet wird. Zu hören ist "Campus-Report" montags bis freitags jeweils um ca. 19.10h im Programm von Radio Regenbogen (Empfang in Nordbaden: UKW 102,8. In Mittelbaden: 100,4 und in Südbaden: 101,1).
Evolution has led to an immense diversity in the form and shape of animals that we can observe today. As a result of an evolutionary trend called cephalization, most animals develop a head as a separate entity than the trunk. Foundations of head as an isolated body part become evident while the animal body plan is established during the early development. Mechanisms of head and trunk separation, however, are poorly understood. Studying how and which developmental programs contribute to the divergence of head-trunk separation mechanism is, therefore, essential. As our understanding of morphogenesis, the making of morphology, has drastically changed over the years, we can now tackle such phenomenon in greater detail. In my thesis, I interrogated the head-trunk separation mechanisms during early gastrulation among dipteran flies as a model. Dipteran tree of flies present a valuable diversity in head-trunk separation strategies. While derived cyclorrhaphan fruit fly Drosophila melanogaster and basal cyclorrhaphan scuttle fly Megaselia abdita embryos employ a head fold called the cephalic furrow, which physically separates embryonic head from trunk; basal non-cyclorrhaphan midge fly C. riparius embryos, like most insects, do not form a head fold. In D. melanogaster, the cephalic furrow formation is a deep epithelial infolding event, that invariably appears in the same position, critically requires the overlapping expression of two transcription factors, even-skipped and buttonhead. My findings suggested that the absence of a head fold in C. riparius coincides with non-overlapping expression patterns these two genes, while M. abdita has a similar overlap with some differences. I further identified that in the absence of such a visible separator, differential arrangement of subcellular contractile actomyosin networks in the anteroposterior axis has a pivotal role in head- trunk separation in C. riparius. Furthermore, uncovering prominent out-of-plane divisions in the C. riparius’ head development allowed me to speculate a putatively analogous function to the cephalic furrow in higher flies, as a number of cells sink below the embryo surface in both cases. Taken together, my thesis shed light onto the variation of head-trunk separation strategies, underlying genetics, and its implementation at the cellular, tissue and embryonic level in dipteran flies.
Over the last decade, the field of gene and cell therapy has experienced a major turning point and has finally begun to fully realize its potential as a very attractive, versatile and innovative platform for the development of gene-based drugs. Gene therapy encompasses a spectrum of approaches, ranging from supplying missing genes to the correction of diseases at their molecular level, that all have in common the need of a vehicle (“vector") for specific and efficient delivery of therapeutic DNA or RNA. One branch of small viruses - the parvoviruses - have gained increasing attention as such vectors due to their non-pathogenicity, ease of engineering and low genotoxicity. Particularly, the adeno-associated virus (AAV) emerged as a top candidate, culminating in the authorization of three AAV-based gene therapy products, Glybera, Luxturna and Zolgensma. However, despite all the successes using recombinant (r)AAVs, there is still a demand for more specific vectors with larger DNA cargo capacity and lower immunogenicity. This need defined the scope of this doctoral thesis, which aimed at the construction and evaluation of new parvoviral vectors (derived from bocaviruses [BoVs]) and to increase the safety of vector application in humans. The first part of this work was fueled by a seminal study by Ziying Yan and colleagues in 2013, who used parvovirus cross-genera pseudotyping to combine an oversized rAAV2 genome of 5.5 kilobases (kb) with the capsid of the human bocavirus 1 (HBoV1). As reported, the rAAV2/HBoV1 vector could be produced efficiently and potently transduced primary human airway epithelial cells (pHAE). Here, we have validated and expanded on these intriguing findings by more comprehensively exploring the upper DNA packaging limit of the HBoV1 capsid. Notably, we found that up to 6.2 kb single-stranded (ss) - or 3.2 kb self-complementary (sc) - AAV genomes can be efficiently packaged into the HBoV1 capsid, as compared to only 5.1 (ssAAV) and 2.8 kb (scAAV) for AAV2, which has important ramifications for the delivery of complex rAAV vector DNA. Next, we further expanded this system to other primate BoV serotypes - three from humans (HBoV2, 3 and 4) and one from Gorilla (GBoV) - that have not been studied as vectors before. To this end, we successfully assembled the capsid genes of HBoV2-4/GBoV and produced chimeric rAAV/BoV vectors of all studied serotypes. With the help of reporter genes, we subsequently started to study and unravel the so-far unknown tropism of the new viral vectors. Strikingly, our screens on various primary cells and cell lines revealed that BoVs (especially GBoV) have a much wider tropism in vitro than previously anticipated. We found a wide range of primary and therapeutically relevant cells to be amenable to BoV infection, including human hepatocytes, T-cells and skeletal muscle cells. In addition, we obtained the first evidence that pseudotyped rAAV/BoV vectors also differ in their reactivity to pooled human antibodies (intravenous immunoglobulin, IVIg), which implies the possibility of vector re-dosing in rAAV/BoV-treated human gene therapy patients. Finally, we aimed to increase the fitness of BoV vectors and therefore employed a high-throughput diversification method called DNA family shuffling (DFS), to create the first library of chimeric BoV capsids. As hoped for, the library was packaging-competent, increased in titer over selection rounds and acquired a unique footprint when cycled in pHAE. Despite an excellent safety record of rAAV vectors, undesirable toxicity resulting from permanent gene expression represents a clinical concern. So far, the ensuing need to gain temporal control over vector persistence or expression has been addressed by using Cre recombinase or inducible systems that necessitate complex vector re-engineering. Thus, in the second part of this work, we aimed to overcome these limitations by introducing novel rAAV vectors that harbor a kill-switch (KS) based on the bacterial CRISPR II system (clustered regularly interspaced short palindromic repeats). This approach has two major components: (i) a (g)uide RNA expressed from the rAAV vector and (ii) the CRISPR/Cas9 endonuclease, which is supplied in trans and directed by the gRNA to a target site in the vector/transgene itself. We tested our KS system extensively in vitro and show a 10- to 100-fold reduction in transgene expression (Firefly luciferase) after supplying Cas9 in trans using ss and scAAV vectors for the expression of full-length and split Cas9, respectively. Moreover, we expanded our study to an in vivo application in mice, where we could recapitulate our findings in cell culture and trigger an up to 50% reduction in transgene expression. Finally, we devised a universal approach to inactivate any rAAV vector without further modifications. Therefore, we developed and experimentally validated self-inactivating (SIN) CRISPR vectors based on split Cas9 and ssAAVs that harbor the anti-target and anti-Cas9 gRNA and hence allow concurrent targeting of both. Moreover, we utilized different RNA polymerase III promoters (Pol III) to study and eventually optimize the effect of differential gRNA expression on the kinetics of both processes. Collectively, this work has yielded original BoV helper constructs and chimeras that represent valuable new tools to investigate fundamental and applied aspects of bocaviral biology, from the discovery of antigenic domains to the construction of designer viral vectors. Concomitantly, we have implemented and validated novel concepts to increase the safety of recombinant vectors including rAAV KS or SIN constructs that can be harnessed in future work, either alone or in combination with BoV capsids, to form the next generation of parvoviral vectors.
In clinical routine, a case-adapted CT examination is usually conducted for each medical indication in order to allow for a comprehensive high-quality diagnosis of a patient. Therefore, image reading requires the transition between various image stacks, since each medical question implicitly requires organ-dependent reconstructions, display settings, multi planar reformations and image analysis tools. In particular, if dual or multi energy CT data are available, various spectral evaluation methods yield material-specific or functional information. However, the interpretation of this large amount of data is a time-consuming and tedious task. Hence, the purpose of this thesis is to evaluate the potential benefit of the incorporation of patient-specific anatomical priors, which are gained from an automatic multi-organ segmentation, in order to discover novel opportunities to improve the clinical workflow. In this thesis, a new paradigm is proposed which combines competing image properties resulting from different reconstruction algorithms and display settings into a context-sensitive CT imaging by means of anatomical prior information. With the incorporation of anatomical prior knowledge, which is obtained using an automatic multi-organ segmentation approach, various desired image characteristics are combined into a single context-sensitive CT image formation and presentation. The comparison with conventional CT images reveals an improved spatial resolution in highly attenuating materials as well as in air-filled body regions. Simultaneously, the compound image maintains a low noise level in soft tissue resulting in a superior soft tissue contrast compared to conventional images. Furthermore, the novel CT imaging framework allows for the combination of mutually exclusive display settings for the presentation of context-sensitive images to the radiologists. By exploiting anatomical prior information, numerous DECT applications can be integrated into one single DE analysis tool. Moreover, the tools can be chosen and applied to different organs simultaneously without any user interaction. The prior-based DE scheme performs all organ-specific feasible methods instantaneously without the need of a manual selection. Exploiting the anatomical priors, DECT analysis and evaluations are automated and standardized. The iodine quantification accuracy is significantly improved using patient-specific calibrations. The evaluation method and the presentation of the data to the radiologist can be realized via color overlays, pop up menus, volume rendering etc. Furthermore, the method can readily be generalized to the cases of multi energy CT data as it is not limited to the processing of DECT data. The principle of incorporation anatomical prior knowledge is then extended to provide a novel pseudo material decomposition that decomposes dual energy data into more than three basis materials. The method consists of multiple three-material decompositions, where the basis materials are automatically adjusted to the organ of interest based on the automatic segmentation. Moreover, a patient-specific calibration is introduced to improve the volume fraction and material quantification accuracy. An organ-adapted basis material triplet is automatically assigned to each anatomical region resulting in overlapping triangles in the dual energy space. The basis materials are calibrated by evaluating ROIs to improve the volume fraction accuracy. Besides presenting evermore increasing material images to the radiologists, the volume fractions are rescaled to organ-dependent material scores and visualized via pie charts to be later correlated with different diagnoses. The prior-based pseudo multi material decomposition is evaluated using phantom and patient data. The materials are quantified according to the anatomical structure they belong to. Overall, the proposed method provides physically plausible volume fractions that bear the potential to improve the material quantification for diagnosis and e.g. tumor treatment monitoring. In addition, the iodine quantification accuracy and the volume fraction accuracy are evaluated depending on different material calibration methods in conventional DECT applications as well as in the novel pseudo multi material decomposition. The accuracy using default parameters or simulation-based calibrations is compared against the accuracy obtained using patient-specific ROIs. All patient-specific calibrations can be performed directly from the patient data itself, such that almost no user interaction is required. It turns out that a patient-specific calibration is superior compared to a default or simulation based calibration. The new paradigm offers the possibility to display evermore complex information in CT imaging in order to significantly improve the workflow of radiologists. In the clinical routine, e.g. during case presentations and discussions, the fast switching between different image stacks is time-consuming and can be avoided in the future since the CS images merge advantageous image properties resulting from various reconstructions and display settings. The results of the DE evaluation can be dynamically superimposed by color overlays. This superposition provides a comprehensive quantitative analysis of the patient data that can be interpreted as an additional image dimension. By means of the combined DECT evaluation scheme, the radiologists might be assisted in finding a precise diagnosis. In summary, diagnostic accuracy could be increased with the CS imaging by improving the sensitivity for incidental findings: e.g. small nodules can be diagnosed in the lung parenchyma, even if the radiologist is mainly focused on assessing soft tissue. The possibility to robustly decompose DECT data into more than three basis materials opens up for novel clinical evaluation to quantify e.g. fat content and iodine content in the liver simultaneously and to assess long term material scores using pie chart visualizations.
This thesis describes LINC00920, a tumor-associated lncRNA identified in the transcriptome dataset of the International Cancer Genome Consortium-Early Onset Prostate Cancer (ICGC-EOPC) cohort. SiRNA-mediated knockdown of LINC00920 negatively affected proliferation, colony formation, and migration of PC-3 prostate cancer cells. Gene set enrichment analysis of microarray expression data revealed perturbation of pathways related to cell cycle, cell division, apoptosis, and cell movement. Focused pathway analysis of the top LINC00920-deregulated genes showed an inverse relationship between the lncRNA expression and FOXO signaling. Furthermore, as measured by qPCR, knockdown of LINC00920 activated canonical FOXO targets GADD45A, BCL2L11, and PMAIP1 while overexpression of the lncRNA reversed this effect.
In both The Cancer Genome Atlas-Prostate Adenocarcinoma (TCGA-PRAD) and ICGC-EOPC cohorts, LINC00920 positively correlated with ERG overexpression. The regulatory influence of ERG on the lncRNA was then established using cell line models of ERG overexpression, chromatin immunoprecipitation (ChIP) of ERG at the LINC00920 promoter, and promoter luciferase assays using wild-type and mutant promoter fragments.
To address the question of how LINC00920 elicits its associated cellular phenotypes with consideration to its presence across cytosolic, nucleoplasmic, and chromatin compartments, chromatin isolation by RNA purification (ChIRP) followed by high throughput DNA sequencing (ChIRP-seq) and mass spectrometry (ChIRP-MS) were conducted. At the chromatin level, LINC00920 was found primarily associating with heterochromatin regions. LINC00920 occupancy was also be detected in a subset of promoter regions and putative enhancer loci. Interestingly, the lncRNA trace across the mappable genome bore a resemblance to that of the enhancer-associated histone mark H3K4me1, suggesting a role for LINC00920 at enhancer elements. At the protein level, most of the identified LINC00920 interacting partners are well established RNA binding proteins typically associated with the process of transcription. Among the LINC00920-precipitated proteins robustly identified in three biological replicates were two 14-3-3 isoforms—14-3-3ε and 14-3-3ζ. Binding of LINC00920 to 14-3-3ε but not to 14-3-3ζ was validated by RNA immunoprecipitation (RIP) and affinity purification of recombinant 14-3-3ε on streptavidin beads using biotinylated LINC00920.
FOXO activity is mitigated by AKT phosphorylation. FOXO phosphorylation triggers 14-3-3/FOXO complex formation, leading to nuclear exportation. Current results indicate the repressive influence of LINC00920 on FOXO signaling as well as the positive interaction between the transcript and 14-3-3ε. Considering these observations, a rational hypothesis emerged wherein LINC00920/14-3-3ε binding further stabilizes the 14-3-3ε/FOXO complex, resulting in a more efficient sequestration and consequent deactivation of FOXO.
Altogether, this thesis contributes a novel mechanism for a tumor-associated lncRNA in the context of ERG-overexpressing prostate cancer cells. Beginning with the transcriptome analysis of the ICGC-EOPC cohort, and later the TCGA-PRAD dataset, LINC00920 was identified to be an ERG-driven transcript. Ultimately, molecular characterization of LINC00920 by ChIRP-MS has revealed its apparent role in modulating FOXO in conjunction with 14-3-3ε, resulting in reduced expression of a subset of tumor suppressive FOXO targets. Since ERG fusions are clonal events while PTEN deletions are subclonal, driving LINC00920 transcription could be a strategy, in part, for ERG-positive cells to alleviate the influence of an intact PTEN, paving the way for tumorigenesis.
In recent years network methods have gained great popularity in the analysis of resting state data obtained by functional magnetic resonance imaging (fMRI). Network alterations have been found to be induced by various psychiatric disorders as well as drug induced states. Depression, being one of the most prevalent psychiatric disorders, is of special interest for psychiatric research. Brain networks have been found to be affected by depression in many clinical studies examining a variety of patient cohorts (different ages, types of depression, treatment status).
Ketamine, an N-methyl-d-aspartate (NMDA) receptor antagonist, was found to act antidepressantly at sub anaesthetic doses. In contrast to conventional antidepressants (e.g. selective serotonin reuptake inhibitors), ketamine acts very quickly (within hours) and reliably even in treatment resistant patients. However ketamine also induces dissociative effects, often percieved as aversive. Since this discovery was made, other NMDA receptor antagonists are examined as potential glutamatergic antidepressants sharing ketamine's antidepressant action but not its dissociative effects. Two candidate drugs were investigated here: Lanicemine (AZD7665), a low trapping NMDA channel blocker, and traxoprodil, a non-competitive antagonist targeting the NR2B subgroup of the receptor.
One objective of this work was the translation of network methods mostly developed for human studies to preclinical fMRI and the demonstration of their translational potential in a study comparing effects of ketamine across species. Network alterations were supposed to be similar in humans and rats.
As expected, we found largely congruent effects. In both species ketamine induced a more segregated network structure as well as large-scale connectivity alterations. These results show the direct translatability of network methods across species and therefore underline their potential for preclinical neuroscientific research.
Furthermore the network effects of traxoprodil and lanicemine were investigated in another fMRI study in rats. Based on to the effects of ketamine found in previous studies we expected increased connectivity in the hippocampal-prefrontal (Hc-PFC) network as well as reduced network integration. For traxoprodil results were expected to be generally similar to those found for ketamine. Due to its similar mechanism of action we expected similar but less pronounced results for lanicemine compared to ketamine. Nevertheless, deviating results between different NMDA antagonists might help explaining the mechanisms underlying their effects.
Like ketamine, traxoprodil induced an increase in Hc-PFC coupling, however its effect was less pronounced. Lanicemine hardly had any significant effect on this subnetwork. The extent to which Hc-PFC coupling was induced by the three drugs corresponds well to their antidepressant efficacy in clinical trials, which suggests a central role of Hc-PFC coupling in the action of glutamatergic antidepressants.
Approximate simulation methods of quantum many-body systems play an important role for better understanding quantum mechanical phenomena, since due to the exponentially scaling Hilbert space dimension these systems cannot be treated exactly. However, a general simulation approach is still an outstanding problem, as all efficient approximation schemes turn out to struggle in different regimes. Hence, a detailed knowledge about the limitations is an important ingredient to approximation methods and requires further studies. In this thesis we consider two simulation approaches, namely the discrete truncated Wigner approximation as a semi-classical phase-space method, and a quantum Monte Carlo method based on a quantum state parametrization via generative artificial neural networks. We benchmark both schemes on sudden quenches in the transverse-field Ising model and point out their limitations in the quantum critical regime, where strong long-range interactions appear. Furthermore, we study the combination of the quantum state representation in terms of artificial neural networks with the neuromorphic chips present in the BrainScaleS group at Heidelberg University. The goal of this combination is to simulate entangled quantum states on a classical analog hardware. We then expect a more efficient way to approximately simulate quantum many-body systems by overcoming the limitations of conventional computation architectures, as well as further insights into quantum phenomena.
Summary The centrosome is a small non-membranous organelle composed of two centrioles and surrounded by the pericentriolar material. The two primary functions of the centrosome are first, to act as the main microtubule-organizing center in interphase and mitosis and second, to generate the primary cilium. The primary cilium is a microtubule-based structure that projects from the plasma membrane, where it acts as a signaling hub to transfer extracellular signals into intracellular responses. Thereby, the cilium coordinates diverse signaling pathways implicated in development, tissue homeostasis and disease. The primary cilium originates from the older centriole of the pair, called the mother centriole. This mother centriole is decorated at its distal tip with a nine-fold symmetric ring of distal and subdistal appendage proteins. Both centrioles duplicate once per cell cycle to generate one copy of themselves and hence one of the centriole contains the oldest appendages. Studies in model organisms proposed that those inherently asymmetric centrosomes potentially work as a scaffold for asymmetric distribution of cell fate determinants during mitosis and thereby acting as an intrinsic cue for asymmetric cell division. Yet, how centrosome asymmetry is established and how, if at all, influences asymmetric cell division in human stem cells remains unclear. Thus, one aim of this study was to characterize centrosome asymmetry in somatic and stem cells. I observed centrosomal asymmetry during mitosis for a subset of appendages, while others dispersed from the centrosome upon the G2/M transition. My data show that an appendage core (composed of ODF2, Cep83 and SCLT1) remained at the mother centriole from interphase to mitosis, whereas a sub-set of appendages including Ninein, Centriolin, Cep123, Cep164 and LRRC45 detached from the mother centriole during mitosis. The behavior of appendages was similar in differentiated cells and human stem cells. The second aim was to unravel whether centrosome asymmetry regulates asymmetric stem cell division. I found that ODF2 can be used as a marker for centrosome asymmetry during mitosis in human hematopoietic stem and progenitor cells (HSPCs). Here, I correlated the position of the daughter and mother centrosome, visualized by ODF2, with the asymmetric distribution of the stem cell marker CD133 in dividing HSPCs using imaging flow cytometry. Although a role for centrosomes in the asymmetric cell division of HSPCs cannot fully be excluded, I could not observe a clear correlation between centrosome age and CD133 segregation. Further, reversion of centrosome asymmetry by ODF2 depletion had no impact on the differentiation potential of HSPCs. The third aim concentrated on the regulation of appendage behavior. Distal appendages, which are required for initial steps of ciliogenesis, are released from centrosomes before mitosis by a mechanism that is currently unknown. Therefore, I aimed to elucidate the regulation of the cell cycle-dependent behavior of appendages and the consequence of perturbed appendages regulation with special regards to their function in ciliogenesis. Here, I show that the mitotic kinase Nek2 regulates the distal appendage removal at the mother centriole at the onset of mitosis. Ectopic overexpression of Nek2 but not kinase-dead Nek2 prematurely displaced those appendages in interphase, indicating a kinase-specific function. This phenotype was recapitulated in breast cancer cells with high levels of Nek2. Conversely, in Nek2 knockout (KO) cells, appendages remained associated with the older centrosome during mitosis. I could show that persistence of distal appendages on the mother centrosome in mitotic Nek2 KO cells did not allow the cells to fully disassemble their cilia before mitosis, resulting in a ciliary remnant during mitosis. This triggered asymmetric inheritance of ciliary signaling components and asynchronous cilium reassembly after cell division. Asynchronous cilium growth may have consequences for cell fate determination by allowing sister cells to differentially detect environmental signals. Therefore, a ciliary remnant during mitosis might be restricted to asymmetrically dividing stem cells, which need asymmetric cilium re-assembly as a tool for differential responding to environmental signals after cell division. Together, my data established the kinase Nek2 as a central regulator of distal appendages.
Astrocytes are organized in what can be regarded as parallel networks to local neuronal networks. On the cellular level astrocytes immediately react to stimulated neuronal activity by transient increases in intracellular calcium concentration ([Ca2+]i) which results in vitro in a vesicular release of so-called gliotransmitters such as ATP, D-serine and glutamate. In turn these gliotransmitters directly modulate synaptic transmission. Those synapses are regarded as tri-partite connections composed of the pre- and post-synapse enveloped by astrocyte processes. Several mouse models suggested astrocytes as key entities to tune synaptic transmission to vigilance states of synchronized neuronal activity oscillations. However, due to conflicting findings it is still under debate whether gliotransmission takes place under physiological conditions and, in particular, whether and how the activity of a single astrocyte or the general astrocyte population is an indicator for altered coordinated activity of a neural network. In the presented study the bidirectional communication of astrocytes and neurons was investigated. First, by a glia-neuron tracing approach in vivo and second by the simultaneous recording of neuronal and astrocytic activity during pharmacologically-induced oscillatory activity of cultured hippocampal slices. The tracing approach demonstrated in multiple independent experiments, that the rAAV-delivered retrograde tetanus toxin heavy chain tracer (eGFP-TTC) in hippocampal neurons is transferred to retrogradely connected neurons but is not translocated to, or between astrocytes. This excludes the existence of the neuron-like presynaptic target structures in astrocytes, exploited by eGFP-TTC. Likewise, eGFP-TTC is not transferred from astrocytes to neurons. Thus, the eGFP-TTC experiments cannot be used for tracing of neurons in putative networks build of tripartite synapses and moreover do not support the gliotransmission hypothesis. For the population analysis during neuronal oscillations, astrocyte [Ca2+]i and neuronal [Ca2+]i transients were recorded simultaneously by neuron- and astroglia-transduced Ca2+ indicators jRGeco and GCaMP6f, respectively. In both cell types the frequency and the kinetics of [Ca2+]i transient were not affected by the pharmacologically induced oscillations. Temporal cross-correlation analysis of the activity failed to identify any correlations in astrocytes and neuronal [Ca2+]i signals and could not identify any preferred [Ca2+]i transient sequence patterns. From these observations it can be concluded, that in the pharmacologically induced oscillation model either the [Ca2+]i transients in both cell populations are independent from the conditions and from each other, or that the applied virus delivered activity indicators combined with epifluorescence imaging are insufficient to allow detection of subtle correlations in these cells.
The genetic basis of many muscle diseases is known but an understanding of the mechanism underlying muscle weakness is often missing hence a gap remains for the development of effective treatments of these pathologies. Since the process of muscle development as well as function is highly conserved throughout evolution, the skeletal muscles of zebrafish (Danio rerio) show remarkable structural and molecular similarities to those of humans. At the same time, they also make up a considerable portion of its body. Therefore, investigating the developmentally relevant motility genes in zebrafish could help to decipher essential but poorly understood aspects of myogenesis. In this project, I adopted two distinct experimental approaches. The first part of the thesis deals with creating a genetic knockout model to understand the molecular function of the genes apobec2a and 2b which were shown to be relevant for muscle development by our lab. In the course of creating knockouts, we applied a novel, efficient and cost-effective method to predict guide RNA efficiency in zebrafish. The quantitative assessment of gRNAs was provided by the web tool, PCR-F-SEQ which was developed and optimized by us especially for the zebrafish model system. This tool represents a simple but powerful method to screen injected batches of embryos before sending them for raising. Although apobec2a/2b morphants show a dystrophic phenotype, the genetic knockouts do not exhibit any muscle phenotype indicating a possible genetic compensation. In addition, an unbiased approach of investigating the motility mutants isolated from Tübingen screens was used. The rate of retrieval of mutant couples from sperm samples of the Ist and IIIrd Tübingen screens were around 66% and 40% respectively. Following the revival and phenotypic characterization of these lines, we developed a pipeline using next generation sequencing to accurately identify the disease-causing alleles. Mapping of mutations and validation of candidate genes were successfully done for all the six revived lines. Amongst which, we reported a missense mutation in choline-O-acetyltransferase a (chata) gene, encoding an enzyme essential for the synthesis of a major neurotransmitter, acetylcholine (ACh). The in-silico analysis showed that the substitution of serine to arginine might affect the protein stability disrupting the catalysis of acetyl CoA and choline to form ACh. In conclusion, this thesis showcases the challenges and strengths of both reverse and forward genetic approaches to study vertebrate development and also highlights the importance of strategies and tools now available for making genetic models.
Cancer is worldwide the leading cause of death in the 21st century. Liver cancer represents the fourth most common cancer with hepatocellular carcinoma (HCC) being the most frequent primary liver cancer. HCC is the second most common cause of cancer-related deaths with a 5-year survival rate below 12% in the US. These sobering numbers illustrate the need for bio-medical research to identify novel target structures and to develop respective therapeutic strategies. The evolutionarily conserved Hippo signaling pathway is frequently deregulated in human hepatocarcinogenesis. Indeed, overexpression of the transcriptional co-activators yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) is associated with pro-proliferative and anti-apoptotic effects in liver tumor cells. However, among the identified direct YAP/TAZ target genes, no functional groups or protein families have been described, which facilitate the biological properties of YAP and TAZ in HCC cells. Since these groups may represent relevant molecular hubs that mediate the tumor-supporting properties of the Hippo pathway, this study aimed to define YAP/TAZ-regulated protein families, which may represent promising therapeutic target structures. Based on comprehensive transcriptome analysis after siRNA-mediated inhibition of YAP and TAZ in HCC cells, the minichromosome maintenance (MCM) protein family, which is crucial for DNA replication, and members of the polycomb repressive complex 2 (PRC2), which inserts epigenetic repression marks (trimethylation of histone H3; H3K27me3), were identified as YAP and/or TAZ target genes. For the MCM family members (MCM2-7), different molecular approaches confirmed the direct transcriptional regulation via YAP/TAZ and the transcription factor TEA domain transcription factor 4 (TEAD4) in independent liver cancer lines. In contrast, the PRC2 components Enhancer Of Zeste 2 (EZH2) and Suppressor Of Zeste 12 Protein Homolog (SUZ12) were predominantly regulated by YAP and TEAD4. Functionally, RNAi-mediated MCM reduction as well as YAP and TAZ perturbations decreased cell proliferation in vitro. Additionally, YAP induces the expression of PRC2 components and their epigenetic mark H3K27me3 in vitro, leading to the repression of target gene transcription. In a YAP-dependent HCC mouse model (inducible expression of constitutively active YAPS127A)) a clear positive association between YAP overexpression and especially abundance of MCM family members confirmed the in vitro results. Statistical correlations between tumor progression, YAP expression and MCM2-7, EZH2, and SUZ12 abundance in primary patient tissues supported the hypothesis that both protein groups are cooperatively induced by YAP and TEAD4 in human HCC. Together, these results illustrate that the Hippo pathway contributes to tumor progression via the regulation of protein groups that induce biological functions. The transcriptional regulation of MCM helicase by the Hippo pathway introduces a promising surrogate marker for YAP driven proliferation. Furthermore, YAP induced transcription of PRC2 members illustrates a new possibility of YAP mediated gene silencing and offers a novel approach to understand oncogenic Hippo/YAP feedback loops. Interestingly, both YAP and TAZ are important for the regulation of all MCM family members, while data for PRC2 constituents point to a predominant role of YAP. This differential dependency of target genes might represent a molecular mechanism of how the Hippo pathway regulates specific biological processes, as epigenetic changes and replication. This is of special importance since direct perturbation of the Hippo/YAP/TAZ-axis could also inhibit pro-regenerative properties (e.g. proliferation), which might be of relevance for patients with chronically damaged livers. Thus, targeting these important downstream hubs could represent one strategy to selectively impair YAP and/or TAZ-dependent HCC cell functionality while saving physiological hepatocellular proliferation in regenerative livers, which could maintain residual liver function. Still, further studies are needed to test if MCM proteins and PRC2 are suitable points-of-interference in HCC patients with YAP and/or TAZ activation.
The tumor microenvironment (TME) is now recognized as an important factor in breast cancer progression and is crucial in determining the response to anti-cancer therapies. Fibroblasts are one component of the TME that are activated in the early stages of oncogenesis and remodel the TME as an early tissue repair response. In the later stages of tumor progression, these activated fibroblasts get transformed into cancer associated fibroblasts (CAFs) that promote tumor progression. The mechanisms underlying this transformation of fibroblasts from normal activated fibroblasts to CAFs remain largely unknown. To address this, bone marrow derived mesenchymal stromal cells (MSCs) that are known to be a significant source of CAFs were treated with conditioned media of a breast cancer cell line, namely, MDA-MB-23, to induce a CAF-like - phenotype. Activation of MSCs was confirmed by assessing their contractility and expression of CAF markers. Gene expression and secretome analysis was performed to identify differentially expressed genes and differentially secreted factors upon activation. Stanniocalcin 1 (STC1), a CAF-secreted protein implicated in metastasis and therapy resistance, was found to be upregulated upon conditioned media treatment. Therefore, the aim of this study was to understand the mechanism of STC1 upregulation and its involvement in the activation of fibroblasts. STC1 knockdown using RNAi both in conditioned media treated MSCs and patient derived activated CAFs showed inhibitory effects on activation status of these cells. Moreover, this down-regulation resulted in a decrease in the expression of several proteins such as NOTCH3, NOTCH4 and β-catenin suggesting possible mechanisms by which STC1 regulates the activation of fibroblasts. Furthermore, the upregulation of STC1 upon conditioned media treatment was found to be dependent on RELA and RELB transcription factors indicating that NF-κB signaling might be involved in its transcriptional regulation. In conclusion, this study demonstrates STC1 as an important factor in the activation of CAFs. Investigating the mechanism of the involvement of STC1 in the unabated activation of CAFs will enable a deeper understanding of the tumor microenvironment and its role in the tumor progression.
Breast cancer is the most frequently diagnosed malignancy among women. Due to its molecular heterogeneity, a generalized therapy is not possible. Nuclear estrogen receptor-α (ER-α) is overexpressed in the majority of breast tumors. ER-positive patients benefit from endocrine therapy that abrogates estrogen-induced tumor growth. However, approximately half of the patients do not respond or relapse due to de novo or acquired resistance against therapy. Occurrence of resistance is a drawback for long-term efficacy of endocrine therapy and leads to poor prognosis. The mechanisms underlying acquisition of endocrine therapy resistance, however, remain elusive. Recently, epigenetic reprogramming has been proposed as a means to render the tumor cells refractory to treatment. Therefore, the aims of this project were to uncover novel targets that confer resistance and to elucidate the involvement of epigenetics in this process. To this end, two ER-positive cell lines (MCF7 and T47D) were utilized to recapitulate endocrine therapy resistance in vitro by treating them either with tamoxifen (TAMR) or depriving them of estrogen (LTED). I identified GLYATL1 (glycine-N-acyltransferase like 1) as a highly de-regulated gene as revealed by RNA-seq and ATAC-seq integrative analysis comparing resistant cell lines to the sensitive parental. GLYATL1 encodes for an enzyme that catalyzes the transfer of an acyl group to glutamine. I showed that knockdown of GLYATL1 sensitizes resistant cell lines while GLYATL1 overexpression renders sensitive luminal cells resistant to endocrine therapy. Furthermore, I found GLYATL1 is involved in acetylation of histone residues H3K9 and H3K14 since the knockdown of GLYATL1 led to a decrease in these two histone marks in resistant MCF7 cells. Moreover, I showed the expression of GLYATL1 to be regulated in these cells by methylation, growth factor receptor HER2, and transcription factors ERα, GATA3 and p300. CRISPR/dCas9-mediated epigenetic editing method was adopted to validate the involvement of methylation in GLYATL1 regulation. This method is a repurposed version of CRISPR/Cas9 system where Cas9 is catalytically inactive and fused to catalytic domain of epigenetic enzymes. Combined with sgRNAs, these effectors can be recruited to target regions to modulate the epigenetic landscape, thereby altering gene expression. Furthermore, I utilized this method of epigenetic editing to investigate endocrine therapy resistance involvement of other genes such as CD44 and BAMBI, expression of which were also found to be elevated in resistant cells compared to parental. I showed that targeting promoter regions of CD44 and BAMBI with dCas9-p300 yielded upregulation of both genes whereas dCas9-G9a combination led to a downregulation which resulted in retarded proliferation in LTED cells. Moreover, altering expression of BAMBI elicited similar changes in CD44 expression further proving CD44 as a direct target gene of the Wnt signaling pathway, for which BAMBI acts as an activator. In conclusion, my results demonstrate the importance of GLYATL1 in initiation and maintenance of endocrine therapy resistance and identify its involvement in H3K9 and H3K14 acetylation. This study demonstrates the potential of epigenetic reprogramming mediated regulation of target gene expression as a novel method of therapeutic intervention.
The current work is devoted to the mathematical modelling of the development of fish respiratory organs, called gills or branchiae. The model organism chosen for the task is the Japanese rice fish (Oryzias latipes), more colloquially known as medaka. Their gills are analysed in the attempt to answer three main developmental questions via mathematical modelling, with possible applications beyond the scope of this thesis. Firstly, how many stem cells are needed to build the organ? What kind of heterogeneities exist among these stem cells? And, finally, what properties and relations with each-other do these stem cells have, that give the organ its shape?
Relying on experimental data from our collaborators in the group of Prof. Lazaro Centanin, Centre for Organismal Studies, Heidelberg University, we use a variety of methods to study the aforementioned aspects. These methods were selected, adapted and developed based on the goal of each project and on the available data. Thus, a combination of stochastic and deterministic techniques are employed throughout the thesis, including Gillespie-type simulations, Markov chains theory and compartmental models.
The study of stem cell numbers and heterogeneities is approached via stochastic simulations extended from the algorithm of Gillespie, and further improved by Markov chains methods. Results suggest that not only very few stem cells are sufficient to build and maintain the organ but, more importantly, these stem cells are heterogeneous in their division behaviour. In particular, they rely on alternating activation and quiescence phases, such that once a stem cell has divided, it becomes activated and divides multiple times before allowing another one to take the lead.
For the study of growth and shape of gills, multiple deterministic models based on different assumptions and investigating various hypotheses have been developed. All these models have a compartmental structure, with increasing number of compartments governed by indicator functions which, in turn, depend on explicit or implicit algebraic equations. For each model, the existence, uniqueness and non-negativity of solutions are proved, the analytical solutions are found and their regularity is discussed. The models are compared based on their ability to reproduce part of the data, and the best one is selected. The chosen model is then applied to further data and speculations on hypotheses supporting the model are made. Results suggest that the main stem cell types, responsible for growing the organ, slow down their proliferation in time, either due to ageing or to the lack of sufficient nutrients.
The main results and strengths of this thesis consist of the high variety of models developed and methods employed, their capability to answer important biological questions and, even more, to uncover new insights on mechanisms previously unknown.
Der Klimawandel ist eine der größten Herausforderungen der Menschheit. Wir wissen alle, dass sich etwas ändern muss. Aber was genau? Was können wir tun und wie sollen wir Fakten und Fake unterscheiden? Antworten geben die besten Mathematiker und Informatiker der Welt beim 7. Heidelberg Laureat Forum. Vom 22. bis zum 27. September treffen die Koryphäen auf 200 Nachwuchswissenschaftler. Campus Reporter Nils Birschmann hat sich das Programm angeschaut.
Der Beitrag erschien in der Sendereihe "Campus-Report" – einer Beitragsreihe, in der über aktuelle Themen aus Forschung und Wissenschaft der Universitäten Heidelberg, Mannheim, Karlsruhe und Freiburg berichtet wird. Zu hören ist "Campus-Report" montags bis freitags jeweils um ca. 19.10h im Programm von Radio Regenbogen (Empfang in Nordbaden: UKW 102,8. In Mittelbaden: 100,4 und in Südbaden: 101,1).
The hypothalamic neuropeptide oxytocin (OT) promotes social communication via its central release in the mammalian brain. However, how social interaction affects electrical activity of OT neurons is still unclear. To address this question, I used cell-type specific viral vectors in combination with optoelectrode-based techniques. I performed the in vivo single-unit recording of optically identified OT neurons in the paraventricular nucleus (PVN) of hypothalamus in adult female rats during their social interactions with unfamiliar female conspecifics. Simultaneously, we monitored behavior and recorded ultrasonic vocalizations. The results showed that active social interactions events induce an increase of PVN OT neurons spiking activity as well as a re-organization of the firing pattern from regular to bursting. The action potentials of simultaneously recorded OT neurons were synchronized and phase-locked with the PVN theta oscillations precisely at the time of social interactions, but not during non-social exploratory behavior. To decipher which sensory stimuli trigger OT neuron activity, I performed experiments with partial deprivation of specific sensory modalities. Direct physical contact between rats, or even gentle skin stimulation, led to a profound increase in OT firing rates. In contrast, presentation of visual, auditory and olfactory social-relevant stimuli alone did not significantly alter OT neuron activity. This led to the conclusion somatosensory component of social interaction drives OT neurons synchronous activity. To further explore the effects of tactile stimuli on the OT system, I examined the expression of the marker of neuronal activity c-Fos after repetitive somatosensory stimulation; it appeared to be significantly increased in a particular subpopulation of OT neurons named parvocellular OT neurons. Employing in-vivo calcium recording via fiber photometry, I investigated the role of parvocellular OT neurons in regulating the activity of the general population of PVN OT neurons, finding that parvocellular OT neurons mediate the activation of the OT system in response to somatosensory stimuli. Next, I selectively modulated the activity of parvocellular OT neurons in awake freely moving rats via pharmacogenetics: activation of this population of neurons resulted in increased social interaction, while inhibition leaded to decrease of social interaction. Finally, I studied the effect of intracerebral infusion of an OT receptor antagonist which induced a substantial reduction of social interaction time, even when parvocellular OT neurons were activated. Altogether, these results indicate that somatosensory stimulation is essential to activate OT neuron ensembles and, hence, can induce central neuropeptide release in socially interacting female rats. This opens perspectives for studying functional and anatomical connectivity between the somatosensory and OT systems in normal and psychopathological conditions.
Spiking neural networks (SNNs) have been proposed both as models of cortical computation and as candidates for solving problems in machine learning. While increasing recent works have improved their performances in benchmark discriminative tasks, most of them learn by surrogates of backpropagation where biological features such as spikes are regarded more as defects than merits. In this thesis, we explore the enerative abilities of SNNs with built-in biological mechanisms. When sampling from high-dimensional multimodal distributions, models based on general Markov chain Monte Carlo methods often have the mixing problem that the sampler is easy to get trapped in local minima. Inspired from traditional annealing or tempering approaches, we demonstrate that increasing the rate of background Poisson noise in an SNN can flatten the energy landscape and facilitate mixing of the system. In addition, we show that with synaptic short-term plasticity (STP) the SNN can achieve more efficient mixing by local modulation of active attractors and eventually outperforming traditional benchmark models. We reveal diverse sampling statistics of SNNs induced by STP and finally study its implementation on conventional machine learning methods. Our work thereby highlights important computational consequences of biological features that might otherwise appear as artifacts of evolution.
Olfaction, the sense of smell, is one of the most important sensory stimuli for any organism, from unicellular bacteria to highly complex metazoans like humans. Volatile chemicals constituting olfactory cues can carry a vast range of information, enabling animals to detect and locate food, mates or shelter. The structure of the olfactory system has remained remarkably conserved, indicating that the precise anatomy is integral to the detection and discrimination of odours. Previous studies have shown that the connectivity of olfactory bulb mitral and granule cells plays a crucial role in odour discrimination in mice, yet several aspects of granule cell function remain unclear. To investigate the impact of modified granule cell function on odour discrimination in mice, we first established a novel automated operant conditioning setup. This setup allowed us to train large (>20 animals) groups of mice on a go/no-go odour discrimination task with minimal experimenter interference. It was used for detailed analysis olfactory behaviour by assessing parameters such as the discrimination time, the speed at which animals were able to discriminate and odour and initiate a corresponding behavioural response. As neurobiological function follows form, we focussed on the specialized dendrodendritic connection between the principal output neurons of the mammalian olfactory bulb, the mitral cells, and the most abundant inhibitory interneurons of the olfactory bulb, the granule cells. As inhibition of mitral cells from granule cell has been shown to directly affect the discrimination of odour mixtures, we sought to modulate the strength of this inhibition using two different approaches. The first strategy was based on increasing the inhibitory output through the overall increase of granule cells. Towards this end, a novel transgenic mouse line was used, in which the pool of neuronal stem cells can be temporarily increased resulting in an olfactory bulb specific increase of physiologically normal and functionally integrated granule cells. Utilizing the automated behaviour setup, we were able to show that discrimination accuracy, but not the discrimination time of highly similar mixtures of two enantiomers is increased in mice harbouring an increased number of granule cells. The second approach was based on reducing the inhibitory output of granule cells by reducing the global activation of granule cells following local dendritic activation, a feature which is thought to be gated by T-type calcium channels. Therefore, we established a simultaneous triple knock-down of all three T-type subunits using adeno-associated virus-based shRNA expression in granule cells. We found that mice with reduced T-type expression did not show the typical increase in discrimination times when comparing simple with complex odours, suggesting that a lack of global activation affects discrimination of simple odours. In summary, these findings highlight the importance of granule cell-derived inhibitory input onto mitral cells for the discrimination of highly similar odour stimuli. Taking into account the subtle nature of the molecular modifications and the flexibility of the novel approach to behavioural phenotyping, these results clearly outline the path to a large-scale, systematic investigation into the limits of olfaction.
Since in trypanosomes most protein-coding genes are constitutively transcribed by RNA polymerase II in a polycistronic manner, gene expression is mainly regulated at the post-transcriptional level. It is therefore interesting to investigate the relevant regulatory factors. In a previous genome-wide tethering screen hundreds of putative mRNA-fate regulators were found, including the proteins BFR1L, an up-regulator of gene expression, and ZC3H5, a down-regulator of gene expression. BFR1L has some similarities to yeast Bfr1p, an ER- and polysome-associated protein. BFR1L displays in vivo mRNA binding although it lacks canonical RNA-binding domains. Double-knockout bloodstream form trypanosomes displayed a slight growth defect. By immunofluorescence microscopy, a tagged version was located in the cytoplasm and overlapped partially with an ER marker. RNA pull-down analysis suggested that most of the BFR1L-bound mRNAs encode ribosomal proteins, but no common RNA motif could be found by in silico analysis. The mRNAs encoding ribosomal proteins are known not to sequester in granules upon starvation stress or heat shock. Similarly, BFR1L protein did not go to granules under starvation stress. It is tempting to speculate that the interaction remains active during stress, and targeting of the mRNAs to the ER could prevent sequestration into granules and could keep the mRNAs in ribosomes. The attachment of BFR1L to the ER could be mediated via the putative interaction partner Tb927.9.9550, which has a transmembrane domain. ZC3H5 knock-down led to a fast growth defect, killing the cells after 48 h of RNAi induction. We therefore analyzed the RNAi effect on growth kinetics, protein levels, nuclei/kinetoplasts ratios and the transcriptome at different time points. After RNAi induction, the proportion of 2N2K cells increased rapidly. In addition, the ZC3H5 RNAi cells often possessed abnormal and higher numbers of nuclei and kinetoplast. While short-term down-regulation of ZC3H5 showed only a minor effect with respect to the transcriptome, an increase of mRNAs encoding ribosomal proteins, the increase of the monosomal peak without an increase of mRNAs in this fraction, the occurrence of half-mers as well as an increase of mRNAs encoding ribosomal proteins in the free fraction were observed with respect to the polysomal profiles. This suggest that the ribosome assembly is disturbed upon knock-down of ZC3H5. However, this seems to be a secondary effect. RNA pull-down analysis suggested that ZC3H5 binds mRNAs encode cytoskeleton proteins. Tandem Affinity Purification of ZC3H5 followed by MS analysis revealed three putative interaction partners which were validated by co-immunoprecipitation (Tb927.8.1500, Tb927.7.3040 and Tb927.11.4900). In addition, tethering of ZC3H5 and its interaction partners to a CAT reporter showed that the proteins are repressors; thus, we have identified a novel repressor complex that may regulate genes required for cell cycle progression. The exact mechanism of action is not known at the moment, but it is tempting to speculate that the function of Tb927.11.4900 as a G protein is responsible for the association and dissociation of the complex. This could be cell cycle dependent, because the target mRNAs peak in S-phase. Maybe the ZC3H5 complex represses its targets during the rest of the cell cycle and targets are de-repressed in S-phase to produce the proteins needed for cytokinesis.
This dissertation concerns the concept of PT symmetry as a replacement of the conventional symmetry of the Dirac Hermiticity of the operators, in particular, for the Hamiltonian. After solving a controversial issue surrounding the claimed violation of the special relativity by PT-symmetric quantum mechanics, we move further to discuss PT symmetry for the systems whose associated time reversal symmetry is odd, i.e., T^2 = −1. Our results, as published papers, are incorporated in the subsequent chapters. Then we conclude by suggestions for future work.
In this thesis, the first high-precision measurement of the ground-state $g$-factor of a boronlike ion, $^{40}$Ar$^{13+}$, with a fractional uncertainty of \SI{1.4e-9}{}, is presented. The measurement has been performed on a single boronlike argon ion with the double Penning-trap setup of the newly developed ALPHATRAP experiment. Within this work, the trap tower of the experiment has been developed, assembled and tested prior to commissioning it together with the rest of the ALPHATRAP setup. The resulting measurement presented here corresponds to the most precise $g$-factor determination of a five-electron system to date. Not only does it allow testing the currently available theoretical predictions for the many-electron, QED and nuclear-recoil contributions, but also distinguishes between calculations that are in disagreement. The $g$-factor obtained here is in agreement with the most recent and most precise theoretical prediction, which has a relative uncertainty of \SI{9e-7}{}. This level of agreement constitutes one of the most accurate tests of many-electron QED contributions in strong fields. The sensitivity of this test will improve in the future with anticipated improvements on the theoretical $g$-factor, which includes higher-order QED contributions. Furthermore, this measurement paves the way towards the independent determination of the fine-structure constant with heavier highly charged ions in ALPHATRAP, where a specific difference of the boron- and hydrogenlike ions' $g$-factors will be used to cancel nuclear structure effects.
In mammals, the process of neurogenesis consists in the generation of various types of neuronal and glial cells from neural stem cells (NSCs). It begins intensively at the embryonic stage and continues through the whole adulthood. In adult rodents, neurogenesis is mainly located in two regions: the ventricular-subventricular zone (V-SVZ) of the lateral ventricles and the dentate gyrus of the hippo-campus. In the last two decades huge progress has been made to characterize the process in detail and to get further insights of its regulation. However, still some fundamental questions remain unanswered. Among those, whether post-transcriptional regulation plays a critical role in NSC activation and differentiation. In this project, I investigated protein synthesis and its modulation upon activation of NSCs using the mouse adult brain as an experimental model. The analysis of the nascent synthesized peptides in NSCs and early neuroblasts (ENBs) of the same lineage revealed that the level of global protein synthesis decreases upon the transition from NSCs to ENBs. The transcriptome and translatome analysis of NSCs and ENBs clearly showed an active involvement of post-transcriptional regulation in gene expression at the onset of NSC differentiation. In particular, translation of neuronal specification transcripts such as Sp8 and Dusp4 was enhanced. On the contrary, the translation of some mRNAs carrying the Terminal Oligo Pyrimidine (TOP) and the Pyrimidine Rich Motif (PRM) such as Sox2 and Rpl18 were selectively repressed. At this transition, we also observed a drop of mTOR activity upon cell cycle exit that was causally linked to repression of TOP- and PRM-transcripts. Altogether, our study underscored the role of protein synthesis and its regulation in NSC differentiation. It also demonstrated a causal link between cell cycle exit, TOR activity and exit of the stem cell state.
Dengue virus (DENV) has emerged as major human pathogen. Despite the serious socio-economic impact of DENV-associated diseases, antiviral therapy is missing. DENV replicates in the cytoplasm of infected cells and induces a membranous replication organelle, formed by invaginations of the endoplasmic reticulum membrane and designated vesicle packets (VPs). Nonstructural protein 1 (NS1) of DENV is a multifunctional protein. It is secreted from cells to counteract antiviral immune responses, but also critically contributes to the severe clinical manifestations of dengue. In addition, NS1 is indispensable for viral RNA replication, but the underlying molecular mechanism remains elusive. In this study, we employed a combination of genetic, biochemical and imaging approaches to dissect the determinants in NS1 contributing to its various functions in the viral replication cycle. Several important observations were made. First, we identified a cluster of amino acid residues in the exposed region of the β-ladder domain of NS1 that are essential for NS1 secretion. Second, we revealed a novel interaction of NS1 with the NS4A-2K-4B cleavage intermediate, but not with mature NS4A or NS4B. This interaction is required for RNA replication, with two residues within the connector region of the NS1 "Wing" domain being crucial for binding of the NS4A-2K-4B precursor. By using a polyprotein expression system allowing the formation of VPs in the absence of viral RNA replication, we show that the NS1 -NS4A-2K-4B interaction is not required for VP formation, arguing that the association between these two proteins plays a more direct role in the RNA amplification process. Third, through analysis of polyproteins containing deletions in NS1, and employing a trans-complementation assay, we show that both cis and trans acting elements within NS1 contribute to VP formation, with the capability of NS1 mutants to form VPs correlating with their capability to support RNA replication. In conclusion, these results reveal a direct role of NS1 in VP formation that is independent from RNA replication, and argue for a critical function of a previously unrecognized NS4A-2K-NS4B precursor specifically interacting with NS1 and promoting viral RNA replication.
In this thesis we develop a numerical solution method for the instationary incompressible Navier-Stokes equations. The approach is based on projection methods for discretization in time and a higher order discontinuous Galerkin discretization in space. We propose an upwind scheme for the convective term that chooses the direction of flux across cell interfaces by the mean value of the velocity and has favorable properties in the context of DG. We present new variants of solenoidal projection operators in the Helmholtz decomposition which are indeed discrete projection operators. The discretization is accomplished on quadrilateral or hexahedral meshes where sum-factorization in tensor product finite elements can be exploited. Sum-factorization significantly reduces algorithmic complexity during assembling. In this thesis we thereby build efficient scalable matrix-free solvers and preconditioners to tackle the arising subproblems in the discretization. Conservation properties of the numerical method are demonstrated for both problems with exact solution and turbulent flows. Finally, the presented DG solver enables long time stable direct numerical simulations of the Navier-Stokes equations. As an application we perform computations on a model of the atmospheric boundary layer and demonstrate the existence of surface renewal.
Um die Bildgebung von gastrointestinalen Stromatumoren mittels Positronen-Emissions-Tomo¬graphie zu verbessern, wurden in dieser Arbeit neue 18F-markierte PET-Radiotracer entwickelt und evaluiert. Dafür wurde einerseits ein Radiotracer ([18F]Fluor-Norimatinib) entwickelt, der die Sensitivität des Tumors gegenüber Imatinib darstellen sollte und andererseits ein Radiotracer ([18F]Fluor-DOG1), mit dem sich gastrointestinale Stromatumore von anderen Sarkomen ohne invasive Biopsie unterscheiden lassen sollten. Literaturbekannte 18F-markierte Imatinib-Derivate weisen eine hohe Lipophilie und damit eine starke Akkumulation in der Leber auf, die die Bildgebung von gastrointestinalen Stromatumoren erschwert, weshalb in dieser Arbeit der hydrophilere Metabolit Norimatinib als Grundstruktur verwendet wurde. Das Radiolabel wurde am Pyridin-Ring eingeführt, da an dieser Position keine metabolischen Prozesse bekannt waren und [18F]Fluorpyridine über Nitropyridin-Vorläufer einfach zugänglich sind. Die Darstellung des Nitropyridin-Vorläufers und des Fluor-Standards von FNI wurden zunächst in einer 6-stufigen Synthese geplant, die allerdings nur für den Standard Fluor-Norimatinib (FNI) erfolgreich durchgeführt werden konnte, da die harschen Reaktionsbedingungen des letzten Reaktionsschrittes nicht mit dem Nitro-Derivat kompatibel waren. Eine neue 7-stufige Synthese, bei der weitestgehend auf mildere Bedingungen zurückgegriffen wurde, konnte jedoch den Nitropyridin-Vorläufer in guten Ausbeuten liefern. Die zweistufige Radiomarkierung konnte erfolgreich etabliert werden, sodass [18F]FNI nach Radiofluorierung, Entschützung und Aufreinigung in guten Ausbeuten erhalten wurde. Die Eva¬luierung in vitro wies aufgrund der Lipophilie nur eine geringe spezifische Bindung auf, jedoch zeigte FNI ein zu Imatinib analoges Bindungsprofil bezüglich verschiedener KIT-Mutanten. In den in vivo-Studien konnte zwar die renale Ausscheidung des Radiotracers stark erhöht werden, wodurch eine vorteilhafte, geringere Akkumulation in der Leber resultierte, allerdings erfolgte keine hohe Anreicherung in den Tumoren, vermutlich aufgrund deren geringer Vaskula¬risierung. Des Weiteren wurde durch die Metabolisierung des Radiotracers [18F]Fluorid freigesetzt, welches zu erhöhter Knochenanreicherung führte. Dies konnte jedoch durch Administra¬tion kleiner Mengen Ketoconazol reduziert werden, wobei zudem die Anreicherung in den Tumoren leicht stieg. Für die Radiomarkierung von [18F]Fluor-DOG1 wurden zunächst verschiedene Vorläufer mit Nitro-, Triazen- bzw. Borsäureester-Abgangsgruppen erfolgreich synthetisiert. Dabei zeigte sich, dass der Nitro-Vorläufer keine ausreichende Stabilität unter den harschen Markierungsbedingungen besaß, weswegen auf den reaktiveren Triazen-Vorläufer zurückgegriffen wurde. Dessen hohe Reaktivität führte allerdings zu einer Vielzahl an Reaktionsprodukten, die sowohl die Analytik, als auch die Aufreinigung derart erschwerten, dass auch dieser Vorläufer keine sinnvolle Option darstellte. Über die Kupfer-katalysierte Radiofluorierung des Borsäureester-Vorläufers hingegen konnte der gewün¬schte Radiotracer [18F]Fluor-DOG1 in guten Ausbeuten erhalten werden. In ersten in vivo-Studien in Mäusen zeigte der Radiotracer keine zufriedenstellende Pharmakokinetik, da die Anreicherung im Tumor nur unzureichend stattfand, jedoch eine starke Akkumulation in der Leber beobachtbar war. Dies wurde auf die geringe Stabilität von [18F]Fluor-DOG1 in murinem Blutserum zurückgeführt, weswegen eine Optimierung dahingehend vorgenommen werden sollte.
Förster resonance energy transfer (FRET) is a quantum effect of energy transfer from the donor chromophore to the acceptor one via non-radiative dipole-dipole coupling when those chromophores are positioned close enough and in the right orientation to each other. Genetically encoded FRET sensors consist of donor and acceptor fluorescent protein pair (usually CFP/YFP) and a protein based sensing domain in between which responds to the presence or the activity of desired molecule via conformational change resulting in change of FRET efficiency. In this thesis, I developed a genetically encoded FRET sensor for detecting double stranded RNA (dsRNA) during viral infection. The response of eukaryotic cells to infection by RNA viruses is based to a large extent on the regulation by protein kinase R. Protein kinase R (PKR, RNA-regulated protein kinase, eIF2α kinase 2) becomes activated via homodimerisation in the presence of double-stranded RNA produced during replication of RNA viruses (or if endogenously present). Active PKR phosphorylates its target – mostly α subunit of eIF2 – and inhibits the translation of viral proteins. PKR is a two domain protein which consists of an N-terminal double-stranded RNA binding domain (RBD) and a C terminal catalytic domain separated by a 100-amino acid unstructured region. The idea exploited in the current project is based on the ability of the N-terminal PKR domain to undergo a conformational change when binding double-stranded RNA, hence functioning as a sensor for double-stranded RNA. The fluorescent sensor is completed by the addition of the fluorescent proteins mTurquoise and cp173Venus forming a FRET pair. I successfully tested the sensor termed KPR1 in vitro as well as in HeLa Kyoto cells against double-stranded RNA and found a high FRET increase upon binding. The sensor responded well to the presence of self-replicating subgenomic Hepatitis C Virus RNA replicon in Huh7 cells. The data on detection of full Hepatitis C infection was inconclusive.
Bispecific T cell engagers (BiTEs), artificial antibodies designed to cross-link T cells to tumor cells, and chimeric antigen receptors (CARs), expressed on the surface of modified T cells and transferring activating signals via intracellular domains, can re-direct T cells to tumor surface antigens for efficient treatment of hematological malignancies. In solid tumors, however, these novel immunotherapies face fundamental challenges: Physical exclusion of T cells and an immunosuppressive microenvironment prevent efficacy while systemic administration is associated with severe toxicities. Oncolytic viruses have emerged as ideal agents for combination immunotherapies, as lytic replication in tumors does not only induce tumor debulking, but also immunogenic cancer cell death and local inflammation. We therefore hypothesized that oncolytic virotherapy, by eliminating physical barriers and reversing local immunosuppression, can promote anti-tumor T cell responses and thus provide a potent treatment option for solid tumors in combination with T cell re-direction. We furthermore hypothesized that tumor-targeted expression of a virus-encoded BiTE could achieve local BiTE activity without systemic side effects. To test these hypotheses, we engineered oncolytic measles viruses (MV) encoding BiTEs targeting CD20 or carcinoembryonic antigen, respectively, as model antigens (MV-BiTE). Kinetics of viral replication and virus-mediated cytotoxicity showed minor differences compared to unmodified virus, and functional BiTEs could be obtained from the supernatant of virus-infected cells. A newly established syngeneic solid tumor model of B16-CD20-CD46 murine melanoma cells stably expressing human antigens CD20 and CD46 as BiTE target antigen and measles virus entry receptor, respectively, engrafted in immunocompetent C57BL/6 mice when implanted subcutaneously. In this model, treatment with MV-BiTE targeting CD20 significantly prolonged survival compared to control treatments with unmodified MV, MV encoding CEA-specific control BiTE, purified BiTE, or carrier fluid only, respectively. UV irradiation of MV-BiTE completely inhibited viral replication but did not abrogate efficacy in this model. Treatment efficacy was furthermore not impaired in mice previously immunized with MV. Increased T cell infiltration into tumors and an effector T cell phenotype characterized by high intratumoral CD8+ T cell levels and low relative abundance of regulatory T cells was observed upon MV-BiTE treatment. Targeted transcriptome analysis revealed upregulation of genes associated with T cell activation, proliferation, and differentiation, but also with inhibition and exhaustion, providing a rationale for combination with checkpoint inhibitors. Combinations of MV, BiTE, and CAR T cell therapies were investigated in a pancreatic cancer model. Cytotoxic potential of each treatment alone towards human pancreatic adenocarcinoma cells was observed in vitro. However, no significant benefit of combinations was observed in a pilot experiment in immunodeficient mice, and T cell persistence was limited. For future in vivo monitoring of T cells by magnetic resonance imaging, labeling with iron oxide nanoparticles was established. In addition, we describe a mathematical model for in silico predictions of treatment outcome to optimize scheduling of combination therapies. Taken together, this study shows for the first time efficacy of a BiTE-encoding oncolytic virus in an immunocompetent mouse model. This warrants further investigations towards future translation. We furthermore introduced a mathematical model of combination immunovirotherapy to further support the development of novel treatment options for cancer patients.
Diborane(4) compounds, exhibiting an electron-precise boron-boron bond, are extremely versatile reagents in organic synthesis, therefore the preparation and reactivity of these compounds is of great interest in modern boron chemistry. The introduction of such boron-boron bonds is limited to very few examples in contrast to the variety of carbon-carbon coupling reactions. Among the established strategies to boron-boron bond formation, dehydrocoupling reactions constitute an elegant, “atom- economic” pathway. However, these reactions are still restricted to a very few selected examples. The aim of this thesis was to establish new strategies to boron-boron bond formation via dehydrocoupling reactions; therefore various bidentate ligands, such as neutral bisphosphanes or anionic amidine or guanidine compounds were introduced. The respective bisphosphane borane adducts were isolated and fully characterized. Furthermore, the reactivity of these diboranes towards carbonyl complexes, hydrid abstraction and potential dehydrocoupling catalysts was investigated. Reaction with iodine selectively replaced one hydride in each borane by iodine, leading to the isolation and characterization of mono iodoboranes, as well as a doubly phosphane stabilized boron dication. However, boron-boron dehydrocoupling could not be achieved from this approach. In contrast, introduction of anionic ligands gave rise to a new route to boron-boron bond formation. The dehydrocoupling strategy developed in this thesis enables access to new nucleophilic diborane(4) compounds. Reaction of the borane adducts (L+H)-BH3 (L+H = 1,2,3,4,4a,5,6,7-octahydro-1,8- naphtyridine) as well as hppH-BH3 (hppH = 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-α]pyrimidine) with iodine at room temperature lead to formation of [IB(μ-L)]2 and [IB(μ-hpp)]2. Both diboranes(4) were isolated and fully characterized. Based on a strong set of experimental data and quantum chemical computations, a reaction pathway for this unusual reaction was proposed. In difference to traditional pathways using reducing reagents, the reduction from BIII to BII is paradoxically achieved by the addition of the oxidation reagent iodine. Furthermore, quantum chemical computations were carried out on the diboranes(4) [IB(μ-L)]2 and [IB(μ-hpp)]2, as well as the hydrogen analogues [HB(μ-L)]2 and [HB(μ-hpp)]2, to determine various parameters, such as proton affinity and HOMO energies. All compounds were classified due to their nucleophilicity. The well documented, intensely studied [HB(μ-hpp)]2 constitutes the strongest nucleophilic character among the analysed compounds while the nucleophilicity of [IB(μ- L)]2 is quite low. Overall this thesis established access to an efficient and unprecedented boron-boron dehydrocoupling reaction under mild conditions, applicable to diverse borane adducts.
Die tropischen Ökosysteme sind zunehmend steigenden Risiken durch Landnutzungsdruck ausgesetzt. Für die Quantifizierung und Bewertung der ökologischen Vulnerabilität dieser Ökosysteme fehlen allgemeingültige Konzepte und praktisch anwendbare Modelle. Zudem sind die tropischen Waldökosysteme Afrikas wenig erforscht. Im Rahmen dieser Arbeit erfolgt eine konzeptionelle Entwicklung eines räumlich hochauflösenden, multifaktoriellen Landschaftsvulnerabilitätsmodells als Ausdruck für die ökologische Vulnerabilität tropischer Ökosysteme. Das Modell der Landschaftsvulnerabilität (LV = Anfälligkeit der Landschaft für anthropogene Gefährdungen) wird am Fallbeispiel des tropischen Inselökosystems von São Tomé umgesetzt. Die international kaum bekannte Insel São Tomé (859 km²) liegt im Atlantik vor der Westküste des tropischen Zentralafrikas. Aufgrund des Status als Hotspot der Biodiversität mit vielen endemischen Arten sowie großer Landschaftsästhetik besitzt São Tomé einen hohen ökologischen Wert. Die Gesamtfläche des Primär- bzw. Altwaldes und des Sekundärwaldes beläuft sich auf ca. 50 %. Hinsichtlich einer schnell ansteigenden Einwohnerzahl auf São Tomé erhöht sich kontinuierlich der Landnutzungsdruck in Form von Walddegradation und Biodiversitätsgefährdung. Die methodischen Grundlagen der Forschungsarbeit basieren auf einem integrierten GIS- (Analyse bzw. Modellierung der LV) und Fernerkundungs-Konzept (LULC-Klassifikation). Das LV-Modell, gekennzeichnet durch eine linear-hierarchische Struktur, stützt sich auf bodenkundliche, topographische, fernerkundungsbasierte, statistische und infrastrukturelle Ausgangsdaten. Die Bewertungsanalyse erfolgt multifaktoriell mit einer anschließenden räumlichen Überlagerungsanalyse und gewichteter Summe. Die Ergebnisse sind nach der Intensitätsklassifizierung der LV räumlich-differenziert und geben Auskunft über die Intensität der Vulnerabilität in verschiedenen Landschaftsbereichen. Dadurch können Landschaftsabschnitte identifiziert werden, die für potentielle anthropogen verursachte Gefährdungen anfällig sind. Die gewonnene Information kann das Landmanagement optimieren und zum Biodiversitätsschutz auf São Tomé beitragen. Dank des exemplarischen Ansatzes ist dieses Konzept auch auf andere regional und klimatisch ähnliche tropische Systeme übertragbar. Darüber hinaus können die aus dem Modellansatz gewonnenen Erkenntnisse für die Bewertung der Vulnerabilität tropischer Ökosysteme auch zur Disaster Risk Reduction (DRR) beitragen.
Über zwei Dinge kann man immer schimpfen: Übers Wetter – und über die Wettervorhersage! Dabei werden die Wetterberichte immer besser. Glauben Sie nicht? Ein Mathematiker am HITS, dem Heidelberger Institut für Theoretische Studien, hat nachgewiesen, dass wir bei Vorhersagen oft einem Denkfehler unterliegen. Campus Reporter Nils Birschmann ist der Sache nachgegangen und sprach dabei mit Dr. Sebastian Lerch.
Der Beitrag erschien in der Sendereihe "Campus-Report" – einer Beitragsreihe, in der über aktuelle Themen aus Forschung und Wissenschaft der Universitäten Heidelberg, Mannheim, Karlsruhe und Freiburg berichtet wird. Zu hören ist "Campus-Report" montags bis freitags jeweils um ca. 19.10h im Programm von Radio Regenbogen (Empfang in Nordbaden: UKW 102,8. In Mittelbaden: 100,4 und in Südbaden: 101,1).
This thesis evaluates three novel synthesized conjugated polymer series with respect to their sensitivity and selectivity in the detection of nitroaromatic analytes, especially in aqueous environments. Central building blocks of the polymers are functionalized truxene or tetraphenylethene centers, which are polycondensed with different diethinylaromatics by Sonogashira cross- coupling. In a first explorative study, Truxene-based hyper-branched polymers (HCPs) were compared with related linear rod-shaped poly(p-phenylenethynylenes) (PPEs) with respect to their sensor performance of nitroaromatics (Chapter 2). By encapsulation with amphiphilic F-127 in water-soluble micelles, 14 nitroaromatics could be distinguished with 100% accuracy in fluorescence quenching experiments. The HCPs display a better sensor performance than the sensor system for nitroaromatics previously known from PPE (Chapter 2). To increase the sensor performance, tetraphenylethene cores (TPEs) replaced the truxene units in the polymers in a follow-up study (Chapter 3). TPEs use aggregate-induced emission to detect the analytes. A sensor field based on four TPE polymers was able to detect and distinguish nitroaromatics in water with excellent sensitivity (ppm range) compared to HCP or PPE systems (Chapter 3). This improved system is able to detect the regioisomers of nitroaromatics in a specific way. Finally, the performance of the TPE system was improved by varying the TPE moiety with amino or nitro groups for aqueous detection (Chapter 4). The functional TPE polymers (F-TPEPs) also exhibit aggregate-induced emission, with enhanced pH-specific (amino groups) or solvatochrome (nitro groups) sensor sensitivity. The presence of amino groups improves the sensory properties of F-TPEPs in the aqueous nitroaromatic system. The challenge of the detection of nitroaromatics in water could be mastered by the systematic development of F-TPEPs sensor system from the well-known PPE sensor.
With the help of a patient-derived clear cell renal cell carcinoma (ccRCC) model system previously established in our laboratory, which recapitulates the heterogeneity of the originating tumor, we were able to study ccRCC on a functional level. In five rounds and in four biological replicates of an in vivo selection, we transplanted lung metastases of orthotopically transplanted tumor cells into the renal capsules of NOD scid gamma (NSG) mice. The tumor was enriched for cells with increased growth and higher metastatic potential compared to the initial heterogeneous population. Comparative gene expression analysis revealed candidate genes associated with enhanced malignant growth and metastasis. Least absolute shrinkage and selection operator (LASSO) regression identified a gene signature that can robustly predict cancer specific patient survival. The prognostic power of our signature was additionally verified in independent patient cohorts suggesting that this approach leverages efficient stratification of patients into distinctive risk groups. Intra- and intertumor heterogeneity remains a clinical challenge as estimated survival rates could vary substantially when comparing different tumor regions. Tetraspanin-8 (TSPAN8) was identified as one of the hallmark genes in the generated ccRCC signature and is known to alter cellular signaling. Therefore, we hypothesized that TSPAN8 contributes to tumor aggressiveness and thus to growth and metastasis of ccRCC. In fact, in knockdown and overexpression xenografts experiments, we could confirm an essential role for tumor aggressiveness in vivo suggesting that TSPAN8 is an attractive target for treatment of clear cell renal cell carcinoma.
Cell migration plays an essential role in development and homeostasis. This extraordinarily complex process, which is dynamically regulated by a coordinated interplay between the extracellular environment and intracellular signaling pathways, can also cause the emergence of disease states when dysregulated. A prominent example is cancer metastasis, which is the leading cause of cancer-related mortality. Despite an increasing body of literature focused on cell migration, many aspects remain unclear. In the current work, two complementary systematic in vitro cell migration studies were performed to gain insights into both healthy immune cell migration and pathological cancer cell migration.
Temperature-dependent neutrophil migration was analyzed using differentiated HL-60 cells as a model cell line. Neutrophils are the first cells to be activated during inflammation and subsequently migrate toward an injured tissue or infection site. This response is dependent on both biochemical signaling and the extracellular environment, one aspect of which includes increased temperature in the tissues surrounding the inflammation site. The results of this study reveal a positive correlation between the average speed of randomly migrating cells and temperatures from 30 ˚C to 42 ˚C. Higher temperatures also induce a concomitant increase in cell detachment. Further analysis of the migration data showed that persistence time is higher at low temperatures, while migration persistence length remains constant throughout the temperature range. Coupled with the increased speed observed at elevated temperatures, the obtained results indicate the ability of neutrophils to adapt their migration characteristics to the temperature in order to maintain relatively constant persistence length. As temperature gradients exist on both cellular and tissue scales, the ability of the HL-60 cells to sense and react to the presence of temperature gradients, a process known as thermotaxis, was also investigated. Towards this aim, a two-dimensional temperature gradient chamber was developed. In a gradient with the range of 27-43 ˚C both positive and negative thermotaxis was observed with cells migrating both to the high and the low temperature sources. To date, thermotaxis in neutrophil differentiated HL-60 cells has never been reported.
Next, an in-depth in vitro migration study was carried out on Panc1 pancreatic cancer cells to elucidate the role of Protein Kinase D (PKD) in cancer cell migration. CRISPR-mediated knockouts of PKD1, 2, and 3 in Panc1 cells were used to investigate isoform-specific effects on cancer cell invasion-related motility. The data presented in this project reveals that absence of each PKD isoform plays a significant role in cell speed and migration persistence. To investigate the pancreatic cancer PKD isoform-dependent contact guidance, a panel of more complex PDMS-based substrates containing microgrooves of different heights and widths was utilized. These experiments further demonstrate pancreatic cancer cell shape, speed, and contact guidance dependence on the presence of different PKD isoforms. Based on these findings, in combination with previous works showing that contact guidance is affected by cell viscoelasticity, mechanical properties of each PKD isoform knockout cell line were also measured using a laser-based optical cell stretcher. These experiments showed a significant increase in Panc1 cell deformability in the absence of PKD1, demonstrating for the first time PKD involvement in regulation of cellular mechanical properties. Finally, these basic mechanical properties were correlated to MMP - independent confined migration, which is required for cells to metastasize in vivo.
Source of the HiRISE Mars dataset: https://www.uahirise.org/dtm/dtm.php?ID=PSP_001513_1655
Bei Früh- und Neugeborenen treten häufig Infektionen, eine Sepsis oder ein septischer Schock auf, verbunden mit einer hohen Rate an Morbidität und Mortalität. Aufgrund der Tatsache, dass die Frühgeburtlichkeit der wichtigste Risikofaktor für neonatale Morbidität und Mortalität ist und aktuell mehr als jede zehnte Geburt eine Frühgeburt ist, besteht ein großes Interesse daran die Inflammationszustände in den Feten sowie Neugeborenen zu lindern und somit die Frühgeburtlichkeit, Morbidität und Mortalität zu verbessern. Zunächst wurde das murine Amnioninfektionsmodell so weiterentwickelt, dass verschiedene potentiell anti-inflammatorische/immunmodulierende Substanzen in vivo getestet werden können. Durch eine intraperitoneale LPS-Injektion wurde in der schwangeren Maus eine Inflammation ausgelöst, die auf die Feten übertragen wurde. Nachdem der Phänotyp und das Ausmaß der dadurch verursachten Inflammation des Feten ausreichend untersucht war, wurde das anti-inflammatorische Potential der natürlichen Gallensäure Tauroursodeoxycholsäure (TUDCA) in Bezug auf die einzelnen Facetten der fetalen Inflammation untersucht: 1. Einfluss auf die Überlebenswahrscheinlichkeit sowie die Frühgeburtenrate 2. Modulation der Leukozytenrekrutierung (Rollen, Adhäsion, Infiltration) 3. Beeinflussung von Inflammationsmarkern im Gewebe und im Blut 4. Untersuchung molekularer Mechanismen von TUDCA in verschiedenen Zelltypen (Neutrophile Granulozyten und Endothelzellen)
Von makroskopischer bis mikroskopischer Ebene war der pro-inflammatorische Effekt von LPS und der anti-inflammatorische Effekt von TUDCA zu detektieren. Durch die zusätzliche Applikation von TUDCA zum LPS konnte eine signifikante Reduzierung der Frühgeburtenrate und eine signifikante Steigerung der Überlebenswahrscheinlichkeit im Vergleich zu LPS beobachtet werden. Der anti-inflammatorische Effekt von TUDCA spiegelte sich auch in der reduzierten Anzahl an adhärenten und infiltrierten Neutrophilen sowie reduzierten Expression pro-inflammatorischer Marker im Gewebe und im Plasma wider. Die Ergebnisse der Arbeit deuten darauf hin, dass Neutrophile Granulozyten und Endothelzellen anti-inflammatorische Effekte von TUDCA vermitteln. Der zugrunde liegende molekulare Mechanismus konnte noch nicht vollständig aufgeklärt werden. Da eine konsistente signifikante Linderung des ER-Stresses im Feten nicht zuverlässig nachgewiesen werden konnte, ist ein zytokinvermittelter anti-inflammatorischer Effekt durch die Hemmung des maternalen ER-Stresses denkbar. Auch andere Signalwege sind zu diskutieren. Beispielsweise könnte die Bindung von TUDCA an den TGR5-Rezeptor zur Aktivierung eines anti-inflammatorischen Signalwegs führen. Angesichts dieser Ergebnisse ist die Basis für weiterführende Studien an anderen Spezies und ggf. höheren Säugetieren geschaffen bis hin zur potentiellen Behandlung von septischen Früh- und Neugeborenen.
Exchange of macromolecules between the nucleo- and cytoplasm is provided by the nuclear transport system (NTS). Karyopherins represent essential components of NTS) by serving as nuclear transport receptors/adaptor proteins. Dysregulation of karyopherins in (hepato ) carcinogenesis, including the pivotal nuclear import factor karyopherin-α2 (KPNA2), has been previously reported. However, the functional and regulatory role of KPNA2 in hepatocellular carcinoma (HCC) remains incompletely understood. To further characterize KPNA2 in this context, mass spectrometry-based proteomics was combined with functional/mechanistic cell-based approaches and data derived from murine and human HCC samples. Quantitative mass spectrometry upon siRNA-mediated KPNA2 knockdown in HCC cells revealed the microtubule-related oncoprotein stathmin among the most downregulated proteins. KPNA2 depletion resulted in impaired colony formation and tumor cell migration in HCC cells, which could be recapitulated by direct knockdown of stathmin. Having identified stathmin as functional relevant “downstream” target of KPNA2 the underlying molecular mechanism of KPNA2-dependent stathmin regulation was dissected. Out of several candidates the transcription factors E2F1 and TFDP1 were identified as transport substrates of KPNA2 and to be retained in the cytoplasm upon KPNA2 ablation followed by reduced STMN1 expression. Finally, significant correlations of STMN1 with E2F1/TFDP1 and KPNA2 expression were found based on data derived from murine HCC models and human HCC cohorts with high KPNA2 and STMN1 expression being associated with poorer patient outcome. Taken together, these data suggest that KPNA2 regulates STMN1 by mediating nuclear import of E2F1/TFDP1 and thereby provide a functionally relevant link between the NTS and microtubule-interacting proteins in HCC. Though further studies are required, interfering with nuclear import factors such as KPNA2 could represent a promising therapeutic approach in liver cancer.
Many applications nowadays rely on statistical machine-learnt models, such as a rising number of virtual personal assistants. To train statistical models, typically large amounts of labelled data are required which are expensive and difficult to obtain. In this thesis, we investigate two approaches that alleviate the need for labelled data by leveraging feedback to model outputs instead. Both scenarios are applied to two sequence-to-sequence tasks for Natural Language Processing (NLP): machine translation and semantic parsing for question-answering. Additionally, we define a new question-answering task based on the geographical database OpenStreetMap (OSM) and collect a corpus, NLmaps v2, with 28,609 question-parse pairs. With the corpus, we build semantic parsers for subsequent experiments. Furthermore, we are the first to design a natural language interface to OSM, for which we specifically tailor a parser. The first approach to learn from feedback given to model outputs, considers a scenario where weak supervision is available by grounding the model in a downstream task for which labelled data has been collected. Feedback obtained from the downstream task is used to improve the model in a response-based on-policy learning setup. We apply this approach to improve a machine translation system, which is grounded in a multilingual semantic parsing task, by employing ramp loss objectives. Next, we improve a neural semantic parser where only gold answers, but not gold parses, are available, by lifting ramp loss objectives to non-linear neural networks. In the second approach to learn from feedback, instead of collecting expensive labelled data, a model is deployed and user-model interactions are recorded in a log. This log is used to improve a model in a counterfactual off-policy learning setup. We first exemplify this approach on a domain adaptation task for machine translation. Here, we show that counterfactual learning can be applied to tasks with large output spaces and, in contrast to prevalent theory, deterministic logs can successfully be used on sequence-to-sequence tasks for NLP. Next, we demonstrate on a semantic parsing task that counterfactual learning can also be applied when the underlying model is a neural network and feedback is collected from human users. Applying both approaches to the same semantic parsing task, allows us to draw a direct comparison between them. Response-based on-policy learning outperforms counterfactual off-policy learning, but requires expensive labelled data for the downstream task, whereas interaction logs for counterfactual learning can be easier to obtain in various scenarios.
Light is an important environmental factor used by light-sensitive proteins, photoreceptors, as a source of information or energy. In several classes of photoreceptors, photon absorption triggers the isomerization around a double bond in the light-sensitive chromophore. In rhodopsins, the isomerization of the chromophore retinal is one of the fastest light-triggered processes and it initiates different, protein-specific functions, spanning from vision and sleep regulation to light-energy conversion and phototaxis. The best characterized rhodopsin is bacteriorhodopsin (bR), a light-activated proton pump. Spectroscopic techniques were used to characterize both the ultrafast processes related to the isomerization, but also the later steps when the translocation of protons occurs. Structural knowledge of the later intermediates was provided mostly by X ray crystal structures of cryo-trapped bR or its mutants, contributing to a good understanding of the proton pumping steps. However, the ultrafast processes evolving on a sub-ps and ps time-scale are not amenable for time-resolved X-ray crystallography using synchrotron radiation, thus no structures of the ultrafast bR intermediates could be obtained. This changed with the advent of novel X-ray sources, the X-ray free-electron lasers, which make time-resolved serial femtosecond crystallography (TR-SFX) on the sub-ps time-scale possible. This enables to address the most puzzling questions about the isomerization not only spectroscopically and computationally, but also structurally. Why is the isomerization of retinal in rhodopsins highly bond-specific and efficient, whereas it is neither specific nor efficient when retinal is free in solution? Is the protein affecting the isomerization reaction not only sterically, but also actively? This work used TR-SFX to obtain structures of the ultrafast intermediates in bR in order to observe the structural changes in the retinal and in the protein on the sub-ps and ps time-scale. Large quantities of well-diffracting bR microcrystals were prepared in very viscous lipidic cubic phase. Time-resolved SFX experiments were performed only with liquid samples before the start of this thesis, therefore methods used for delivery of viscous samples in SFX needed to be adapted specifically for this time-resolved experiment. The crystal structures obtained in the TR-SFX experiment on bR visualize the torsion of the isomerizing double bond in retinal. They also show oscillatory motion in the retinal and in specific protein residues and their distances to other residues or to ordered, functionally relevant water molecules. Changes in the distances in the internal hydrogen-bonded network of water molecules and protein residues are also observed. Similar to many TR-SFX experiments, this experiment was performed at very high pump laser excitation intensity, which can induce multiphoton processes, complicating the functional interpretation of the observations made. Unlike other TR-SFX experiments, this work acknowledges and addresses this caveat. Since the TR-SFX experiment could not be repeated at lower laser excitation intensity, additional spectroscopic and computational studies were carried out instead to gain more insight into multiphoton processes. These indeed provide new findings about decay channels in the multiphoton regime. Yet, it still remains open what the implications for the observations made in the TR-SFX structures are. In spite of that, a comparison of the TR-SFX observations with published spectroscopic and computational work performed in the single-photon regime shows remarkable similarities. The insights obtained in this work pave the way for future TR-SFX experiments using optimal excitation conditions, which will clarify the relation of the observed structural changes to single-photon processes. This will allow judging whether the concerted motions observed in the retinal, residues and water molecules are part of the mechanism by which the protein actively controls the isomerization reaction of the chromophore. Furthermore, the methodological advances established here with the model system bR can now be directly applied to study other more challenging rhodopsins, such as the new family of anion-conducting channelrhodopsins (ACR). This work established insect-cell expression of an ACR protein and identified crystallization conditions yielding showers of microcrystals, which is the first step towards a future TR-SFX experiment.
Most organisms on earth are able to sense light, to which they adapt their behavior by using photoreceptor proteins containing light-absorbing chromophores. Phytochrome photoreceptors contain a covalently-attached tetrapyrrole chromophore and switch between two thermally stable forms, a red-absorbing (Pr) and a far-red-absorbing (Pfr) state. Although phytochromes have been studied for more than fifty years, the molecular mechanisms defining their photoinduced properties are not fully understood, hampering the efficient engineering of phytochrome-based molecular tools. The computational study presented in this thesis combines quantum chemical calculations and molecular dynamics simulations in order to elucidate the molecular mechanisms of spectral tuning and excited-state decay in phytochromes. The calculations have demonstrated that the spectral red shift of the Pfr state is induced by the hydrogen bond formation between the chromophore and a highly conserved aspartate. Here it is also shown how the formation of this hydrogen bond is coupled to dynamics of other active-site interactions. In addition, the chromophore deprotonation by a protein residue is proposed to contribute to the absorption at the Q-band blue shoulder in the Pr-state spectrum. For the first time, the photoinduced electron transfer coupled to proton transfer was characterized in phytochromes. These charge transfer pathways may contribute to the excited-state decay by quenching fluorescence and influencing photoproduct formation. The discoveries provided in this thesis will facilitate further phytochrome investigations and the rational design of phytochrome-based fluorescent markers and optogenetic tools.
Somatic mutations accumulate in tissues primarily through cell divisions. This observation opens the opportunity to use somatic mutations as clonal markers for inferring the past dynamics of cell turnover during tissue growth and homeostasis. In this thesis, I develop mathematical approaches to the inference problems that are stimulated by deep genome sequencing data of malignant growth and physiological tissue turnover. In the first part of this thesis I reconstruct the evolutionary history of adult glioblastoma, a highly aggressive brain cancer, prior to and after standard therapy. To this end, I develop a likelihood-based multinomial model that jointly infers genetic subclones and their phylogenetic relationships from whole genome sequencing data. Applied to 21 sample pairs from primary and recurrent glioblastomas, the model infers a common path of early tumorigenesis characterized by three pervasive copy number changes on chromosome 7 (gain), chromosome 9p (loss) and chromosome 10 (loss). TERT promoter mutations are subclonal in one third of the tumor pairs and are thus placed at a later stage of tumorigenesis. Our data indicate that recurrent tumors typically re-grow from multiple subclones of the primary tumor with no evidence for a 'resistance genotype' induced by therapy. Combining the results from phylogenetic inference with population dynamics models of tumor growth, I estimate that glioblastomas originate several years prior to initial diagnosis but reach detectable sizes only after TERT promoter mutations stabilized cellular survival. This project provides new insights into the evolutionary history of glioblastoma that may ultimately aid early diagnosis. In the second part I analyze the mutation frequency distribution in normal tissues. To this end, I extend existing theory on mutation accumulation in exponentially growing tissues to a two-stage situation of initial embryonic expansion and subsequent homeostasis during adulthood. Based on stochastic simulations I show that the theoretical framework recovers the average mutation frequency spectrum in stem cell populations. Whole genome sequencing data from murine granulocytes and human leukocytes from subjects of different ages without diagnosed leukemia confirm the model prediction in the majority of cases but reveal an unexpectedly high mutational burden in a smaller subgroup. These cases were associated with one or several leukemic driver mutations, suggesting that perturbed hematopoiesis or pre-leukemic expansions caused the deviation of the mutation frequency spectrum from neutrality. The comprehensive analysis of the mutation frequency spectra in normal and perturbed hematopoiesis may aid the understanding of tumor initiation in vivo.
In the present thesis we are interested in modelling the behaviour of actors in a network in dependence of explanatory variables which give information about every pair of actors. The behaviour is here expressed in interactions which the actors may cast amongst each other. Our model is based on a survival analysis idea: We assume that the interaction times between any two actors are encoded in a counting process such that we observe a counting process for any pair of actors. The intensity functions of these counting processes are then assumed to depend on the covariates in a certain parametric way. We allow that the parameters are time dependent functions, thereby the model becomes non-parametric. We present a rigorous analysis of the asymptotics of a non-parametric estimator based on a local likelihood approach. This includes point-wise asymptotics of the estimated parameter curves as well as asymptotics for an $L^2$-type test statistic.
In order to carry out the mathematical analysis of these terms we introduce three ideas to handle the complex dependence structure on the network. These provide different tools for handling covariances and proving concentration inequalities which might be of independent interest.
The theoretical analysis is complemented with an application to real-world data: We investigate the impact of different network quantities on a bike sharing network.
In this thesis, the development of a novel magnetic resonance imaging (MRI) pulse sequence based on magnetic resonance fingerprinting (MRF) is presented. The proposed technique, termed “Flow-MRF”, allows time-resolved velocities and relaxation constants to be quantified simultaneously, in shorter acquisition times than conventional MR-based velocimetry. The simultaneous quantification of both sets of parameters was achieved by formulating the combined problem in the MRF framework. An MRF pattern was designed to create minimal coupling between the relaxometric and velocimetric parameter encoding. Flow-MRF was validated and tested in simulations, phantom experiments, and an in vivo study targeting the popliteal artery and the gastrocnemius muscle. In each investigation, Flow-MRF quantified relaxation constants and flow velocities in strong agreement with literature and reference measurements. Furthermore, the use of high velocity encoding moments (Δm1 = 60 mT/m·ms^2) was demonstrated while maintaining a range of correctly quantifiable velocities beyond 800 cm/s. In the volunteer study, Flow-MRF determined an average longitudinal relaxation time of (1384±75) ms and a transverse relaxation time of (26±4) ms in the gastrocnemius muscle. The average velocity deviation over all three volunteers between Flow-MRF and the reference was (-2.6±5.2) cm/s. Lastly, the potential to quantify the complete Reynolds stress tensor with Flow-MRF was investigated and shown in a stenotic flow phantom experiment. Flow‐MRF presents a novel method of quantifying velocities in up to fourfold shorter measurement times than conventional velocity mapping techniques, while simultaneously providing relaxometric maps of static tissue. These improvements can potentially be helpful in the assessment of pathologies such as arteriosclerosis.
Nonribosomal peptide synthetases (NRPSs) are modular mega-enzymes found in bacteria and fungi that produce nonribosomal peptides (NRPs) in an assembly line fashion. Each module is in charge of adding a specific amino acid (AA) to the growing peptide chain. Three basic domains constitute one NRPS module: the adenylation (A), peptidyl carrier protein (PCP) and condensation (C) domains. The A domain recognizes and activates the AA. An external enzyme, the PPtase, attaches a phosphopantetheine (PPant) arm to the PCP domain which then picks up the activated AA and delivers it to the C domain. The C domain recognizes the growing peptide chain (donor) as well as the new AA (acceptor) and fuses the two together. A special feature of NRPSs is their ability to recognize and incorporate not only proteinogenic AAs, but also other building blocks like fatty acids (FAs) or non-proteinogenic AAs. All building blocks can be further modified through the action of additional domains: epimerization (E), methylation (M) and oxidation (Ox) domains, among others. In this manner a great variety of different NRPs can be synthesized, many of which are bioactive and exhibit anti-microbial or anti-cancer properties. Thus, it is highly desirable to understand how NRPS domains and modules function and find ways to genetically re-engineer them for custom NRP production. Since the discovery of NRPSs, many efforts have already been made to engineer these enzymes in order to create custom NRPs, but general design rules yet remain elusive. The successful attempts to re-create functional NRPS for the production of novel NRPs include: (i) mutations of the A domain, (ii) subdomain modifications and (iii) rearrangements on the module level. Yet, many engineered NRPSs exhibit only slow reaction rates and low product yields. In some cases, the desired NRP products cannot be detected at all, possibly due to additional control mechanisms that have not been taken into account during the engineering process, such as substrate specificity of C domains. Hence there is still a great need to identify the general rules for successful NRPS engineering in order to exploit the ever-growing molecular toolbox of newly discovered NRPSs for recombinant production of novel bioactive compounds. In this work I present my attempts to develop an approach to easily monitor the outcome of NRPS manipulation using a pigment-producing synthetase as a genetic tag. To this end, I first investigated two homologous synthetases, IndC and BpsA, which produce the blue pigment indigoidine, and mutants thereof and revised the proposed biosynthesis mechanism. I then created a series of fusion constructs between modules coming from different NRPSs and IndC/BpsA to test if indigoidine-tagged peptides could be produced. I identified a promising construct for which point mutations in the upstream module resulted in weaker or null pigment production. However, the expected indigoidine-tagged AA was not detectable, which could be due to the fact that indigoidine production inevitably leads to the separation of the donor AA. These results raised further questions as to whether in a native NRPS, the same modifications lead to congruent effects in neighboring modules. I addressed this question using a fragment of a non-engineered NRPS to monitor the activity of the native and modified versions in an in vitro assay, which I present in the last part of the results. Surprisingly, the effects of the same set of modifications on neighboring modules did not only differ between the engineered NRP-pigment synthetase and the native NRPS, but also between different modules within the native NRPS. These results hint at highly individual behavior of NRPS modules, depending on the context they are in.
Air sided flow profiles were measured in the linear wind-wave-facility of the Heidelberg Institute for Environmental Physics (IUP) to assess the wind-induced momentum transport to a water surface. Therefore, a recently developed technique of particle streak velocimetry (PSV) was used, which allows the time-resolved imaging of two-dimensional flow fields. This measurement technique, that uses the camera-imaging of particles seeded in the flow field, could thereby be tested and approved in another wind-wave-tunnel. From the flow fields, the mean total drag and its partition into turbulent and viscous shear stresses could be determined for different flow-conditions. From these, also the acting pressure forces were estimated. The measurements were made at three different fetches from 75 to 220 cm and four windspeeds each, ranging from about 1 to 6 m/s. The obtained stress-profiles match those of similar studies and furthermore allow the assessment of the influence of different fetches on the partitioning of the shear stress for the first time. During the evaluation problems occurred in the detection of particles in certain velocity-ranges. Their impact on the data could be minimized, but the problems could not be solved within the scope of this study.
ALICE is one of the four major experiments at the Large Hadron Collider (LHC). It is the dedicated heavy-ion experiment and therefore primarily examines the Quark–Gluon Plasma. In order to prepare for the running conditions of 50 kHz lead-lead interactions at the LHC after the Long Shutdown 2 (2018–2021), an extensive upgrade program is carried out. The goal of the upgrade is a continuous readout of the TPC without the need of a trigger. It is essential to reduce the enormous data rate of 3.7 TB/s, generated by the upgraded detector, already during the data taking by a factor of about 60. Otherwise the data volume would exceed the expected available bandwidth and storage capabilities. In this thesis, an online Cluster Finder (CF) was developed and implemented for FPGAs which processes the whole data volume in real-time during the read out. This is the first step in the data reduction sequence which achieves already a factor of about 5 by keeping only physically relevant information and making use of a better suited data format. In addition to the CF, also the whole data preparation chain was designed and implemented to decode the input data stream, to resort the individual channels to allow for cluster finding and to correct the detector effects in the input signals. All modules which were implemented were extensively simulated to verify their proper functionality. With this, the complete processing chain within the FPGAs was prepared and validated.
Postembryonic growth is a highly coordinated and delicate process, during which shape and function of complex organs have to be maintained. Anamniotes, such as fish and amphibia, grow lifelong. This property is mediated by stem cells, which must ensure full organ functionality along with proper scaling with the increasing body size. In the anamniote retina, postembryonic neurogenesis and growth is mediated by retinal stem cells in the ciliary marginal zone (CMZ). The family of Rx transcription factors is highly conserved among vertebrates and plays a key role in early eye development. In the teleost medaka (Oryzias latipes), the retina-specific homeobox transcription factor 2 (Rx2) has been shown to be a marker for multipotent retinal stem cells. Its closely related paralogue Rx1 has not been studied in detail so far. I hypothesise a role for rx1 and rx2 genes in establishment of the retinal stem cell domain and therefore in mediating postembryonic growth and shape of the eye. I used novel CRISPR/Cas9-mediated gene tagging approaches, developed in the framework of this thesis, to visualise endogenous medaka rx2 and, for the first time, rx1 expression. I carried out clonal loss-of-function studies of medaka rx1 and rx2, indicating a role in balancing stem cell fate decisions. Systemic loss-of-function studies of rx1-/-/rx2-/- double mutants revealed a role in early retinal morphogenesis, as well as in establishment and maintenance of the CMZ by conferring retinal stem cell identity. Furthermore, I established tools necessary to generate a labelled allelic series of rx1 and rx2 mutants. Eventually, I characterised an rx2 conditional allele, which will be instrumental to address late function of rx2 in the CMZ, thereby deciphering whether the retinal stem cell niche is dynamic or rather deterministic. Taken together, my results reveal a function for rx1 and rx2 in establishment and maintenance of the medaka CMZ and indicate a role in regulation of growth and scaling of eye size.
Measurements of airway surface liquid (ASL) on primary airway epithelial cultures (AECs) grown at air-liquid interface (ALI) identified ASL depletion as a characteristic abnormality in cystic fibrosis (CF) and may be used as an endpoint for preclinical testing of strategies to improve airway surface hydration in patients with CF. Traditionally, ASL height has been determined by confocal fluorescence microscopy after addition of fluorescently labelled liquid to the apical side of the epithelium, however, several hours are required to restore steady state ASL depth after this volume challenge. Moreover, the current protocol requires fluorescently labelled liquid addition to the apical side of the epithelium resulting in non-physiological conditions. The aim of this project is to determine the suitability of confocal reflection microscopy as a novel approach to study ASL regulation without requirement of fluorescent labelling/volume. Reflection microscopy detects the inherent differences in refractive indices between the permeable membrane, cytosol of cells and liquid layer. Primary airway epithelial cells were prepared from wild-type mice and βENaC-overexpressing mice as a model of CF lung disease, and also from non-CF and CF patients, and were cultured under physiological conditions at air-liquid interface. ASL homeostasis was investigated by confocal fluorescence and reflection microscopy. Using the fluorescence-based volume challenge protocol, directly after the addition of the fluorescent dye in a volume of 20 µl, ASL height on AECs from wild-type mice determined by confocal reflection microscopy did not differ from values obtained by fluorescence microscopy. Two hours after volume challenge, ASL height was reduced and at 24 hour time point ASL height recovered to normal levels that did not differ between the two techniques. In AECs from βENaC-overexpressing mice, ASL height was similar at early time points, but remained reduced at 24 hour time point. Similar to studies in wild-type AECs, ASL height data did not differ between reflection and fluorescence based measurements. Moreover, continuous measurements of unperturbed steady state measurements using reflection confocal microscopy revealed a stable ASL height measured in both wild-type and ENaC-overexpressing AECs for 5 hours. Similar to comparison studies wild-type mice derived AECs showed higher ASL height than ENaC-overexpressing cells. In addition to murine primary AECs, measurements were performed also with human primary AECs from non-CF and CF patients. Steady state measurements of non-CF patient derived AECs showed a higher ASL height than CF patient derived AECs.With the basolateral addition of benzamil, due to blocked Na+ and liquid absorption ASL height of non-CF AECs was increased, showing that reflection confocal microscopy is capable of measuring even the small changes in ASL height. However, due to lack of functional CFTR channels, ASL height did not change in CF AECs. In conclusion, these results support that reflection confocal microscopy can be used for accurate measurements of ASL height (dys)regulation without the need of addition of fluorescently labelled dye. This approach may facilitate preclinical evaluation of novel drugs designed to improve airway surface hydration in patients with CF under more physiological steady state conditions.
The terminal cells of the tracheal system in Drosophila melanogaster are one of the few known cell types that form stable, long branches. Like all epithelial cells, terminal cells have an apical and a basal membrane domain. Throughout larval life, each terminal cell's basal membrane repeatedly bifurcates and elongates to establish an extensive branched network, and the apical membrane is invaginated to form a subcellular tube that expands a lumen in each branch. The lumen is contiguous with the tracheal system's lumen and carries air to deliver oxygen to internal organs. To achieve this elaborate structure, terminal cells expand their volume and surface area at least hundredfold over the course of a few days. The synthesis and distribution of membrane and proteins must thus pose a particular challenge. In addition to the necessary bulk of production, all material needs to be delivered to the appropriate membrane domain. In particular, the specialised apical extracellular matrix within the tracheal lumen must be supplied with the structural molecules that confer the tracheal system its physical stability and the ability to contain and exchange gas. My hypothesis was thus that terminal cells possess a customised membrane trafficking system that allows them to transport large amounts of newly synthesised proteins and lipids through secretory pathways to the apical and basal domains as appropriate. This directed the focus of my research to the Rab family of small GTPases, a group of proteins known as master regulators of membrane trafficking. Rabs bind to the plasma membrane or to intracellular membrane compartments and subsequently function as platforms to recruit effector proteins. These effectors execute virtually all mechanisms that direct membrane-bound and secreted molecules from one membrane compartment to the next, as well as to and from the cell's exterior environment. To explore membrane trafficking systems in terminal cells, I first conducted a systematic quantitative analysis of normal and abnormal terminal cell phenotypes to better understand how morphological defects should be interpreted. This was necessary because of the variability with which mutant phenotypes manifest in terminal cells. My results provide a reference for the range of natural variability in TC phenotypes, and identify phenomic analysis, currently only applied to plants, as a powerful method for extracting meaning from partially penetrant loss-of-function phenotypes. The main part of my work was a tracheal knockdown screen targeting all Drosophila Rabs. Rather than using specific RNAi constructs against each gene, I utilised a collection of endogenously YFP-tagged Rabs and knocked down the YFP tag in animals where all copies of a Rab are tagged. This indirect approach confers more confidence in its results because unlike conventional RNAi, its risk of false positives is minimal. Through this and supplementary experiments, I pinpointed a secretion route from endoplasmatic reticulum through Golgi and endosomes to the plasma membrane that ensures proper organisation of the extracellular matrix and allows the cell to grow. I identified only one Rab whose phenotype suggested a role in establishing the shape and size of the subcellular tube: Rab8. In my final experiments, I therefore investigated how Rab8 is involved in apical morphogenesis in terminal cells. While not definitive, my results suggest that Rab8 acts on apically-directed secretory endosomal traffic to retain basally-destined cargoes, thereby preventing their erroneous apical secretion.
This thesis investigates the biopharmaceutical knowledge of different liposomal formulations using two different phosphatidylcholines (EPC and S 90) in the presence and absence of gelatine. The study aimed to characterize the oral lipid-based formulations in terms of physiochemical features, X-ray diffraction, dissolution, in vitro lipolysis and in vivo pharmacokinetic studies. The prepared liposomes were used to investigate the physical features of vesicular phospholipid gels (VPGs) and the light-scattering assessed the size and polydispersity of the liposomes. Fenofibrate-loaded liposomes had remarkably high entrapment efficiency (95%–99%). The dissolution behaviour of the samples was evaluated in a paddle model and the results showed a negative correlation between the rate of liposome release and gelatine concentration. The prepared liposomes were used in a dynamic in vitro lipolysis model to study the digestion and solubilisation of the drug formulations in biorelevant media simulating the gastrointestinal environment. The release profile of fenofibrate for all liposomal formulations showed a general increasing trend (regardless of the type of phosphatidylcholine and amount of gelatine) towards the release of fenofibrate from the formulations in the aqueous phase. For the in vivo studies, EPC and S 90 liposomes were administered orally to fasted rats for pharmacokinetic characterization. The in vivo studies demonstrated that Soluthin S 90 formulations, regardless of the presence or absence of gelatine, showed significantly higher (p<0.01) pharmacokinetic parameters in terms of AUC and Cmax. All formulations showed a similar Tmax, and the presence or absence of gelatine had no significant effect on AUC, Cmax or Tmax for both EPC- and Soluthin S 90-type liposomal formulations. In this study, the absence and presence of gelatine in the in vitro rat lipolysis model correlated with the results of the in vivo pharmacokinetic studies, and the data from the NaOH titration, corresponding with the digestion and FA release, virtually correlated with the in vivo plasma concentration. However, in term of the drug solubilisation profile, no significant correlation was observed between the in vitro and in vivo studies, particularly in the case of the in vitro lipolysis model. In conclusion, the addition of gelatine did not have a significant effect on pharmacokinetic parameters. The new phosphatidylcholine mixture, Soluthin S 90, strongly improved the absorption and bioavailability of fenofibrate-loaded liposomes. Further studies should be performed to investigate the effect of fenofibrate-loaded liposomes prepared by Soluthin S 90 on the intestinal permeability of the API and solubilisation capacity of the formulations in an in vitro lipolysis model combined with an absorption step. It is also recommended to combine the formulation with different surfactants to choose the optimal formulation. In addition, it is necessary to examine the bioavailability of different poorly water-soluble drugs (PWSDs) formulated in Soluthin S 90 liposomes to elucidate the pharmacokinetic effect of such a formulation.
The aim of this thesis is to improve our understanding of the fragmentation behavior of Population III protostellar disks under the influence of rotation, turbulence, and magnetic fields. We further evaluate consequences that may be inferred for the later evolution of the star-forming halo and its surroundings with respect to protostellar ejections and in terms of the impact of radiative feedback on later chemical enrichment of neighboring halos. In the main part of this thesis, we follow the collapse of a primordial gas cloud until the formation of the first protostar and the creation of a highly gravitationally unstable protostellar disk system. We find that turbulence promotes the fragmentation of the protostellar disk and both rotation and magnetic fields can provide some stabilization against it. While the total mass growth of the collection of protostars is only mildly affected by rotation and turbulence, magnetic fields can have such a strong impact on the dynamical evolution of the disk system that accretion onto the protostars is highly disturbed and their mass growth is significantly reduced. In spite of all the differences, the disk generally breaks up into a protostellar cluster that develops a top-heavy mass function. Interactions between protostars in the cluster are highly dynamical and lead to a considerable number of protostellar ejections. We demonstrate that some of these ejected Population III protostars, even if they continue to accrete for some longer period after they have left the disk environment, continue to have masses of M < 0.8 solar mass. Hence, they have lifetimes longer than the current age of the Universe and thus describe Population III candidates that could still be observable today. In another project, we assess the role of photoevaporation of a pristine halo by a near-by Population III star prior to the supernova explosion of that star. We demonstrate that it is crucial for realistic simulations of metal enrichment to account for the photoevaporation as the radiation ablates and thins out the outer halo layers and thus makes the halo more susceptible to mixing with the metals from the supernova ejecta. In this thesis, we use both analytical estimations and numerical simulations. Further tests are conducted to investigate the performance of our numerical methods and the sensitivity of our results to the numerical resolution. We demonstrate that general trends, in particular when effects of turbulence are examined, cannot be reliably deduced from only a single numerical run. Instead a statistical analysis of an ensemble of realizations based on the same initial conditions needs to be considered.
Clearance of apoptotic cells by professional, tissue resident macrophages is crucial during development, maintenance of tissue homeostasis and disease. Phagocytic cells can engulf and process several dying cells with high efficiency while still maintaining their dynamic behaviour and morphology. Effective intracellular processing of ingested cells is likely to be crucial for the phagocyte to function, but the underlying cellular mechanisms are poorly understood. Here, we investigate this by focusing on microglia, the tissue resident macrophages of the Central Nervous System (CNS). In particular, we take advantage of the optical transparency of the zebrafish embryo and apply a combination of genetic, chemical and imaging approaches to study phagocytosis of neurons in vivo. In this study we focus on bubblebrain (blb), a zebrafish mutant that has bloated microglia. The cell body of blb microglia is occupied by a single large vesicle that grows progressively. By comparing wild type and blb embryos we discovered that efficient processing of engulfed neurons depends on the shrinkage and packaging of phagosomes into the gastrosome, a unique cellular compartment with distinct ultra-structural and molecular features. Moreover, we show that the blb phenotype is caused by lack of Slc37a2, a putative glucose-6-phosphate transporter localized on phagosomes. Loss of Slc37a2 prevents phagosomal shrinkage, and this in turn results in the expansion of the gastrosome and in the dramatic bloating of the cell. Interestingly, we show that gastrosomal defects impact on microglial activities and affect the ability of these cells to engulf neurons and to migrate towards brain injuries. Thus, this work provides experimental evidence for the existence of the gastrosome, a critical new component of the phagocytic pathway that allows easy manipulation of key microglial activities in vivo.
The photo-induced isomerization of retinal protonated Schiff base (RPSB) inside the protein pocket is one of the fastest (<ps) and most stereo-selective photochemical reactions in nature. The ground state structure of the RPSB and its surrounding protein constructions are thought to be the two most crucial factors to drive this reaction. The investigation of each factor individually was the main goal of this thesis. Anabaena Sensory Rhodopsin (ASR), a recently discovered microbial retinal protein, serves as an ideal system for this study as it binds two structural isomers (all-trans: AT and 13-cis: 13C) of the RPSB within the same protein constructions in its photocycle. In the present work, the photo-induced dynamics of the RPSB in ASR has been explored with the help of time resolved coherent vibrational spectroscopic methods, which monitor the photo-induced sub-ps structural changes of the RPSB. These studies have helped to shed light on the intricate relationship between electronic and vibrational dynamics of the RPSB. In the first half of this thesis, a comparative study showed both electronic and vibrational dynamics are widely distinct for the AT and 13C isomers of the RPSB in ASR. In particular, the 13C isomer exhibited more than five folds faster dynamics than the AT isomer. One possible molecular origin behind this dynamical difference was found by comparing the ground state Raman spectra of the two isomers. It depicted an increase in the amplitude of hydrogen-out-of-plane (HOOP) modes for the 13C isomer, which is usually considered to be an evidence of distortion in the ground state structure for the retinal system. The ground state pre-distortion has been reported as a potential element for the acceleration of the isomerization reaction for the 13C isomer, in analogy with the cis isomers of visual rhodopsin and bacteriorhodopsin. The second half of this work explored the role of the part of protein helix inside the retinal pocket as well as that far away from the pocket. In particular, the replacement of the amino acid residues in vicinity of the RPSB by point mutation caused an acceleration of the reaction rate for the AT isomer, but it had only a minor effect for the 13C isomer of the RPSB. Furthermore, the truncation of the part of the protein, embedded into the cytoplasmic region, affected the formation of the primary photoproduct. All these experimental results lead to two major conclusions of this thesis: (i) the protein constructions govern the retinal isomerization dynamics and (ii) the same protein cage exerts differential interactions on two structural isomers of the RPSB.
In this thesis, several aspects of nuclear structure effects and corrections from quantum electrodynamics (QED) in the spectra of hydrogen-like systems are investigated. The first part is concerned with the structure of bound states between a muon and an atomic nucleus, so-called muonic atoms. Here, precise calculations for transition energies and probabilities are presented, using state-of-the-art numerical methods. QED corrections, hyperfine interactions, and the interaction with atomic electrons were evaluated and finite nuclear size effects were incorporated non-perturbatively. Furthermore, new methods for the calculation of higher-order corrections for the hyperfine structure are presented, including a complete calculation of the second-order hyperfine structure and leading-order vacuum polarization corrections for extended electric quadrupole distributions inside the nucleus. In connection with recent x-ray spectroscopic measurements on muonic atoms, the nuclear quadrupole moment of Re-185 and Re-187 is extracted. The second part of this thesis is about the g factor of a bound electron and its dependence on the shape of the nuclear charge distribution. A numerical, non-perturbative approach for the calculation of the corresponding nuclear shape correction is presented and implications for the uncertainties of theoretical predictions are discussed. In particular, the model-uncertainty of the finite-nuclear-size correction to the g factor can be reduced due to the more realistic model of the nuclear charge distribution. Finally, calculations of finite-size and vacuum-polarization corrections to the g factor of a muon bound to a He-4 nucleus significantly contribute to the theoretical prediction on the 10^−9 uncertainty level. As shown in an earlier work, an experimental value of the same accuracy could give access to an improved value of the muon’s mass or magnetic moment anomaly.
In this work we propose new methods for the design of economic Nonlinear Model Predictive Control (NMPC) feedback schemes for Average Output Optimal Control Problems (AOCPs). AOCPs are Optimal Control Problems (OCPs) defined on infinite time horizons with averaging performance critera as objective functionals. Such problems arise frequently for continuously operating systems such as for example power plants. Due to the infinite time horizon and the resulting intrinsic nonuniqueness of solutions, the design of appropriate NMPC schemes for AOCPs is challenging. Often, the analysis of the closed-loop behavior of economic NMPC schemes depends on dissipativity conditions on the dynamical system and the associated performance criterion, which sometimes can be hard to check. The methods we develop are based on the observation that periodic solutions exhibit excellent approximation properties for AOCPs, which is exploited by splitting the time horizon and the objective functional of the NMPC subproblems into a transient and a periodic part. For the analysis of the closed-loop behavior of the resulting controller we develop new methods that essentially work by showing that the (appropriately defined) difference of two subsequent NMPC subproblem solutions vanishes asymptotically. Complementary to many other economic NMPC schemes, this approach is not based on dissipativity assumptions on the dynamical system and the associated performance criterion but rather on assumptions on existence of periodic orbits, controllability of the dynamical system, and uniqueness of the NMPC subproblem solutions itself. As a result, we can show that the economic performance of the closed-loop system is equal to the economic performance of the optimal periodic solutions. Furthermore, the approach is extended in two directions. First, we consider the general setting of a parameter dependent dynamical system where the parameter can be subject to change during operation. This parameter change can lead to a change in the optimal periodic behavior, in particular also to a change of the optimal period, which we take into account by including the period as an optimization variable in the NMPC subproblem. Second, we show that the approach can also be applied to systems with time-dependent periodic performance criteria. All the described methods are implemented within the MATLAB NMPC toolkit MLI and are applied to a number of demanding applications. The simulation results confirm that the generated closed-loop trajectories perform economically equally well as the optimal periodic trajectories.
Throughout evolution most of the mitochondrial genes have been transferred to the nuclear genome. Thus, proteins targeting one of the four mitochondrial compartments have to be translocated after their synthesis in the cytosol. This process is essential for mitochondrial biogenesis in all eukaryotes. Nevertheless, mitochondrial protein import machineries of parasitic protists differ significantly from the established models in opisthokonts (e.g. mammals and yeast), although import signals are functionally conserved. In yeast and mammals, Mia40/CHCHD4 and the sulfhydryl electron transferase Erv1/ALR are the essential components for the import and oxidative folding of sulphur-containing proteins in the mitochondrial intermembrane space. Substrates of this pathway carry conserved cysteine motifs that are recognised by Mia40 and subsequently oxidized to the formation of intramolecular disulphide bonds. In a disulphide relay system, electrons are transported from Mia40 to Erv1 and on to cytochrome c, which finally introduces them into the respiratory chain. However, a Mia40 homologue could not be identified in the genome of important apicomplexan and kinetoplastid parasites, while Erv1 is ubiquitously conserved. Both the Erv homologues from the kinetoplastid parasite Leishmania tarentolae (LtErv) and the malaria parasite Plasmodium falciparum (PfErv) were imported into yeast mitochondria but failed to replace their yeast counterpart. Here I analyse structure-function relationships of LtErv in the endogenous as well as the opisthokont model system Saccharomyces cerevisiae. I characterise particularities of the overall structure and the cysteine motifs that distinguish the protein from other homologues that actually interact with Mia40. The most obvious features comprise an additional C-terminal domain (KISS) restricted to this group of parasites and a partially conserved cysteine residue close to the N-terminus (C17). Yeast complementation assays suggest that the presence of the KISS domain does not inactivate the protein in the yeast system. In contrast, LtErv gained functionality when C17 was removed or the yeast shuttle arm was N-terminally fused to the protein. Residue C17 is therefore most likely the reason why LtErv does not interact with yeast Mia40 and interrupts the oxidative protein folding machinery in yeast. However, the translocation of LtErv itself into yeast mitochondria is not affected. Unlike soluble ScErv1, the protist protein is presumably imported due to its membrane association. Whether the translocation of LtErv is structurally connected to its dysfunction in yeast and controlled by a conserved trans side receptor or by protein-lipid interactions remains to be further investigated. Additionally, the data strongly support the existence of a Mia40 replacement in parasitic protists because both LtErv and PfErv were unable to initiate substrate import on their own in yeast mitochondria. Potential replacement candidates were enriched by trapping mixed disulphide intermediates with a model substrate and have to be evaluated in the future.
Secretion of insulin in response to extracellular stimuli, such as elevated glucose levels and small molecules that act on G-protein coupled receptors (GPCRs), is the hallmark of β-cell physiology. Sufficiently high blood insulin levels are ensured by the coupling of the secretory activity within pancreatic islets. Intercellular and inter-islet coordination are partly mediated by small diffusible ligands of GPCRs within the extracellular space of pancreatic islets. Therefore, insulin release is considered a synchronized multi-cellular process. We show herein that β-cell activity and insulin secretion essentially rely on the presence of extracellular endogenous (autocrine) signaling factors, exemplified by two classes of small cellular metabolites. Trace amines (TAs) are small aromatic metabolites that were identified as low-abundant ligands of the trace amine-associated receptor 1 (TAAR1) in the central nervous system (CNS). In the presented work, we identify TAs as essential autocrine signaling factors that maintain and regulate oscillations of the intracellular Ca2+ concentration ([Ca2+]i oscillations) along with insulin secretion from β-cells via TAAR1. We found that the modulation of endogenous TA levels by the selective inhibition of TA biosynthetic pathways directly translated into changes of [Ca2+]i oscillations and insulin secretion. Application of aromatic amine-withdrawing β-cyclodextrin temporarily reduced [Ca2+]i oscillations. This demonstrates the essential role of TAs for β-cell activity as well as their high metabolic turnover rates. Notably, herein applied inhibitors and synthetic TAAR1 (ant-)agonists are partly approved for the therapeutic modulation of biogenic amine levels within the CNS, and hence for the treatment of common neurological disorders. According to our findings, these drugs even affect β-cell activity and insulin secretion through pancreatic TAAR1. With the discovery of the free fatty acid (FA) receptor GPR40 in β-cells, FAs have come into focus as exogenous insulin secretagogues. However, the role of FAs as endogenous (local) signaling factors of β-cells has not been considered so far. We show herein that lowering endogenous FAlevels in the presence of FA-free bovine serum albumin (FAF-BSA) immediately reduced [Ca2+]i oscillations and insulin secretion. [Ca2+]i oscillations resumed upon exchange of FAF-BSA by buffer or upon restoration of extracellular FA pools. The latter was accomplished by the photolysis of caged FAs on plasma membranes, by the addition of a recombinant lipase or of FA-loaded BSA. Our approach to subordinate β-cell activity and insulin secretion to the presence of autocrine signaling factors of the yet underestimated receptors TAAR1 and GPR40 in the pancreas contributes to a more detailed and complete understanding of the fundamental regulation of β-cell activity and insulin secretion.
Lipid droplets (LD) are intracellular storage organelles that are found in most of all cell types and play an important role in the pathology of various human diseases like diabetes type II, cardiomyopathy and atherosclerosis. The core of the LD consists of different neutral lipids, mainly triglycerides and cholesteryl esters. This hydrophobic core is surrounded by a phospholipid monolayer embedding specific proteins and enzymes. One of these enzymes is the long chain acyl-CoA synthetase 3 (ACSL3) that belongs to the family of acyl-CoA synthetases (ACS). Proteins of the ACS family catalyze the esterification of fatty acids with coenzyme A, which is an essential prerequisite for further metabolism. Among all ACS family members, ACSL3 is the only ACS enzyme that is consistently found on LD. ACSL3 is characterized by a dual localization on LD and the endoplasmic reticulum (ER). It translocates from the ER to the LD upon fatty acid supplementation and is already present on nascent LD. Based on the current data, ACSL3 localization on LD and the ACS activity mediated by ACSL3 is presumed to be biologically relevant. This work investigated the relevance of ACSL3-mediated ACS activity and ACSL3 localization in anabolic and catabolic lipid metabolism. The main approach was based on the depletion of ACSL3 in COS-7 cells by RNA interference or CRISPR/Cas9 knockout. ACSL3-knockout cells were transduced with genetically modified enzyme variants and lipid metabolism was investigated. A mutated ACSL3 variant lacking ACS activity was applied to identify potential ACSL3 functions independent of ACS activity. An ACSL3 variant localized to the ER but excluded from the LD was supposed to elucidate the importance of ACSL3 localization on LD. Experimental approaches included radiolabelling and quantification of intracellular lipid species by 14C-oleic acid and 14C-acetate, quantification of newly synthesized LD in starved cells and intracellular triglyceride measurement during basal lipolysis. Molar quantification of ACSL3 in A431 cells was carried out to calculate the metabolic capacity of LD-associated ACSL3 for triglyceride synthesis. ACSL3-knockout reduced ACS activity by 93 % indicating that ACSL3 is the dominant ACS family member in COS-7 cells. Incorporation of 14C-oleic acid was significantly decreased by 33 % in the ACSL3-knockout cells compared to wildtype COS-7 cells. Triglyceride synthesis was markedly reduced by approximately 50 % and LD biogenesis was decreased by 65 % in the ACSL3-knockout cells compared to wildtype COS-7 cells. The basal lipolytic rate was increased in the ACSL3-knockout cells resulting in intracellular triglyceride levels that were 46 % lower than the triglyceride levels measured in the control cells after 6 h of starvation. Cells expressing the ACSL3 variant lacking ACS activity were slightly reduced in basal lipolysis and thus exhibited triglyceride levels elevated by 24 % compared to the ACSL3-knockout cells after 6 h of starvation. Cells expressing only ER-localized ACSL3 were increased in triglyceride synthesis, but decreased in LD biogenesis by 54 % compared to cells expressing also LD-associated ACSL3. Model calculations in A431 cells estimated that local triglyceride synthesis on LD accounted to 3.3 % of total cellular triglyceride synthesis. In conclusion, this study revealed that ACSL3 is essential for triglyceride synthesis and LD biogenesis by activating fatty acids and delivering them to enzymes involved in lipid synthesis. However, the metabolic capacity of LD-associated ACSL3 is too low to significantly contribute to LD growth. Thus, LD expansion is probably dependent on lipid transfer from the ER to the LD. Moreover, ACSL3 decreases lipolysis maybe by re-esterification of fatty acids deliberated during lipolysis but also possibly by recruitment of antilipolytic proteins to the LD. ER-localized ACSL3 is superior in triglyceride synthesis probably due to the proximity of lipogenic enzymes harboured in the ER membrane. Furthermore, ACSL3 localization appears to be important for LD formation. ACSL3 is an important key player involved in lipid metabolism. Future studies of its underlying functional mechanism are promising to advance the understanding of diseases associated with lipid metabolism and to develop new therapeutic strategies.
Haematopoiesis, the process by which blood cells are formed, is extensively studied because of its relevance for animal life. Uncovering the mechanisms of blood formation and its regulation is fundamental to cope with anomalies or illnesses such as anaemia and leukaemia, or massive blood loss. Haematopoiesis is driven by the haematopoietic stem cells, HSCs. HSCs are able to reconstitute, upon transplantation, all blood lineages of an animal deprived of its haematopoietic cells (multipotency), and to generate one or two HSCs upon division (self-renewal). However, it is unclear how often they self-renew or differentiate into more mature compartments, according to which differentiation pathways, and how physiological and stressed conditions differ. Similarly, the kinetic properties of the progenies of the stem cells are mostly unknown. Here we present an approach to quantify the kinetics of the haematopoietic system via a deterministic mathematical model. The model is driven by two different sets of in vivo measurements: fate mapping of HSCs and BrdU accumulation data. In the first experiment we consider, an inducible, inheritable label is switched on in the stem cells without altering the physiological conditions. The fraction of labelled cells in the stem and in the downstream populations is measured over time. We build a model of population dynamics, which describes the time course increase of the labelled cells fraction in the progenies. The model has only one parameter, the time a cell resides in a population. Fitting reveals that the immediate progenies of stem cells have a long residence time, which suggest a small role of stem cells in normal haematopoiesis, sustained rather by early progenitors. We then infer the differentiation rate of a cell into its progeny by incorporating in the model the ratio of population sizes, and again confirm an infrequent contribution of stem cells. In the second experiment we consider, the thymidine analogue BrdU is fed to mice over time. BrdU labels the cells that undergo DNA replication. The fraction of labelled cells in the stem cells and in the downstream populations is measured over time. We adapt the population dynamics model of the previous part, incorporating the simplified assumption that cells are BrdU positive if and only if they have divided at least once. We fit the adapted model to BrdU and fate mapping data simultaneously and infer the rate at which cells divide, as well as the frequency at which division of different types (symmetric or asymmetric) happen. This analysis reveals infrequent and mainly symmetric divisions of the stem cells. Moreover, we investigate whether a subdivision of the stem cells and their immediate progeny into several heterogeneous sub-populations is compatible with the parameters inferred as described above. We adapt the model to again fit data that consider this subdivision. We find coherent estimates for quantities that are model-invariant, which supports the robustness of our approach. Finally, we adapt our model to describe fate mapping and cell-cycle-related data in non-stationary conditions, namely after irradiation. Contrary to normal conditions, stem cell proliferation and differentiation are significantly activated, demonstrating their importance in reconstituting a severely compromised system. In conclusion, we suggest via data-driven deterministic modelling that HSCs fuel but do not majorly sustain normal haematopoiesis, role played by their immediate progenies. On the contrary, they are very responsive in stressed conditions, rapidly replenishing the depleted cells via enhanced proliferation and differentiation.
"An old city like Heidelberg illustrates how its public space survives the renewal phases of its buildings and makes places permanently distinguishable. It is above all the built public space of a city that carries sustainability and identity in its structure – a goal to which Heidelberg feels particularly committed.” (STADT HEIDELBERG 2005). The great challenge of climate change requires solutions, especially in cities, which are equally responsible for a large part of the anthropogenic greenhouse effect. Current questions on climate change adaptation have a high practical relevance for sustainable urban development on the one hand and for current scientific aspects on the other. Sustainable future models of the modern city primarily aim at reduction, with two essential factors: energy saving and energy efficiency. Increasing heavy precipitation and overheating events are examples that affect all fields of action of the city and require adaptation strategies. The main objective of this project is to collect data, evaluate various climate parameters and to survey citizens on their perception of climate change and the design of public spaces in Heidelberg. The comparison of locations in the historical old town with newly created areas makes it possible to evaluate the key factors of urban planning in order to generate both climatic and social benefits. Following on from this, the research question is: What impulses can new urban climate data and data on the perception of climate change in public places in Heidelberg provide for sustainable urban development? The transdisciplinary approach is based on the combination of different methods of physical as well as human and social geography, the involvement of urban actors and citizens and the concept of research-oriented teaching. The latter creates a learning environment in which students can experience the current problems of urban development in times of climate change and actively participate in the development of solution and adaptation strategies. In cooperation with the Environmental Office of the City of Heidelberg, current issues of urban development adaptation to climate change are to be researched transdisciplinarily and also prepared for teaching in teacher training. The evaluation of the overall data set shows current trends in climate change development. In the summer of 2018, many parts of the city exhibit severe heating and drought. Measures that have a regulating effect on the microclimate are in co-benefit with the respondents' wishes for shading, greening, diversity and a general improvement in the quality of life in public places such as the University square or the Schwetzinger Terrasse. These adaptation measures are essential from a scientific point of view.
Tay-Sachs disease (TSD) is an autosomal-recessive genetic disorder which results in the dysfunction of the metabolic enzyme hexosaminidase A (HexA). It leads to severe lysosomal storage of acidic glycosphingolipid, namely ganglioside GM2, and early fatalities for humans with the infantile on-set form. Despite fifty years of research, to date there is no effective treatment beyond palliative care. It was found that mouse models of HexA deficiency display only moderate GM2 accumulation, which was connected with a late onset neuronal phenotype. Therefore neuraminidases were investigated as possible bypass enzymes for the degradation of GM2 and offer a new opportunity for therapeutic approaches in humans. However, to assess the extent of side effects for such a therapeutic bypass, the substrate specificity and ganglioside (GG) turnover has to be defined in detail. This work presents the development of an HILIC based LC MS2 method as well as mass spectrometry imaging (MSI) using DESI (QqQ)MS2 and MALDI TOF to monitor GG pattern changes in mouse brains. The HILIC MS2 analysis of mouse brain tissue with neuraminidase 3 or 4 deficiency in the background of TSD as well as combined knockouts of GG synthesis enzymes revealed an overlapping but distinct substrate processing for the neuraminidases Neu3 and Neu4. MSI of the same tissue samples displayed similar patterns in spatial neural GM2 accumulation that suggest rather a broad distribution of these sialidases in mouse brain. Proposed neuroinflammation and demyelination in mouse brains of TSD led to a modulated HILIC MS2 method with which hexosylceramide isomer separation of GG precursor β-glucosylceramide (β-GlcCer) and prominent myelin sheath component β-galactosylceramide (β-GalCer) was achieved. Decreased levels of β-GalCer as a marker for demyelination in brains of TSD combined with neuraminidase deficiency could not be observed at the age of 6 month. Furthermore, proof of concept study and screening of various WT mouse tissues revealed the adaptability of this method. Even α-anomeric HexCers could be separated from mammalian β-anomers. In contrast to the mentioned β-HexCers, invariant natural killer T cells are activated most effectively when recognizing galactosylceramide with an α-glycosidic linkage appearing on the cell surface receptor CD1d of antigen presenting cells. One natural bacterial source of this compound in contact with our body is Bacteroides fragilis, a bacterial member of the human gut microbiome. This work highlights the detection and separation of α-GalCer(d17:0;h17:0) in B.fragilis and three other bacteria of the human gut microbiome β-HexCers. Very recent preliminary studies indicate the identification of an α-glycosidic GalCer in the mouse microbiome with the proposed structure of BdS α-GalCer(d18:0;h16:0).
It is the aim of this thesis to analyze the initiation and regulation of the fast brassinosteroid response pathway Arabidopsis thaliana and its role for cell elongation in an integrative fashion using mathematical modeling. Brassinosteroids are plant steroid hormones that mediate various physiological and developmental processes. One of these processes is cell elongation, which is the major mechanism for organ growth in plants. As sessile organisms, plants have to rely on growth to open up new resources. However, growth is an energy consuming process that has to be tightly regulated. Therefore, it is necessary to understand the activation and regulation of the fast brassinosteroid response. The computational modeling and analysis of the fast brassinosteroid signaling focused on several different aspects. Because of the importance of compartmentalization in biological systems, I first studied the different modeling approaches to describe multi-compartment processes in models consisting of ordinary differential equations and how these modeling approaches react to changes in cell morphology. This analysis shows that including the membrane as interaction area can be crucial to proper modeling behavior depending on the modeled system. Second,I used molecular modeling to clarify the interactions between receptor, co-receptor and a negative regulator of the fast brassinosteroid response. Here, the simulated complexes show that the negative regulator acts by blocking the catalytic domain of the co-receptor, which is then unable to participate in propagating the signal. Third, I used a dynamic model consisting of ordinary differential equations to simulate the fast brassinosteroid response on a cellular scale. The parameters of this model were fitted to dose-response data of the membrane potential change. Furthermore, this model includes the BR-induced increase in cell wall volume. I validated this model with respect to the behavior in the meristematic root zone and the behavior in a deletion of a negative regulator. Based on the model behavior and the quantification of model species, we hypothesize that H+-ATPase levels in the different root zones determine the response to brassinosteroid stimulation in the fast response pathway. Finally, I expanded the ordinary differential equation model for the fast brassinosteroid response to include the process of cell elongation. This model can describe the experi- mentally observed elongation behavior of an epidermis cell from the meristematic zone to the final cell length in the maturation zone. I combined this model with an agent-based representation of the root. This model provides an integrative view on cell elongation. While this multi-scale model is currently limited to one cell type and a maximal cell length of 25µm, this shows that it is a valid approach to modeling root elongation.
Glycodelin (Genname: progesterone associated endometrial protein, PAEP) wurde initial als ein Immunsystemsuppressor während der Implantation des Embryos und in der Schwangerschaft beschrieben. Heute ist dessen Expression ebenfalls in unterschiedlichen Tumorerkrankungen wie unter anderem dem nicht-kleinzelligen Lungenkarzinom (non-small cell lung cancer, NSCLC), einer Form des Lungenkarzinoms, bekannt. In dieser Karzinomart wurde Glycodelin nicht nur als ein möglicher Verlaufsmarker beschrieben, sondern auch als potentielles Zielmolekül zukünftiger Immuntherapien. Signalwege, welche die PAEP/Glycodelin-Expression in Tumorerkrankungen regulieren, sind bislang weitestgehend unbekannt. Im Rahmen dieser Arbeit wurde der Einfluss einiger Signalweginduktoren, nachgeschalteter Signalwege und Transkriptionsfaktoren auf die Regulation der PAEP/Glycodelin-Expression untersucht. Als Modell hierfür wurde eine Adenokarzinom (adenocarcinoma, ADC)- sowie eine Plattenepithelkarzinom (squamous cell carcinoma, SQCC)-Zelllinie verwendet und anschließend die Ergebnisse in NSCLC-Patientengeweben validiert. Die Expression von PAEP/Glycodelin wurde hauptsächlich durch die kanonische TGF-β-Signalkaskade in SQCC-Zellen sowie durch den PKC-Signalweg in beiden Zelllinien induziert. Eine etwas schwächere Stimulation der PAEP/Glycodelin-Expression wurde mit dem MEK/ERK-Signalweg assoziiert. Die Aktivierung der Transkriptionsfaktoren JUNB und STAT3 folgte auf eine Stimulation der PKC- und MEK/ERK-Signalkaskade, während NFκB ausschließlich durch eine starke Induktion der PKC stimuliert wurde. Der PI3K/AKT-Signalweg inhibierte die Expression von PAEP/Glycodelin in ADC-Zellen mit aktivierenden EGFR- sowie PIK3CA-Mutationen und lässt diesbezüglich einen antagonistischen Einfluss gegenüber den anderen untersuchten Signalwegen folgern. Die Validierung dieser Ergebnisse in NSCLC-Patientenproben ergab eine hohe Übereinstimmung mit den in vitro Daten. Verschiedene zugelassene oder in aktuellen Studien verwendete NSCLC-Medikamente hemmen den MEK/ERK- sowie den PI3K/AKT-Signalweg. Die Ergebnisse dieser Arbeit lassen darauf schließen, dass die Inhibierung spezifischer Signalwege in NSCLC-Patienten die Expression von Glycodelin und damit dessen immunsuppressive Wirkung mutationsbedingt beeinflusst. Darüber hinaus ermöglichen Kenntnisse über die Regulation der PAEP/Glycodelin-Expression weitere therapeutische Strategien, um die Immunabwehr von NSCLC-Tumoren zu schwächen.
Tumor heterogeneity is a term that refers to differences between tumors of the same type in distinct patients as well as to differences observed between cells within a tumor. The later is known as intratumoral heterogeneity (ITH) and is of high clinical relevance, since it directly affects the robustness of prognostic, diagnostics and prediction of biomarkers. Up to date ITH has been mainly investigated at the genomic level. Sequencing of multiple regions from the same cancer specimen have revealed that within a single tumor several clones of cells with distinct mutational landscapes exist, likely as a consequence of clonal evolution. However, ITH can also be driven by differences in the microenvironment that may rather be reflected in differential gene expression or protein turnover than in genomic changes. Nevertheless, to what extent the ITH is manifested on a proteome-wide scale remains largely unknown, mainly due to technical limitations. To overcome these limitations an efficient protocol that allows for proteomic analysis of limited amounts of formalin-fixed and paraffin-embedded (FFPE) material was developed and employed to characterize the proteomic changes in hepatocellular carcinoma (HCC). First, by comparing neoplastic to the adjacent, non-neoplastic tissues, I defined proteomic features that distinguish tumor from peritumoral tissues. The analysis revealed a decrease in abundances of various mitochondrial proteins including components of the NADH dehydrogenase complex I, possibly indicating the metabolic rearrangement in HCC. Subsequently, by analyzing different regions of HCC, I demonstrated the existence of a proteomic heterogeneity, beyond genetic variations, even in morphologically homogenous specimens, which affects various biological processes. Several clinically relevant proteins were identified as differentially expressed across the analyzed tumors or subject to ITH, thus underlying the importance of ITH studies for biomarker discovery and diagnostic applications. In the second part of my thesis, I focused on the functional characterization of gp210 – a transmembrane component of the nuclear pore complex (NPC). In eukaryotic cells the nuclear envelope constitutes a barrier separating the nucleoplasm and cytoplasm. The transport of macromolecules between these compartments occurs through NPCs which form channels across the inner and outer membrane of the nuclear envelope. Apart from regulating the nucleocytoplasmic transport, NPCs are also involved in the other cellular processes such as chromatin organization, regulation of gene expression or differentiation. The NPC is comprised 8 of multiple copies of around 30 proteins called nucleoporins (~1000 protein in total). While the stoichiometry of scaffold components is constant across cell lines, differences in the composition of peripheral sites have been observed. One example of a nucleoporin with a cell-type specific expression is gp210. It is a transmembrane nucleoporin that associates with the NPC via its short C-terminal domain. The remaining larger part of the protein is localized within the perinuclear space and it is not required for the interaction with the NPC. The luminal function of gp210 so far has been linked to muscle cell differentiation but apart from this, its role remains largely unknown. In order to investigate the luminal function of gp210, I attempted to draft a map of potential interacting proteins. This was achieved by in-situ proximity labeling combined with mass spectrometry-based proteomics using the so-called BioID approach. Data obtained in BioID experiments indicate a functional link between gp210 and endoplasmic reticulum (ER) related biological functions. I have identified multiple factors involved in the regulation of ER stress and several proteins involved in glycophosphatidylinositol anchor attachment.
African trypanosomes are flagellated protozoan parasites that cause sleeping sickness in humans and nagana in cattle. During their life cycle, they change their morphology and metabolism through robust gene regulation processes. Trypanosomes have a unique polycistronic gene arrangement and have to rely almost entirely on post-transcriptional regulation mechanisms, which include mRNA processing, export, stability and translation, in order to regulate gene expression. RNA binding proteins and translation factors are involved. Trypanosomes have six eIF4Es of varying cap-binding affinities and five eIF4Gs, suggesting numerous possibilities for translation regulation. TbEIF4E1 does not interact with any of the eIF4Gs but instead interacts with 4E-interacting protein, 4EIP, which is the first focus of this thesis. Both TbEIF4E1 and Tb4EIP repress a reporter mRNA in a tethering assay, but suppression by TbEIF4E1 requires Tb4EIP. Bloodstream form Trypanosoma brucei lacking Tb4EIP have only a mild growth defect. At high parasitemia, bloodstream forms stop dividing, suppress translation and become stumpy forms, which are adapted to differentiate to procyclic forms when taken up by a tsetse fly. Interestingly, lack of Tb4EIP compromises stumpy formation, and the defect can be rescued by a truncated Tb4EIP that is unable to bind TbEIF4E1. Tb4EIP knockout stumpy forms have abnormally high protein synthesis rates indicating that Tb4EIP is required for translation suppression during differentiation to the stumpy form. RNA binding protein PUF3 is among 11 T. brucei pumilio domain containing proteins. Little was known about PUF3 save for its repression of a reporter mRNA and that it co-purifies with poly(A) mRNA. It is therefore the second focus of this thesis. PUF3-depleted monomorphic and pleomorphic bloodstream cells have a marginal growth defect but PUF3 knockout cells strangely lack this defect. Interestingly, when put to differentiate to stumpy and procyclic forms, PUF3-depleted pleomorphic bloodstream cells experience a delayed differentiation manifested by a low expression of the stumpy form marker PAD1 and procyclic surface coat proteins EP/GPEET. Nevertheless these cells eventually differentiated to viable procyclic forms. Surprisingly, pleomorphic bloodstream cells without PUF3 later seemed to adapt and lack this differentiation defect. The defect however persists in monomorphic cells, suggesting an adaptation mechanism in pleomorphic cells that equilibrates the cellular metabolism to life without PUF3. Using TRIBE (Targets of RNA binding proteins Identified by Editing) 295 putative targets of PUF3 were identified in stumpy-like cells. 79 of these targets are enriched in bloodstream forms while only 12 are enriched in procyclic forms, consistent with a role of PUF3 as a repressor during differentiation to procyclic forms. These targets include mRNAs encoding cytoskeleton proteins, protein kinases, RNA binding proteins, leucine rich repeat proteins, expression-site associated genes, chaperones and translation factors. The results here suggest that Tb4EIP and PUF3 fine-tune gene expression in readiness for differentiation.
Profilin is an actin binding protein playing a central role in cell motility. Interestingly, the group of Apicomplexa that contains a number of important pathogens like Toxoplasma and Plasmodium has a very unusual type of profilin. In these parasites, profilin contains three additional structural motifs that are not found in other organisms like yeast, plants or animals. Additionally, apicomplexan parasites have a unique way of cell locomotion that is based on an unusual actin-myosin motor. This motor enables the invasive stage of the parasite to move with high speeds. In Plasmodium the sporozoite, the stage transmitted to the skin of the host by a mosquito, displays the highest speed. One of the additional structural motifs of apicomplexan profilin, a β-hairpin extension called the arm motif, has been suggested to be involved in actin binding. During this work, I could show through a combination of biochemical and computational analyses of proteins, molecular genetics of Plasmodium berghei parasites and biophysical measurements on sporozoites that this assumption is true. Not only could we show that the arm motif is involved in actin binding, but we could show that the arm motif is crucial for 2D gliding motility of sporozoites. Deletion of the arm motif produced sporozoites that were less virulent than wild type sporozoites. Certain mice were able to survive and clear sporozoites-induced infections with the arm deletion parasites and were later protected against severe malaria and able to clear wild type infections. This indicates, that it should in principle be possible to generate a genetically attenuated parasite for use in sporozoite-induced blood stage vaccinations. We found indications that another additional motif, the acidic loop, is involved in defining the arm motif orientation and thus influences actin binding. Even though profilin was suggested to be essential I was able to generate a profilin knockout. This revealed that profilin is not essential in the blood stage, although the growth rate of the profilin knockout was significantly reduced. However, profilin was necessary during mosquito development, as loss of profilin led to impaired crystalloid body integrity and most significantly, a lack of salivary gland sporozoites.
The number of people in the modern world being affected by allergic diseases and asthma has reached epidemic proportions. With over 1 out of three people requiring some sort of treatment for allergic disease, the burden placed onto industrialized nations healthcare systems is increasing. Mild forms of allergic diseases, such as allergic rhinitis, can be treated with anti-histamines or immunological desensitization. However, more severe forms of disease, such as asthma and atopic dermatitis, require a reduction of systemic inflammation by administration of broad acting systemic immunosuppressants, to effectively alleviate symptoms. However, systemic immunosuppression often results in susceptibility to infection. Mast cells, which are evidently involved in numerous allergic pathologies, have not been adequately targeted by conventional therapeutics. Specific removal of mast cells, by means of antibody-dependent depletion, would interrupt the allergic cascade and might yield significant benefit for patients. In order to develop such a mAb- mediated mast cell ablation approach we sought to established proof of principle in a well-controlled system with our newly developed transgenic mouse model. The Cpa3hCD4 mouse model expresses a truncated human CD4 receptor from a knockin into the mast cell specific Cpa3 locus. With this model system, employing an artificial mast cell marker cell surface protein, we could show that several tissue-resident mast cell compartments can be safely and efficiently depleted after intravenous α- hCD4 administration. To identify a physiologically relevant target on the surface of mast cells, we underwent extensive mass spectrometry-assisted proteomic profiling of primary mouse and human mast cells. Analysis of the data revealed a cross- species conserved mast cell protein signature, among which we found several drugable receptors. Quantitative analysis, paired with flow cytometry-based verification of the cell surface expressed mast cell proteins allowed us to identify several highly expressed mast cell specific cell surface markers. Targeting of one of these receptors with an α-CD63-Immunotoxin resulted in the efficient depletion of mast cells in vitro. In vivo however, efficacy of mast cell depletion was limited by excessive on-target toxicities. Along these lines, we are currently evaluating the herein identified physiological mast cell targets for cellular depletion by different antibody-mediated mechanisms.
Human papillomaviruses (HPVs) cause cervical cancer and are also closely linked to other malignancies in the anogenital and oropharyngeal regions. It is widely assumed thatHPV-positive cancer cells are ‘oncogene addicted’ in that they depend on the continuous expression of the two viral oncogenes, E6 and E7, to maintain their malignant phenotype and to avoid tumor-suppressive senescence induction. However, recently it was shown that hypoxic HPV-positive cancer cells efficiently shut down E6/E7 expression and enter a growth arrest without inducing senescence. This process is reversible upon reoxygenation and can be counteracted by high glucose concentrations. These surprising findings shed new light on the crosstalk between the viral oncogenes and the host cell, particularly under hypoxia, a condition frequently found in cervical cancer that is associated with poor patient prognosis. The present work aimed to delineate the regulatory mechanisms underlying hypoxic E6/E7 repression. It was revealed that E6/E7 mRNA and protein expression is impaired in hypoxic HPV-positive cancer cells by a combination of transcriptional repression and reduction of mRNA and protein half-lives. Moreover, hypoxic repression of E6/E7 was shown to be not only glucose-sensitive, but also dependent on intracellular calcium. There was no indication for a role of HIF transcription factors, considered master orchestrators of the cellular response to hypoxia, or for involvement of transcription factors from the glucose-responsive Mlx network. However, the PI3K/AKT signaling pathway was identified as being crucial for the hypoxic repression of E6/E7. AKT is activated by hypoxia which can be counteracted by high glucose concentrations. Blocking of hypoxia-induced AKT activation by chemical inhibitors, including clinically applied drugs that target AKT or its upstream activators PI3K and mTORC2, counteracts repression of E6/E7 under hypoxia. Knockdown and overexpression experiments showed that the AKT1 and AKT2 isoforms act redundantly in hypoxic repression of E6/E7. Reactivation of E6/E7 expression under hypoxia upon inhibition of the PI3K/AKT pathway does not induce proliferation and can sensitize HPV-positive cells to therapeutic effects of chemotherapy in a cell line-dependent manner. Finally, comparison of the proteome composition of HPV-positive cancer cells under normoxia and hypoxia as well as proteome analyses of hypoxic cells treated with an AKT inhibitor or high glucose concentrations were performed. They identified several novel candidate proteins possibly linked to hypoxic repression of E6/E7 providing an experimental basis for future investigations. Collectively, these findings provide new insights into the regulation of the viral oncogenes in hypoxic HPV-positive tumor cells and have implications for the development of novel treatment strategies.
Adaptive Cone-Beam Scan-Trajektorien für interventionelle Anwendungen Die interventionelle Röntgenbildgebung stellt Ärzten während minimalinvasiven Eingriffen Informationen über die Patientenmorphologie bereit. Sie muss aber aufgrund der gewebeschädigenden Wirkung mit Bedacht eingesetzt werden. Derzeit können Ärzte nur zwischen dosisarmen Röntgenprojektionen ohne Tiefeninformation und strahlungsintensiven Cone-Beam- Computertomografien mit Tiefeninformation wählen. Viele medizinische Anwendungen wie Positionskontrollen erfordern zwar Tiefeninformation, aber keinen vollständigen 3D-Datensatz. Adaptive Scan-Trajektorien können diese Lücke schließen, indem sie Objekte gezielt unterabtasten und die relevanten Informationen so dosiseffizient in Erfahrung bringen. In dieser Arbeit wird eine Methode präsentiert, die eine Implementierung von neuen adaptiven Scan-Trajektorien an einem C-Bogen-System erlaubt. Am Beispiel einer Klasse von Scan-Trajektorien, der zirkulären Tomosynthese (ZT), wurde die Realisierbarkeit der Methode demonstriert. Streustrahlenmessungen ergaben, dass die ZT eine vorteilhaftere Streustrahlenverteilung als die klassischen 3D-Trajektorien aufweist. In kritischen Körperpartien wie oberer Torso und Gesicht, wurde eine geringere relative Dosis von 75% und 46% (ZT) als bei klassischen Trajektorien (100% und 63%) gemessen. Die Scan-Trajektorien wurden mit einer Kalibrierung kombiniert, die auch eine retrospektive Kalibrierung an beliebigen Positionen im Interventionsraum erlaubt. In Streßtests konnten die Positionen von Metallkugeln eines Evaluierungsphantoms mit einer mittleren Genauigkeit von (0,01 ± 0,08) mm und einer mittleren Radiusabweichung von (0,13 ± 0,07) mm bestimmt werden. Bei einer Voxelgröße von 0,48 mm sind die Abweichungen kleiner als die Messgenauigkeit des bildgebenden Systems. Die untersuchten Trajektorien verwenden nur ein Viertel bis ein Fünftel der Projektionen herkömmlicher 3D-Trajektorien. Die Unterabtastung des Objekts und die Dosiseinsparung verursachen Artefakte in den Bilddaten. Mithilfe eines vorwissenbasierten Ansatzes konnten diese Artefakte minimiert und die Bildqualität auf die eines konventionellen 3D-Datensatzes verbessert werden. Die Ergebnisse dieser Arbeit zeigen, dass adaptive Scan-Trajektorien die interventionelle Röntgenbildgebung um einen neuen Bildgebungsmodus erweitern können, der gegenüber derzeitigen Bildgebungsmodi relevante Bildinformationen bei reduzierter Dosis akquiriert.
In this study we aimed at studying the tumor aggressiveness and differentiation potential of Ramos Burkitt’s lymphoma cells upon mitogenic stimulation in light of a novel regulator of immune cell metabolism and activation, ADPGK. We have identified the role of ADPGK in regulation of aerobic glycolysis in Burkitt’s Lymphoma cells and shown that its knock-out leads to reduced tumor aggressiveness, as measured in-vitro via co-culture, migration experiments and metabolic profile, and in-vivo Zebrafish. We found significantly reduced MYC transcription in ADPGK knock-out Burkitt’s lymphoma cells and importantly, several folds reduction in accumulated random mutations in translocated MYC in these cells. We additionally observed a stalled pathway to differentiation of ADPGK knock out B-cells into plasma cells upon stimulation by mitogenic signals. Overall, the study provided the first insights into the role of a novel ER resident protein acting as a regulator of two complementary phenomenon, cell-differentiation and cancer aggressiveness, and thereby opens up new possibilities of therapeutic interventions for hematopoietic malignancies.
In mammals, X-chromosome inactivation (XCI) re-establishes the dosage balance between male and female gene expression levels. However, up to a third of the X-linked genes escape this phenomenon with varying degrees of consistency across tissues, cell lines, or individuals. Here, I take advantage of a new extensive developmental dataset for several organs and species to explore the developmental dimension of sex chromosomes expression levels. I find that only a small fraction of genes escaping XCI show a consistent female overexpression across development, organs, and species. The consistently sex-biased genes are almost exclusively either directly involved in the establishment of XCI, or are protein-coding genes with a broadly expressed Y homolog. The conservation of the sex-bias of these genes across species suggests that they might be responsible for the evolution of escape from XCI. I also present and test a model of allelic contribution to the total expression levels of X-linked genes using marsupial bulk tissue RNA sequencing data. Finally, I describe my contribution to a study of sex-biased micro-RNAs in mammals.
A remarkable feature of cancer-associated Human papillomavirus (HPV) types (e.g. HPV16/18) is that the open reading frame (ORF) of the major capsid protein L1 comprises alternative translation initiation codons, thereby potentially encoding different isoforms. Notably, the same can be found within the genome of Mastomys natalensis papillomavirus (MnPV), an etiological agent for non-melanoma skin cancer in the rodent Mastomys coucha.
Capsid formation of these L1 variants was tested using both baculovirus and pseudovirus production systems. L1 translated from the first and second ATG (referred as L1LONG and L1MIDDLE) inefficiently induced capsid formation. In contrast, virions could be efficiently formed with capsomeres derived from L1SHORT protein starting from the third ATG. Even after adding L1SHORT and L2 proteins, L1LONG and L1MIDDLE were still unable to form the spherically correct virus particles.
Analysis of MnPV-infected animals during early infection revealed strong serological responses against MnPV L1LONG and L1MIDDLE protein, but these antibodies were not protective. Contrary, neutralizing antibodies against conformational epitopes of L1SHORT only appeared during the late phase of infection, apparently enabling the virus to accumulate. Therefore, the additional N-terminal part of L1LONG might play a decisive role in capsid formation and form a loop which prevents the assembly of correct capsomeres in vivo. Such a transitory conformational epitope is apparently only recognized during early infection, allowing the virus to escape from humoral immune surveillance. Moreover, using competition assays during pseudoviruses formation revealed that L1LONG protein could interfere with the capsid forming process in the presence of L1SHORT. This could be also shown by changes in intracellular localization when L1LONG or L1MIDDLE is co-expressed with L1SHORT.
In summary, these data provide evidence of an adaptive immune escape initial after infection by avoiding the production of protective antibodies. Since other “high-risk” mucosal and cutaneous HPVs can also encode such L1 isoforms, these results may have important implications in the establishment of a persistent viral infection and the outcome of the disease.
TGF-β1 is a key player in the onset, the progress, and end stages of CLD promoting fibrogenesis and tumorigenesis. To date, the expression and function of TGF-β2 have not been investigated thoroughly in liver disease. In this thesis, we provide evidence that TgfB2 and not, as formerly known, only TgfB1 correlate with fibrogenesis and liver cancer development. In a comparative analysis, we analyzed TgfB2 and TgfB1 expression and secretion in murine and human HSCs, hepatocytes, and HCC/hepatoblastoma cell lines. In various mouse models reflecting regeneration, acute and chronic liver disease, and human HCC sample cohorts, we demonstrated that both isoforms are expressed in different types of liver cells and that expression is elevated during the progression of CLD in mouse models in most cases. Although TgfB2 is mostly secreted at lower levels than TgfB1, its expression patterns largely follow similar profiles. However, the secretion of TgfB2 exceeded that of TgfB1 in some HCC cell lines. Our data indicates a more prominent implication of TgfB2 in biliary-derived liver disease models. In this thesis, the anti-fibrotic and immunoregulatory effects of TgfB2 silencing in cholestatic MDR2-KO mice have been delineated for the first time. TgfB2 silencing by AONs specifically reduced collagen deposition and αSMA expression, but induced anti-fibrotic PparG expression. Accumulation of TGF-β2-specific AON was detected in macrophage-activated fibroblasts, LSEC, and activated HSCs in mice. This was in accordance with TgfB2 expression in these cell types. CD45-positive immune cell infiltration was reduced upon TGF-β2-specific AON treatment in the livers of MDR2-KO mice. Furthermore, TGF-β2 levels were found to be elevated and correlated with CD45-positive immune cell infiltration in PSC and PBC patients. In summary, the data presented, provides a strong rationale to examine anti-TgfB2-directed treatment in patients with cholestatic liver damage as PSC or PBC. Taken together, this thesis points towards TGF-β2 as a promising therapeutic target in CLD especially of biliary origin. It provides a direct rationale for TGF-β2-directed drug development an further suggests to initiate a clinical trial testing TGF-β2 inhibition in PSC and PBC patients.
We study the evolution of star clusters, starting from their birth in molecular gas clumps until their complete dissolution in the Galactic tidal field. We have combined the “local-density-driven cluster formation” model of Parmentier and Pfalzner (2013) with direct N-body simulations of star clusters following instantaneous expulsion of their residual star-forming gas. Our model clusters are formed with a centrally peaked star-formation efficiency (SFE) profile, that is, the residual gas has a shallower density profile than stars. We build a large grid of simulations covering the parameter space of global SFEs, cluster masses, sizes and galactocentric distances. We study the survivability of our model clusters in the solar neighborhood after instantaneous gas expulsion and find that a minimum global SFE of 15 percent is sufficient to produce a bound cluster. Then studying their long-term evolution we find that our simulations are able to reproduce the cluster dissolution time observed for the solar neighborhood, provided that the cluster population is dominated by those formed with a low global SFE (about 15%). Finally, we find that the cluster survivability after instantaneous gas expulsion, as measured by cluster bound mass fraction at the end of violent relaxation, is independent of the Galactic tidal field impact.
We study physical systems composed of at least two immiscible fluids occupying different regions of space, the so-called phases. Flows of such multi-phase fluids are frequently met in industrial applications which rises the need for their numerical simulations. In particular, the research conducted herein is motivated by the need to model the float glass forming process. The systems of interest are in the present contribution mathematically described in the framework of the so-called diffuse interface models. The thesis consists of two parts.
In the modelling part, we first derive standard diffuse interface models and their generalized variants based on the concept of multi-component continuous medium and its careful thermodynamic analysis. We provide a critical assessment of assumptions that lead to different models for a given system. Our newly formulated class of generalized models of Cahn-Hilliard-Navier-Stokes-Fourier (CHNSF) type is applicable in a non-isothermal setting. Each model belonging to that class describes a mixture of separable, heat conducting Newtonian fluids that are either compressible or incompressible. The models capture capillary and thermal effects in thin interfacial regions where the fluids actually mix.
In the computational part, we focus on the development of an efficient and robust numerical solver for a specific isothermal model describing incompressible fluids. The proposed numerical scheme, which is based on the finite element method, partly decouples the system of governing equations on the level of time discetization. We carefully discuss the advanced design of the preconditioner for the computationally most demanding part of the scheme, given by the system of incompressible Navier-Stokes equations with variable coefficients. The numerical scheme has been implemented using the FEniCS computing platform. The code capable of running parallel 2D and 3D multi-phase flow simulations is available in the newly developed FEniCS-based library MUFLON.
Fall is a leading cause of injuries in older adults, which mostly occurs during walking and under a challenging condition. Examples of challenging conditions are an unexpected perturbed walking, stair walking, or walking while positional transitions. A better understanding of postural control to maintain balance during similar tasks can help in reducing the risk of fall. For this aim, one approach is to examine the effects of the challenged walking on gait stability. This dissertation consists of three studies focusing on postural responses under four different challenging circumstances including 1) walking while performing a manual and a cognitive secondary task, 2) stair walking at different inclinations (i.e. different levels of complexity), 3) sudden mechanically perturbed walking, and 4) gaze-shift walking. Firstly, the postural responses of healthy adults under the mentioned conditions were assessed. Secondly, different representative measures in order to quantify balance during perturbed walking were evaluated. Thirdly, the postural responses of young and old healthy adults during walking while gaze-shifting in terms of gait parameters and their variability were contrasted. The first study examined how secondary cognitive and manual tasks interfere with stair gait when a person concurrently performed tasks at different levels of complexity. Gait kinematic data and secondary task performance measures were obtained from fifteen healthy young males while ascending and descending a four-step staircase at three inclinations (17.7°, 29.4°, and 41.5°) as well as level walking. They performed a cognitive task, ‘backward digit recall’, a manual task, ‘carrying a cup of water’ and a combination of the two tasks. Gait performance and dynamic stability were assessed by gait speed and whole body center of mass (CoM) range of motion in the medial-lateral direction, respectively. No significant effect of the gait task on the cognitive task performance was observed. In contrast, stair walking adversely affected the performance of the manual task compared to level walking. Overall, more difficult postural and secondary tasks resulted in a decrease in gait speed and variation in CoM displacement within a normal range. Results suggest that CoM displacement and gait alterations might be adopted to enhance the stability, and optimize the secondary task performance while walking under challenging circumstances. The findings of this study are useful for balance and gait evaluation, and for future falls prediction. The second study examined changes in spatiotemporal gait and stability parameters in response to sudden mechanical perturbations in mediolateral (ML) and anterior-posterior (AP) direction during treadmill walking. Moreover, the most representative parameters to quantify postural recovery responses were evaluated. Ten healthy adults (mean=26.4, SD=4.1 years) walked on a treadmill that provided unexpected discrete ML and AP surface horizontal perturbations. Participants walked under no perturbation (normal walking), and under left, right, forward, and backward sudden mechanical perturbation conditions. Gait parameters were computed including stride length (SL), step width (SW), and cadence, as well as dynamic stability in AP- (MoS-AP) and ML- (MoS-ML) directions. Gait and stability parameters were quantified by means, variability, and extreme values. Overall, participants walked with a shorter stride length, a wider step width, and a higher cadence during perturbed walking, but despite this, the effect of perturbations on means of SW and MoS-ML was not statistically significant. These effects were found to be significantly greater when the perturbations were applied toward the ML-direction. Variabilities, as well as extremes of gait-related parameters, showed strong responses to the perturbations. The higher variability as a response to perturbations might be an indicator of instability and fall risk, on the same note, an adaptation strategy and beneficial to recover balance. Parameters identified in this study may represent useful indicators of locomotor adaptation to successfully compensate sudden mechanical perturbation during walking. The third study was aimed to determine the gait characteristics of healthy young and older adults during gaze-shifting while treadmill-walking. Eleven young (age: 25 ± 4.5 years, 3 females) and 13 older (age: 72 ± 3.9 years, 6 females) adults performed normal treadmill-walking (no visual-triggers) and then treadmill-walking while rapidly gaze-shifting to randomly presented visual-triggers. A multilevel linear regression model was used to assess changes in a set of gait parameters between subject groups and walking conditions: normal walking, one gait cycle before (Pre-Cycle), and after (Post-Cycle) each triggering during gaze-shift walking. Comparing Pre-Cycle to normal walking, young adults showed no instability-related changes in their gait but older adults showed a more cautious gait with shorter step length (Est. = -1.59cm [95% CI: -2.2cm; -0.9cm]), reduced step width (Est. = -0.8cm [95% CI: -1.1cm; -0.6cm]), increased step frequency (Est. = 0.04 1/s [95% CI: 0.03 1/s; 0.05 1/s]), decreased maximum toe clearance (Est. = -0.3cm [95% CI: -0.4cm; -0.2cm]), and 30% higher minimum toe clearance variability. During Post-Cycle compared to Pre-Cycle, direct effects of gaze-shifts on gait parameters were significant but rather small. This experiment shows an influence of gaze-shifts on gait in both groups, although, the effect is larger in the older which might therefore need more compensation compared to the young adults. Present insights may facilitate the development of specific training paradigms to improve the oculomotor-locomotor interaction.
Cancer drugs have among the lowest response rates across all diseases. Combining the wealth of omics data and machine learning is a promising way to reach this goal. In this thesis, we addressed the following aspects of precision oncology: (i) We used Macau, a bayesian multitask multi-relational algorithm to explore the associations between the drugs’ targets and signaling pathways’ activation. We applied this methodology to drug synergy prediction and stratification. (ii) We leveraged through a collaborative machine learning competition to understand the association between genome, transcriptome and proteome in tumors. The main focus of this thesis is to use machine learning to generate actionable insights, for more personalized therapies.
The Epstein-Barr virus (EBV) is a γ-herpesvirus that establishes lifelong infection in the majority of the human population. Whilst EBV infection is asymptomatic in most cases, the global disease burden of EBV is substantial. EBV is the primary cause of infectious mononucleosis and approximately 200,000 cases of EBV-associated malignancies are annually diagnosed. EBV has multifaceted life cycle that comprises lytic replication and latency, with the establishment of latency enabling lifelong EBV carriage. The majority of EBV vaccine candidates have previously focused on the major glycoprotein gp350. However, the interrogation of a gp350 vaccine in a phase II clinical trial showed that it was unable to prevent EBV infection. Our laboratory has recently developed a vaccine candidate in the form of defective EBV particles. These defective particles lack viral DNA, are non-infectious and are composed of several dozen EBV lytic proteins. In the present work, the antigenic spectrum of defective EBV particles was enlarged to include immunodominant latent proteins. The introduction of latent proteins into the defective particles enabled the stimulation of several lytic protein- and latent protein-specific CD4+ T cells. Polyclonal T cells specific for the modified particles were shown to be far superior to gp350-specific T cells at controlling EBV-infected B cells ex vivo. Furthermore, the modified particles afforded significant protection against EBV-infection in humanised mice. This suggests that EBV vaccines can be enhanced through the inclusion of additional antigens. By incorporating the most immunodominant EBV antigens into defective particles, it would prime the immune system against EBV antigens that are expressed at all stages of viral infection and in all EBV-associated malignancies. Such a multipronged approach is likely increase the possibility of achieving sterile immunity after prophylactic vaccination.
State-of-the-art cosmological hydrodynamical simulations have succeeded in modelling realistic Milky Way (MW) type galaxies with spatial resolution of the order of a few hundred parsec, similar to the scale-height of MW's stellar disc and the half-light radius of classical satellite galaxies. I divide the present study into two parts, the build-up of MW's stellar disc and bulge and the formation and evolution of its satellites and dwarf galaxies.
In the first part I show that observed clumpy stellar discs in the early phases of the formation of the Galaxy are dynamically unimportant for its further evolution. This confirms recent observational results where a non linear mapping between stellar mass and light causes stellar discs to appear clumpy. I turn then to explore the formation mechanism of a peanut bulge in cosmological context. I study the kinematical properties of the central stellar populations of a model galaxy using a kinematical decomposition technique and find that the observed kinematic features of the (MW) bulge can only be explained if it consists of both, a peanut bulge and a spherically symmetric bulge both formed via disc instabilities. Observing and disentangling both components will soon be possible thanks to large scale Galactic surveys like Gaia.
In the second part I study the dwarf galaxy population of (MW) mass galaxies. The simulations presented here are among the first to be able to study the formation of dwarf satellite galaxies in a realistic cosmological environment. The employed sub-grid models of the simulations reconcile simulated and observed Local Group satellite mass functions and produce dwarf galaxies whose central stellar velocity dispersion agrees with observations. Using the dwarf galaxies, I test the observational prospects of identifying tidally affected dwarfs in the Local Group using three observables: their distance, line-of-sight velocity and central velocity dispersion. Finally, I investigate the evolution of planes-of-satellites in the framework of the Cold Dark Matter model with a cosmological constant (ΛCDM). These planes quickly dissolve because they consist of a large fraction of chance aligned satellites as recently confirmed with the proper motions of the classical satellite galaxies derived from Gaia data.
Over time, viruses have evolved several strategies to infect host cells, which can be roughly categorized into two distinct modes of viral transmission, namely cell-free and cell-to-cell. Various studies have opted for the latter to play the dominant role in infections with human immunodeficiency virus (HIV) and hepatitis C virus (HCV). How to quantify the exact contribution of each transmission mode to the infection dynamics, without biasing results due to the experimental protocol, remains to be answered to allow for more efficient drug and vaccine development.
Previously, mathematical models based on mass-action kinetics were fitted to bulk measurements obtained from in vitro cell cultures cultivated in aqueous media. This combination of experiment and modeling is very specific and might not be appropriate to describe other situations such as in vivo studies. To allow generalization, the effect of modeling strategy and experimental protocol on the predicted contributions of cell-to-cell spread to the infection dynamics should be studied in more detail. In this thesis, I therefore analyzed possible consequences of varying extracellular microenvironments and types of target cells on the different transmission modes during an infection and how certain modeling strategies affect predictions.
In a first step, I studied the impact of varying extracellular microenvironments on in vitro spread of HIV. To this end, I developed a detailed mass-action kinetics model combining experiments and mathematical modeling to describe HIV infection dynamics in aqueous media and two types of collagen. My results show that for HIV infection of motile target cells the contribution of transmission modes varies dependent on the environment.
Next, I moved from motile to stationary cells analyzing cell-to-cell transmission of HCV among immobile hepatocytes. To account for the local nature of cell-to-cell spread in stationary target cells, I developed an agent-based model, which combines deterministic intracellular viral processes with stochastic intercellular infection events. This model was applied to spatially-resolved data, describing HCV infection dynamics obtained from an in vitro experiment conducted on hepatocytes in a monolayer. I found that the contribution of cell-to-cell transmission to the infection dynamics is dominant in the early stages of the experiment. However, depending on the composition of the used serum, the contribution can shift towards a stronger role of cell-free spread during the experiment.
Moreover, I investigated how to reconcile bulk measurement data with mass-action kinetics models in the context of viral cell-to-cell transmission among stationary target cells. Fitting complex models to describe various aspects of viral infections such as cell-to-cell transmission usually requires sufficiently resolved data. Nonetheless, experimental protocols often only allow bulk measurements, which are generally combined with mass-action kinetics models that assume a well-mixed system. In the context of locally confined movement of target cells infected by viruses, which are capable of infecting via cell-to-cell transmission, this assumption is likely violated. Therefore, I developed an extension of a previously published model, incorporating the decreasing proportion of cells contributing to cell-to-cell transmission with progression of infection. The extended model then allowed correct quantification of the infection dynamics.
In summary, I developed different modeling approaches to analyze the contribution of cell-to-cell transmission to the infection dynamics of HIV and HCV under varying conditions. Furthermore, I provide an extension to a mass-action kinetics model to allow correct description of cell-to-cell transmission in the context of stationary cells while still keeping a simple structure, which can be fitted to population-level data. All approaches can be adapted to other viruses to allow quantification of transmission modes, which will help to guide drug and vaccine development more efficiently.
Heterogeneity is a hallmark of biological systems at every conceivable scale. In this work, I develop computational methods for describing various interacting types of biological heterogeneity. I apply them to explore two scenarios of biomedical interest: the evocation of protective B cell responses by vaccination and the growth dynamics of an aggressive brain tumour.
In the vast majority of currently licensed vaccines, antibody titres are strong correlates of vaccine-induced immunity. However, diseases like influenza, tuberculosis and malaria continue to escape efficient vaccination, and the mechanisms behind many established vaccines remain incompletely understood. In the first part of this work, I therefore develop a data-driven computational model of the B cell memory response to vaccination based on an ensemble of simulated germinal centres. This model can address immunisation problems of different difficulty levels by allowing both pathogen- and host-specific parameters to vary. Using this framework, I show that two distinct bottlenecks for successful vaccination exist: the availability of high-quality precursors for clonal selection and the efficiency of affinity maturation dependent on binding complexity. Together with experimental collaborators, we have used these results to interpret single-cell immunoglobulin sequencing data from a vaccination trial targeting the malaria parasite Plasmodium falciparum (Pf ). As predicted for a complex antigen, after repeated immunisation with Pf sporozoites, the clonal selection of potent germline and memory B cell precursors against a major surface protein outpaces affinity maturation because the majority of immunoglobulin gene mutations are affinity-neutral. These findings have implications for the design of potentially personalised vaccination strategies to induce potent B cell responses against structurally complex antigens.
A quantitative understanding of functional cell heterogeneity in tumour growth promises insights into the fundamentals of cancer biology. In the second part of this work, I correspondingly develop mathematical models of glioblastoma growth. Employing a Bayesian approach to parameter estimation and incorporating a large body of experimental data from mouse models, I show that brain tumour stem cells drive exponential tumour growth while more differentiated tumour progenitor cells, although fast cycling, are unable to sustain expansion by themselves. Comparing a three-dimensional simulation of tumour growth to experimental growth curves, I derive that glioblastoma stem cells are highly migratory. Based on single-cell clonal tracing data and a combination of deterministic and stochastic modelling approaches, I identify their migration rate and explain experimentally observed clone size distributions. Finally, I employ the resulting fully quantified model of tumour growth to predict the response to two therapeutic interventions. These predictions were verified experimentally by our collaborators, suggesting that quantitative knowledge on the hierarchical subpopulation structure of a tumour may provide valuable guidance for treatment.
The organization of cells within tissues allows them to work together. Tight spatiotemporal control over cell-cell interactions is essential for individual cells to self-assemble and function as tissues. In addition, during many biological processes, such as embryogenesis and cancer development, cell-cell interactions undergo dynamic changes to alter their function. Analogously, in the context of bottom-up synthetic biology it is of interest to dynamically control the interactions between minimal synthetic cells and assemble them into precise multi-compartment prototissues with high spatiotemporal resolution. The aim of the first part of this thesis was to reversibly self-assemble different types of micrometer-sized colloids, which were used as synthetic cell-mimics, with high spatiotemporal resolution using visible light. Light provides a dynamic, non-invasive, and biocompatible control with high spatiotemporal precision. In order to control the self-assembly of cell-mimics, I functionalized them with photoswitchable proteins that specifically interact with each other under blue or red light. For this purpose I used several combinations of photoswitchable proteins that are dimerizing under blue light: heterodimerizing iLID and Nano proteins, nMagHigh and pMagHigh proteins, and homodimerizing VVDHigh protein. For the red light switchable proteins I used both the heterodimerization of phytochrome B (PhyB) and phytochrome interaction partner (PIF6) proteins and the homodimerization of Cph1 protein. All of these light dependent protein interactions enabled controlling the self-assembly of cell-mimics with light. Additionally, blue light dependent protein interactions are reversible in the absence of light with red light dependent interactions reversing under far-red light illumination. Consequently, the self-assembly of cell-mimics mediated by these protein interactions was also reversible. Additionally, the high specificity and the independent response of these protein interactions to blue or red light offers the potential to self-assemble a specific population of cell-mimics in the presence of others on demand. In multicellular organisms, cells do not just self-assemble but they also self-sort into precise arrangements in order to work together. As part of this thesis, I also mimicked the self-sorting behavior with synthetic compartments inspired by the observations in nature. Self-sorting is defined by the ability to distinguish between self and non-self, and happens in different modes depending on the interactions between the particles. One mode is social self-sorting, which leads to the separation of colloids into independent colloidal families and requires heterophilic and orthogonal interactions. In this part of the thesis, I used heterodimerization of two blue light switchable protein pairs, iLID/Nano and nMagHigh/pMagHigh, for the social self-sorting between four different populations of colloids within one mixture. Each protein pair specifically and orthogonally brings together two different subpopulations of colloids providing tight and reversible control over their self-sorting into two distinct families using blue light. On the other hand, asocial sorting is another mode of self-sorting, which requires homophilic interactions to bring together compartments of the same type into isolated aggregates. This could potentially be achieved by combining homodimerization of VVDHigh and Cph1 proteins under blue and red light respectively. Eventually, all the versatile and orthogonal light-switchable proteins and different dimerization modes have the potential to be incorporated together in different combinations to achieve the desired self-sorting outcome in complex prototissues. In the second part of the thesis, I addressed the spatiotemporally controlled formation of protein patterns on synthetic cell-mimics. Protein patterns and gradients on cell membranes are important during many biological processes to locally trigger events in multicellular structures with high spatiotemporal precision. To create and control protein patterns on synthetic membranes such as giant unilamellar vesicles (GUVs) with light, I used the blue light switchable heterodimerization of iLID and Nano proteins. For this purpose, the GUVs were functionalized with iLID. This allowed for the blue light mediated, reversible recruitment of a fluorescent protein (mOrange) fused to Nano using blue light with high spatiotemporal resolution. Further, this approach allowed scaling the size of protein patterns from the level of a single GUV to the level of a tissue-like GUV carpet. Hence, these photoswitchable proteins offer a versatile, reversible, dynamic, and non-invasive method to photopattern proteins with high spatiotemporal control that operates under mild conditions. Overall, photoswitchable proteins are important building blocks in the bottom-up synthetic biology toolbox. Incorporating them onto minimal synthetic cells can be used to self-assemble and self-sort different types of cell-mimics and to generate protein patterns, thus mimicking complex processes that occur in nature. Most importantly, these protein interactions provide high spatiotemporal precision and specificity to control these biomimetic processes. Ultimately, this concept can be transferred to assemble prototissues using various types of cell-mimics that host different functionalities, which would allow for controlling, how different synthetic cells work together in a prototissue.
A long-standing aim in biology is to elucidate how the genome is tightly compacted inside the eukaryotic nucleus while still retaining its capacity to orchestrate the correct functionality of the cell. While years of research have revealed that this three-dimensional structuring of DNA plays a major role in the transcriptional regulation, most of the existing studies have focused on long-range chromatin interactions, which are mainly established by the CCCTC-binding factor (CTCF), rarely centring at the gene level. Furthermore, our current knowledge on the interplay between structure and function remains largely descriptive with little mechanistic insight. In this dissertation I present three distinct computational studies which integrate multiple levels of molecular phenotype data in an attempt to gain further insights into the influence of chromatin organisation in (i) splicing regulation, (ii) in how distal genetic variants convey their signal, (iii) and an overall view of the misregulation of chromatin compaction in ageing stem cells. Firstly, I describe a novel splicing mechanism whereby CTCF-mediated DNA- loops that are formed within genes facilitate exon inclusion. My results provide substantial evidence that intragenic loops regulate exon usage and that CTCF binding can be affected either by genetic variation across individuals or by the epigenomic landscape in different cell lines. Those exons being CTCF-regulated frequently overlap annotated protein domains and are enriched for being involved in cellular stress-response and signalling pathways. In summary, this study provides strong evidence for alternative exon usage being regulated by chromatin structure, and thus increases our understanding of functional consequences underlying variation in chromatin architecture. In a second study, I show initial efforts to unravel the mechanisms that allow a genetic variant (distal-QTL) to confer its effect at distant regions through long-range interactions. By measuring allele-specific biases of various molecular phenotypes occurring along chromatin interactions, I propose two models that intend to explain the propagation of this signal. In the “touch-and-act model” functionality is transmitted through the physical contact of both anchors, independent of the region inside the loop, while in the “spreading model” the function is propagated along the entire loop resulting in a coordinated activation or repression of the whole local neighbourhood. There is evidence for both models occurring at varying proportions, which are partially explained by transcription factor co-enrichments. Finally, I present a study on how chromatin accessibility impacts the transcriptome and the proteome in mesenchymal stem cells (MSC) from human donors of multiple ages. I also observed a profound misregulation of chromatin organisation occurring with age, possibly due to a decrease in chromatin-related proteins such as histones, CTCF, CENPB, and lamins, which ultimately affect heterochromatin at centromeres and telomeres contributing to genomic instability. By subtle but significant changes in the transcription factor landscape of young and old MSCs, I observe a bias in the differentiation potential. Additionally, I show a loss of bivalent modifications at enhancer and promoter regions that correlate with DNA methylation changes and that could possibly contribute to a decrease in stemness with age. In summary, I describe a novel splicing mechanism mediated by chromatin intragenic interactions, propose models of how distal-QTLs propagate histone marks, and advance the understanding of chromatin accessibility changes occurring with age in stem cells.
Viral vectors based on Adeno-associated viruses (AAV) have a broad application spectrum including gene therapy and basic research. However, because naturally occurring AAV capsids are rarely sufficiently efficient and/or specific for a given application, techniques were developed to broaden the existing capsid repertoire. A prototype technology is DNA family shuffling where, in a first step, homologous cap genes encoding capsid subunits are fragmented and recombined, yielding a viral library which can then be subjected to selection in order to enrich promising variants. The aim of the present study was to dissect and improve four critical steps along this procedure. Firstly (1), two different methodologies for production of cap gene fragments were compared, resulting in the identification of DNase I based fragmentation as the most robust approach. Interestingly, cap DNA concatamer formation during nested PCR was observed, leading to amendment of the PCR purification protocol. Next (2), we studied the impact of chimerism on the essential assembly-activating protein (AAP) that is encoded in an alternative open reading frame within cap and is recombined as well during DNA family shuffling. Importantly, by performing a battery of complementary experiments, we were able to show that shuffling of AAP is not impairing its function, i.e. the support of particle assembly. Furthermore, no influence on titers was observed for wild-type and most chimeric vector productions, altogether relieving long-standing concerns about a potential rate-limiting role of AAP for AAV vector generation and evolution. Thirdly (3), we established a pioneering in vivo AAV library selection strategy in which, unlike most previously reported schemes, we selected novel capsids in specific cell types within an organ instead of the organ as a whole. Specifically, we were motivated by the facts that liver disease is wide-spread in humans and that hepatic stellate cells (HSC) are known to drive liver fibrosis, thus contributing to disease progression. Alas, tools to genetically manipulate HSCs are limited. Therefore, a library encompassing 10 capsid variants was selected in HSC by AAV injection into mice, HSC isolation and PCR rescue using purified total DNA. Following multiple selection rounds, in vivo bulk validation was performed based on next-generation sequencing. In total, 157 capsid variants were screened in parallel and again, the liver was segregated into the single cell types, i.e. hepatocytes, HSC, Kupffer cells and liver sinusoidal endothelial cells. Notably, this revealed that the selection was successful as hepatocyte-detargeted vectors were identified that showed a strong co-transduction of HSC and Kupffer cells. Intriguingly, we noted differences in vector specificity and efficiency on the DNA versus the RNA level. In order to even further restrict the new vectors to a given cell type, vector cassettes were generated bearing cell-type specific promoters and miRNA binding sites to suppress off-targeting in cells expressing these miRNAs. Testing of these constructs in vitro gave promising results especially for the miRNA-based detargeting strategy. Finally (4), we implemented improvements during the selection and analysis steps, including the use of PacBio/SMRT sequencing technology to monitor AAV sequence enrichments throughout the course of selection. Additionally, we managed to increase the stringency of the PCR rescue of cap genes, by incorporating sample-specific barcodes, i.e., short, unique nucleotide stretches, into the AAV library genomes. By using these barcodes as a primer during sample recovery, we could isolate single libraries out of a complex library mixture, as validated in vitro. In the future, this original strategy could be exploited to track individual libraries in vivo upon injection of a mixture of libraries, which should in turn help to accelerate the identification of top-performing variants for validation studies. In summary, different steps along the powerful methodology of DNA family shuffling were improved advancing future vector development and the lingering concern about AAP impairment upon shuffling was dispersed.
Mitotic phosphatases play crucial roles in anaphase regulation and mitotic exit by annulling the kinase-mediated protein phosphorylation. In budding yeast Saccharomyces cerevisiae, Cdc14 (cell division cycle 14) phosphatase antagonizes key Cdk1 (cyclin-dependent kinase 1) functions to drive cells out of mitosis. Despite the presence of highly conserved catalytic domains, human CDC14s are dispensable for cell cycle progression. Nevertheless, to decipher the molecular mechanisms of human CDC14s functions, we have investigated the knockout cellular models aided by various imaging and proteomics approaches. Phenotypic analyses of the generated hCDC14A knockout in human retinal pigment epithelium (hTERT-RPE1) cells have exhibited the occurrence of longer primary cilia upon serum starvation. The intermediate longer cilia in haploid-insufficient cells, as well as the extended cilia observed upon siRNA-mediated acute depletion of hCDC14A, have further confirmed the phenotype. Primary cilia are microtubule-based structures that control various aspects of growth and development through sensing extracellular signals. Defects in this regulation lead to a host of pathological conditions collectively known as ciliopathies. Indirect immunofluorescence and electron microscopy have revealed that the disassembly pathways, as well as the crucial structures like axoneme and basal body, were intact in the elongated cilia. Inducible expression of hCDC14A has indicated its presence in the proximal end and subdistal appendage of the basal body. Global phosphoproteome along with proximity-based interaction proteomics approaches under conditions that favor ciliation have identified substrates for hCDC14A. Some of the identified substrates are involved in actin cytoskeleton reorganization with a function in cilia length control. The actin bundling protein drebrin (DBN1) was one of the identified hCDC14A substrates that have recently been reported to be associated with ciliogenesis. We show that the counteracting phospho-regulation of DBN1 at serine residue 142 by the proline directed kinase CDK5 and hCDC14A phosphatase regulates cilia length. However, significantly longer cilia in hCDC14A knockout cells than those from DBN1 knockout cells indicate that there are more substrates for hCDC14A that might be involved in ciliogenesis. The longer cilia phenotype in hCDC14A knockout cells could be explained by the enhanced recycling endosomes (transferrin) as well as increased ciliary vesicle docking (smoothened) in the pericentrosomal areas.
The reliable statistical inference of epigenetic regulatory networks that govern mammalian cell fates is very challenging. In this thesis we study this question for the differentiation decisions of T-helper (Th) cells, which have recently been shown to adopt a continuum of differentiated states in response to cytokine signals. To infer the underlying regulatory networks we introduce a novel framework for the inference of epigenetic regulatory network topologies based on statistical learning. First, we infer, via a Hidden Markov Model, chromatin states based on histone modification patterns in naïve Th cells and differentiated Th1, Th2 and mixed Th1/2 states; these states are controlled by external cytokine stimuli and the gene dose of the Th1 master transcription factor Tbet (Tbx21). We then introduce a linear multivariate correlation measure for mapping enhancers to their target genes, which is parametrized on a training set of known enhancers. This analysis is refined further by the application of partial correlations to distinguish direct from indirect effects. Applying this approach to our data, we recover known enhancers and obtain a genomewide enhancer-gene mapping. We also extend this to the correlation of repressive regulatory elements with gene expression. Next, we focus on the enhancers that regulate differentially expressed Th1 and Th2 specific transcripts. Building machine learning based predictors, we identify Th1 and Th2 specific enhancer and repressive state classes characterized by their response patterns to cytokine stimuli and Tbet dose. In turn, we use chromatin immunoprecipitation data of transcription factors to define the transcriptional regulatory logic governing the activities of the enhancer classes. Finally, we combine enhancer-target gene maps and enhancer regulatory logic as well as inhibitory elements to infer a bipartite epigenetic network. The network architecture builds on enhancer and repressive state classes as well as on genes and transcription factors leading to a weighted multidigraph. The network topology reveals distinct community structures related to Th1, Th2 and hybrid functionality. We furthermore analyse multiplex networks resulting in condition-specific topologies. From these analyses we obtain unique contributions of distinct network nodes. Utilizing random walks on multidigraphs we extract metastable processes underlying the observed system. In conclusion we present a robust quantitative framework for mapping chromatin states to gene activity, and, by factoring in transcription factor regulation of enhancers, inferring epigenetic regulatory networks. This methodology is applicable to a wide range of systems.
DNA mutation, epigenetic alteration, and gene expression are three major molecular components that distinguish cancer from normal cells. Although it is widely accepted that epigenetic modifications can greatly affect the expression of the target genes, because of the complex combinations of epigenetic marks, together with the interactions between multiple non-coding regulatory elements, measuring the epigenetic effects on gene expression is not an easy task. Nevertheless, it is estimated that epigenetic modifications have a greater effect than DNA mutations on tumorigenesis. In addition, epigenetic alterations are the initiating factor in some chromosome abnormalities and aberrant gene expression, making the study of epigenetic alterations a central aspect in understanding the underlying mechanisms in cancer and cell development. The aim of this thesis is to conduct qualitative and quantitative analyses of differential epigenetic modifications. To this end, a variety of existing approaches were applied in the ChIP-Seq analyses of six histone marks on glioblastoma data from four distinct subtypes. The results depict a comprehensive landscape of active and poised regulatory elements specific to glioblastoma subtypes, which describes the different aspects of tumor progression. However, the descriptive model of multiple histone marks (ChromHMM and peak calls) was also shown to be prone to various biases and artifacts. Moreover, some models also neglect the quantitative information of ChIP-Seq data, making it inadequate in addressing the magnitude of changes between epigenetic modification and gene expression levels. Therefore, in the second part of my work, I designed an integrative, network-based approach, in which I integrated two levels of epigenetic information: the signal intensities of each epigenetic mark, and the relationships between promoters and distal regulatory elements known as enhancers. Applying this approach to a variety of test cases, it predicts a number of candidate genes with significant epigenetic alterations, and comprehensive benchmarking validated these findings in cancer and cell development. In summary, as increasing amounts of epigenetic data become available, the computational approaches employed in this study would be highly relevant in both comparative and integrative analysis of the epigenetic landscape. The discovery of novel epigenetic targets in cancers, not only unfolds the fundamental mechanisms in tumorigenesis and development, but also serves as an emerging resource for molecular diagnosis and treatment.
Pluripotent stem cells are considered a prime source of cells for regenerative therapies and gene therapy applications because of their extensive proliferation, the potential for self-renewal and their capability for multi-lineage differentiation. A great advantage of induced pluripotent stem cells (iPSCs) is their derivation from a patient’s somatic cells, which can be isolated using non-invasive techniques, thus eliminating not only ethical concerns associated with embryonic stem cells but also the risk of immune rejection. Therefore, iPSCs are an attractive tool for personalised medicine, drug screening and to generate disease models. Typically, the modification of pluripotent cells is done by using integrating viral vectors. Although vectors based on modified viruses are unquestionably the most effective gene delivery systems in use today, their efficacy at gene transfer is, however, tempered by their potential integration and genotoxicity. Non-viral DNA vectors are attractive alternatives to viral gene delivery systems because of their low toxicity, relatively easy production and great versatility. However, their efficiency is still regarded as below the requirements for realistic in vivo gene therapy due to deficient delivery exacerbated by the merely transient gene expression of plasmid DNA in vivo.
Thus, the development of safer, more efficient and easily and economically prepared persistently expressing genetic vectors remains one of the main strategic tasks of gene therapy research and is the crucial prerequisite for its successful clinical application. An ideal vector for the genetic modification of cells should deliver sustainable therapeutic levels of gene expression without compromising the viability of the host in any way. Permanently maintained, episomal and autonomously replicating DNA vectors, which comprise entirely human elements, might provide the most suitable method for achieving these goals.
This thesis presents the development of a non-viral, non-integrating and autonomously replicating DNA vector system based on the use of a Scaffold Matrix Associated Region (S/MAR), for the persistent genetic modification of differentiating and dividing cells, including but not limited to murine and human Stem Cells (SCs). Although this DNA Vector is among the best of its class, one of its limitations is that as it is produced in bacteria it comprises a large proportion of bacterial sequences which are unnecessary and undesirable for clinical application. Accordingly, the vector system has been refined, updated and all aspects of its functionality have been improved whilst also reducing its impact on cells following its delivery, resulting in higher levels of more sustained expression than previous versions. Molecular and genetic analysis of S/MAR-labelled cells revealed that the vectors are kept at low copy numbers, are present in their episomal forms and do not modify or genetically damage the cells or their progeny, as the cells fully retain their pluripotent capabilities and are able to generate chimeric mice. This new vector system is also used to generate iPSCs from murine or patient-derived fibroblasts.
For the first time, this work shows that genetic modification with this DNA vector system provides robust transgene expression which is sustained through the reprogramming and differentiation process in vitro and in vivo.
In eukaryotic cells, DNA transcription, replication and repair events are controlled by the regulation of DNA compaction mechanisms that determine the open and closed chromatin states. Nucleosomes are the basic DNA packaging units of chromatin. The nucleosome core (NC) consists of a core histone protein octamer with approximately two tight superhelical turns of DNA wrapped around it. The NC is extended at its entry and exit points by linker DNA (L-DNA) and a linker histone (LH) protein binds between the two L-DNA arms to form a chromatosome. The dyad is the single DNA base pair between the nucleosome entry and exit points determining the symmetry axis and is used to define the position of LH binding to a nucleosome. For LH - nucleosome binding, previous studies indicate both on- and off-dyad binding modes, as well as different LH orientations. Thus, the molecular determinants of the structure of LH – nucleosome complex and the dynamics of LH – nucleosome binding are not fully understood. The aim of the research described here was to obtain an atomic-detail level understanding of chromatosome formation. Analysis of the experimentally determined structures of LH – nucleosome complexes showed that instead of a single 3D structure, an ensemble of structures of LH – nucleosome complexes exists. To understand the distribution of these ensembles, normal mode analysis (NMA), standard and accelerated molecular dynamics (MD & AMD) and Brownian dynamics (BD) simulations were applied to LH, nucleosome and chromatosome systems. MD and AMD simulations showed that the globular domain of the LH (LH GD) prefers to be in its closed form in solution. Upon nucleosome binding, the LH GD structure transformed to an open structure due to hydrophobic interactions with the L-DNA of the nucleosome. Additionally, LH GD binding constrained the flexibility of the L-DNA and affected the directions of movement of the L-DNA arms. BD simulations indicated that various chromatosome configurations were possible depending on LH GD sequence and L-DNA opening angles. These findings suggest that LH – nucleosome binding is mediated by a combination of conformational selection and induced fit mechanisms. Further BD simulations show that chromatosome configurations were affected by single point mutations in the LH GD and varied for different LH isoforms. My results indicate that by making specific single point mutation exchanges, it is possible to swap LH – nucleosome configurations among different LH GD isoforms. Similar shifts were observed in chromatosome configuration upon introduction of post translational modifications (PTMs) in the LH GD. I applied BD simulations to compute dissociation rate constant (koff) values and compare them with previously reported fluorescence recovery after photobleaching (FRAP) data on the binding of various LH mutants to chromatin. The results of the BD simulations correspond with the relative trends in measured FRAP recovery half-times (t50) of LH – chromatin binding of various LH mutants. The results thus enable the interpretation of the FRAP data in terms of a physical model of LH – nucleosome binding. My thesis provides detailed insights into the structure, dynamics and kinetics of chromatosome formation in eukaryotes. The results presented in this work can guide further experiments on the sequence determinants of LH – nucleosome binding.
The Epstein-Barr virus (EBV) infects the majority of the population. The EBV M81 strain isolated from a nasopharyngeal carcinoma (NPC) efficiently infects and transforms primary B cells, but it also induces potent virus lytic replication in a minority of these cells. We used recombinant viruses to reveal the function of the EBER RNAs. We found that the number of cells in which lytic replication takes place is increased both in vitro and in vivo by the non-coding RNA EBER2, but not by its homolog in the genome of the B95-8 strain. M81 and B95-8 EBER2 homologs displayed a limited number of polymorphisms, some of which influence their half-life and expression levels. M81 EBER2 modified the expression of a large number of cellular genes including CXCL8. This chemokine was able to compensate the absence of EBER2, suggesting that it represents the main target of this non-coding RNA. We found that the exosomal fraction of B cells infected with wild type M81 carries the EBER molecules, are able to increase CXCL8 and BZLF1 production. The effect of EBER2 on EBV lytic replication required a functional TLR7, a sensor of viral single- stranded RNA (ssRNA). Therefore, we propose a model in which EBERs are vehicled into the exosomal fraction of infected B cells to initiate lytic replication in a paracrine manner through CXCL8 secretion induced by TLR7 stimulation. These results indicate that EBERs NPC-derived virus variant contribute to lytic replication in B cells and activate production of a chemokine involved in carcinogenesis.
The development of advanced discretization methods for the radiation transport equation is of fundamental importance, since the numerical effort of modeling increasingly complex multidimensional problems with increasing accuracy is extremely challenging. Different expressions of this equation arise in several science fields, from nuclear fission and fusion to astrophysics, climatology and combustion.
Mathematically, the radiation intensity is usually a rapidly changing function, causing a considerable loss in accuracy for many discretization methods. Depending on the coefficient ranges, the equation behaves like totally different equation types, making it very difficult to find a discretization method that is efficient in all regimes. Computationally, the huge amount of unknowns involved demands not only extremely powerful computers, but also efficient numerical methods and optimized implementations. Today, solvers covering all the coefficient ranges and still being robust in the diffusion dominated case are very scarce.
In the last 20 years, Discontinous Galerkin (DG) methods have been studied for the monoenergetic problem, unsuccessfully, due to lack of stability for diffusion-dominated cases. Recently, new mathematical developments have fully explained the instability and provided a remedy by using a numerical flux depending on the scattering cross section and the mesh size. The new formulation has proven to be stable and allows the application of multigrid, matrix-free methods, reducing the memory needed for such an amount of unknowns.
We use these numerical methods to address the solution of a energy dependent problem with a multigroup approach. We study the diffusion approximation to the transport problem, obtaining convergence proofs for the symmetric scattering case and advances in the nonsymmetric case, using field of values analysis.
For the full transport case, we discretize by means of an asymptotic preserving, weakly penalized discontinuous Galerkin method that we solve with a multigrid preconditioned GMRES solver, using nonoverlapping Schwarz smoothers for the energy and direction dependent radiative transfer problem.
To address the local thermodynamic equilibrium (LTE) constraint, we use a nonlinear additive Schwarz method to precondition the Newton solver. By solving full local radiative transfer problems for each grid cell, performed in parallel on a matrix-free implementation, we achieve a method capable to address large scale calculations arising from applications such as astrophysics, atmospheric radiation calculations and nuclear applications.
To the best of our knowledge, this is the first time this preconditioner combination has been used in LTE radiation transport and in several tests we show the robustness of the approach for different mesh sizes, cross sections, energy distributions and anisotropic regimes, both in the linear and nonlinear cases.
Environmental systems are nonlinear, multiscale and non-separable. Mathematical models describing these systems are typically high-dimensional and always have missing physics. Therefore, determining the system’s state and its future development relies on in situ observations. Information from models and observations are combined using data assimilation methods, which are mainly developed for divergent systems as they arise from weather prediction. Applying them also to convergent systems requires modifications of these methods. I investigated the differences of data assimilation in convergent and divergent systems and found that parameter estimation is essential in convergent systems. In this work, I enhanced the particle filter, an ensemble-based data assimilation method. In contrast to other methods, the particle filter is able to handle nonlinear systems and to describe the resulting non-Gaussian probability density functions. However, for parameter estimation modifications of the resampling, i.e. the renewal of the ensemble, are necessary. I developed a resampling method that uses the weighted covariance information calculated from the ensemble to generate new particles. This method correlates observed with unobserved dimensions and can effectively estimate state and parameters in a convergent system. To be applicable in high-dimensional systems, particle filters need localisation. The introduced resampling allows localisation, which further increases the efficiency of the filter.
This thesis aims at the investigation and development of the control of waste heat recovery systems (WHR) for heavy duty trucks based on the organic Rankine cycle. It is desired to control these systems in real time so that they recover as much energy as possible, but this is no trivial task since their highly nonlinear dynamics are strongly affected by external inputs (disturbances). Additionally, nonlinear operational constraints must be satisfied. To deal with this problem, in this thesis a dynamic model of a WHR that is based on first principles and empirical relationships from thermodynamics and heat transfer is formulated. This model corresponds to a DAE of index 1. In view of the requirements of the employed numerical methods, it includes a spline-based evaluation method for the thermophysical properties needed to evaluate the model. Therewith, the continuous differentiability of the state trajectories with respect to controls and states on its domain of evaluation is achieved. Next, an optimal control problem (OCP) for a fixed time horizon is formulated. From the OCP, a nonlinear model-predictive control (NMPC) scheme is formulated as well. Since NMPC corresponds to a state feedback strategy, a state estimator is also formulated in the form of a moving horizon estimation (MHE) scheme. In this thesis, we make use of efficient numerical methods based on the direct multiple shooting (DMS) method for optimal control, backward differentiation formulae for the solution of initial value problems for DAE, and the corresponding versions of the real-time iteration (RTI) scheme in order to approximately solve the OCP and implement the MHE and NMPC schemes. The simultaneous implementation of NMPC and MHE schemes based on RTI has been already proven to be stable in the control literature.
Several numerical instances of the DMS method for the proposed OCP, NMPC and MHE schemes are tested assuming a given real-world operation scenario consisting of truck exhaust gas data recorded during a real trip. These data have been kindly provided by our industry cooperation partner Daimler AG. Additionally, the PI and LQGI control strategies, of wide-spread use in the literature of control of WHR, are also considered for comparison with the proposed scheme. An important result of this thesis is that, considering the highest energy recovery obtained from both strategies as a reference for the given operation scenario, the proposed NMPC scheme is able to reach an additional energy generation of around 3% when the full state vector is assumed to be known, and its computational speed allows it to update the control function in times shorter than the considered sampling time of 100 [ms], which makes it a suitable candidate for real-time implementation. In a more realistic scenario in which the state has to be estimated from noisy measurements, a combination of both aforementioned NMPC and MHE schemes yields an additional energy generation of around 2%.
Concretely, this thesis presents novel results and advances in the following areas:
• A first principles DAE model of the WHR is presented. The model is derived from the energy and mass conservation considerations and empirical heat transfer relationships; and features a tailored evaluation method of thermophysical properties with which it possesses the property of being at least continuously differentiable with respect to its controls and states on its whole domain of evaluation.
• A new real-time optimization control strategy for the WHR is developed. It consists of an NMPC strategy based on efficient simulation, optimization and control tools developed in previous works. The scheme is able to explicitly handle nonlinear constraints on controls and states. In contrast to other NMPC instances for the WHR found in the literature, our scheme's efficient numerical treatment make it real-time feasible even if the full nonlinear WHR dynamics are considered.
• To the author's knowledge, this is the first implementation that considers both the NMPC and the MHE approaches used simultaneously in the control of the WHR. The combination of NMPC and MHE produces a closed-loop, model-based implementation that can treat realistic measurements as inputs and calculates the corresponding control functions as outputs.
Die Chemie des Bors wird fast ausschließlich von seinem elektrophilen Charakter geprägt. Daher lassen sich viele der seit Jahrzehnten etablierten Hydroborierungs- und Borylierungs-Reaktionen lediglich auf ungesättigte organische Substrate anwenden. Eine der größten Herausforderungen der Bor-Chemie ist deshalb die Darstellung von nukleophilen Bor-Reagenzien. Trotz großer Fortschritte der letzten Jahre ist die Anzahl der isolierbaren nukleophilen Bor-Verbindungen überschaubar. Durch die starke Lewis-Basizität bizyklischer Guanidinate wird ein außerordentlich hoher Elektronenreichtum der Diborane erreicht. Zusätzlich wird durch den verbrückenden Bindungsmodus der Schritt der Dehydrokupplung über eine Vororientierung der Boratome begünstigt. Das doppelt guanidinatstabilisierte Diboran(4) verfügt über eine einzigartige Reaktivität, die durch den nukleophilen Charakter der B–B-Bindung bestimmt wird. In der vorliegenden Arbeit gelang durch Einführung neuer Substituenten die Isolierung und Charakterisierung einer Reihe von nukleophilen Diboran(4)-Verbindungen. Bei diesen und bereits literaturbekannten Diboranen wurde der Einfluss der Substituenten auf die Eigenschaften der B–B-Bindung auf Basis experimenteller und theoretischer Methoden systematisch untersucht. Dabei konnte gezeigt werden, dass eine hohe Nukleophilie des Moleküls nicht zwingend eine nukleophile B–B-Bindung zur Folge hat. Die Substituenten der beiden Diborane [HB(μ-hpp)]2 und [nBuB(μ-hpp)]2 verfügen über keinen +M-Effekt und weisen nach eingehender Analyse quantenchemisch berechneter Parameter ausschließlich die B–B-Bindung als nukleophile Position auf. Im Gegensatz dazu wird die Nukleophilie von [(Me2N)B(μ-hpp)]2 primär von den freien Elektronenpaaren der Amin-Gruppen geprägt. Weiterhin wurden mehrere Synthesemöglichkeiten asymmetrischer Diboran(4)-Verbindungen untersucht. Dabei erwies sich die Darstellung von [(PhCC)B(μ-hpp)2BH] über das phosphoniumstabilisierte Diboranyl-Kation [HB(μ-hpp)2B(PCy3)]+ als die beste Methode. Zusätzlich konnte durch die Darstellung von [(PhCC)B(μ-hpp)2B(PCy3)]+ die Bildung des intermediären Diboranyl-Kations [(PhCC)B(μ-hpp)2B]+ bestätigt werden. Oxidationsexperimente an [HB(μ-hpp)]2 führten zum Dimerisierungsprodukt [H4B4(μ-hpp)4]2+. Als Mechanismus zur Bildung wurde eine Einelektronen-Oxidation der B–B-Bindung mit Umlagerung zu einem Bor-zentrierten Radikal-Kation postuliert, welches anschließend über Radikalkupplung dimerisiert. Das intermediär auftretende Radikal-Kation [HB(μ-H)(μ-hpp)2B]•+ konnte mit TEMPO abgefangen werden. Die gewonnen Erkenntnisse gewähren ein grundlegendes Verständnis des nukleophilen Charakters von B–B-Bindungen. Dadurch wird die Basis für weitere Folgeexperimente geschaffen, mit denen der nukleophile Charakter der Diborane(4) weiter ausgebaut und für breitere Anwendungsgebiete geöffnet werden kann.
This thesis presents several experiments investigating the regimes of behaviour of a proto-typical open quantum many-body system far away from equilibrium. The experimental platform is based on ultracold atoms laser-excited to Rydberg states, which we engineer to emulate a strongly interacting, driven-dissipative quantum spin system. The high degree of control over the relevant microscopic processes and their parameters, as well as the ability to widely tune interaction and driving strengths provides the means to address fundamental questions on how different regimes of dynamical behaviour emerge in complex open quantum systems and how they can be characterised.
In the first part, we discuss how control over the relevant properties of Rydberg spin systems, like temperature, density, long evolution times, strong interactions, tunable driving, and dissipation can be achieved. We introduce the new experimental apparatus developed during this thesis, which combines both single-photon and two-photon Rydberg excitation schemes with high atom densities and long excitation times using an optical dipole trap. Additionally, we investigate theoretically how long coherence times exceeding motional timescales combined with strong many-body interactions can be achieved in such a system. We identify an optimal parameter regime for two-photon excitation where such conditions can be generated with coherence times and interaction strengths comparable to what is achievable for single-photon excitation.
In the second part we explore the macroscopic non-equilibrium behaviour of our system and devise suitable observables for characterising different regimes of behaviour. We discover that the slow atom loss inherent to our system provides a convenient observable for the many-body state of the system. Focussing on evolution times where the effect of the atom loss on the evolution is small, we discover that the rate of atom loss exhibits powerlaw scaling with the driving strength over several orders of magnitude. The measured scaling exponents reveal the non-equilibrium phase structure of the many-body system and allow us to distinguish dissipation-dominated, paramagnetic and critical regimes, as well as an instability. In the observed critical regime, collectively enhanced driving dominates over dissipation, leading to scaling associated to the critical point of the non-dissipative equilibrium Rydberg spin system. Based on the known microscopic processes of our system, we perform classical many-body rate equation simulations, which agree well with the observed phase structure as well as the position and associated scaling exponents of the individual regimes. These findings open up new means to study and classify out of equilibrium systems based on slow particle loss and powerlaw scaling.
In the last part of this thesis we reveal a second type of criticality in our system, which is genuinely non-equilibrium in character and arises from an absorbing state phase transition. The critical state of this phase transition dominates the system evolution at late times where particle loss is no longer negligible. We identify self-organisation as the mechanism driving the system to this critical state, which we observe through powerlaw scaling of the non-equilibrium steady-state. These experiments establish Rydberg atoms as a well controlled platform for implementing and exploring models of absorbing state phase transitions and self-organised criticality with unprecedented access to the underlying microscopic properties of the system.
The sensitivity of intensity-modulated proton therapy to uncertainties requires case-specific uncertainty assessment and mitigation. As an alternative to scenario-based methods, this thesis describes the implementation, application and conceptual extension of the Analytical Probabilistic Modeling (APM) framework introduced by Bangert, Hennig, and Oelfke (2013). APM represents moments of the probability distribution over dose in closed-form, providing a probabilistic analog to nominal pencil-beam dose calculation subject to range and setup uncertainties that further enables probabilistic optimization. First, APM was implemented in MITKrad, a treatment planning plugin for MITK built completely from scratch. APM’s computations were validated against sample statistics, showing nearly perfect agreement. Run-times within minutes could be realized for uncertainty assessment and probabilistic optimization on patient data. Reformulation of APM enabled linear separation of the computations into random and systematic uncertainty components. Uncertainty over the full fractionation spectrum could then be modeled and optimized with a single pre-computation. It could be shown that fractionation is exploited in optimization with APM for additional organ at risk sparing. APM was then extended to propagation of uncertainties from dose to clinically relevant plan quality metrics. Expectation and variance could be modeled accurately for organ mean dose and dose-volume histograms. However, approximations for equivalent uniform dose and minimum and maximum dose values did not provide reliable results. Finally, the closed-form plan metrics were used to conceptualize constrained probabilistic optimization. Besides novel probabilistic objectives, confidence constraints could be established. Due to increased computational complexity of the new models, the proof-of-concept was provided through evaluations on a one-dimensional prototype anatomy. In conclusion, the herein extended APM framework is able to provide probabilistic analogs to established nominal concepts of dose calculation, plan quality metrics, and constrained optimization. If computational hurdles can be overcome in the future, clinical application would be within reach.
The identification of novel tumor-specific ligands as transport vehicles for both diagnostic and therapeutic radionuclides represents a major objective in nuclear medical oncology research. The use of selective compounds with high affinity to their target structure results in an increased tumor uptake of the radionuclide, while minimizing undesired radiation exposure of healthy tissue. Based on increasing knowledge about tumor physiology as well as ongoing technological progress, numerous tumor-targeting compounds have been identified and transferred into clinical practice. However, the majority of these compounds comprises antibodies, which feature serious deficiencies, such as poor membrane permeability and tissue penetration, slow clearance and immunogenicity. Peptides, instead, represent a favorable alternative with regard to size, pharmacokinetics and the possibility to be produced cost-effectively in large quantities by automated solid phase synthesis.
Phage display technology represents a powerful tool for the de novo identification of target-specific peptides. This technique is based on the presentation of large polypeptide libraries on the surface of bacteriophages. To this end, the phage genome is manipulated to display the foreign peptides, which are physically linked to their encoding nucleic acids. Following several iterative rounds of incubation with any target protein of interest, the enriched target-specific ligands can therefore be easily identified by DNA sequencing.
Tumor growth and malignancy are not only affected by the characteristics of cancer cells but strongly depend on the attributes of several different cell types within the local tumor microenvironment. Cancer-associated fibroblasts (CAFs) represent an important subpopulation of these stromal cells and are known to promote tumor growth, inflammation and metastasis. They account for a large part of the tumor mass and are genetically more stable and therefore less susceptible to the development of therapy resistance than cancer cells per se. In contrast to normal fibroblasts, CAFs entail a number of tumor-specific marker proteins such as the integral membrane peptidase Fibroblast Activation Protein (FAP). This protein is highly expressed in the microenvironment of more than 90% of epithelial tumors including breast, lung and pancreatic carcinoma. Overexpression of FAP is associated with a poor prognosis in a wide range of different cancers. Due to very low expression levels in normal tissues, FAP represents an attractive molecular target for the development of tumor-specific diagnostics and therapies.
To identify novel FAP-specific peptides using phage display, two combinatorial libraries based on the scaffold structures Sunflower Trypsin Inhibitor-1 (SFTI-1) and Min-23 were built. They comprise a cyclic, disulfide-bridged backbone and a variable sequence consisting of six to ten random amino acids. After incubation of the libraries with the target protein in four rounds of biopanning, the FAP-specific ligands were isolated and their sequence determined using next-generation sequencing. A selection of suitable peptides was synthesized by solid phase synthesis, purified and characterized in different cell-based radioligand assays.
In contrast to the SFTI-1 based biopanning, which did not lead to any enrichment of FAP-specific peptides, the use of the Min-23 library was successful in identifying several FAP-targeting ligands. However, these peptides showed a very low affinity to their target protein, which most likely results from a rapid degradation. It is conceivable that the enzymatic function of the membrane protein effectuates a rapid hydrolysation of the small peptides upon initial binding. A solution to this is the use of an alternative scaffold, which impedes enzymatic cleavage or ensures cell internalization after hydrolysation.
In this context, the cyclotide-based FAP-specific miniprotein MC-FA-012, identified by BionTech AG, was characterized regarding target affinity, specificity and pharmacokinetic profile in radioligand binding assays as well as in FAP-positive tumor-bearing mice. In contrast to the Min-23-based peptides, MC-FA-012 and its trimerized version DOTA-(MC-FA-012)3 demonstrated high affinity to human FAP, while no substantial binding to the structurally related protein CD26 was observed. In addition, DOTA-(MC-FA-012)3 rapidly accumulated in FAP-positive tumor xenografts in vivo and showed negligibly low unspecific binding in healthy tissue, except for the kidneys. A first clinical analysis of the radiolabeled compound in patients with metastatic pancreatic carcinoma revealed a robust accumulation of the tracer in the primary tumor as well as in lymph node and bone metastases. In contrast, tracer uptake into normal tissue was very low. The radioactivity was cleared rapidly from the blood stream and excreted predominantly via the kidneys, resulting in high contrast PET images.
Since the high accumulation of DOTA-(MC-FA-012)3 in the kidneys could not be sufficiently reduced by different competition strategies, further development of the radiotracer regarding a potential therapeutic application was not pursued. The investigation of the structure-activity relations of MC-FA-012 revealed that both the scaffold and certain functional groups within the FAP-specific binding sequence account for high FAP affinity. Introducing even minor changes in the molecule's conformation resulted in an almost complete loss of binding.
Consequently, a different approach towards the development of a FAP-specific compound was adopted by designing the small molecules FAPI-01 to FAPI-15 based on a potent FAP inhibitor. Each of the compounds demonstrated high specific binding to human and murine FAP and internalized rapidly into FAP-expressing cells. Imaging and biodistribution studies in xenotransplanted mice proved a rapid tumor uptake of the tracers in a genetically modified FAP-overexpressing tumor model as well as in human FAP-negative tumors due to endogenous murine FAP expression. Notably, the radiotracers demonstrated rapid renal clearance without substantial binding to non-cancerous tissue. Due to their advantageous pharmacokinetics and an excellent stability in human serum, the compounds FAPI-02 and FAPI-04 were selected for further clinical investigation. Using PET/CT, biodistribution of the radiotracers was analyzed in patients with metastasized epithelial cancers, including breast, lung, pancreatic and colon carcinoma as well as in high-grade glioblastomas. Both tracers rapidly accumulated in the primary tumor as well as in soft tissue, lymph node and bone metastases. In contrast, tracer uptake into normal tissue was very low, resulting in favorable tumor-to-organ ratios and high contrast images.
Comparative imaging in one patient with locally advanced lung adenocarcinoma revealed an obvious advantage of FAPI-02 compared to the commonly used PET tracer 18F-FDG. FAPI-02 showed a higher tumor uptake and significantly lower activities in blood and liver, resulting in higher image contrast with better visibility of metastatic lesions. In contrast to FDG, which highly accumulates in cells with high glucose consumption, e.g. the brain or inflammatory tissue, FAPI-02 selectively targets FAP-expressing carcinomatous lesions. This opens up new perspectives for the precise diagnosis of malignant lesions in tissues with high metabolic activity, such as liver or brain.
Furthermore, various approaches for a potential therapeutic application of the FAP-ligands are currently being investigated, including different radionuclides and alternative effector molecules, such as chemotherapeutics or immunomodulators. Given the possibility to use either diagnostic or therapeutic nuclides with the same molecule, FAPI-02 allows simple stratification of patient cohorts likely to benefit from a therapeutic intervention.
The thesis is devoted to the analytical and numerical studies of high-order harmonic generation and super-intense single attosecond pulse emission via ultra-relativistic laser-plasma interaction. In the ultra-relativistic regime, the laser radiation pressure induces plasma ion motion through the so called hole-boring effect, resulting in frequency widening of the harmonic spectra. This widening, analyzed analytically and validated by particle-in-cell simulations, produces a quasi-continuous frequency spectrum, a prerequisite for generating an intense single attosecond pulse. Based on the results and physical considerations, parameter maps highlighting the optimum regions for generating a single intense attosecond pulse and coherent XUV radiation are presented. Moreover, a robust plasma gating is developed to generate a super-intense phase-stabilized single attosecond pulse. The hole-boring effect limits the most efficient high-frequency emission in one laser cycle making it possible to isolate a single attosecond pulse. The generated pulse is characterized by a stabilized spectral phase ψ(ω) ≈ ±π/2 and an ultra-broad exponential spectrum up to keV region bounded by ROM scaling and CSE scaling. The unprecedented intensity highlights the potential of the isolated attosecond pulse for performing attosecond-pump attosecond-probe experiments.
Desinfektion ist im Bereich der Schwimmbadwasseraufbereitung obligatorisch, um aus Sicht des Infektionsschutzes die Sicherheit der Badegäste zu gewährleisten. Das am häufigsten eingesetzte Desinfektionsmittel ist freies Chlor (FAC), das aufgrund seiner hohen Reaktivität auch mit Verunreinigungen in verschiedener Form reagieren kann. Dabei entsteht eine Vielzahl an sogenannten Desinfektionsnebenprodukten (DNP). Bisherige Forschungen haben sich mit der Identifizierung der unterschiedlichen DNP, deren Entstehung bei Verwendung verschiedener Aufbereitungstechnologien, den absoluten Konzentrationen im Wasser und in der Luft sowie den Auswirkungen auf den Menschen befasst. Dementgegen gibt es zu der Chemie hinter der Entstehung kaum Untersuchungen. Die vorliegende Arbeit ist die erste umfassende und systematische Studie, die die Entstehung von DNP aus verschiedenen Quellen und darüber hinaus die konkrete Kinetik auf molekularer Ebene untersucht. Zunächst wurde der Eintrag von Verunreinigungen durch Badegäste bestimmt, der im Schnitt 230 mg gelösten organischen Kohlenstoff (DOC), 40 mg partikulären organischen Kohlenstoff (PaOC), 1010 Bakterien (KBE) und 240 mL Urin pro Person beträgt. In weiteren Untersuchungen wurde das Trihalogenmethan-Bildungspotential aus den o. g. Verunreinigungen zu 100 mg Chloroform pro Person bestimmt, das zu 63 % Urin und zu 35 % Verunreinigungen von der Haut zugeordnet werden konnte. Anschließend wurden die Kinetiken von 13 potentiellen Vorläufer-Verbindungen sowie von Gemischen untersucht und Zitronensäure als wichtigster Vorläufer für die Bildung von Chloroform identifiziert. Die Ergebnisse zeigten zudem, dass die Chloroform-Bildung aus Zitronensäure ein verhältnismäßig langsamer Prozess ist (Ausbeute < 60 % nach vier Tagen). Diese Ergebnisse, insbesondere die Kinetiken, wurden im Rahmen des EU-Projekts „Intellipool“ in ein Modell zur Vorhersage von DNP-Konzentrationen in Schwimmbädern implementiert, das bereits zur Optimierung der Technik von Schwimmbadanlagen genutzt wird.
We use a novel kinetic field theory approach to investigate structure formation [1] in vastly different classical systems ranging from cosmic large-scale structures to many-body systems of Rydberg atoms or spins. The interaction laws governing the dynamics of these systems greatly differ from one another.
In the application of the formalism to the formation of cosmic large-scale structures, we address the question whether the shape of the gravitational potential has any influence on the shape of the non-linear density-fluctuation power spectrum on small scales. Since the non-linear power spectrum is a convolution of the NFW halo density profile with the mass function according to the halo model, we are interested in finding out whether the NFW profile depends on the potential shape. However, we find that the balance between the attractive force due to particle interactions and the damping due to momentum-diffusion is very finely tuned and is broken easily when Newtonian gravity is replaced by any different power-law interaction potential. This prevents us from drawing definitive conclusions and requires further analysis.
The applications of the kinetic field theory formalism to classical laboratory systems such as Rydberg gases and spin-system is a much less evolved field. We therefore present first results as a test of the applicability of KFT to such systems which, so far, are encouraging.
We combine a novel approach to cosmic structure formation called Kinetic Field Theory (KFT) with resummation methods to investigate the small-scale structure formation in dark and baryonic matter. For pure dark matter, we compute the first nonlinear corrections to the density contrast power spectrum and compare these to the results obtained in standard Eulerian perturbation theory (SPT). We find that we can precisely reproduce the SPT 1-loop result if we adopt the same expansion scheme as SPT. However, we also show that the natural expansion scheme of resummed KFT is actually fundamentally different and might thus be able to overcome the problems of SPT when trying to describe nonlinear structure formation. To describe the effect of baryons, we demonstrate how isothermal and adiabatic gas dynamics can be implemented consistently into resummed KFT and how a system of gravitationally coupled dark and baryonic matter can generally be treated in this framework. In the isothermal case, we further investigate the linear evolution of this coupled system in a cosmological setting. Our results show a suppression of the baryonic power spectrum on scales below approximately 1 Mpc/h which is qualitatively comparable to simulation results but underpredicts the effect by nearly an order of magnitude. Furthermore, simulations show an enhancement of the dark matter power spectrum absent in our results. We expect these deviations to decrease once adiabatic gas dynamics and nonlinear corrections are taken into account.
Polar Mesospheric Clouds (PMCs) are H2O ice clouds occurring at high latitudes in the summer mesopause of Earth. They have a counterpart in the mesosphere of Mars consisting of CO2 ice particles. Both types of clouds most likely form via heterogeneous nucleation on nanometer-sized meteoric smoke particles. However, the onset conditions for ice particle formation are only poorly known. Therefore, I investigated the microphysical formation process of these clouds in the laboratory. Experiments on adsorption, nucleation and growth processes of H2O and CO2 molecules on meteoric smoke analogue particles were performed using the MICE-TRAPS setup. For Earth, it was found that amorphous solid water is the primary phase which forms under the extreme temperatures of the summer mesopause. The vapor pressure of this ice phase was measured and is 2 to 4 times higher than previously assumed. Nevertheless, ice formation is activated at low supersaturation, which is described by a newly developed activation model. In contrast, CO2 ice formation on Mars initiates at high supersaturation, which is described using classical nucleation theory with the parameters determined in this work. The results presented in this dissertation significantly enhance the understanding of mesospheric ice cloud formation on Earth and Mars.
The ’rigorous coupled-wave analysis’ (RCWA) is advanced in performance and extended in its application range. First, the RCWA framework is adapted to the treatment of structured incident and transmitted regions in order to enable the exact modeling of axially extended objects such as integrated optical components, long waveguides or fibers without incurring unwanted back-reflections from distant interfaces to homogeneous regions. Furthermore, a method to determine the propagation direction of eigenmodes is derived. Second, the treatment of coherent bidirectional light incidence is introduced and applied to the simulation of sample-induced aberrations in 4π-microscopy. In this context, a consistent formulation to describe arbitrary polarization states of structured incident light sources is derived. Third, the ’fast rigorous iterative method’ (FRIM) is developed, an algorithm based on an iterative approach, which enables the rigorous simulation of structures such as certain diffractive optical elements with a significantly higher mode count than presently possible. This is achieved by replacing the computationally complex eigenmode decomposition inherent to standard modal methods by a sequence of efficient matrix multiplications. Thereby, the numerical cost is reduced from O
Nowadays, an increasing number of numerical modeling techniques, notably by means of the finite element method (FEM), are involved in the industrial design process and play a vital role in the area of the biomedical engineering. Particularly, the computational fluid dynamics (CFD) has become a promising tool for investigating the fluid behavior and has also been used to study the cardiovascular hemodynamics to predict the blood flow in the cardiovascular system over the recent decades.
However, simulating a fluid in rotational frames is not trivial, as the classical fluid calculation considers that the geometry of the fluid domain does not alter along the time. In the meanwhile, due to the high rotating speed and the complex geometry of the ventricular assist device (VAD), a turbulent flow must be developed inside the pump housing. The Navier-Stokes equations are not applicable in respect of our available computing resource, additional assumptions and approaches are often applied as a means to model the eddy formation and cope with numerical instabilities.
For many applications, there is still a big gap between the experimental data and the numerical results. Some of the discrepancies come especially from uncertain data which are used in the physical model, therefore, Uncertainty Quantification (UQ) comes into play. The Galerkin-based polynomial chaos expansion method delivers directly the mean and higher stochastic moments in a closed form. Due to the Galerkin projection’s properties, the spectral convergence is achieved.
This thesis is dedicated to developing an efficient model to simulate the blood pump assuming uncertain parametric input sources. In a first step, we develop the shear layer update approach built on the Shear-Slip Mesh Update Method (SSMUM), our proposition facilitates the update procedure in parallel computing by forcing the local vector to retain the same structure. In a second step, we focus on the Variational Multiscale method (VMS) in order to handle the numerical instability and approximate the turbulent behavior in the blood. As a consequence of utilizing the intrusive Polynomial Chaos formulation, a highly coupled system needs to be solved in an efficient manner. Accordingly, we take advantage of the Multilevel preconditioner to precondition our stochastic Galerkin system, in which the Mean-based preconditioner is prescribed to be the smoother. Besides, the mean block is preconditioned with the Schur-Complement method, which leads to an acceleration of the solution process. Hence, by developing and combining the proposed solvers and preconditioners, dealing with a large coupled stochastic fluid problem on a modern computer architecture is then feasible. Furthermore, based on the stochastic solutions obtained from the previous described system, we obtain valuable information about the blood flow accompanied with certain level of confidence, which is beneficial for designing a new blood-handle device or improving the current model.
In dieser Arbeit wurden detaillierte Untersuchungen zu generellen Aspekten der massenspektrometrischen Lipidanalytik durchgeführt. Dies umfasste das Ermitteln von für die Quantifizierung geeigneten Konzentrationsbereichen in komplexen Mischungen, die Untersuchung des Lipid-Ionisierungsverhaltens in Zwei-Komponenten-Mischungen, den Vergleich der Effizienz gängiger Lipidextraktionsverfahren und Untersuchungen zu Nicht- Lipid-Kontaminanten in RP-UPLC-MS-Analysen. Unter der Bedingung des Vorliegens einer direkten Proportionalität waren Quantifizierungen in einem Konzentrationsbereich von 1 nM bis 30 μM pro Lipidklasse möglich. Es wurden Hinweise für mögliche Abweichungen von der direkten Proportionalität abhängig von Flussrate gefunden. Die Untersuchungen der Zwei-Komponenten-Mischungen lieferten keine Hinweise auf eine intermolekulare Beeinflussung der Ionisierung. Die Verwendung von Glas im Vergleich zu Plastik lieferte keine signifikanten Unterschiede des Kontaminanten-Gehalts. In dieser Arbeit durchgeführte vergleichende Untersuchungen zur Spezifität von verschiedenen gemischten Full-MS/Fragmentierungsscans zeigten, dass Full-MS/AIF-Scans für erste Charakterisierungen von komplexen Proben geeignet sind und bei chemisch reinen Proben ohne Koelution von Spezies Ergebnisse qualitativ gleichwertig zu denen von targeted-MS/MS-Scans liefern. Die Software MassMap hat sich aufgrund der Möglichkeit einer benutzerfreundlichen Bewertung identifizierter Signale als geeignetes Software-Tool für die Auswertung von Datensätzen mit unterschiedlichen Scanmethoden erwiesen. Des Weiteren wurde eine neuartige Methode für die dGPL-Regioisomer-Analyse mit Hilfe von UPLC-targeted-MS/MS im Negativmodus etabliert. Hiermit konnten die Regioisomer-SICs mit Hilfe eines dedizierten Auswerte-Moduls der Software MassMap rekonstruiert und die Regioisomer-Anteile in Mischungen ermittelt werden. In dieser Arbeit wurden außerdem E. coli-Lipidomveränderungen in Abhängigkeit von der Wachstumszeit und die Wirkung der Roemerin-Behandlung auf E. coli K12 TB1 untersucht. PE-Spezies und PG-Spezies mit gleicher Fettsäurezusammensetzung zeigten ein paralleles Verhalten der Speziesanteiländerungen mit zunehmender Wachstumszeit. Innerhalb der exponentiellen Phase wurden fast keine signifikanten Speziesanteiländerungen gefunden, während zwischen der exponentiellen und der stationären Phase eine Vielzahl signifikanter Unterschiede gefunden wurde. Roemerin wies keinen rein bakteriostatischen Effekt auf E. coli auf. Bei der Behandlung von B. subtilis 168 mit Berberin, Boldin und Roemerin wurde nur für Roemerin eine antibakterielle Aktivität gefunden, die mit signifikanten metabolischen und morphologischen Veränderungen der Zellen einherging.
The aim of this thesis was to allow for the processing of large-scale atmospheric 14CO2 samples into graphite targets for high-precision analysis on an accelerator mass spectrometer (AMS) and to further develop the sampling itself, which takes place throughout Europe within the Integrated Carbon Observation System (ICOS) network. For the first part, a largely automated Extraction and Graphitisation Line (EGL) was developed at the ICOS Central Radiocarbon Laboratory(CRL); the construction at the institute workshop was guided and process behaviour and target quality was characterised. Process fractionation in δ13C from the whole-air sample flask to the graphite target cannot be distinguished from zero with (0.04 ± 0.09)‰. The deviation from the absolute canonical Δ14C scale was determined to (0.7 ± 0.5)‰. It was shown that the reproducibility of Δ14C results from processed air samples is at ±1.9‰ or below for the final graphitisation parameters. Compatibility tests provided a deviation of the results of samples processed with EGL and analysed by the AMS at the Curt-Engelhorn-Centre Archaeometry (CEZ) from the results of the CRL Low-Level Counting (LLC) laboratory of (2.2 ± 0.9)‰. The reason for the deviation is currently unknown. For the further development of 14CO2 sampling, a new trajectory-triggered strategy was simulated in an atmospheric forward modelling system. It was shown that weak fossil CO2 signals from emission hotspots at four German ICOS stations can be amplified by a factor of up to 7, while the signal background is estimated with parallely taken samples.
Near-surface flow patterns and their influence on local transport processes are investigated in a horizontal plane with visualization techniques. For this, a new method was developed that produces tracer particles with high effective Schmidt number (Sc = O(10^6) ) by chemical reaction directly at the water surface. While different chemical systems were tried, best results were achieved using the precipitate AgCl, formed by AgNO 3 in the water body and a controlled influx of HCl gas. Trace amounts of precipitate are made visible by scattering laser light. Using the new method, the influence of wind induced turbulences at the water interface can be investigated with previously unachieved sensitivity. Illumination of the wavy water surface leads to shadowing and lensing effects that become apparent in the image data and cannot fully be compensated for with image processing. The results are qualitatively compared to data from active thermography (heat, Pr ≈ 7) and boundary layer imaging (gas, Sc ≈ 600), showing a close resemblance of the flow pattern. With higher Schmidt number of the tracer, the structure becomes more pronounced and higher sensitivity to surface convergence is observed. Experiments with glass spheres (d = 70 μm-2 mm, Sc → ∞) were conducted for further comparison. Tracking of individual particles allows for a precise measurements of the surface velocity and investigations of the local momentum transfer.
Seit der Intensivierung der Glazial-Interglazial-Zyklizität ab dem späten Pliozän reagierte das Klimasystem sehr sensibel auf Veränderungen in der Atmo-, Kryo- und Ozeanosphäre. Diese führten nicht nur zu ausgeprägten Klimaschwankungen über orbitalskalige Zeitintervalle, sondern resultierten auch in massiver kurzfristiger (d.h. jahrhundert- bis jahrtausendskaliger) Klimavariabilität. In Anbetracht des erheblichen Einflusses des Menschen auf das Klimasystem ist ein Verständnis dieser kurzzeitigen Klimavariabilität dringend erforderlich. Zeitlich hochauflösende Analysen des 1,35 Myr umfassenden Klima- und Vegetationsarchivs von Tenaghi Philippon (NE-Griechenland, NE Mittelmeerraum) können einen wichtigen Beitrag zu einem verbesserten Verständnis kurzzeitiger Klimavariabilität leisten. Das Marine Isotopenstadium 19 (MIS 19; 790–761 ka) ist hierbei von besonderem Interesse, da es als bestes orbitales Analogon des heutigen Holozän-Interglazials gilt. Die in der vorliegenden Arbeit durchgeführten Pollen- und Röntgenpulverdiffraktometrieanalysen des MIS-19-Interglazials von Tenaghi Philippon dokumentieren eine massive kurzzeitige Klimavariabilität. Die zeitliche Einordnung der Vegetationsveränderungen wurde durch magnetostratigraphische Methoden ermöglicht. Klimawechsel hin zu einem kalttrockenen Klima während zum Teil vollinterglazialer Bedingungen führten fünf Mal zu einer Kontraktion temperater Wälder und der Ausbreitung von Kältesteppen innerhalb weniger Jahrzehnte bis Jahrhunderte. Die Erholung der Wälder mit der Rückkehr warmgemäßigter Bedingungen erfolgte ähnlich abrupt. Ein Vergleich der Klima- und Vegetationsdynamik von Tenaghi Philippon mit Klimaveränderungen im Nordatlantik zeigt, dass die insolationsgetriebenen Temperatur- und Eisschild-Veränderungen der hohen nördlichen Breiten und des Nordatlantiks sowie Verschiebungen der Innertropischen Konvergenzzone die Klimavariabilität im nordöstlichen Mittelmeerraum steuerten. Die genannten Faktoren beeinflussten über die Klimasysteme des Nordatlantiks und der hohen nördlichen Breiten indirekt die Vegetation in Tenaghi Philippon. Bei einer starken „bipolar seesaw“ im Atlantik gelangten warmfeuchte Luftmassen über die Westwinde in den Mittelmeerraum und führten zu warmgemäßigten Phasen in Tenaghi Philippon. Demgegenüber sind „ice-rafted debris events“ im Nordatlantik synchron mit kalttrockenen Events in Tenaghi Philippon, da südliche Ausbrüche des Sibirienhochs gleichzeitig kalttrockene Luftmassen in die Mittelmeerregion leiteten. Eine Ausnahme repräsentiert das kalttrockene Event CDE-19/1, das als massives intra-interglaziales Event in Tenaghi Philippon während des MIS-19c-Interglazials nur ein schwach ausgeprägtes Analogon im Nordatlantik und im Mittelmeerraum besitzt. Die abnehmende boreale Sommerinsolation führte in diesem Zeitintervall zur Ausdehnung des Meereises und des Polarwirbels der hohen nördlichen Breiten. Während der Nordatlantik und der westliche Mittelmeerraum jedoch nur eine geringfügige Abkühlung verzeichneten, führten intensive Ausbrüche des Sibirienhochs zu kälteren und trockeneren Bedingungen in Tenaghi Philippon. In einer Gegenüberstellung der Pollendaten von MIS 19 und dem Holozän von Tenaghi Philippon wird deutlich, dass sich im derzeitigen Interglazial temperate Wälder deutlich stärker ausdehnten als während MIS 19. Dies spricht für lokal warmgemäßigtere Bedingungen und somit eine stärkere Intensität des Holozäns gegenüber MIS 19. Zudem unterscheidet sich das heutige Interglazial von MIS 19 durch das Fehlen von Analoga zum intra-interglazialen kalttrockenen Events CDE-19/1 während MIS 19c und zum kalttrockenen Event CDE-19/2, wie es das Ende von MIS 19c in Tenaghi Philippon markiert. Hätte das Holozän ein weniger warmgemäßigteres Klima ähnlich dem des MIS-19c-Interglazials charakterisiert, dann wäre bei einer Übereinanderlegung der Präzessions-Kurvenverläufe beider Zeitintervalle ein Äquivalent zum kalttrockenen Event CDE-19/2 vor ca. 1.600 Jahren eingetreten. Eine Ursache für das im Holozän humidere Klima und die deutlich schwächere kurzzeitige Klimavariabilität könnten die im Vergleich zu MIS 19 deutlich kleinerennördlichen Eisschilde des heutigen Interglazials sein, die möglicherweise aufgrund von Veränderungen in der Glazial-Interglazial-Zyklizität im Zuge der Mittelpleistozänen Übergangsphase und dem „Mid-Brunhes Event“ nicht die Größe des älteren Analogons erreichten. Abgesehen von den kleineren Eisschilden verhinderte auch die spätholozän ansteigende atmosphärische CO2-Konzentration das Kippen des Klimasystems und das Eintreten abrupter Klimavariabilität.
We observed a declining trend in malaria transmission at Nouna, Burkina Faso during the rainy season from 2009-2011. Single and mixed infections for Plasmodium falciparum and Plasmodium malariae were significantly reduced compared to the baseline data (rainy season 2000) and a lower prevalence of P. malariae was associated with a reduced transmission intensity. Microscopic examination has a lower detection limit and is associated with under-estimation of parasite burden, which suggested the use of molecular diagnosis such as PCR as a more sensitive method in determining the prevalence of malaria infections.
Amodiaquine (AQ) is an antimalarial compound chemically and functionally related to chloroquine (CQ). Currently, it is used in combination with artesunate (AS) as the first choice to treat uncomplicated P. falciparum malaria in some countries in Africa and South America. Chloroquine-resistant parasites and cross-resistance between CQ and AQ or its active metabolite Desethyl-amodiaquine (DQ) have been observed in Burkina Faso. pfcrt and pfmdr1 are genes genetically associated with the resistance mechanism and the underlying mechanisms for cross-resistance are still under debate. The main aim of this study was to assess the susceptibility patterns of clonal field isolates P. falciparum compared to reference laboratory strains (Dd2 and HB3). Blood samples from 402 patients from the village Bourasso, Nouna, Burkina Faso were analysed. Genomic DNA was extracted from filter papers using the Chelex-100 method and different Plasmodium species were analysed by microscopy and species-specific nested-PCR.
The mutation in pfcrt which was associated with CQ and AQ resistance was analysed using pyrosequencing, and in vitro susceptibility of clonal parasites to CQ, AQ and DQ was determined. Three different phenotypes of P. falciparum (S9, S47, S173) based on the IC50 values were cultured and the clonal lines were obtained. All clones from S9 and S47 harboured pfcrt CVIET haplotypes while all clones from S173 were CVMNK haplotype. The clones from S9 showed higher IC50 values on average to CQ and AQ compared to S47 clonal parasites. Some clones were sensitive to DQ for S9 and all were sensitive in clonal lines for sample 47. Clonal parasites from S173 has lower IC50 values towards CQ. Responses to CQ, AQ and DQ varied between the clones. Clear cross-resistance were observed in clonal lines S9, eg. 9C9, 9C7, 9H8 and in clone S47E8. There was a moderate correlation between AQ and DQ and a weaker correlation between AQ and CQ. Cross resistance exists but not high in the clonal lines.
After a consistence result of IC50, 9C6, 47C7 and 173D3 were selected for the drug accumulation study. Accumulation of CQ and AQ does not always correlate with IC50. It appeared that clones accumulating high levels of CQ and AQ were susceptible to CQ and AQ respectively, while clones accumulating lower CQ and AQ were resistant. High CQ and AQ IC50 values were associated with lower amounts of drug uptake and the two response parameters reciprocally correlated to each other. Long-term and continuous culture can cause loss of the resistance phenotype. From the observations and findings of this study, we concluded that Pfcrt plays a major role in the resistance to CQ and AQ and we suggest that one or more genes or SNPs may be involved in AQ drug resistance.
There were no resistant clones isolated from the in-vitro selection strategy after challenge with 60 nM DQ but suggested dormant parasites to DQ developed after the exposure. After investigation using quantitative recrudescence assays, the parasites were not dormant but were in fact dead. Several strategies and processes could be improved for future studies to select for AQ resistant parasites.
In many animals, a specialized cytoplasm forms within the oocyte that harbors all the molecular factors required for germ cell fate specification and is defined as the germ plasm. In Drosophila melanogaster, the germ plasm (or pole plasm), is assembled at the posterior pole of the oocyte in a stepwise process triggered by Oskar protein. oskar mRNA transcribed in the nuclei of nurse cells is actively transported into, and to the posterior pole of, the oocyte. At the posterior pole, oskar mRNA is translated into Oskar protein, which recruits the other pole plasm components required for germline specification and posterior patterning in the embryo. Recently, it was shown that Oskar binds polyadenylated mRNAs in vivo and that the C-terminal domain of the protein binds RNA in vitro. In that study, by using UV crosslinking and immunoprecipitation experiments, I showed that Oskar associates in vivo with three mRNAs involved in posterior patterning and germ cell fate specification: nanos, polar granule component and germ cell-less. In order to identify Oskar’s binding site(s) on its target transcripts I applied the iCLIP method to early Drosophila embryos. To this end, I analyzed the efficiency of three key steps of the iCLIP protocol: RNAse digestion, 3’ end dephosphorylation, and adapter ligation. I found that, while the 3’ end dephosphorylation was generally efficient, the ligation of an adapter to RNA was a limiting step in the protocol. By performing a series of optimization experiments, I established new reaction conditions for adapter ligation that increased the efficiency of the reaction significantly. Even after optimization of the iCLIP protocol, the data produced in the Oskar iCLIP were inconclusive, due at least in part to the low affinity of Oskar for RNA. Hence, positional information regarding Oskar’s interaction with specific RNAs in vivo is still lacking. Such information would enable validation and further characterization of the RNA-binding activity of Oskar, providing important new insight into the molecular mechanisms underlying Oskar’s unique pole plasm inducing activity. The optimized iCLIP protocol I developed should be applicable to other RNA-binding proteins in Drosophila and in other model organisms.
Clathrin is a unique scaffold protein, which forms polyhedral cages at the plasma membrane. Adaptor proteins recruit clathrin to the plasma membrane where the triskelion-shaped clathrin units interact with each other and assemble into flat and curved lattices. The function of the curved clathrin-coated pits in forming clathrin-coated vesicles via dynamin-dependent scission during endocytosis is well studied. On the contrary, the role of the flat hexagonal clathrin arrays remains ambiguous and has been a controversial topic for decades. In this PhD thesis, we used different microscopic techniques (live-cell confocal microscopy, stimulated emission depletion nanoscopy, transmission electron microscopy, correlative light and electron microscopy) combined with mathematical modelling or micrometre-scale manipulation of substrates to unravel the role of flat clathrin lattices during two cellular processes: 1) Endocytosis and 2) Cell migration. 1) We describe a novel clathrin-coated pit formation mechanism in which clathrin arrays first assemble as flat clathrin-coated structures until they reach around 70 % of the final clathrin content. At that point a change in the adaptor/clathrin ratio marks the conversion from a flat to curved lattice and further addition of clathrin triskelia leads to the creation of a complete and invaginated clathrin-coated pit. We could show that the flat-to-curved transition of the clathrin coats is sensitive to the biophysical properties of plasma membrane and can be block by elevated plasma membrane tension. 2) We found a so far undescribed spatial relation between disassembling focal adhesions and newly forming larger flat clathrin lattices, called clathrin-coated plaques, during cell migration. We demonstrated that clathrin-coated plaques containing the extracellular matrix receptors integrins are generated at topographical cues of remodelled extracellular matrix and regulate cell migration as novel adhesive unit. These specific functions put a new focus on the poorly understood flat clathrin coats and highlight the multiple cellular applicability of clathrin arrays.
The mathematical representation of soil water movement exhibits uncertainties in all model components. Data assimilation methods, like the ensemble Kalman filter (EnKF), combine models and measurements into an improved representation and can – at least in principle – account for all uncertainties. However, a proper description of the uncertainties is required, which is particularly difficult in soil hydrology, where model errors typically vary rapidly in space and time. Inflation methods can account for unrepresented model errors. To improve the EnKF performance, I designed an inflation method specifically for soil hydrology, that is capable of adjusting inflation factors to spatiotemporally varying model errors. For the application on a real-world case, I assessed the key uncertainties for the specific hydraulic situation of a 1-D soil profile with TDR (time domain reflectometry)-measured water contents. With the EnKF, I directly represented and reduced all key uncertainties (initial condition, soil hydraulic parameters, small-scale heterogeneity, and upper boundary condition), except for an intermittent violation of the local equilibrium assumption by the Richards equation. To bridge this time, I introduced a closed-eye period, which ensures constant parameters and improves the EnKF towards the goal of knowledge fusion – the consistent aggregation of all information pertinent to some observed reality.
The Mu3e experiment is a proposed experiment to probe for new physics by searching for the charged lepton-flavour violating decay μ+ → e+e+e− with a branching ratio sensitivity of 10^−16 , improving the current limit by four orders of magnitude. To search for such rare events, extremely high muon decay rate, good background suppression and high detector efficiencies are required. This demands an excellent momentum, vertex and timing resolution from the detector systems. Furthermore, the experiment will be running at a muon stopping rate of more than 10^9 Hz in order to observe enough muon decays in a reasonable experiment running time. This poses another challenge to the detectors and readout electronics, which have to be designed to cope with the present event rate.
This thesis presents the development of a dedicated Silicon Photomultiplier (SiPM) readout Application-Specific Integrated Circuit (ASIC) for the Mu3e timing detectors. It provides the precise timing measurement while being capable of working with the high event rates. Fully differential analog front-end channel and 50 ps time binning TDC are utilized to achieve excellent timing resolution. The customized Low-Voltage Differential Signaling (LVDS) transmitter cell and double data rate serializer provides gigabit data rate to transfer data out of the chip. Detailed measurements have been preformed to characterize the timing performance and to verify the digital functionalities of the chip.
The need for quantitative analysis is crucial when studying fundamental mechanisms in cell biology. Common assays consist of interfering with a system via protein knockdowns or drug treatments. These very often lead to important response variability that is generally addressed by analyzing large populations. Whilst the imaging throughput in light microscopy (LM) is high enough for such large screens, electron microscopy (EM) still lags behind and is not adapted to collect large amounts of data from highly heterogeneous cell populations. Nevertheless, EM is the only technique that offers high-resolution imaging of the entire subcellular context. Correlative light and electron microscopy (CLEM) has made it possible to look at rare events or addressing heterogeneous populations. Our goal is to develop new strategies in CLEM. More specifically, we aim at automatizing the processes of screening large cell populations (living cells or pre-fixed), identifying the sub-populations of interest by LM, targeting these by EM and measuring the key components of the subcellular organization. New 3D-EM techniques like focused ion beam - scanning electron microscopy (FIB-SEM) enable a high degree of automation for the acquisition of high-resolution, full cell datasets. So far, this has only been applied to individual target volumes, often isotropic and has not been designed to acquire multiple regions of interest. The ability to acquire full cells with up to 5 nm x 5 nm x 5 nm voxel size (x, y referring to pixel size, z referring to slice thickness), leads to the accumulation of large datasets. Their analysis involves tedious manual segmentation or so far not well established automated segmentation algorithms. To enable the analysis and quantification of an extensive amount of data, we decided to explore the potential of stereology protocols in combination with automated acquisition in the FIB-SEM. Instead of isotropic datasets, a few evenly spaced sections are used to quantify subcellular structures. Our strategy therefore combines CLEM, 3D-EM and stereology to collect and analyze large amounts of cells selected based on their phenotype as visible by fluorescence microscopy. We demonstrate the power of the approach in a systematic screen of the Golgi apparatus morphology upon alteration of the expression of 10 proteins, plus negative and positive control. In parallel to this core project, we demonstrate the power of combining correlative approaches with 3D-EM for the detailed structural analysis of fundamental cell biology events during cell division and also for the understanding on complex physiological transitions in a multicellular model organism.
Mechanical allodynia is a type of neuropathic pain in which innocuous touch evokes severe pain. Despite several years of research, the primary sensory neurons that mediate allodynia still remain unidentified. In this study, we demonstrate that a population of low threshold mechanoreceptors expressing TrkB mediates the input that produces pain from light touch after nerve injury. Using an inducible Cre line driven from the TrkB gene locus, we show that TrkB is expressed in Dhairs and RA Ab-mechanoreceptors. Ablation of TrkB positive neurons leads to reduction in sensitivity to the gentle touch under naïve conditions, and failure to develop mechanical allodynia in a nerve injury model of neuropathic pain. Also, selective optogenetic activation of these neurons evokes marked nocifensive behavior after nerve injury. Furthermore, we develop a phototherapeutic strategy based on the ligand for TrkB to perform ligand-mediated photoablation of TrkB positive afferents. Using this approach we show that selective photo-ablation of TrkB afferents in the skin results in a pronounced rescue of mechanical allodynia in multiple models of neuropathic pain. We thus identify the peripheral neurons which transmit pain from light touch and establish a novel therapeutic strategy for its treatment.
Addiction is a chronically relapsing brain disorder, involving compulsive drug seeking and taking. Enduring vulnerability to relapse is a challenging feature to manage in substance use disorder, with devastating effects to those who suffer from it, as well as at familial and public health levels. Incubation of drug craving characterized by gradual increases in cue-induced drug craving following halting of drug use, may contribute to heightened relapse risk, even after prolonged abstinence. Addictive drugs act upon and usurp the mesolimbic circuit, with long-term drug abuse leads to reward processing, cognitive and decision-making deficits. Drug-driven neuronal plasticity within the Prefrontal cortex (PFC) to Nucleus Accumbens (NAc) pathway is a known key substrate and mediator of addictive behavior. Here we performed longitudinal in vivo local field potential (LFP) recordings in freely behaving rats throughout an incubation of drug seeking paradigm. This approach proved suitable to assess both evoked and spontaneous LFP activity at distinct behavioral stages of the addiction cycle, in a within subject manner. Chronic cocaine self-administration induced strengthening of the PFC-NAc pathway, accompanied by enhanced glutamate release, when compared to drug naïve conditions. Compellingly, the degree of synaptic adaptation correlated with the cocaine intake and incubation severity of individual rats. At the network level, accumbal oscillatory profile of rats that underwent CSA was also altered, with suppression of high gamma and enhanced alpha and beta waves. Throughout withdrawal, persistent pre-synaptic release subsisted, while network changes proved to be transient. Yet, rats with history of cocaine intake did showed altered LFP patterns, upon a cocaine challenge, when compared to saline yoked counterparts, suggesting impaired corticostriatal network dynamics that endures after long-term abstinence. During reinstatement, i.e. relapse-like conditions, distinct frequency components were found to be differentially modulated by drug seeking behavior. Drug-driven adaptations to synaptic transmission and concomitant alterations of oscillatory landscape of functionally connected areas, such as the PFC and NAc, represent multiple-leveled dysregulation exerted by addictive drugs. Concerted maladaptive changes may contribute to the development of a de novo homeostatic threshold that is both driven by and drives drug abuse, craving and relapse in a spiraling cycle of addiction.
Um das Messnetz für Globalstrahlung auszubauen, wird in dieser Arbeit das Verfahren PV2RAD vorgestellt. PV2RAD ermöglicht die Ableitung von direkten und diffusen Strahlungsflussdichten aus Leistungsdaten von Photovoltaikanlagen. Zunächst werden die Photovoltaikanlagen mit Hilfe von vier anlagenspezifischen Parametern an klaren Tagen charakterisiert. Im Anschluss werden auf Grundlage der Leistung von Photovoltaikanlagen und den anlagenspezifischen Parametern, unter Anwendung der linearen Inversion, die direkte und die diffuse Strahlungsflussdichte in Abhängigkeit der atmosphärischen Parameter abgeleitet. Auf Grundlage der aus der Inversion abgeleiteten direkten Strahlungsflussdichten kann der atmosphärische Parameter Aerosol Optische Dicke an klaren Tagen standortgenau bestimmt werden. PV2RAD wird an einer Testanlage am Standort Hochschule Bonn-Rhein-Sieg getestet und validiert. Für sieben klare Tage werden die direkte und die diffuse Strahlungsflussdichte ermittelt. Dabei liegt die Differenz zwischen gemessener und aus der Inversion abgeleiteter Strahlungsflussdichte im Tagesgang zwischen 2% und 9%. Anschließend wird PV2RAD auf sechs Photovoltaikanlagen des Versorgungsgebiets der AllgäuNetz GmbH & Co. KG angewandt. Die Differenz zwischen gemessener und aus der Inversion abgeleiteter Strahlungsflussdichte im Tagesgang liegt durchschnittlich zwischen 2% und 16%. Die aus der Inversion abgeleiteten Strahlungsflussdichten sind im Vergleich zu den etablierten Verfahren (Satellit, Bodenmessstation und Wettermodelle) zeitlich und räumlich höher aufgelöst.
State-of-the-art methods for the calculation of electronic structures of molecules predominantly use Gaussian basis functions. The algorithms employed inside existing code packages are consequently often highly optimised keeping only their numerical requirements in mind. For the investigation of novel approaches, utilising other basis functions, this is an obstacle, since requirements might differ. In contrast, this thesis develops the highly flexible program package molsturm, which is designed in order to facilitate rapid design, implementation and assessment of methods employing different basis function types. A key component of molsturm is a Hartree-Fock (HF) self-consistent field (SCF) scheme, which is suitable to be combined with any basis function type.
First the mathematical background of quantum mechanics as well as some numerical techniques are reviewed. Care is taken to emphasise the often overlooked subtleties when discretising an infinite-dimensional spectral problem in order to obtain a finite-dimensional eigenproblem. Common quantum-chemical methods such as full configuration interaction and HF are discussed providing insight into their mathematical properties. Different formulations of HF are contrasted and appropriate SCF solution schemes formulated.
Next discretisation approaches based on four different types of basis functions are compared both with respect to the computational challenges as well as their ability to describe the physical features of the wave function. Besides (1) Slater-type orbitals and (2) Gaussian-type orbitals, the discussion considers (3) finite elements, which are piecewise polynomials on a grid, as well as (4) Coulomb-Sturmians, which are the analytical solutions to a Schrödinger-like equation. A novel algorithmic approach based on matrix-vector contraction expressions is developed, which is able to adapt to the numerical requirements of all basis functions considered. It is shown that this ansatz not only allows to formulate SCF algorithms in a basis-function independent way, but furthermore improves the theoretically achievable computational scaling for finite-element-based discretisations as well as performance improvements for Coulomb-Sturmian-based discretisations. The adequacy of standard SCF algorithms with respect to a contraction-based setting is investigated and for the example of the optimal damping algorithm an approximate modification to achieve such a setting is presented.
With respect to recent trends in the development of modern computer hardware the potentials and drawbacks of contraction-based approaches are evaluated. One drawback, namely the typically more involved and harder-to-read code, is identified and a data structure named lazy matrix is introduced to overcome this. Lazy matrices are a generalisation of the usual matrix concept, suitable for encapsulating contraction expressions. Such objects still look like matrices from the user perspective, including the possibility to perform operations like matrix sums and products. As a result programming contraction-based algorithms becomes similarly convenient as working with normal matrices. An implementation of lazy matrices in the lazyten linear algebra library is developed in the course of the thesis, followed by an example demonstrating the applicability in the context of the HF problem.
Building on top of the aforementioned concepts the design of molsturm is outlined. It is shown how a combination of lazy matrices and a contraction-based SCF scheme separates the code describing the SCF procedure from the code dealing with the basis function type. It is discussed how this allows to add a new basis function type to molsturm by only making code changes in a single integral interface library. On top of that, we demonstrate by the means of examples how the readily scriptable interface of molsturm can be employed to implement and assess novel quantum-chemical methods or to combine the features of molsturm with existing third-party packages.
Finally, the thesis discusses an application of molsturm towards the investigation of the convergence properties of Coulomb-Sturmian-based quantum-chemical calculations. Results for the convergence of the ground-state energies at HF level are reported for atoms of the second and the third period of the periodic table. Particular emphasis is put on a discussion about the required maximal angular momentum quantum numbers in order to achieve convergence of the discretisation of the angular part of the wave function. Some modifications required for a treatment at correlated level are suggested, followed by a discussion of the effect of the Coulomb-Sturmian exponent. An algorithm for obtaining an optimal exponent is devised and some optimal exponents for the atoms of the second and the third period of the periodic table at HF level are given. Furthermore, the first results of a Coulomb-Sturmian-based excited states calculation based on the algebraic-diagrammatic construction scheme for the polarisation propagator are presented.
It is commonly accepted that oxidative stress, caused by increased metabolic flux and subsequent activation of the four major pathways of hyperglycemic damage are a causative factor for the development of diabetic nephropathy. However, therapies inactivating these pathways do not improve the symptoms of nephropathy. As such it was analysed, whether oxidative stress is present at the onset of nephropathy. Streptozotocin-induced diabetic mice (BL6-STZ) and genetically diabetic db/db mice, characterized by incipient albuminuria, were screened for the effects of hyperglycaemia on mitochondrial function, ROS production and the activation of the major pathways of hyperglycaemic damage in the kidney. Despite increased intracellular glucose, substrates of the tricarboxylic cycle were not increased. ATP production and mitochondrial oxygen consumption were increased in the BL6-STZ model, whereas mitochondrial oxygen consumption was decreased in the db/db model. This was not associated with either increased oxidative stress or activation of the protein kinase C and advanced glycation endproduct pathways. The polyol and the hexosamine pathways were increased in the BL6-STZ model but decreased in the db/db model. These differences, despite increased intracellular glucose, could not explain the symptoms of nephropathy. The finding that the Nrf2-NQO1 axis was increased in both models would suggest that the onset of nephropathy is associated with ROS-independent cellular stress.
CD8+ T cells are an important part of the adaptive immune system. They are not only able to efficiently kill infected cells, but can also provide protection against subsequent infections. Recently, T cell-based vaccines, which are able to elicit protection-mediating CD8+ T cell responses, have shown their potential against various infectious diseases, including malaria or HIV. However, the efficient implementation of these approaches is currently hampered by a lack of knowledge regarding the dynamical processes generating the protective responses. In this thesis, we combined experimental data and mathematical modelling to analyse and quantify CD8+ T cell dynamics in different infectious diseases. First, we study a specific immune responses elicited by cytomegalovirus (CMV) infection, known as 'memory in ation'. We used mathematical modelling to test different hypotheses regarding the processes that generate and maintain memory in ation and analysed how viral dynamics shape the corresponding CD8+ T cell responses. Since CMV has already been used as a vaccine vector expressing foreign epitopes, our findings are relevant for improving the efficacy of these vector-based vaccination approaches. To generate sufficient protection, vaccination strategies usually require the application of one or more booster injections. Here, factors such as dosage, frequency and timing of injections can influence the protective levels reached. To determine the impact of current vaccination strategies against malaria, we analysed the in uence of vaccination regimens, differing in dosage and frequency of injections, on the generation of organ-specific CD8+ T cell responses. We identified the underlying cellular differentiation and migration pathway and determined the impact of different vaccination doses on the build-up and maintenance of protection-mediating liver-resident memory cells. Our results do not only provide a quantitative understanding of CD8+ T cell responses elicited by immunisation, but can also be used to improve existing vaccination approaches. Understanding the impact of T cell-based vaccination regimes on the immune system requires knowledge about the underlying cellular dynamics. While mathematical modelling allows the determination of differentiation pathways and the quantification of cellular turnover, the reliability of these analyses depends strongly on the quality of the available data. While the labelling of cells has been a useful method to increase the amount of information within cellular data, it has not been analysed so far how the design of applied labelling strategies aspects the mathematical estimation of cellular turnover. To this end, we determined the robustness of different labelling strategies to infer cellular dynamics including data suffering from experimental limitations. Our fndings can be used as a guideline to determine cellular dynamics more accurately in future experiments. In summary, by combining experimental data and mathematical modelling our results do not provide a quantitative understanding of CD8+ T cell responses in infectious diseases, but can also be used to improve the efficiency and efficacy of T cell-based vaccines.
Cell wall recalcitrance is a major limitation hindering the exploitation of the enormous potential of lignocellulosic biomass as a renewable resource for energy and bio-based products. In the last decades, C4 grasses from the genus Miscanthus have emerged as a most promising energy crop for the production of lignocellulosic biomass in temperate climates. Secondary cell walls, which represent the largest proportion of lignocellulosic biomass are formed in specialised cells after cessation of growth. In higher plants, the formation of secondary cell walls is tightly regulated, both spatially and temporally, by a complex network of transcription factors. However, in Miscanthus only little is known about molecular players regulating secondary cell wall formation and lignin biosynthesis. In this study, application of the herbicide isoxaben was shown to trigger ectopic lignification in Miscanthus seedling roots. The chemical treatment may be exploited in the future to identify regulators and biosynthetic genes involved in lignification in Miscanthus. In addition, Miscanthus sinensis transcription factors related to SECONDARY WALLASSOCIATED NAC DOMAIN1 (SND1) and SECONDARY CELLWALL MYBs 1-4 (SCM1-4) were identified in the Miscanthus transcriptome, which act as regulators of secondary cell wall formation and lignin biosynthesis. During Miscanthus development, expression of MsSND1 and MsSCMs coincided with the onset of secondary cell wall formation and lignification of vascular tissue and sclerenchyma fibers. MsSND1 and MsVND7 were capable to fully restore growth in a SCW-deficient Arabidopsis thaliana mutant, suggesting they are the Miscanthus orthologues of the well characterised Arabidopsis determinants of SCW formation in fibers and vessels. Ectopic expression of MsSND1 and MsVND7 in tobacco leaves prompted the formation of patterned deposition of lignin, cellulose, and hemicellulose reminiscent of xylem elements. This observation was in sharp contrast to uniform lignification after transient expression of various MsSCMs. Expression of particular Miscanthus TFs led to specific cell wall compositions, providing attractive targets for biomass improvements. Transgenic Arabidopsis lines carrying an inducible system of MsSND1 revealed that MsSND1 regulates directly or indirectly the expression of a broad range of genes involved in secondary cell wall formation.
This thesis is concerned with the development of methodological and mathematical foundations of plane wave ultrasound particle image velocimetry. This innovative technology, also known as Echo PIV, is a non-invasive imaging technique developed to estimate the blood flow in the vascular system.
In this thesis, we address two relevant problems related to in-vitro Echo PIV. First, we consider the image reconstruction problem which amounts to estimating the location of small isolated scatterers, embedded in a homogeneous medium, from plane wave ultrasound measurements. We link the Delay and Sum method with the leastsquares solution for the linearized inverse acoustic scattering problem. Numerical studies show that the least-squares approach provides accurate reconstruction results for synthetic and in-vitro data.
In the second part of the thesis, we focus on the motion estimation problem for pipe flow. For laminar and steady flow, we develop a global dictionary-based approach that estimates the model flow parameter via an converging iterative refinement scheme. Furthermore, we show that the flow parameter can be the determined from the geometry of the image sequence spectra. To discriminate between the steady and turbulent flow, we design a filter-bank that is sensitive to all flow directions and extract the local velocity field by analyzing the phase component of the response function for combined filters. We validate our methods on synthetic and in-vitro data.
Computer sind dumm. Es wird zwar ständig über Künstliche Intelligenz geredet, aber bislang gilt für die Geräte, mit denen wir zu tun haben, noch überwiegend die alte goldene IT-Regel: „shit in = shit out“. Was schon ganz gut funktioniert, ist das maschinelle Lernen. Da geht es um Software, die lernt, immer besser spezielle Aufgaben zu lösen. Wie das funktioniert, berichtet Campus Reporter Nils Birschmann und hat dabei mit Prof. Dr. rer. nat. Fred A. Hamprecht gesprochen.
Der Beitrag erschien in der Sendereihe "Campus-Report" - einer Beitragsreihe, in der über aktuelle Themen aus Forschung und Wissenschaft der Universitäten Heidelberg, Mannheim, Karlsruhe und Freiburg berichtet wird. Zu hören ist "Campus-Report" montags bis freitags jeweils um ca. 19.10h im Programm von Radio Regenbogen (Empfang in Nordbaden: UKW 102,8. In Mittelbaden: 100,4 und in Südbaden: 101,1)
2018 ist das Wissenschaftsjahr. In hunderten Projekten und Events geht es um „Arbeitswelten der Zukunft“. Ohne die Unterstützung von Unternehmen und Stiftungen wäre so eine Initiative unmöglich. Überhaupt sähe die Wissenschaft ziemlich alt aus, wenn sich nur der Staat darum kümmern würde. Allein die Heidelberger Klaus Tschira Stiftung hat seit der Gründung 1995 mehr als 500 Mio. Euro Projekt- und Fördermittel vergeben. Was die Stiftung im Wissenschaftsjahr vorhat, berichtet Campus Reporter Nils Birschmann und spricht dabei mit Beate Spiegel, Geschäftsführerin der Klaus-Tschira-Stiftung.
Der Beitrag erschien in der Sendereihe "Campus-Report" - einer Beitragsreihe, in der über aktuelle Themen aus Forschung und Wissenschaft der Universitäten Heidelberg, Mannheim, Karlsruhe und Freiburg berichtet wird. Zu hören ist "Campus-Report" montags bis freitags jeweils um ca. 19.10h im Programm von Radio Regenbogen (Empfang in Nordbaden: UKW 102,8. In Mittelbaden: 100,4 und in Südbaden: 101,1)
A fundamental requirement for embryonic development is tight spatiotemporal coordination between a steadily increasing number of cells. A notable example of highly coordinated cellular activity occurs during body axis segmentation of the vertebrate embryo. Organized oscillatory signaling activity produces regular waves of gene expression that traverse the tissue. These oscillations are thought to define the timing of segmentation and the positioning of embryonic segments, the somites. The striking rhythmicity of the process is ascribed to a molecular oscillator in undifferentiated cells, the “segmentation clock”, involving the Notch, Wnt and FGF signaling pathways in mouse. While the segmentation clock and its intercellular coordination mechanism have been studied in some detail, the start of the process, the establishment of an initial synchrony between several hundreds of presomitic cells, remains largely unknown. I chose a live imaging approach to examine the onset of synchronous signaling oscillations in the developing embryo. To this end, I established a mounting and culture method to enable long-term multi-sample post-implantation mouse embryo imaging on a light-sheet microscope. This new setup enables routine embryo culture and imaging from early gastrulation on embryonic day 6.5 for up to two days, closely recapitulating in utero development. Using genetically encoded signaling reporters to visualize the cellular oscillation status, I was able to capture the very first observable coherent oscillations of the cyclic gene Lfng as well as the preceding dynamics. I found concurrent upregulation of Lfng expression across nascent mesoderm and the primitive streak at mid gastrulation, marking the first synchronous activity pattern of this segmentation clock gene. This expression pulse was also seen for the FGF signaling target Dusp4 but not the Wnt target Axin2. Despite Lfng being a Notch signaling target in the context of somitogenesis, I found the Lfng pulse to occur in the presence of Notch signaling inhibitor DAPT as well as in the absence of Notch transcription factor RBP-J κ and the Notch signaling core oscillator HES7. These findings argue for an initial synchronization signal independent of the Notch pathway. Quantification of early wave dynamics identified a gradual buildup of phase waves and revealed that a period gradient, which is present from the very first observable oscillation onwards, functionally underlies the establishment of waves in the gastrulating embryo. Taken together, this study provides detailed and quantitative insight into the dynamics of the early segmentation clock in the mouse embryo, pioneering future studies on the molecular mechanism of the synchronization process. The post-implantation embryo culture and imaging method described here opens up new possibilities for the study of the gastrulation-to-organogenesis stages, which in the past have been difficult to follow in real time. Further technical developments of multi-sample mammalian embryo microscopy, which I also discuss in this work, will continue to make an important contribution to the investigation of the establishment of the segmentation clock, and beyond can help to better understand many aspects of mammalian embryonic development.
Lungenkrebs verursacht weltweit die meisten krebsbedingten Todesfälle. Aufgrund der späten Diagnose und frühen Metastasenbildung sind die meisten Patienten inoperabel und systemische Therapien wie Chemotherapie müssen angewendet werden. Patienten mit aktivierender Mutation in dem epidermalen Wachstumsfaktor-Rezeptor (EGFR) können zielgerichtete Therapien mit Tyrosinkinaseinhibitoren (TKIs) gegen EGFR erhalten. Viele Chemotherapie-Patienten leiden an Anämie und müssen z.B. mit Erythropoese-stimulierenden Substanzen (ESAs) behandelt werden, die möglicherweise jedoch Resistenz gegen die Chemotherapie induzieren. Auch viele Lungenkrebs-Patienten, die mit EGFR-TKIs behandelt werden, entwickeln schnell Therapieresistenz, die durch verstärkte Expression einer verwandten Rezeptor-Tyrosinkinase, des Hepatozytenwachstumsfaktor (HGF)-Rezeptors MET, vermittelt werden kann. Um die komplexen Mechanismen der Therapieresistenz zu verstehen, wurde in dieser Arbeit ein systembiologischer Ansatz angewendet. Um die unterschiedlichen Antworten von ESAs auf gesunde oder Tumor-Zellen zu untersuchen, wurde ein mathematisches Modell entwickelt, das Zelltyp-spezifische Unterschiede identifizieren und mögliche Zielstrukturen vorhersagen kann, die Tumorzellen, aber nicht gesunde erythroide Vorläuferzellen inhibieren. Es wurde nachgewiesen, dass der Erythropoietin-Rezeptor von Endothelzellen bei ESA-Stimulation phosphoryliert wird, was zu einer erhöhten Vaskularisierung und Zugänglichkeit von Chemotherapeutika in Xenograft-Mausmodellen führt. Um eine optimierte ESA-Dosis für unter Anämie leidende Krebspatienten zu bestimmen, wurde ein mathematisches Modell des ESA-Abbaus und der induzierten Signalaktivierung von STAT5 entwickelt. Es wurde gezeigt, dass ESAs mit einer niedrigen Bindungsaffinität Tumorzllen weniger induzieren als gesunde erythroide Vorläuferzellen. Sie wurden daher als sicherere Behandlungsoption von Chemotherapie-bedingter Anämie vorgeschlagen. Um den Mechanismus der MET-vermittelten Resistenz gegen EGFR-TKIs zu identifizieren, wurde die Aktivierung von Signaltransduktionskomponenten in zwei Lungenkrebszelllinien nach Stimulation mit EGF, HGF oder Co-Stimulation zeit- und dosisaufgelöst gemessen. Eine Zelltyp-spezifische verstärkte Aktivierung von MET unter Co-Stimulation wurde beobachtet. Unter Verwendung eines mathematischen Modells zur Beschreibung der zellspezifischen Dynamik und unter Verwendung von Einzelmolekül-Mikroskopie konnte die Bildung von EGFR:MET-Heterodimeren mit einer reduzierten Internalisierungsrate als zugrunde liegender Mechanismus identifiziert werden. Weiterhin wurde gezeigt, dass das EGFR zu MET Expressionsverhältnis den Umfang der Verstärkung bestimmt und dass dieser Effekt die Wirksamkeit von EGFR-TKIs durch Inhibierung der Heterodimere beeinflusst. Es wurde gezeigt, dass die Wirksamkeit von EGFR-TKIs durch eine Verringerung der Expression von MET durch den pharmakologischen Antikörper MM-131 erhöht wird. Dieser Mechanismus legt eine Verwendung des EGFR/MET-Expressionsverhältnises zur Patientenstratifizierung nahe und sagt neuartige Kombinations-Behandlungen zur Reduzierung von Therapieresistenz vorher. Zusammengefasst liefert diese Arbeit verschiedene neue Einblicke in Mechanismen der Signaltransduktion, welche die Wirksamkeit von EGFR-TKIs und ESAs beeinflussen, und schlägt Strategien vor um das Ansprechen auf die Therapien zu erhöhen.
Magnetic Resonance Imaging (MRI) is an important imaging modality in both the clinic and in research. MRI technology has been trending toward increasing field strengths to improve the signal-to-noise ratio of the MR signal and fast excitation/encoding strategies to more flexible target anatomical regions during excitation to reduce the total imaging time. While largely successful, both strategies rely on the application of increasingly strong and rapidly switched magnetic fields: the radio frequency (RF) field for excitation and the gradient field for encoding. The technology for generating these fields (and rapidly switching them) has advanced to the point that we are limited by biological responses to the switching fields. For the gradient field, the electric field generated in the tissue causes peripheral nerve stimulation (PNS) causing mild but bothersome sensations at low levels, up to pain or cardiac malfunction at higher levels. The electric fields created by the much faster time-varying RF cause heat deposition, ultimately denaturing proteins and causing tissue damage. In this thesis, methods are presented to characterize and minimize these two problems associated with the switched magnetic fields in MRI. The deposited RF energy (Specific Absorption Rate, SAR) incurred during shaped excitations can be significantly reduced by optimizing gradient and RF waveforms for inner-volume excitations that allow imaging of a sub-volume of the body without wrapping artifacts. The adverse effects of the switching gradient fields are addressed by designing time-optimal gradient encoding waveforms and by developing a method to predict and characterize PNS using field simulations and a full-body nerve model allowing these critical effects to be addressed at the gradient coil design stage. In the first part, time-optimal gradient trajectories are demonstrated that use the gradient hardware at the maximum available performance. The skeleton of the trajectory is defined by a set of k-space control points. The method optimizes gradient waveforms that traverse the k-space control points in the minimum possible amount of time. By using an analytic representation of the gradients (piece-wise linear), the design process is fast and numerically robust. The resulting trajectories sample k-space efficiently while using the gradient system at maximum performance. Compared to the leading Optimal Control method, the proposed method generates gradient waveforms that are 9.2% shorter. The computation process is ∼100x faster and does not suffer from numerical instabilities such as oscillations. In the second part, a method is developed that jointly optimizes parallel transmission RF and gradient waveforms for fast and robust 3-D inner-volume excitation of the MRI signal in minimal time and with minimal energy deposition. The optimization of the k-space trajectories is based on a small number of shape parameters that are well-suited for joint optimization with the RF waveforms. Within each iteration of the trajectory optimization, a small tip-angle least-squares RF pulse design problem is solved. Using optimized 3-D cross (shells) trajectories, a cube shape (brain shape) region was excited with 3.4% (6.2%) NRMSE in less than 5 ms using a 7 T scanner with 8 Tx channels and a clinical gradient system (Gmax = 40 mT/m, Smax = 150 T/m/s). Incorporation of off-resonance robustness in the pulse design significantly altered the k-space trajectory solutions and improved the practical performance of the pulses. In the final part, a framework is presented that simulates PNS thresholds for realistic gradient coil geometries and thus allows, for the first time, to directly address PNS in the coil design process. The PNS framework consists of an accurate body model for simulation of the induced electric fields, an atlas of peripheral nerves, and a neurodynamic model to predict the nerve responses to imposed electric fields. With this model, measured PNS thresholds of two leg/arm solenoid coils and three commercial actively-shielded MR gradient coils could be reproduced with good accuracy. The proposed method can be used to assess the PNS capability of gradient coils during the design phase, without building expensive prototype coils.
Plasmodium sporozoites, the motile forms that are transmitted from the mosquito to the mammalian host, use a unique type of locomotion called gliding motility. This motility is powered by an actin-myosin motor underneath the plasma membrane: Myosin pulls on actin-filaments that are connected to adhesins of the thrombospondin-related anonymous protein (TRAP) family including TLP (TRAP-like protein). These membrane-spanning proteins interact with host cell receptors thereby transmitting the generated force to the substrate resulting in a forward movement of the cell. As a consequence, the filamentous actin-adhesin complexes are driven back to the rear of the cell, which is known as retrograde flow. The role of TLP and actin filaments in force production and retrograde flow of Plasmodium berghei sporozoites was investigated using reverse genetics, live cell imaging and optical tweezers. This instrument allows manipulation of microscopic objects by exerting forces in the piconewton range via a highly focused laser beam. In our experiments, we positioned polystyrene particles onto the gliding sporozoites, which actively translocated these beads towards the posterior end of the cell. We found that transport speeds were significantly higher than the sporozoite forward movement (1-2 μm/s) independent of particle size or functionalization. This bead transport most likely indirectly reflects retrograde flow in sporozoites. Wild type sporozoites and transgenic sporozoites lacking the surface protein TLP with and without different concentrations of actin-modulating drugs (cytochalasin D and jasplakinolide) were challenged to pull beads from the optical traps at different forces. These experiments revealed a role of TLP in controlling the retrograde flow by converting it into optimal force transmission for gliding motility. Further, force experiments on mutant sporozoites with altered TLP C-termini refined our hypothesis and suggested a function for the extracellular domain in recruiting surrounding surface proteins and possibly stabilizing them laterally for force transmission. In a second project, I assessed parasite-induced surface protrusions called knobs on P. falciparum-infected erythrocytes using Atomic Force Microscopy (AFM). This analysis revealed that the knobs on infected erythrocytes carrying heterozygous sickle cell- traits were larger but fewer.
Persistent infections with high-risk human papillomaviruses (hr-HPVs) may cause cervical and other types of cancer. The key event for the transformation of hr-HPV-infected cells into malignant tumor cells is the deregulated expression of the hr-HPV oncogenes E6 and E7. Both gene products interfere with cell cycle checkpoints, inhibit DNA damage repair and induce chromosomal instability. Deregulation of hr-HPV oncogene expression as well as transformation of the host cells are driven by the hypermethylation of specific CpG dinucleotides in the viral and host cellular genome. Thereby, the HPV E2-mediated control of E6 and E7 transcription is disrupted and the expression of host cellular tumor suppressive genes and microRNAs is prevented. Therefore, we hypothesized that the application of demethylating agents might re-establish these regulatory mechanisms reducing the hr-HPV oncogene expression and inhibiting cell proliferation.
To test this hypothesis, the demethylating agent 5-aza-2’-deoxycytidine (DAC) was applied to a panel of six HPV-transformed cell lines. DAC treatment significantly decreased the expression of the HPV oncogenes. As a consequence, the levels of E6 and E7 target proteins, including p53 and p21, increased repressing cell proliferation and colony formation. In addition, the application of DAC strongly induced the expression of tumor suppressive miR-375, which was shown to target and degrade E6 and E7 transcripts. In conclusion, the presented data demonstrate the effectiveness of DAC in the treatment of HPV-transformed cells and suggest its testing in clinical trials.
In addition to the evaluation of treatment strategies, the present thesis aimed to study the effects of HPV oncogene expression on chromosomal stability, gene expression patterns and DNA methylation levels. For this, chromosomally stable HCT116 cells were used as a model system to generate clones allowing the inducible HPV 16 E6 and E7 expression. Immortalization of HCT116 cells, which is characterized by microsatellite instability, can be clearly distinguished from HPV-driven immortalization, which depends on the induction of chromosomal instability. Therefore, HCT116 cells represent an ideal model system to study the HPV 16 oncogene-mediated induction of chromosomal instability.
Induction of HPV 16 oncogene expression affected the chromosomal stability of HCT116 cells by causing abnormal centrosome and spindle pole numbers, by inducing DNA damage and by increasing the number of aneuploid cells. Furthermore, a panel of genes was found to be differentially expressed after induction of HPV 16 oncogene expression potentially representing candidate genes and indicating pathways that might play a role during the transformation of HPV-infected cells. Taken together, HPV 16 oncogene expression seems to increase the genomic variability in the cell population presumably elevating the risk for the generation of highly proliferative subclones over time.
The mucin-like sialoglycoprotein podoplanin (PDPN) is widely expressed throughout the human and rodent body. Although numerous studies have revealed its essential function in development, especially of the lymphatic system, the lungs and heart, the overall picture of its physiologic function is still incomplete. Emerging evidence of the past decade has associated PDPN de novo or overexpression with numerous cancer entities including glioblastoma, and in particular with the invasive behavior of tumor cells. As the infiltrative growth of tumor cells is one major challenge in glioblastoma therapy, the identification of novel candidates in tumor cell migration remains an essential pre-requisite for the development of new and effective therapeutic means. However, the postulated pro-tumorigenic and pro-invasive function of PDPN in glioblastoma has never been validated in vivo. Moreover, the underlying mechanism of a potential malignant effect of PDPN has not been addressed. Thus, the aim of this study was to close this gap of knowledge by the combination of correlative and functional assays. Descriptive in vivo approaches involving patient-derived xenografts were primarily taken to confirm the previous correlations of PDPN expression and malignant progression and to establish a model that enables the investigation of underlying mechanisms. For the functional validation of the hypothesis that PDPN is a major driver of glioblastoma progression and especially invasion, the gene was deleted by the novel CRISPR/Cas9 technology. Xenotransplantations of control and knockout cells indicated the dispensability of PDPN for glioblastoma growth and progression. The reliable analysis of the postulated pro-invasive function of PDPN required the optimization of a three-dimensional invasion assay based on organotypic brain slice cultures. The usage of adult murine brain slices and a red emitting fluorescent membrane dye significantly improved the assay quality. The application of this advanced technique identified PDPN as a non-rate limiting component in glioblastoma cell invasion. These data and the detailed analysis of further malignant features including proliferation, apoptosis and angiogenesis have rebutted the previous assumption of a tumor promoting effect of PDPN. Despite the dispensability of PDPN for tumor development and tumor cell invasion, the obtained results suggest PDPN as a marker for malignant glioblastoma cells. In conclusion, this study represents an important contribution in the process of preclinical drug development, as the results object the frequently suggested development of a PDPN blocking therapeutic agent. Instead, this work suggests PDPN as a marker for prognosis or targeted delivery of cytotoxic compounds into glioblastoma tumor cells.
The phytohormone auxin controls a wide spectrum of biological processes by regulating the activity of AUXIN RESPONSE FACTORS (ARFs) transcription factors. ARFs are also post-transcriptionally regulated by TRANS-ACTING SIRNA3 (TAS3)-derived trans-acting small-interfering RNAs (tasiARFs). The tasiARFs pathway is highly conserved in land plants, regulating functions ranging from developmental timing to lateral roots formation. This pathway is also present in primary root tip and in embryo, where its function(s) remains elusive. A modifier genetic screen using a transcriptional reporter for MIR390A, a tasiARFs pathway element, identified a mutant with no expression in the primary root tip. The mutation was mapped to AT1G75860, a gene of unknown function. Here, we tried to assign a function for this pathway in embryo and primary root, we further characterized the AT1G75860 mutant, and we also tested a possible interaction with the miR156/SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) pathway in the control of lateral root development. AT1G75860-mutant and -T-DNA lines were characterized regarding MIR390A presence in the primary root tip. We performed an allelism test between the mutant and the T-DNA lines; we tried to complement the mutant and, after more rounds of backcross, we tested if there was still linkage between mutation and phenotype. The results could not link the mutation with the phenotype, and indicated that the MIR390A transcriptional reporter might not faithfully reflect MIR390A expression pattern. We tested for functions of the tasiARFs pathway in the primary root. We used mutants, gain-of-functions, or overexpressors of this pathway and looked for a primary root growth or meristem size phenotype in normal and abiotic-stress conditions. However, no primary root phenotype could be identified. To check if there is an interaction between the tasiARFs and miR156/SPLs pathways in lateral root development, we tested, by qPCR and using the MIR390A transcriptional reporter, the response of one pathway to perturbations in the other. Due to inconsistences between experiments and methods for miR156 detection, only miR390 response could be examined, but no conclusive proof of interactions could be obtained. Finally, regarding the role of the tasiARFs pathway in embryo, we could show that elements of the tasiARFs pathway are expressed and actively repress the expression of ARF3. Furthermore, using mutants of this pathway, we confirmed its role in control of seed number, and we propose a possible novel role in endosperm development.
In dieser Studie sollte untersucht werden, inwieweit gängige Laborparameter in der Alkoholdiagnostik (GOT, GPT, GGT, MCV und CDT), auch innerhalb der Normwertgrenzen Aussagen über den Alkoholkonsum im Rahmen des „sozialen Trinkverhaltens“ erlauben. Insgesamt 23 Proband/innen haben sich an der insgesamt viermonatigen Studie beteiligt, Diese war in vier Phasen von jeweils vier Wochen aufgeteilt. In der ersten Phase sollte das normale Trinkverhalten genau protokolliert werden. Diese Angaben ermöglichten eine Berechnung der Alkoholdosis pro Woche/kg Körpergewicht. Die untersuchten Laborparameter konnten dann in Korrelation zu diesen Alkoholmengen gesetzt werden. (Querschnittsdarstellung) Hier zeigte sich kein signifikanter Zusammenhang zwischen Konzentration bzw. Aktivität der Laborparameter und Höhe der eingenommenen Alkoholmenge. Die zweite Phase war eine komplette Alkoholabstinenzphase. Der erwartete Abfall der Laborparameter GOT, GPT, GGT und CDT konnte nicht nachgewiesen werden. Der Mittelwert des CDT lag knapp über 1%, also über dem Cut-off-Wert, der oft zur Bestätigung einer Alkoholabstinenz herangezogen wird. In Phase 3 und 4 wurden jeweils genau definierte Alkoholmengen eingenommen. In Phase 4 war dies die doppelte Menge im Vergleich zu Phase 3. Es waren aber immer nur geringe Alkoholmengen. Auch hier zeigte sich kein eindeutiger Anstieg der Laborparameter. Es konnte also weder eine Korrelation von Transaminasen, MCV und CDT zu den Alkoholmengen, die in den Trinkprotokollen angegeben waren, hergestellt werden, noch gerichtete Reaktionen der Parameter auf Änderungen des Trinkverhaltens aufgezeigt werden. Vor dem Hintergrund der hohen Anforderungen an die Aussagekraft der Veränderung der Laborparameter, die im Rahmen von Begutachtungen gestellt werden, kann man von der Höhe der Parameter und ihrer Entwicklung in der Längsschnittbeobachtung keine Rückschlüsse auf das Trinkverhalten ziehen. Am Ende von Phase 4 lag der Mittelwert des CDT-Wert mit 1,246% des Gesamt-transferrins deutlich über dem Cut-off-Wert. CDT-Werte im sogenannten Graubereich zwischen 1,0% und 1,7% lassen also nicht auf einen übermäßigen Alkoholkonsum schließen. Selbst nach einer Abstinenzphase lag der Mittelwert noch über 1%, sodass ein CDT-Wert im Graubereich auch nicht unbedingt darauf rückschließen lässt, dass eine Alkoholabstinenz nicht eingehalten wurde. Ein verkehrsmedizinisches Gutachten, das darlegen soll, dass kein übermäßiger Alkoholkonsum erfolgt ist, kann sich nicht allein auf einen CDT-Wert stützen. Wichtig ist die Verlaufsbeobachtung verschiedener Parameter über einen längeren Zeitraum.
Despite the tremendous progress in the fight against malaria during the last two decades, it remains one of the most important infectious diseases worldwide, leading to approximately 500 000 lethal cases annually, mostly among young children. The emergence and spread of resistance of the Plasmodium parasites to all the drugs currently available on the market are a major threat to its control and eradication. It moreover emphasizes the dire need for new antimalarial agents with distinct modes of action. Previously, the medicinal chemistry team at the biotechnology company 4SC AG, Munich, presented a series of promising antimalarial compounds, optimized around an amicarbalide backbone. Two agents were selected out of this series as lead-compounds for further studies, namely SC81458 and SC83288. The work presented here aims to characterize the in vitro activity of the SC-lead compounds. First, it revealed them as potent inhibitors of P. falciparum blood stage parasites, acting preferentially on late stages. The lack of activity on the ring stages is reflected in their fast speed of action, yet not as fast as artemisinin, the fastest compound described so far, that acts on all blood stages. Importantly, the SC-lead compounds were unaffected by the most common resistance mechanisms to antimalarial drugs used in the clinic. Particularly, no cross-resistance mechanism between artemisinin and its derivatives and the SC-lead compounds was observed, and their antiplasmodial modes of action appeared to be distinct from each other. The second part of this work focused on the mode of action of the SC-lead compounds and the mechanisms of resistance that the parasite could develop. Although the Ca2+ ATPase pump PfATP6 was disproved as a direct molecular target of the SC-lead compounds, it was demonstrated to be implicated in a resistance mechanism. The F972Y mutation and the overexpression of the A108T, A109T variant led to a drastic decrease in the SC-lead compounds responsiveness. The F972Y substitution correlated with an in vitro fitness cost for the parasite, which was linked to a lower intracellular calcium resting concentration compared to its parental line. The molecular details of the disturbed calcium homeostasis and its correlation with the resistance to the SC-lead compounds remain to be unraveled. Overall, the findings of this work demonstrate the promising in vitro potency of the SC-lead compounds, particularly SC83288, and highly support its further development into (pre)clinical trials.
Alle reden von künstlicher Intelligenz, es ist das heiß diskutierte Zukunftsthema schlechthin. Manche warnen davor, andere freuen sich auf ein leichteres Leben durch KIs. Dabei haben wir bislang noch nicht einmal verstanden, wie unsere eigene, menschliche Intelligenz überhaupt funktioniert. Um das zu ändern, arbeiten Forscher der Uni Heidelberg daran, einen Schaltplan des Gehirns zu entwickeln. Campus-Reporter Nils Birschmann hat sich das angeschaut.
Der Beitrag erschien in der Sendereihe "Campus-Report" - einer Beitragsreihe, in der über aktuelle Themen aus Forschung und Wissenschaft der Universitäten Heidelberg, Mannheim, Karlsruhe und Freiburg berichtet wird. Zu hören ist "Campus-Report" montags bis freitags jeweils um ca. 19.10h im Programm von Radio Regenbogen (Empfang in Nordbaden: UKW 102,8. In Mittelbaden: 100,4 und in Südbaden: 101,1)
N-terminal acetylation is a conserved co-translational protein modification that is highly abundant among eukaryotes. In Saccharomyces cerevisiae, at least five enzymes with distinct substrate specificities (N-terminal acetyl transferase Nat A to E) act to acetylate 50–70% of the yeast proteins. Despite being one of the most common protein modifications, its biological significance remains largely ambiguous. I set out to study the role of N-terminal acetylation in yeast cells by employing quantitative proteomics and ribosome profiling for analysis of the consequences of failure of N-terminal acetylation in strains lacking specific N-terminal acetyl transferases. My results revealed a multi-faceted stress response in natB deletion mutant that modulates protein quality control machinery, protein biogenesis capacity, and energy regeneration pathways in order to establish protein homeostasis. Systematic analysis of proteome stability in the natB deletion mutant suggests no global effect of the loss of N-terminal acetylation on the turnover of NatB substrates, but an increase in the level of global protein aggregation. SILAC-based mass spectrometry analysis of aggregated proteins isolated from the natB deletion mutant shows no significant enrichment of NatB substrates, indicating that protein aggregation in the natB deletion mutant cannot be solely explained as a direct consequence of the loss of N-terminal acetylation. In contrast, these protein aggregates show strong enrichment for components of specific biological pathways, in particular of the translation apparatus, suggesting an underlying selective sequestration mechanism. Consistently, quantitative proteomics revealed that, on average, approximately 40% of each of the quantified ribosomal proteins is sequestered into protein aggregates in the natB deletion mutant. Moreover, the aggregated proteins showed significantly higher interaction between each other and overlapped with aggregated proteins generated upon environmental stress, suggesting a common mode of sequestration of proteins into aggregates. Interestingly, the aggregated proteins in the natB deletion mutant strongly overlap with those identified upon deletion of the genes encoding the ribosome-associated Hsp70 chaperone Ssb. In addition, deletion of SSB in the natB deletion mutant leads to synthetic growth defects. Moreover, isolation of radiolabeled protein aggregates after 5 min 35S pulse labeling showed that a fraction of the newly synthesized proteins is readily sequestered into aggregates. These findings together suggest a new link between N-terminal acetylation by NatB and co-translational protein folding activity by Ssb. Parallel analysis of natA deletion mutant revealed similar protein aggregation patterns, suggesting a general role of N-terminal acetylation in the maintenance of proteome integrity.
In der vorliegenden Arbeit werden physikalische und elektrochemische Eigenschaften von potentiellen Elektrodenmaterialien für Lithium-Ionen-Batterien untersucht. Die elektrochemischen Untersuchungen wurden mittels zyklischer Voltammetrie und galvanostatischer Zyklierung durchgeführt. Zur physikalischen Charakterisierung der Ausgangsmaterialien und elektrochemisch zyklierter Proben kamen Röntgendiffraktometrie, Elektronenmikroskopie und Magnetisierungsmessungen zum Einsatz. Zur Untersuchung der magnetischen Eigenschaften einzelner mikro- bis nanoskaliger Partikel wurde ein Mikro-Hall-Magnetometer aufgebaut und getestet. Die dargestellten Ergebnisse zeigen insgesamt, dass die elektrochemischen Eigenschaften von Anodenmaterialien durch Nanoskalierung in Kompositen mit Kohlenstoffmodifikationen signifikant verbessert werden können. In diesem Kontext wurden sowohl die oxidischen Konversionsverbindungen Mn3O4, MnO2, Fe2O3, CoFe2O4, ZnO und SnO2, die mit Lithium legierungsbildenden Elemente Ge und Sn als auch das Interkalationsmaterial TiO2 untersucht. Im Fall von gefüllten mehrwandigen Kohlenstoffnanoröhren (CNTs) können die reversiblen theoretischen Kapazitäten der Füllmaterialien erreicht und über mindestens 50 Zyklen erhalten werden. Die Ergebnisse von erstmalig untersuchten manganoxidisch gefüllten CNTs (Mn3O4@CNT) bestätigen insbesondere den Konversionsmechanismus im Inneren der CNTs. Auch hierarchisch strukturierte Kompositmaterialien von Kohlenstoffhohlkugeln mit MnO2- beziehungsweise SnO2-Nanopartikeln weisen durch die Beiträge der oxidischen Aktivmaterialien erhöhte spezifische Kapazitäten mit guter Zyklenstabilität auf. Untersuchungen am Kathodenmaterial NH4V3O8 zeigen, dass die reversible Ein- und Auslagerung von über zwei Li+-Ionen pro Formeleinheit über einen mehrstufigen diffusionskontrollierten Interkalationsmechanismus abläuft.
Human cytochrome P450 (CYP) enzymes play an important role in the metabolism of drugs, steroids, fatty acids and xenobiotics. CYPs also catalyze the conversion of some pro-drugs into active drugs. Only about a dozen human CYPs metabolize 70-80% of all drugs. A subset of CYPs is responsible for steroidogenesis, of these CYP17 is a major drug target for prostate cancer therapy. Human CYPs are anchored to the endoplasmic reticulum membrane by their N-terminal transmembrane (TM) helix. However, most crystal structures of CYPs have been resolved after truncating the TM-helix or mutating residues that form contacts with the membrane. Therefore, the structural basis for CYP-membrane interactions and orientation, and the mechanism of substrate entrance into the buried binding pocket and product release is not clearly understood. In order to understand the interactions and orientations of CYPs and their degree of penetration into the membrane, I have optimized a multiscale modeling protocol that involves coarse-grained and all-atom molecular dynamics simulations. The protocol was validated by applying it to several drug-metabolizing CYPs (CYP1A1, 1A2, 2C9, 2C19, 3A4) and CYPs involved in steroidogenesis (CYP17, CYP19) in a lipid bilayer. The simulations revealed that the sequence and structural differences in the protein-membrane interface alter the interactions and orientations of CYPs in the membrane. Furthermore, mutations in the TM-helix of CYP17, particularly W2A and E3L, were seen to disrupt the CYP-membrane interactions and in some cases, obstruct the ligand tunnels between the active site and the membrane, which could lower enzyme turnover. In conclusion, the optimized multiscale simulation protocol has been used to identify different interactions and orientations adopted by the globular domains of CYPs with the membrane that have implications for CYP function. This protocol is also suitable for studying protein-protein-membrane complexes and proteins in membranes with different lipid compositions.
Mechanical forces between cells ensure organic development and homeostasis, but they are also associated with diseases such as cancer or viral infections. The absolute forces in nature can be as high as 1.5 kN, which allows the mantis shrimp to smash oysters, down to a few pN transduced by single cellular receptors. However, these small forces are strong enough for cells to probe their local environment. While the mechanism, which enables cells to investigate the stiffness of their surrounding, is already well elucidated, information on how cells sense spatial distribution of ligands is missing. In this thesis I established a method, which allows to measure cellular traction forces on elastic substrates with varying nano-spacing of extracellular ligands. In contrast to previous studies on stiff substrates, adhesion complexes and tractions were larger for longer distances between extracellular adhesion sites. This can be theoretically explained by the force load on individual integrin receptors, which has to exceed a certain threshold value to promote adhesion growth through conformational changes in a protein of the “clutch complex”. In order to experimentally access the force load per integrin heterodimer, I combined molecular tension fluorescence microscopy (MTFM) with traction force microscopy (TFM). For the first time, I could assess a homogeneous distribution of forces > 19pN underneath the adhesion area of cells on soft substrates. Simultaneously, macroscopic tractions up to 2.7 kPa were observed at the cell edges. Applying stronger tension probes and analyzing tractions in the zdirection will help to cross-validate the results obtained from these two state-of-the-art methods in biomechanics as a next step. In the second part I investigated the mechanical parameters of virus particle uptake by cells. Many intracellular pathogens, such as mammalian reoviruses as employed in this thesis, mimic extracellular motives to interact with host cells and initiate their internalization. This leads to the assumption that host cells sense this specific ligand presentation, engage the endocytic machinery and generate forces, which are able to overcome the bending and tension energy of their plasma membrane. I demonstrated that these forces exerted on single reovirus particles on the basolateral side of cells are strong enough to break down the biotin-NeutrAvidin bond used for virus immobilization on stiff and soft substrates. I quantified the forces to exceed 40pN by kinetic analysis of the tearing of viruses from these surfaces and single MTFM with covalently immobilized reoviruses. The herein presented methods are powerful tools to study forces exerted by individual receptors as well as on single particles e.g. during endocytosis. The involvement of the actin cytoskeleton, specific receptors or molecules of the endocytic machinery was examined. Inhibition of the ligand-receptor interactions between reoviruses and cells did not significantly change the rate of virus uptake. Interestingly, bare nanoparticles of comparable diameter lacking specific binding sites were torn off at a similar rate and thus with the same forces as viruses. Hence, specific receptors seem to be dispensable for virus particle uptake.
Cells interact with neighboring cells and the extracellular matrix forming cell-cell and cell-matrix contacts, which are mainly mediated by cadherins and integrins, respectively. These processes are dynamic and spatially and temporally tightly regulated during many biological events including embryogenesis, wound healing and cancer development. Dynamic control of cell interactions is a key to understanding many underlying cellular processes, to achieving the bottom-up assembly of single cells into tissues and to developing medical implants. The challenge lies in controlling specific cell-cell and cell-material interactions both dynamically and reversibly with high spatiotemporal control in a non-invasive way over a long period of time. The aim of this thesis is to generate platforms where these cell interactions are controlled spatially, temporally, dynamically and reversibly using visible light responsive proteins from plants.
The recent developments in the field of optogenetics provide powerful tools to overcome above-mentioned challenges and they have been employed to control many intracellular signaling pathways. Among others cryptochromes and phytochromes were used in this study. Cryptochrome 2 (CRY2) is a blue light photoreceptor and it heterodimerizes with CIBN upon blue light irradiation. This interaction can be reversed to the ground state passively in the dark. Phytochrome B (PhyB) is a red and far-red light sensing protein and upon red light illuminations it interacts and forms heterodimers with PIF6. This heterodimerization can be reversed under far-red light or passively in the dark.
To control cell-cell interactions with light, the blue light dependent heterodimers, CRY2 or CIBN were expressed on the surfaces of the cells, which do not form any native cell-cell contacts. In equally mixed cultures of CRY2 and CIBN expressing cells, these cells form cell-cell interactions upon blue light illumination, which provides high high spatial and temporal control. These photoswitchable interactions are reversible in the dark, and can be repeatedly and dynamically switched on and off. These genetically encoded interactions can be sustained over a long time as they are genetically encoded and they respond to nontoxic low intensity blue light.
Towards controlling cell-material contacts of multiple cell types, one of the heterodimerization partners (CIBN or PIF6) was immobilized on non-adhesive glass surfaces. By expressing CRY2 or PhyB on the cell surfaces, their adhesion to CIBN and PIF6 functionalized substrates can be triggered under nontoxic low intensity blue and red light illumination, respectively. CRY2/CIBN and PhyB/PIF6 interactions are orthogonal to each other since they respond to only blue and red/far-red light, respectively. This orthogonality provides wavelength selective adhesion of one cell type to its complementary substrate in the presence of the other cell type. The ability of PhyB/PIF6 system to far-red light also makes orthogonal reversion of these adhesions possible while the other cell type (CRY2) remains adhered to its substrate. These photoswitchable cell-material interactions are reversible in the dark or under far-red light, and can be repeatedly and dynamically switched on and off.
Overall, this optogenetic approach to control cell interactions reflects the dynamic and reversible nature of cell-cell and cell-material interactions and provides the desired spatiotemporal control in a noninvasive manner. These blue and red/far-red light responsive proteins are genetically encodable; hence, they can be sustained over a long time. Finally, photoswitchable cell interactions will provide a new way of studying them and assembling cells into multicellular structures in the context of bottom-up tissue engineering.
Thе аntі-vіrаl cytоkіnе іntеrfеrоn аlphа (ІFNα) іs usеd fоr thе trеаtmеnt оf chrоnіc Hеpаtіtіs B аnd C аnd vаrіоus typеs оf cаncеrs. Оftеn pаtіеnts dо nоt rеspоnd tо thе trеаtmеnt whіch hаs bееn cоrrеlаtеd tо prе-аctіvаtіоn оf ІFNα-іnducеd sіgnаlіng, аs аssеssеd by еnhаncеd еxprеssіоn оf ІFNα tаrgеt gеnеs. ІFNα аctіvаtеs thе JАK/STАT sіgnаl trаnsductіоn pаthwаy, whіch іnvоlvеs nеgаtіvе fееdbаck lооps mеdіаtеd by SОCS1, SОCS3 аnd USP18 аnd pоsіtіvе fееdbаck lооps mеdіаtеd by ІRF9, STАT1 аnd STАT2. Thе оbjеctіvе оf thіs study wаs tо іnvеstіgаtе іf thе dynаmіc bеhаvіоr оf ІFNα-іnducеd sіgnаl trаnsductіоn іs аffеctеd іn ІFNα prе-trеаtеd cеlls аnd tо dіssеct thе mеchаnіsm cоntrіbutіng tо а pоssіblе mеmоry еffеct оf prе-аctіvаtіоn оf ІFNα-іnducеd sіgnаl trаnsductіоn. Thеsе аіms wеrе аpprоаchеd by cоmbіnіng quаntіtаtіvе tіmе- аnd dоsе-rеsоlvеd dаtа оf ІFNα-іnducеd sіgnаlіng іn hеpаtоmа cеll lіnеs аnd prіmаry humаn hеpаtоcytеs wіth mаthеmаtіcаl mоdеlіng. Tо еxаmіnе thе еffеct оf prе-аctіvаtіоn оf thе ІFNα-іnducеd sіgnаlіng trаnsductіоn, cеlls wеrе prе-trеаtеd wіth dіffеrеnt dоsеs оf ІFNα, wеrе stіmulаtеd wіth а hіgh dоsе оf ІFNα аnd thе dynаmіcаl bеhаvіоr оf ІFNα-іnducеd sіgnаlіng wаs аnаlyzеd by quаntіtаtіvе іmmunоblоttіng. Thе оbtаіnеd rеsults rеvеаlеd thаt prе-trеаtmеnt wіth а lоw dоsе оf ІFNα rеsultеd іn еnhаncеd ІFNα-іnducеd phоsphоrylаtіоn оf STАT1 аnd STАT2 оbsеrvеd іn thе nuclеus, whіlе prе-trеаtmеnt wіth а hіgh dоsе оf ІFNα rеsultеd іn much rеducеd ІFNα-іnducеd phоsphоrylаtіоn оf STАT1 аnd STАT2 іn thе cytоplаsm аnd іn thе nuclеus, іndіcаtіng а pоsіtіvе аnd nеgаtіvе mеmоry оf ІFNα-іnducеd sіgnаl trаnsductіоn, rеspеctіvеly. Thе аnаlysіs оf ІFNα-іnducеd gеnе еxprеssіоn shоwеd thаt nеgаtіvе mеmоry оf ІFNα-іnducеd sіgnаl trаnsductіоn prеvеntеd nо furthеr іnductіоn оf ІFNα-іnducеd gеnе еxprеssіоn, whіlе pоsіtіvе mеmоry rеsultеd іn fаstеr gеnе еxprеssіоn оf ІFNα-tаrgеt gеnеs. Tо unrаvеl thе mеchаnіsms cоntrіbutіng tо pоsіtіvе аnd nеgаtіvе mеmоry оf ІFNα-іnducеd sіgnаl trаnsductіоn, а mаthеmаtіcаl mоdеl wаs еstаblіshеd аnd cаlіbrаtеd wіth tіmе- аnd dоsе-rеsоlvеd dаtа cоmprіsіng prе-trеаtmеnt wіth dіffеrеnt dоsеs оf ІFNα аnd subsеquеnt ІFNα stіmulаtіоn tо chаrаctеrіzе thе іnductіоn оf thе pоsіtіvе аnd thе nеgаtіvе fееdbаck lооps. Еxpеrіmеnts shоwеd thаt ІRF9, STАT1 аnd STАT2 аrе іnducеd аt lоwеr ІFNα dоsеs cоmpаrеd tо USP18, SОCS1 аnd SОCS3. Tо аnаlyzе thе spеcіfіc rоlе оf USP18, USP18 еxprеssіоn wаs іnducеd tо thе sаmе аmоunt аs prеsеnt іn cеlls prе-trеаtеd wіth а hіgh dоsе оf ІFNα, ІFNα-іnducеd phоsphоrylаtіоn оf STАT1 аnd STАT2 wаs lоwеr cоmpаrеd tо wіldtypе cеlls, but much hіghеr cоmpаrеd tо cеlls prе-trеаtеd wіth а hіgh dоsе оf ІFNα, іndіcаtіng thаt USP18 аbundаncе аlоnе іs nоt suffіcіеnt tо еxplаіn thе nеgаtіvе mеmоry оf ІFNα-іnducеd sіgnаl trаnsductіоn. Mаthеmаtіcаl mоdеl аnаlysіs rеvеаlеd thаt SОCS1 аnd USP18 tоgеthеr synеrgіstіcаlly еnhаncе dеgrаdаtіоn оf аctіvе rеcеptоr cоmplеxеs аnd thеrеby much rеducеd thе ІFNα-іnducеd phоsphоrylаtіоn оf STАT1 аnd STАT2. Mоdеl аnаlysіs оf thе pоsіtіvе mеmоry іdеntіfіеd STАT2 аnd ІRF9 аs kеy cоntrіbutоrs. Mоdеl sіmulаtіоns prеdіctеd thаt thе аbundаncе оf STАT2 аnd USP18 tоgеthеr, but nоt оf SОCS1, іs suffіcіеnt tо prеdіct thе dіrеctіоn аnd еxtеnt оf thе mеmоry оf ІFNα-іnducеd sіgnаl trаnsductіоn. Quаntіfіcаtіоn оf thе аmоunt оf STАT2 аnd USP18 іn prіmаry humаn hеpаtоcytеs frоm thrее dіffеrеnt pаtіеnts іdеntіfіеd hіgh pаtіеnt-tо-pаtіеnt-vаrіаbіlіty іn thе аmоunt оf USP18, but nоt оf STАT2. Thеrеfоrе, USP18 аbundаncе wаs іdеntіfіеd аnd еxpеrіmеntаlly vаlіdаtеd аs pаtіеnt-spеcіfіc іndіcаtоr оf mеmоry оf ІFNα sіgnаl trаnsductіоn, prоvіdіng nеw pеrspеctіvеs fоr оptіmаl dоsіng оf ІFNα thаt wоuld prеvеnt nоn-rеspоnsіvеnеss tо ІFNα trеаtmеnt іn іndіvіduаl pаtіеnts.
Telomeres are nucleoprotein structures that protect and maintain the ends of eukaryotic linear chromosomes. Telomeres shorten at each round of DNA replication due to the end replication problem. The enzyme telomerase, by adding telomeric repeats to chromosome ends, can counteract this process. In the absence of telomerase, telomeres progressively shorten until they reach a critical length that activates the DNA damage response, thereby halting the cell cycle in a condition referred to as replicative senescence. Telomeres are transcribed into a long, non-coding RNA dubbed TERRA, which can hybridize with its template strand, thereby forming R-loops at S. cerevisiae and human telomeres. Recent data implicate telomeric R-loops in the promotion of homologous recombination at telomeres, leading to telomere lengthening events which can partially compensate for telomere shortening in the absence of telomerase. Telomeric R-loops are regulated by RNase H1 and H2 enzymes, which can degrade the RNA moiety of RNA-DNA hybrids. While the accumulation of telomeric R-loops in cells lacking both enzymes delays senescence onset by promoting homologous recombination at telomeres, the depletion of telomeric R-loops by overexpressing RNase H1 leads to premature senescence onset. This PhD thesis aims to better understand how telomeric R-loops are regulated especially during replicative senescence in S. cerevisiae. We found that RNase H2 localizes to long telomeres and physically interacts with the telomere-associated protein Rif2, which is required for RNase H2 recruitment to telomeres. Accordingly, in the absence of Rif2 telomeric R-loops accumulate, indicating that Rif2 and RNase H2 play a pivotal role in restricting R-loops at long telomeres. Importantly, the interaction between RNase H2 and Rif2 is strongest in late S phase, which is reflected in the degradation of telomeric R-loops in this time frame. We propose that this cell cycle regulated telomeric R-loop degradation is required to avoid collisions of the replication machinery, which replicates long telomeres in late S phase, with R-loops, an event that could have detrimental effects on telomere stability. It was previously shown that, as telomeres shorten, Rif2 localization to telomeres is diminished. We show that decreased Rif2 association to short telomeres leads to impaired recruitment of RNase H2, which is functionally reflected in the accumulation of R-loops at short telomeres. Moreover, while RNase H1 could not be detected at long telomeres, we observed its localization to short telomeres, thereby indicating a distinct requirement for the RNase H enzymes. By analyzing the effect of single RNase H enzymes deletion on the kinetics of senescence onset in telomerase negative cells, we revealed an opposing effect of the two enzymes, suggesting that, differently from what was proposed, RNase H enzymes do not have redundant functions at telomeres. In conclusion, we propose that, while at long telomeres R-loops are timely regulated by Rif2-RNase H2 to avoid collisions with the replication machinery, at short telomeres R-loops are allowed to accumulate, thereby promoting homologous recombination-mediated telomere extension.
Viral infections of the kidney, with severe clinical pictures, represent an ongoing threat. The hemorrhagic fever with renal syndrome (HFRS) is caused by several species of pathogenic Old World hantaviruses and is clinically characterized by cellular permeability disorders, leading to acute kidney injury. Previously, it was shown that podocytes and tubular epithelial cells, which play a key role in renal function, are permissive for hantaviruses and further, that the infection was causing a disruption of cell-to-cell contacts correlating with the disease severity. Up until today, the renal-specific pathogenesis mechanisms of hantavirus infections, leading to the cellular leakage, are not well understood. In this study, functional and morphological consequences of hantavirus infection are examined in human renal cells and compared to African green monkey Vero E6 cells, which are commonly used to study hantaviruses. Analysis of podocytes and tubular epithelium in human renal biopsies of hantavirus-infected patients revealed distinct morphological changes known to be involved in functional disorders of various kidney diseases. To investigate how hantaviruses cause cellular disorders, the effects were further examined using in vitro experiments. Infections of podocytes and tubular epithelial cells with either Hantaan (HTNV) or Puumala virus (PUUV) caused cytoskeletal rearrangements in combination with an impairment of adhesion and motility capacity. The nucleocapsid (N) protein was associated with the actin cytoskeleton and, in addition to that, the integrity of filamentous (F-) actin was crucial for viral release. Furthermore, the transfection of podocytes revealed that the expression of N protein alone was sufficient to impair cellular functions. Soluble factors contributing to the cellular disturbances were neither detected in the supernatant of in vitro infected podocytes nor in the serum of hantavirus-infected patients, indicating direct effects of infection on renal cells. According to the severity of the clinical picture, the in vitro effects of hantaviral infections and N protein expression were more pronounced for HTNV than for PUUV. In contrast, the hantavirus infections were clearly different in Vero E6 cells and no functional consequences and actin involvement were observed, demonstrating the importance of choosing relevant cell culture models. These results demonstrate that hantavirus-induced morphological and functional injuries are species- and cell type-specific. The association of N protein with actin and its involvement in the viral release might play a role in the underlying mechanism, leading to functional impairment and cell-to-cell contact disruption. Moreover, the N protein is identified to be a pathogenicity factor of Old World hantaviruses. Conclusively, these findings might contribute to the understanding of the pathogenesis mechanisms leading to the clinical picture of hantavirus-induced acute renal failure.
In this thesis we propose new methods in the field of numerical mathematics and stochastics for a model-based optimization method to control dynamical systems under uncertainty. In model-based control the model-plant mismatch is often large and unforeseen external influences on the dynamics must be taken into account. Therefore we extend the dynamical system by a stochastic component and approximate it by scenario trees. The combination of Nonlinear Model Predictive Control (NMPC) and the scenario tree approach to robustify with respect to the uncertainty is of growing interest. In engineering practice scenario tree NMPC yields a beneficial balance of the conservatism introduced by the robustification with respect to the uncertainty and the controller performance. However, there is a high numerical effort to solve the occuring optimization problems, which heavily depends on the design of the scenario tree used to approximate the uncertainty. A big challenge is then to control the system in real-time. The contribution of this work to the field of numerical optimization is a structure exploiting method for the large-scale optimization problems based on dual decomposition that yields smaller subproblems. They can be solved in a massively parallel fashion and additionally their discretization structure can be exploited numerically. Furthermore, this thesis presents novel methods to generate suitable scenario trees to approximate the uncertainty. Our scenario tree generation based on quadrature rules for sparse grids allows for scenario tree NMPC in high-dimensional uncertainty spaces with approximation properties of the quadrature rules. A further novel approach of this thesis to generate scenario trees is based on the interpretation of the underlying stochastic process as a Markov chain. Under the Markovian assumption we provide guarantees for the scenario tree approximation of the uncertainty. Finally, we present numerical results for scenario tree NMPC. We consider dynamical systems from the chemical industry and demonstrate that the methods developed in this thesis solve optimization problems with large scenario trees in real-time.
With advances in genotyping and cost-effective sequencing technologies, Genome-wide association studies (GWAS) have emerged as approaches to study the genetics of natural variation. GWAS are particularly useful when inbred lines are available (as once they are genotyped, these lines can be phenotyped multiple times) and also with the availability of automated image acquisition and analysis systems for rapid phenotyping. The objective of this thesis is to identify a variety of phenotypic traits from the inbred lines of the teleost fish Medaka (Oryzias latipes) which will then assist in the investigation of the genetic basis for such a variety. Medaka is chosen as the model organism because of the presence of still free living wild populations in Japan and East Asia and for the ability to generate new inbred strains from these wild fish. Moreover, Quantitative Trait Loci (QTL) analysis done so far on craniofacial traits in adult Medaka shows that a substantial genetic component underlies the variance seen between two inbred strains. In this study different southern and northern Japanese Medaka hatchlings at 10 days post fertilization (dpf) and 20 dpf were characterized. The focus is on the two elements that essentially define an organism: morphology and behavior. Gross morphological features were extracted and quantified using custom developed algorithms. In addition, behavioral patterns of the different inbred lines are studied since behavior provides a link and a perspective of how an organism relates to its environment. Specifically, locomotion, feeding, and prey capture behavior were analyzed and quantified. To our knowledge, this is the first characterization of prey capture behavior in Medaka. This behavior reveals interesting prey capture strategies and a comparison with a related teleost fish, the zebrafish, suggests that prey capture is not necessarily conserved. This combination of morphometric and behavioral features provides a large phenotype parameter set that will be used as a basis for genotyping to study the degree of polymorphism and to eventually establish a phenotype-genotype map for the inbred lines.
\nohyphens{Cosmological and astrophysical observations indicate the existence of Dark Matter. Many beyond Standard Model theories predict associated production of Dark Matter particles with a Higgs boson at energies that can be probed at collider experiments. The signature of the presented analysis consists of a $b\bar{b}$ pair from a Higgs boson decay recoiling against missing transverse momentum from Dark Matter particles, \hbbmet. The presented \hbbmet search is performed using proton-proton collisions at a centre-of-mass energy of $13$~TeV with an integrated luminosity of $36.1~\text{fb}^{-1}$ recorded with the ATLAS detector at the Large Hadron Collider. Several new refined event selections were developed, which increase considerably the experimental sensitivity of the search to beyond Standard Model theories resulting in a \hbbmet signature. Discovery significance along with its gains from the new refined event selections are presented. The observed data are found to be in agreement with the Standard Model predictions. Upper limits on the production cross-section of $h+\met$ events times the \htobb branching ratio in a two-Higgs-doublet model with an extra $Z^{\prime}$ boson scenario are presented at $95\%$ confidence level. The improvement of the limits from the new refined event selections is quantified. A new topological algorithm is suggested for future analyses and its associated increase in experimental sensitivity is also presented.}
This thesis is concerned with numerical methods for Mixed-Integer Optimal Control Problems with Combinatorial Constraints. We establish an approximation theorem relating a Mixed-Integer Optimal Control Problem with Combinatorial Constraints to a continuous relaxed convexified Optimal Control Problems with Vanishing Constraints that provides the basis for numerical computations. We develop a a Vanishing- Constraint respecting rounding algorithm to exploit this correspondence computationally.
Direct Discretization of the Optimal Control Problem with Vanishing Constraints yield a subclass of Mathematical Programs with Equilibrium Constraints. Mathematical Programs with Equilibrium Constraint constitute a class of challenging problems due to their inherent non-convexity and non-smoothness. We develop an active-set algorithm for Mathematical Programs with Equilibrium Constraints and prove global convergence to Bouligand stationary points of this algorithm under suitable technical conditions.
For efficient computation of Newton-type steps of Optimal Control Problems, we establish the Generalized Lanczos Method for trust region problems in a Hilbert space context. To ensure real-time feasibility in Online Optimal Control Applications with tracking-type Lagrangian objective, we develop a Gauß-Newton preconditioner for the iterative solution method of the trust region problem.
We implement the proposed methods and demonstrate their applicability and efficacy on several benchmark problems.
Das Ziel dieser Arbeit war es, die In-vivo-23Na-Magnetresonanz(MR)-Bildgebung des Körperstamms bei B0 = 7 Tesla zu ermöglichen. Zur Anregung der 23Na-Kernspins sowie zur Detektion der 23Na-Magnetisierung wurde eine 23Na-HF-Körperspule entwickelt, aufgebaut und optimiert. Drei Spulenkonfigurationen der ovalen, eng anliegenden Birdcage-Spule wurden untersucht: Zur Erhöhung der Homogenität des Sende- und Empfangsfeldes wurde im ersten Optimierungsschritt die herkömmliche Zweikanal-Einspeisung zu einer Vierkanal-Einspeisung erweitert. Im zweiten Optimierungsschritt wurde durch eine Anpassung der Sendephasen der relative Flipwinkelfehler in einem Bereich der Größe (23×13×10) cm³ von 8,6 % auf 4,9 % reduziert. Die 23Na-HF-Körperspule mit vier Empfangskanälen stellt ein relativ homogenes Sendefeld ((11,97 ± 0,59) μT, HF-Sendeleistung 2,4 kW) sowie Empfangsfeld zur Verfügung, welche einen großen Bereich des Körperstamms abdecken. Daher ermöglicht die HF-Körperspule erstmalig die Aufnahme von In-vivo-23Na-MR-Bildern der gesamten Breite des Körperstamms eines Erwachsenen mit einer großen Abdeckung in Längsrichtung bei B0 = 7 Tesla (Sichtfeld FOV = (40 cm)³). In den rekonstruierten 23Na-MR-Bildern treten Verschmierungen aufgrund der Atembewegung auf. Daher wurde das intrinsische Atemsignal retrospektiv aus den 23Na-MR-Daten bestimmt. Basierend auf diesem Atemsignal wurden die aufgenommenen MR-Daten in zwei Atemzustände (eingeatmet, ausgeatmet) aufgeteilt, was zu einer Reduktion der Verschmierungen führt. Die Zuordnung basierend auf dem intrinsischen und dem extrinsischen Atemsignal (Atemgurt) zeigte für drei Probanden eine gute Übereinstimmung von (90,6 ± 2,8) % bei der 23Na-Lungen-MR-Bildgebung und von (82,3 ± 3,8) % bei der 23Na-MR-Bildgebung des Abdomens.
The present doctoral thesis focused on the multimodal imaging investigation of brain mechanisms in neuropsychiatric disorders, emphasizing on the research questions of whether and how neurochemistry is associated with brain anatomical structures and brain functions. The aim of the thesis is to provide a biochemical insight underlying the altered brain morphology and functions in the two disorders studied, which might ultimately offer evidence for novel therapeutic implications. There are two brain imaging projects included in this thesis. In project I, the first aim was to explore the mechanism of partial volume recovery during the first two weeks of abstinence from alcohol at a whole-brain level. The hippocampus was then chosen as a seed region, to investigate the abstinence-induced neurochemical changes and whether the hyperglutamatergic state induced by alcohol withdrawal may affect GM volume recovery in the hippocampus. We found cortical thickness alteration corresponds to the partial cortical volumetric recovery. Moreover, alcohol differentially impacts on sulci and gyri of the neocortex. Sulci are more susceptive to excessive alcohol consumption and abstinence-induced recovery. Lower subcortical volume was found in alcohol dependent patients at withdrawal, and no subcortical volume regain was observed during the initial two weeks of abstinence. In support of a ‘hyperglutamatergic state’ induced by withdrawal, our rat model demonstrated elevated Glu/Gln ratios during acute withdrawal (12h and 60h after stopping alcohol intake) and a trend towards an increase in Glu levels at 12h compared to control rats. The main novel finding of this study was that in both species a negative association was found between Glu markers and GM volume in the hippocampus after alcohol withdrawal (but not during withdrawal), suggesting that this tissue damage is a consequence of withdrawal and results from withdrawal-induced hyperglutamatergic neurotoxicity. In project II, the study emphasized the additional value of multimodal imaging analyses to unravel group differences between BPD patients and HCs which could not be detected by BOLD response and ACC GABA levels per se. The superior aim was to explore the interrelationship between GABA, neural correlates of interference inhibition, and impulsivity traits in BPD. We found task-related functional connectivity and the association of fMRI measures with MRS derived GABA levels are significantly different between the two groups. These analyses give support for a disconnection of the fronto-striatal network during interference inhibition in BPD patients that is related to elevated impulsivity ratings, specifically the UPPS sensation seeking score. Our hierarchical analyses also give first evidence for the hypothesis that the fronto-striatal network during inhibitory control serves to mediate the association between ACC GABA levels and impulsivity symptomatology in patients with BPD. In other words, GABAergic transmission in the ACC drives the inhibitory-related fronto-striatal brain network, whereas the disruption of fronto-striatal connectivity is of core relevance to the sensation seeking symptom in BPD patients. Taken together, multimodal imaging fusion analysis of neurobiochemistry - structure/function relationship can offer opportunities to deepen our understanding of neurobiological mechanisms of brain disorders.
The study of dynamic processes that drive the evolution of planet-forming disks is fundamental to understand the origin and diversity of planetary systems. This requires observations at high spatial resolution and sensitivity, which nowadays typically reveal intriguing disk substructures including gaps, rings, spirals, and shadows. This thesis investigates the capability of polarization observations at multiple wavelengths to trace the earliest stages of planet formation. In-depth radiative transfer calculations are carried out in order to link numerical simulations of dust and gas evolution in disks with their observational indicators. This approach demonstrates that measuring polarization is a powerful tool to identify the shaping effects that possible embedded planets have on the density distribution of different dust grain sizes. On the observational part, this work presents several case studies of individual planet-forming disks that were observed with polarimetric imaging by the VLT/SPHERE instrument and subsequently modeled to quantify their structure. A particular focus is the characterization of spiral and ring/gap structures in the context of dust growth, planet-disk interactions, and dust dynamics near ice lines. Furthermore, a modeling study of marginally gravitationally unstable disks is presented to study the influence of the disk self-gravity on the shape and contrast of planet-induced spiral arms in scattered light images. Additionally, it is demonstrated that polarized emission of disks at millimeter wavelengths can be caused by self-scattered thermal dust emission. It is shown that the latter is a viable method to constrain grain properties and identify dust concentrations of different origin. New ALMA observations are presented that offer the first look at a dust trap in polarized scattered light in the sub-millimeter range.
Advances in semiconductor manufacturing still lead to ever decreasing feature sizes and constantly allow higher degrees of integration in application specific integrated circuits (ASICs). Therefore the bandwidth requirements on the external interfaces of such systems on chips (SoC) are steadily growing. Yet, as the number of pins on these ASICs is not increasing in the same pace - known as pin limitation - the bandwidth per pin has to be increased.
SerDes (Serializer/Deserializer) technology, which allows to transfer data serially at very high data rates of 25Gbps and more is a key technology to overcome pin limitation and exploit the computing power that can be achieved in todays SoCs. As such SerDes blocks together with the digital logic interfacing them form complex mixed signal systems, verification of performance and functional correctness is very challenging.
In this thesis a novel mixed-signal design methodology is proposed, which tightly couples model and implementation in order to ensure consistency throughout the design cycles and hereby accelerate the overall implementation flow. A tool flow that has been developed is presented, which integrates well into state of the art electronic design automation (EDA) environments and enables the usage of this methodology in practice.
Further, the design space of todays high-speed serial links is analyzed and an architecture is proposed, which pushes complexity into the digital domain in order to achieve robustness, portability between manufacturing processes and scaling with advanced node technologies. The all digital phase locked loop (PLL) and clock data recovery (CDR), which have been developed are described in detail.
The developed design flow was used for the implementation of the SerDes architecture in a 28nm silicon process and proved to be indispensable for future projects.
Case-control association studies in human genetics and microbiome pave the way to personalized medicine by enabling a personalized risk assessment, improved prognosis, or allowing an early diagnosis. However, confounding due to population structure, or other unobserved factors, can produce spurious findings or mask true associations, if not detected and corrected for. As a consequence, underlying structure improperly accounted for could explain lack of power or some unsuccessful replications observed in case-control association studies. Besides, points considered as outliers are commonly removed in such studies although they do not always correspond to technical errors. A wealth of methods exist to determine structure in genetic and microbiome association studies. However, there are few systematic comparisons between these methods in the frame of genetic or microbiome association studies, and even less attempts to apply robust methods, which produce stable estimates of confounding underlying structure, and which are able to incorporate information from outliers without degrading estimates quality. Consequently, the aim of this thesis was to detect and control robustly for underlying confounding structure in genetic and microbiome data, by comparing systematically the most relevant standard and robust forms of principal components analysis (PCA) or multidimensional scaling (MDS) based methods, and by contributing new robust methods. Own contributions include robustification of existing methods, adaption to the genetic or to the microbiome framework, and a dimensionality exploration and reduction method, nSimplices. Analysed datasets include a first synthetic example with a low-variance 2-groups confounding structure, a second synthetic example with a simple linear underlying structure, genome-wide single nucleotide polymorphism (SNP) from 860 case and control individuals enrolled in the European Prospective Investigation into Cancer and nutrition (EPIC prostate), and finally, 2 255 microbiome samples from the human microbiome project (HMP). Synthetic or real outliers were added in the second example and in EPIC and HMP datasets. All meaningful existing and contributed methods were applied to the EPIC and HMP datasets, while a restricted set was applied to the synthetic, illustrative examples. The 10 principal components or top axes resulting from each method were kept for further analysis. Quality of a method was assessed by how well these axes summarized the underlying structure (using Akaike's information criterion -AIC- from the regression of the 10 axes on known underlying structure in the data), and by how robust the estimates stayed in the presence of outliers (adjusted R2 from the regression of each outlier-disturbed axis on the original axis). In synthetic example 1, only ICA was able to uncover the low-variance confounding structure, whereas PCA or MDS failed to do so, in agreement with the fact that these methods detect large rather than small variance or distance components. In synthetic example 2, non-metric MDS remained the most representative and robust method when distance outliers are included, while nSimplices combined with classical MDS was the only method to stay representative and robust if contextual outliers are present. In the EPIC dataset, Eigenstrat was the most representative method (AIC of 782.8) whereas sample ancestry was best captured by new method gMCD (unbiased genetic relatedness estimates used in a Minimum Covariance Determinant procedure). Methods gMCD, spherical PCA, IBS (MDS on Identity-by-State estimates) and nSimplices were more robust than Eigenstrat, with a small to moderate loss in terms of representativity (AIC between 789.6 and 864.9). Association testing yielded p-values comparable with published values on candidate SNPs. Further SNPs rs8071475, rs3799631, rs2589118 with lowest p-value were identified, whose known role in other disorders could point to an indirect link with prostate cancer. In the HMP dataset, the new method nSimplices combined to data-driven normalization method qMDS mirrored best the underlying structure. The most robust method was qMDS (with nSimplices or alone), followed by CSS and MDS. Lastly, the original method nSimplices performed in all settings at least comparably (except ancestry in EPIC), and in some cases considerably better than other methods, while remaining tractable and fast in high-dimensional datasets. The improved performance of gMCD and qMDS agrees with the fact that these methods use adapted measures (genetic relatedness, selected model distribution, respectively) and recognized robust approaches (minimum covariance determinant and quantiles). Conversely, wMDS is likely to have failed because variance is not an adequate parameter for microbiome data. More generally, different methods report the underlying structure differently and are advantageous in different settings, for example PCA or non-metric MDS were best in some settings but failed in other. Finally, the original method nSimplices proved useful or markedly better in a variety of settings, with the exception of highly noisy datasets, and provided that distance outliers are corrected. Current genetic case-control association studies tend to integrate several types of data, for example clinical and SNP data, or several omics datasets. These approaches are promising but could be subject to increased inaccuracies or replication issues, by the mere combination of several sources of data. This motivates a reinforced use of robust methods, which are able to mirror accurately and steadily genetic information, such as gMCD, nSimplices or spherical PCA. Nevertheless, results on Eigenstrat show this stays a reasonable method. Results in microbiome confirmed that MDS based on proportions is a suboptimal method, and suggested the exponential distribution should be considered instead of multinomial-based distributions, certainly because the exponential better represents the inherent competitiveness between phylogenies in the microbiome. Moreover, illustrative and real world examples showed that methods could capture relevant, but different information, encouraging to apply several complementary methods when starting to explore a dataset. In particular, a low-variance confounder could stay undetected in some methods. Additionally, methods based on least absolute residuals revealed several shortcomings in spite of their utility in a univariate frame, but their expected benefit in a multivariate setting should motivate the development of more tractable implementations. Finally, SPH, IBS, gMCD are recommended methods in a genetic SNP dataset, while Eigenstrat should perform best if no more than 2% outliers are present. To mirror structure in a microbiome dataset, nSimplices (combined with qMDS, or with CSS) can be expected to perform best, whereas MDS on proportions is likely to underperform. Method nSimplices proved beneficial or largely better in various situations and should therefore be considered to analyse datasets including, but not limited to, genetic SNP and microbiome abundances.
Polo-like kinase 1 (Plk1) is a serine-threonine protein kinase widely accepted as one of the master regulators of cell cycle. Overexpression of Plk1 is a frequent occurrence in an array of different human tumor types and it is usually correlated with poor prognosis in patients. However, very little is known about the exact role of Plk1 in tumorigenesis. Here, we use inducible mouse models to determine the in vivo consequences of Plk1 overexpression. During this study, we established that Plk1 is not an oncogene and overexpression of Plk1 has a strong tumor suppressive effect on Her2 or Kras driven breast cancer. Furthermore, tumors with elevated levels of Plk1 displayed evidence of whole genome doubling coupled with increased levels of aneuploidy. Histological characterization of murine mammary glands prior to tumor development affirmed a correlation between Plk1 overexpression and increase in genomic content at an early stage. Utilizing in vitro culture systems, we demonstrate that overexpression of Plk1 leads to reduced proliferation and causes polyploidy. Time-lapse imaging of mammary organoid cultures and mouse embryonic fibroblasts (MEFs) overexpressing Plk1 revealed that the polyploid cells originate due to impaired chromosome segregation as well as a failure of cytokinesis occurring during mitosis. Increased mitotic aberrations and supernumerary centrosomes accompanied these defective cell division processes. Mechanistically, the observed phenotype can be partially attributed to decrease of Shugoshin 1 (Sgo1) a target of Plk1, which is responsible for maintaining cohesion at the centromeres thus holding the sister chromatids together prior to anaphase. Premature loss of Sgo1 during prometaphase caused a partial or complete loss of cohesion. In this dissertation, we report that one of the major consequences of Plk1 overexpression is a staggering tumor suppressive effect, although this does not necessarily negate the possibility that Plk1 could promote tumor development under a different set of circumstances. Despite these findings, Plk1 inhibition in human tumors could still have a beneficial outcome because the kinase is indispensable for cell division. This work is not aimed at discouraging further research on Plk1 inhibitors; rather it provides a testament to the use of genetically engineered mouse models (GEMMs) that closely mimic human disease for pre-clinical testing. It is imperative to understand the molecular mechanisms and cellular processes leading up to tumor onset prior to the development of therapeutic strategies.
Obesity has reached pandemic proportions, and is associated with several co-morbidities like cardiovascular disorders, hypertension, dyslipidemia, and Type 2 diabetes mellitus. The dysregulated adipose tissue metabolism in obesity leads to an altered adipokine secretion, which can influence energy homeostasis, promote insulin resistance, as well as provide pro-tumorigenic signals to tumor cells. We investigated the link between obesity, and the progression of pancreatic ductal adenocarcinoma (PDAC). Although, obesity has been associated with a poor prognosis in PDAC, the underlying mechanisms are unclear.
At first, we used adipose tissue specific knockout of TBLR1 (ATKO) mice as a model of obesity, to investigate if the transcriptional inactivation of the transcriptional co-factor Transducin beta like related 1 (TBLR1) promoted PDAC progression. Although, subcutaneous (SC) Panc02 tumors grew significantly larger in the ATKO mice, compared to wt littermates, histological examination of the tumors did not reveal increased proliferation, or adipocyte infiltration in the tumors from ATKO mice. Also, gene expression analysis in tumors from wt and ATKO mice did not reveal any significantly differentially regulated genes. Finally, in vivo effects could not be validated by treating PDAC cells with conditioned media (CM) generated from primary adipocytes of ATKO mice, or from Tblr1 knockdown 3T3-L1 adipocytes. Therefore, we could not establish a robust link between the absence of Tblr1 in the adipose tissue, to PDAC progression.
Using in vitro assays, we demonstrated that adipocyte-conditioned media (ACM) from differentiated 3T3-L1 adipocytes, but not fibroblasts, promoted PDAC cell viability, proliferation, migration, and invasion in vitro. Therefore, we hypothesized that secreted factors from adipocytes mediate pro-tumorigenic effects on PDAC. Previously it was shown by our group that SC Panc02 tumors grew significantly larger in db/db mice, compared to wt mice. Microarray analysis of the SC inguinal white adipose tissue (iWAT) of db/db, and wt mice identified ob gene encoding leptin as one of the most significantly differentially regulated genes. Although, recombinant leptin did not affect proliferation, or migration of PDAC cell monolayer cultures, it increased the invasion of BxPC3 spheroids through collagen matrix. Most importantly, leptin increased the viability of PDAC cells upon treatment with gemcitabine. Thus, we hypothesized that leptin improves the chemo-resistance of PDAC cells to gemcitabine. However, knockdown of Lepr in PDAC cells did not affect proliferation, and viability upon gemcitabine treatment. Also, Lepr kd Panc02 tumors implanted subcutaneously into wt mice, displayed similar chemo-sensitivity to gemcitabine treatment in vivo, as compared to wt Panc02 tumors. Finally, pharmacological inhibition of human LEPR with anti-LEPR antibody did not affect the chemo-resistance of PDAC cells to gemcitabine.
In conclusion, although there was a strong effect of ACM from 3T3-L1 cells on PDAC growth in vitro, the effects could not be attributed entirely to leptin. Further investigation is required to identify the secreted factors from dysregulated adipocytes which could play a potential pro-tumorigenic role on PDAC progression, thus providing a link between obesity and PDAC development.
Aging is a complex process that is associated with changes in many parts of the body over the lifespan of an individual. In this work, various aspects of aging in the human bone marrow were investigated. As the regenerative power of a tissue is linked to the potential of its stem cells to replace the accumulated damages, the aging process in somatic stem cells was studied focusing on the influence of the niche in regulating stem cell aging. The hematopoietic stem cells (HSC) together with elements that constitute the bone marrow niche were investigated as a model for somatic stem cell aging as HSCs are accessible in healthy human individuals. The subjects ranged from 20 to 60 years with a median age of 33.2 years. From each bone marrow sample, the CD34+ population as HSCs and four other cell subpopulations, lymphocytes and precursors (LYM), monocytes/macrophages precursors (MON), granulocytic (GRA), and erythroid precursors (ERP) were isolated by flow cytometry. The mesenchymal stromal cells (MSC) were isolated by in vitro culture. We found that the relative proportions, cell size as well as cell granularity of the major bone marrow constituents did not correlate with the biological age of the donors. However, further downstream analysis indicated that age-associated changes were prominent on protein level in HSCs as well as in other cell types of the niche such as MSCs. The interactions between the HSCs and the niche were studied in vitro using a coculture system of CD34+ cells and mesenchymal stromal cells (MSC). As previous studies indicated that the supportive function of MSCs as well as their differentiation potentials towards adipocytes and osteocytes change significantly with age, we have examined the supportive ability of the undifferentiated MSCs versus adipogenically differentiated MSCs (ADI-MSCs) and osteogenically differentiated MSCs (OST-MSCs) for HSCs. We showed that MSCs, ADI-MSCs and OST-MSCs were able to support the proliferation of HSCs and maintain their primitive immunephenotype. Compared to undifferentiated MSCs and OST-MSCs, the co-culture with ADI-MSCs increased the proliferation of HSCs much stronger while still maintaining the HSCs at a high expression level of CD34. As the impact of the MSCs on HSCs might be caused by epigenetic changes, the aging-associated alterations in the marrow niche were studied at the chromatin level. To this end, changes in chromatin accessibility were studied in MSCs by ATAC-seq. After establishing the protocol for performing ATAC-seq using primary MSCs, we studied the MSC samples derived from 16 healthy human subjects of different ages between 21 and 59 years. A set of 122,884 ATAC-seq peaks was identified. We have demonstrated that donor age is associated with alterations in open chromatin profiles. Moreover, at a false discovery rate of 5%, we could identify 4,579 differential chromatin accessible sites upon aging. A functional analysis of these sites showed enrichment of cell development and differentiation processes. Additionally, genes of the hippo signaling pathway, TGF-beta signaling pathway, cancer pathways and cell adhesion pathways were also found to be enriched. A motif enrichment analysis suggested that TATA box motifs and binding sites for transcription factors TFAP2C, KLF16, HIC1.p2, WT1 and MTF.p2 were enriched in promoter regions of differential chromatin accessible sites upon aging. In conclusion, this study showed that the interplay with the stem cell niche controls HSC functions. The differentiation of MSCs affects the proliferation and stemness of HSCs in vitro. Furthermore, we have demonstrated that aging is associated with chromatin accessibility alterations in MSCs, which provides a foundation for further in-depth mechanistic analyses.
Electro-optical deflectors provide a very attractive means of laser scanning in coordinate-targeted super-resolution microscopy due to their high scanning precision and high scanning velocity. Setups equipped with electro-optical deflectors demonstrate especially high resolving powers, fast imaging and reduced photobleaching.
Two major shortcomings limit a widespread application of such devices. Their polarizing properties prevent de-scanning causing either a loss in signal or an increased background signal and the restricted deflection angles severely narrow the field of view.
Herein, I report solutions to both of these problems. The polarization issue is evaded via a passive polarization rectifier that allows unpolarized light to pass the laser scanner. The field of view is nearly doubled through a digital light deflector composed of a Pockels cell and a Wollaston prism. This principle could be extended by N stages of the same kind yielding a field of view enlargement by a factor of 2^N. Thus, the work at hand paves the way for ultrafast electro-optical laser scanning with a large field of view.
In dieser Arbeit werden die chemische Zusammensetzung, die Orientierung und der Ladungstransfer an Grenzflächen mit Hilfe von Infrarotspektroskopie im mittelinfraroten und ferninfraroten Bereich untersucht. An der Anodengrenzfläche wurde der Einfluss der Modifikation von Indiumzinnoxid durch flüssigprozessiertes Nickeloxid (sNiO) und thermisch verdampftes Molybdänoxid (MoO3) auf das Donormaterial, in diesem Fall fluoriertes Zinkphtalocyanin (F4ZnPc), analysiert. Die Messungen wurden in situ während des Aufdampfvorgangs des F4ZnPc durchgeführt. So konnte eine chemische Veränderung des Moleküls an der Grenzfläche zu ITO und sNiO festgestellt werden und ein Ladungstransfer, aber keine chemische Veränderung auf MoO3. Der Ladungstransfer führte zur Bildung des F4ZnPc-Kations, wobei sich eine Raumladungszone mit einer Ausdehnung von 8nm formte. Die Orientierung der Moleküle in der F4ZnPc-Schicht wurde durch die Modifikation für Schichtdicken über 20nm nicht signifikant beeinflusst. Auf der Kathodenseite wurden selbstorganisierende Monolagen (SAMs) aus Dimethylamin- Biphenyl-Phosphonaten dazu verwendet die Austrittsarbeit zu verkleinern, aber nicht den Kontaktwinkel von ITO zu verändern. Die Moleküle der SAM wurden zunächst auf ITO charakterisiert und dabei Neigungswinkel, Austrittsarbeitsänderung und Kontaktwinkel bestimmt. Danach konnte der organische n-Typ Halbleiter N,N’-bis(2- phenylethyl)Perylen-3,4,9,10-bis-(dicarboximid) (BPE-PTCDI) aufgedampft werden und im IR vermessen werden. Eine Korrelation zwischen Orientierung des BPE-PCTDI und der Kontaktwinkeländerung des Substrats konnte gefunden werden. Außerdem wurde die elektronische Wechselwirkung zwischen ITO/ SAM und BPE-PTCDI gemessen, die mit der Austrittsarbeitsänderung durch die SAM einhergeht, und substratabhängig ist.
Nоn-smаll-cell lung cаncer (NSCLC) іs the leаdіng cаuse оf cаncer-relаted mоrtаlіtіes wоrldwіde. NSCLC cаn be further subdіvіded іn lung аdenоcаrcіnоmа (LUАD) аnd lung squаmоus cell cаrcіnоmа (LUSC). Elevаted levels оf the Trаnsfоrmіng Grоwth Fаctоr betа (TGFβ) lіgаnd cоrrelаte wіth а рооr survіvаl оf lung cаncer раtіents. Hоwever, the underlуіng mechаnіsms cоntrіbutіng tо the рrо-tumоrіgenіc effects оf TGFβ sіgnаl trаnsductіоn іn lung cаncer аre рооrlу understооd. Fіrst, the аctіvаtіоn stаtus оf the TGFβ sіgnаl trаnsductіоn wаs exаmіned аt the рrоteіn аnd mRNА level іn tumоr tіssue оf lung cаncer раtіents. Thіs аnаlуsіs reveаled thаt the cоmроnents оf the TGFβ раthwау were substаntіаllу аctіvаted іn humаn lung cаncer tіssue wіth cоncоmіtаnt eріgenetіc sіlencіng оf а negаtіve regulаtоr оf the TGFβ раthwау, the decоу рseudоreceрtоr BАMBІ. Recоnstіtutіоn оf BАMBІ іn NSCLC cells decreаsed TGFβ-іnduced Smаd2/3 рhоsрhоrуlаtіоn аnd TGFβ-іnduced іn vіtrо cell mіgrаtіоn аnd іnvаsіоn. Furthermоre, BАMBІ recоnstіtutіоn reduced the іn vіvо metаstаtіc роtentіаl оf NSCLC cell lіnes аs аssessed bу а mоuse lung cоlоnіzаtіоn аssау. These results demоnstrаted thаt the lоss оf the negаtіve regulаtоr BАMBІ рrоmоtes the аctіvаtіоn оf the TGFβ раthwау аs well аs TGFβ-drіven іnvаsіveness оf lung tumоrs. Secоnd, mоleculаr cоmроnents cоntrіbutіng tо TGFβ-іnduced lung cаncer рrоgressіоn were іnvestіgаted. The dуnаmіcs оf gene exрressіоn іn TGFβ-treаted LUSC cells wаs аssessed bу next-generаtіоn mRNА sequencіng. The exаmіnаtіоns reveаled uр-regulаtіоn оf mоtіlіtу- аnd аctіn cуtоskeletоn-relаted genes іncludіng the nоn-muscle mуоsіn 10 (MYO10). Knоckdоwn оf MYO10 аbrоgаted TGFβ-іnduced cоllаgen gel іnvаsіоn оf LUSC cells. Exаmіnаtіоn оf mRNА exрressіоn іn раіred surgіcаllу resected tіssues оf LUSC раtіents shоwed thаt the mRNА exрressіоn rаtіо оf MYO10 іn tumоr аnd tumоr-free tіssue іs рrоgnоstіc fоr раtіent оverаll survіvаl аnd fаcіlіtаtes the рredіctіоn оf the resроnse оf these раtіents tо аdjuvаnt chemоtherару. Hаvіng estаblіshed thаt the TGFβ sіgnаl trаnsductіоn раthwау іs deregulаted іn lung cаncer, іt іs іmроrtаnt tо understаnd hоw the dуnаmіc рrорertіes оf the раthwау аre cоntrоlled. The аnаlуsіs оf TGFβ-іnduced sіgnаl trаnsductіоn іn three LUАD cell lіnes shоwed а dіstіnct dуnаmіc behаvіоr оf the TGFβ-іnduced Smаd2/3 рhоsрhоrуlаtіоn аnd а dіfferentіаl іmраct оf іnhіbіtоr рerturbаtіоns. These results suggested а dіfferent рrevаlence оf negаtіve feedbаcks thаt іnduce the degrаdаtіоn оf the TGFβ receрtоr оr reduce іts аbіlіtу tо рhоsрhоrуlаte Smаds іn the exаmіned cell lіnes. The mоdel-bаsed аnаlуsіs рredіcted thаt the TGFβ receрtоr рrоteіn undergоes cоnstаnt turnоver: the unstаble receрtоr рrоteіn іs cоnstаntlу degrаded аnd рrоduced аgаіn frоm the stаble receрtоr mRNА. Hіgh stаbіlіtу оf the TGFβ receрtоr mRNА wаs cоnfіrmed bу mRNА hаlf-lіfe аnаlуsіs, whіle the аccumulаtіоn оf the TGFβ receрtоr рrоteіn uроn іnhіbіtіоn оf the рrоteаsоme functіоn wаs vаlіdаted usіng tаrgeted quаntіtаtіve mаss sрectrоmetrу. These fіndіngs hіghlіghted thаt the TGFβ receрtоr іs оne оf the mоst sensіtіve nоdes thаt cоntrоls раthwау аctіvаtіоn. Therefоre, tаrgetіng рrоcesses thаt cоntrоl receрtоr аbundаnce rаther thаn usіng cоnventіоnаl TGFβ receрtоr kіnаse іnhіbіtоrs cоuld be а рrоmіsіng therарeutіc аррrоаch. Tаken tоgether, the рresented wоrk рrоvіdes іnsіghts іntо mоleculаr аlterаtіоns thаt cаuse TGFβ раthwау deregulаtіоn, suggests new роtentіаl bіоmаrkers аnd shоwcаses the роtentіаl оf the mаthemаtіcаl mоdelіng аррrоаch cоmbіned wіth quаntіtаtіve exрerіments tо uncоver generаl рrіncірles оf cell tурe-sрecіfіc regulаtіоn оf TGFβ-іnduced Smаd2/3 рhоsрhоrуlаtіоn іn lung cаncer cell lіnes.
Paleoceanographic reconstructions are fundamental for our understanding of past climate variabilities and have mainly focused on changes of circulation and environmental conditions in the surface or deep ocean. The thermocline (100 – 1000 m), acting as a link and buffer between the well-mixed warm surface and the slow and cold deep water, has been largely overlooked. In this study two cold-water coral (CWC) bearing cores from two depths in the thermocline of the southern Gulf of C´adiz (sGoC) were analysed in a mutli-proxy approach. U-series dated glacial CWCs were analysed for recorded Li/Mg temperatures, water mass 14C reservoir ages and radiogenic Nd isotope signatures (epsilonNd). For the first time, a CWC epsilonNd record was extended by two independent co-located hemipelagic sediment records. A seesaw pattern for the glacial sGoC intermediate waters alternating between predominant Eastern North Atlantic Central Water (ENACW) and Eastern Antarctic Intermediate Water (EAAIW) is proposed. Glacial ENACW and EAAIW both exhibited polar temperatures (~0°C) and more radiogenic epsilonNd signatures (~-9) than nowadays and were distinguishable by their reservoir ages, with better ventilated glacial ENACW than EAAIW. A compilation of existing CWC mound aggregation records allowed for a first estimate of initial CWC settlement in the ocean around 3.4Ma ago, coinciding with the mid-Pliocene warm period and the onset of Northern Hemisphere glaciation. Additionally, Ba isotopes (delta138/134Ba) recently introduced for seawater, was calibrated for the use in CWCs. This lays the foundation for the missing nutrient or surface biological productivity proxy in fossil CWCs.
In vielen Organismen nimmt die Verstoffwechselung von molekularem Sauerstoff eine zentrale Rolle im Energiestoffwechsel ein. Ziel dieser Arbeit war es, die dynamische Magnetresonanztomographie (MRT) des Sauerstoffisotops 17O zur quantitativen und lokalen Bestimmung des zerebralen Sauerstoffumsatzes (CMRO2) mittels eines Inhalationsexperimentes bei einer Magnetfeldstärke von B0=7T weiterzuentwickeln sowie in ihrer Robustheit und Genauigkeit zu evaluieren. Die physikalischen Eigenschaften des 17O-Atomkerns verlangen spezielle radiale Auslesetechniken und erlauben nur geringe räumliche Auflösungen. Die daraus resultierenden Partialvolumeneffekte(PVE) verfälschen die Signalwertbestimmung und vermindern die erreichbare Präzision bei der funktionellen Quantifizierung. Daher wurde ein PVE-Korrekturalgorithmus etabliert, der eine verbesserte Konzentrations- sowie T2*-Bestimmung erlaubt. Für Inhalationsexperimente wurde ein Aufbau bestehend aus einem Gasapplikationssystem und optimierter Hochfrequenz-Sende- und Empfangsantenne konzipiert. Des Weiteren wurde für die 17O-MRT die Korrekturstabilität und erreichbare zeitliche sowie nominelle räumliche Auflösung von (4,5mm)3 verifiziert: eine temporäre Abtastrate von 1:00 min erlaubt eine Genauigkeit der Konzentrationsbestimmung innerhalb von 5–9% des Literaturwertes. Die Analyse des dynamischen Signal- und Korrekturverhaltens mittels Simulationen führte zum Protokoll eines In-vivo-Inhalationsexperiments. Die quantifizierten funktionellen Parameter einer Probandenstudie waren CMRO2,GM=(2,03–2,52)±0,14μmol/g min (graue Substanz, GM) sowie CMRO2,WM=(0,61–0,74)±0,08μmol/g min (weiße Substanz, WM) und lagen damit im GM-Kompartiment höher als in vorherigen Studien. Die Ergebnisse zeigten eine geringe Variation über alle Messungen und demonstrierten dadurch die Reproduzierbarkeit der vorgeschlagenen Methode der dynamischen 17O-MRT.
Alterung ist ein hoch komplexes und streng reguliertes Zusammenspiel zwischen genetischen und umweltlichen Faktoren in lebenden Organismen. Auf zellulärer Ebene ist der Alterungsprozess begleitet von zahlreichen Modifizierungen, einschliesslich Veränderungen der posttranslationalen Proteinmodifikationen (PTM). Mit einem quantitativen proteomischen Ansatz ist das Ziel dieser Arbeit unser jetziges Verständnis von Alterung in der Knochenmarknische zu erweitern, mit einem Fokus auf die Rolle der PTM. In der folgen den Arbeit wird das menschliche Knochenmark in sechs Zellsubpopulationen unterteilt. Veränderungen des Proteoms wurden für 59 Spender untersucht, deren Altersunterschied 40 Jahre beträgt. Modifizierte Proteine wurden identifiziert durch Kombinierung von Masse-tolerant und Standard Datenbank Suchmethoden. Die Analyse zeigte, dass PTM im Knochenmarkproteom weitverbreitet und abhängig vom Zelltyp sind. Zu den unterschiedlichen modifizierten Proteinen gehören Proteine der Glykolyse und Proteine der heterogenen Ribonukleinfamilie. Zusätzlich zeigte die altersabhängige Assoziierungsanalyse der modifizierten Proteine, dass das Modifizierungsniveau für die Mehrheit der modifizierten Proteine während des Alterungsprozesses stabil blieben. Die acetylierten Proteine in den hämatopoetischen Progenitoren korrelierten negativ mit dem Alter, was vielleicht auf das reduzierte Differenzierungspotential der Stammzellen zurückzuführen war. Koregulierungen der modifizierten Proteine während des Alterungsprozesses wurden ebenfalls in den Knochenmarkzellen, vor allem aber in den hämatopoetischen Progenitoren beobachtet. Die Ergebnisse dieser Arbeit könnten als Basis dienen für die Aufklärung biologischer Mechanismen, in denen die beschriebenen PTM die altersassoziierten Prozesse in unterschiedlichen Knochenmarkzellen regulieren.
Galaxies act like engines converting gas into stars, which in turn produce the matter around us, laying the foundations of life. Studying the composition of this interstellar gas informs our understanding of how star formation proceeds, while also providing insight into the structure and evolution of our own Galaxy and the Universe. Research has found a strong connection between star formation and the molecular gas within galaxies. However, the most easily accessed molecular observables, such as carbon monoxide (CO) emission, only probe low-density gas in extragalactic systems, allowing us to scratch the surface of the star-forming structures.
Molecules which trace denser gas, such as hydrogen cyanide (HCN), are more challenging to observe but probe the immediate sites of star formation. In this thesis I analyze data from the first survey mapping the entire star forming disk of a sample of nearby galaxies in a suite of dense gas tracers (EMPIRE), to understand the interplay between dense gas and a wide range of galactic environments, distinct from the Milky Way. By studying the content of this dense gas in nearby galaxies I find surprising results: systematic variations of the dense gas fraction and its efficiency to form stars within and among galaxies. While more dense gas is located in regions of high interstellar pressure, this dense gas is less efficient to form stars.
To characterize the dense gas, I also explore how changes in the optical depth can affect the effective gas densities where the dense gas tracers emit, a new measurement in the disks normal star-forming galaxies. To better understand the bulk, lower-density molecular gas out of which the dense gas eventually forms, I also analyze CO line ratios to constrain carbon isotope abundances. I find the abundance to vary systematically within galaxy disks, likely due to strong fractionation effects. The results obtained in this thesis support a model where star formation depends strongly on host galaxy and the local galactic environment in the disk.
This thesis explores the implementation of a spin-1/2 system to realise quantum simulation of Heisenberg XX and XXZ models. The spins are mapped onto two high-lying atomic levels, so-called Rydberg states, in an ultracold sample of 87Rb and coupled by a microwave field. Efficient synthesis and control of the driving field has been introduced in the setup in order to probe the spin dynamics with NMR sequences. Two- and three-photon excitation schemes are implemented to prepare the Rydberg spins. In order to spatially resolve the Rydberg excitation dynamics, a new imaging technique is employed, which uses the depletion of absorption in presence of Rydberg atoms to detect their distribution in the atomic cloud, revealing the emergence of spatial order of the Rydberg excitations due to strong van der Waals interactions. To benchmark the validity of this platform, the coherence of the spin ensemble is measured by Ramsey techniques in the low-density regime, where the single-spin dynamics accurately describes the observations. Despite the black-body redistribution of Rydberg spins setting a limit for the T2* time of the spin system, the coherence is measured to persist over long timescales on the order of 130 μs. Thus, scaling up the density of spins, first signatures of dipolar many-body effects for |nS⟩ − |nP⟩ (XX) and |nS⟩ − |(n + 1)S⟩ (XXZ) spin combinations have been observed.
The appearance of bubbles was determined in three different wind wave facilities utilizing an optical bright field technique. Based on the bubble detection algorithm by W. Mischler [20], an algorithm has been developed by means of which bubbles and their sizes can be detected automatically. The method is applicable up to moderate to high bubble densities. At the Aeolotron bubble detection up to the water surface is feasible. It has been shown that the density of bubbles correlates to wind speed, water conditions, the distance to the water surface as well as to the tank geometry. Observations show an increased bubble density in salt water compared to fresh water at the same wind conditions. A study on the influence of lower Butanol concentrations of about 50ml/m^3 was carried out. Low Butanol concentrations in the water lead to an increased density of bubbles and additionally, as could be shown in the Aeolotron, a significant increase of very small bubbles (r<65µm). Adding Butanol seems to be a good, non-corrosive substitute for seawater. The influence of the fetch length was systematically researched by carrying out measurement series in the annular wind wave facility in Heidelberg as well as measurement series in linear wind wave facilities in Marseille, France and Kyoto Japan, both of which have different sizes and fetch lengths. Comparing these three wind wave facilities a high correlation between the appearance of bubbles and the fetch length could be detected. Analysis of fresh water measurements at hurricane like wind conditions lead to the discovery of a saturation effect on the appearance of bubbles.
The trypanosomatids are a group of eukaryotic parasites (Trypanosoma brucei, Trypanosoma cruzi and Leishmaniab sp.), which causes disease affecting humans and animals. Since most genes do not have individual promoters and regulating transcription factors, the mode of gene expression regulation is post-transcriptional: almost entirely determined by RNA binding proteins, stabilitzing complexes and the mRNA decay machinery. In my PhD, I worked on two projects in Trypanosoma brucei; a screen to identify the genome wide interactors of the CAF1-NOT complex and the role of RNA binding protein ZC3H30 in stress. The decay-promoting deadenylation machinery, the CAF1-NOT complex, degrades poly (A) tails, an event that marks mRNA for degradation. I did a genome-wide yeast-two-hybrid screen to discover novel proteins that might influence CAF1-NOT complex function in the parasite. I looked at the total pool of genes obtained after screening, and assessed the protein class and biological process of the candidates obtained. To assess interaction specificity I compared the yeast-two-hybrid screen data with those obtained from other trypanosome proteins. African trypanosomes undergo stress in their insect host in the form of heat shock or in their human host from the immune reponse, or during fever or intake of drugs. The parasites have developed differential gene expression to cope with stress using RNA binding proteins, The second part of the manuscript focuses on a zinc finger RNA binding protein ZC3H30, which is inessential for the growth of trypanosomes under normal conditions. However, conditions like heat shock, ER stress, oxidative stress and starvation stress, are lethal for the trypanosomes that lack ZC3H30. The absence of ZC3H30 has no effect on stress markers, except that major cytoplasmic HSP70 mRNA is more abundant. I have shown that ectopically expressed ZC3H30, when tethered to reporter mRNA, can reduce its transcript and encoded protein abundance. ZC3H30 is a bona fide component of stress granule; in presence of heat shock, ZC3H30 re-localises to stress granules from the cytoplasm. It pulls down another protein, which also migrates to stress granules upon heat shock and might also have a role in stress management for trypanosomes. By transcriptome profiling, procyclic cells lacking ZC3H30 showed no difference from wild type in transcript abundance, and specific targets were not found. However, procyclic form trypanosomes have a higher higher overall translation rate than wild type cells. Possibly ZC3H30 affects the mis-reading of mRNAs during translation, since the knockouts of ZC3H30 are less sensitive than wild type to aqminoglycoside translation inhibitors like hygromycin. Nevertheless, I have not been able to identify definitively the role of ZC3H30 in stress resistance.
Microsatellite-unstable (MSI) cancers occurring in the context of the hereditary Lynch syndrome or as sporadic cancers elicit pronounced tumor-specific immune responses. The pronounced immune response was shown to be closely associated with frameshift peptides (FSP) that are generated as a result of deficiency in DNA mismatch repair system leading to insertion/deletion mutations in coding microsatellites (cMS). FSP neoantigens are long antigenic amino acid stretches that bear multiple epitopes to be presented. There is no central tolerance against FSPs, and shared FSPs derived from driver mutations are promising candidates for vaccination approaches to treat or prevent MSI cancers. In the present thesis, the main goals were to set up a mouse model for the immunology of MSI cancers and to systematically identify immune evasion mechanisms in MSI cancers. A murine model is essential to characterize alterations of immune responses over time, in all stages of cancer and pre-cancerous stages. In addition, it allows testing an FSP vaccine for efficacy in tumor prevention and treatment, either as a single agent or on combination with other immune-modulatory drugs. To establish such a model, the complete mouse genome was screened and genes bearing cMS were detected. After mutation and expression analysis by using murine Lynch tumors, epitopes of the most promising potential FSP candidates were predicted by using the Syfpeithi and netMHC algorithms. Immunogenicity of the 10 FSPs with the highest ranks was analyzed by vaccinating C57BL/6 mice and analyzing immune responses using IFNg ELISpot. Four FSPs were identified that were highly immunogenic and inducing spot numbers higher than Ova control peptides: Maz (-1) and Senp6 (-1) induced only CD4 T cell responses, Xirp1 (-1) induced only CD8 T cell and Nacad (-1) induced both CD4 and CD8 T cell responses. Peptide-specific IgG Elisa demonstrated that three of the peptides Senp6 (-1), Maz (-1) and Nacad (-1) also induced humoral immune response. Immunogenic regions of the peptides could be mapped to the C-terminus of Senp6 (-1) and Xirp (-1) and to the N-terminus of Nacad (-1), whereas the antigenic region for Maz (-1) spanned almost the entire peptide. These results suggest that the Lynch mouse model is well suitable for evaluating the efficacy of FSP vaccination to treat and even prevent tumors in Lynch syndrome. II The second aim of this thesis was to systematically analyze immune evasion mechanisms in MSI cancer. We first analyzed mutations of genes related to MHC class I antigen presentation in publicly accessible mutation databases. The mutation data of 91 MSI patients in the DFCI cohort showed that 72% of all MSI CRC tumors had defects in MHC class I presentation; displayed by at least one mutation in the corresponding genes (B2M, TAP1, TAP2, HLA-A, HLA-B, HLA-C and NLRC5). Mutual exclusivity analysis revealed that mutations affecting B2M were negatively related to HLA-B mutations, whereas there was a strong positive correlation between HLA class I heavy chain mutations. These results indicate that there is a strong immunoselection in MSI tumorigenesis, leading to immune evasion through mutations of MHC class I-related genes in more than two-thirds of MSI cancers. We identified NLRC5 mutations as a potential novel immune evasion mechanism in the database analysis; therefore, potential consequences of NLRC5 inactivation were further analyzed in MSI colorectal cancer samples. We detected cMS mutations of NLRC5 in 4 out of 95 tumor samples (4.2%), three of them being one-basepair deletions and one silent mutation. Importantly, we detected low levels of MHC class I antigen expression in NLRC5- mutated tumors. One tumor showed partial reduction of MHC class I expression, which colocalized with the NLRC5 mutation. These results suggest NLRC5 mutations as a novel potential mechanism of immune evasion in MSI cancer. Taken together, the present thesis led to the establishment of the first model to evaluate the immune biology of MSI cancers and Lynch syndrome in the murine system. Moreover, it has established a comprehensive overview of immune evasion in MSI cancers, thus contributing to the development of better treatment strategies and potentially to the first cancer-preventive vaccine for non-viral human cancers.
Despite having survived numerous experimental tests, the standard model of particle physics is not a complete description of nature. The Mu3e experiment tests theories beyond the standard model by searching for the lepton flavour violating decay mu->eee, aiming at a branching ratio sensitivity of 2*10^-15 in a first phase of the experiment. A high precision magnetic spectrometer combined with scintillation detectors will measure the momenta, vertices and timing of the decay products of 10^8 mu/s stopped on a target. In this work, a prototype of the high voltage monolithic active pixel sensor envisaged for the spectrometer was characterised. With an efficiency >99% and a time resolution of 14 ns, it meets the requirements imposed on the final sensor. Furthermore, an online signal selection process was developed and implemented on a graphics processing unit (GPU), keeping 98% of signal decays, while reducing the data rate of 80 Gbit/s by a factor of 140; resulting in a rate that can be stored to disk. With the computing performance achieved on the GPU, the selection process can run on the hardware planned for the experiment. Both the online selection and the silicon sensor are key aspects for the success of Mu3e.
Activating BRAF mutations are frequently found in diverse types of cancer, including multiple myeloma. Moreover, current targeted therapy can abrogate mutant BRAF kinase activity although drug resistance mechanisms are still a challenge. However, this mutation has not been functionally characterized in multiple myeloma and biological consequences along BRAF inhibitor resistance mechanisms are unknown.
Here we show that BRAF V600E expression in multiple myeloma leads to an active MAPK/ERK pathway and eventual DNA damage. This mutation can be targeted with BRAF inhibitors (BRAFi). Nevertheless, we demonstrate that BRAF wild type under BRAFi leads to paradoxical activation. To overcome this effect a MEK inhibitor was employed for an effective pathway abrogation in multiple myeloma.
Furthermore, to identify mechanisms that contribute to BRAFiresistance, whole exome sequencing of a BRAFi-resistance model of MM was performed. We identified a novel mutation in the transcription factor EGR-1 followed by its protein functional characterization. This showed that the mutant protein (p. 332fs) along the wild type protein is overexpressed in our in vitro model and mutant EGR-1 influences its own subcellular localization. Overexpression of the mutant EGR-1 conferred complete resistance to BRAF inhibition.
In over 40% of all cancers, the key tumor suppressor p53 is inactivated via mutation. Mutant (mut) p53 can gain new properties (gain-of-function, GOF), which actively contribute to tumorigenesis. In many tumors, a massive accumulation of mutp53 protein is observed and a prerequisite for the GOF activity. Therefore, tumors often depend on sustained high levels of mutp53, which suggests that interfering with mutp53 accumulation may be exploitable in cancer therapy. However, the mechanisms that control excessive mutp53 stabilization are not fully understood. To this end, the main aim of this study was to identify regulators of mutp53 accumulation in Burkitt’s lymphoma (BL) as a model for a highly aggressive cancer.
Despite the presence of functional MDM2, the main negative regulator of p53, mutp53 was found to be stabilized in BL. To identify proteins regulating mutp53 levels in an unbiased fashion, a flow cytometry-based RNA interference (RNAi) screen was conducted in a mutp53 BL cell line model. The primary screen hit was TRRAP (transformation/transcription domain-associated protein), a constituent of several histone acetyltransferase (HAT) complexes. TRRAP knock-down and knock-out resulted in depletion of mutp53 protein (but not mRNA) in lymphoma and colorectal cancer cell lines with a diverse spectrum of p53 mutations. Conversely, TRRAP overexpression increased mutp53 levels. Mass spectrometric analysis of the mutp53 interactome after TRRAP knock-down indicated that TRRAP silencing caused nuclear export of mutp53 and degradation via the MDM2-proteasome axis, suggesting targeting of mutp53 to the physiological p53 degradation machinery. Gene expression profiling after TRRAP knock-down showed a suppression of cell cycle-related genes and an induction of interferon signaling, which however did not contribute to mutp53 regulation. To map functional regions of TRRAP, a CRISPR/Cas9 mutagenesis approach (“CRISPR scanning”) was applied which identified a 109 amino acid region in the N-terminal HEAT repeat region crucial for mutp53 accumulation and cell survival. In wild-type p53 BL cells, TRRAP silencing attenuated p53 stabilization and activity upon genotoxic stress. Finally, to transfer the results from RNAi screening, a drug-based screening was performed and identified that inhibition of histone deacetylases (HDAC) and specifically HDAC1/2/3 decreased mutp53 levels to a surprisingly similar extent as TRRAP knock-down.
In summary, this study identifies TRRAP as a key regulator of p53 levels and links histone-modifying complexes to p53 protein accumulation. Based on the GOF properties of mutp53, this may provide a basis for therapeutic targeting of mutp53 in lymphoma and other cancers.
Nichtlokale Gravitationstheorien sind Versuche Quantenkorrekturen in die Einstein-Hilbert-Wirkung miteinzubeziehen. Dies ist ein eleganter Weg, um offene Fragen der Allgemeinen Relativitätstheorie anzugehen. In dieser Dissertation haben wir hauptsächlich infrarot-relevante, nichtlokale Modifikationen von Gravitation als mögliche Quelle für die beschleunigte Expansion des Universums zu späten Zeiten untersucht. Wir zeigen, dass, wenn diese Korrekturen in der Effektiven Wirkung mit einer infrarot-relevanten Größenordnung auftreten, sie zu einer validen Kosmologie zu späten Zeiten führen können, die sowohl auf dem Hintergrund- als auch den Störungsniveaus mit allen beobachteten Daten übereinstimmt. Überdies können manche dieser Modelle besser mit den beobachteten Daten übereinstimmen als das LambdaCDM Standardmodell. Wir haben auch Probleme untersucht, die mit der theoretischen Formulierung nichtlokaler Gravitationsmodelle assoziiert werden, wie beispielsweise die Rolle der Anfangsbedingungen für die Lösungsräume. Wir haben herausgefunden, dass eine unvorsichtige Behandlung der Anfangsbedingungen zu einem Übersehen mancher physikalisch sinnvoller Lösungen führen kann. Desweiteren haben wir die Frage nach den klassischen Instabilitäten in tensoriellen nichtlokalen Gravitationsmodelle untersucht, woraus wir schließen, dass neue Mechanismen oder Symmetrien eingeführt werden müssen, um die Gültigkeit dieser Modelle zu etablieren.
Hypertension, a chronic and persistent increase in blood pressure, is a modifiable risk factor for, e.g. coronary heart disease, hemorrhagic stroke and end-stage renal failure. Despite the pathophysiological importance of hypertension, understanding of the cellular mechanisms at the onset of this disease remains elusive. During hypertension, vascular smooth muscle cells (VSMCs) are exposed to increased wall stress. This promotes a phenotype shift of the VSMCs to overcome the undue rise in wall stress and eventually reinstate vascular tone. At the cellular level, excessive stretch induces translocation of the mechanotransducer protein zyxin from focal adhesions to stress fibers and to some extent to the nucleus where it indirectly via myocardin-related transcription factor-A (MRTF-A) fine-tunes the expression of mechanosensitive genes. VSMCs isolated from 3-months old zyxin knockout (Zyxko) mice showed a synthetic phenotype. In contrast, only deoxycorticosterone acetate (DOCA)-salt treated very old (18-months) Zyxko mice revealed a prominent vascular phenotype exemplified by failure to develop hypertension, a lower resistivity index, i.e. peripheral resistance, and an altered composition of the extracellular matrix (ECM). The age dependency of the in vivo phenotype argues for a possible compensation by other LIM-domain proteins of the zyxin family. Therefore, the first aim was to analyze whether LPP (lipoma preferred partner) or TRIP6 (thyroid receptor interacting partner 6) might compensate for the loss of zyxin in hypertension-induced arterial remodeling. Like zyxin, LPP but not TRIP6 in vitro translocated from focal adhesions predominantly to actin stress fibers as well as to the nucleus upon exposure to supraphysiological stretch. Interestingly, only the level of LPP protein significantly declined in arteries of very old Zyxko mice. VSMCs isolated from 3-months old Lppko mice functionally mimicked the synthetic phenotype of VSMCs isolated from age-matched Zyxko mice. Although young Lppko mice (3 months old) failed to reveal a synthetic VSMC phenotype in experimental hypertension, their significantly lower resistivity index may point to the presence of some vascular dysfunction at this young age. Overexpression of either Lpp in Zyxko VSMCs or Zyx in Lppko VSMCs fully compensated for their alternate loss of function in restoring the contractile VSMC phenotype. Hence, these findings characterize zyxin as a novel regulator of the phenotypic shift of VSMCs during hypertension-induced arterial remodeling with LPP capable of reinforcing zyxin in preventing this shift to occur. Distinct from VSMCs, terminally differentiated cardiomyocytes (CMs) respond to arterial hypertension by increasing their cell size hence thickness of the ventricular wall (hypertrophy) thereby normalizing wall stress according to the law of Laplace. Following this compensatory CMs hypertrophy, loss of CMs as well as excessive deposition of ECM proteins by cardiac fibroblasts (CFBs) causes cardiac dysfunction. The mechanotransducer zyxin seems to be important for CMs survival. Therefore, the second aim of this work was to study the consequences of the loss of zyxin for cardiac function in DOCA-salt induced experimental hypertension. In fact, 12 months old Zyxko mice showed cardiac dysfunction possibly due to excessive cardiac fibrosis and the resulting stiffening of the heart. To corroborate this finding, a cardiac fibrosis model, where 3, 6 and 12 months old Zyxko and wild type (WT) mice are treated with a high dose of angiotensin II (Ang II) or saline delivered through implanted osmotic mini pumps, was established. In this model, untreated 3 months old Zyxko mice presented with a reduced diastolic but preserved systolic function as compared to age-matched WT mice. Upon Ang II treatment, systolic cardiac function progressively deteriorated in the Zyxko mice with age and this was paralleled by a prominent age-dependent rise in collagen deposition in the heart. The effects of Ang II on both interstitial and perivascular cardiac fibrosis were significantly less pronounced in age-matched WT mice, indicative of the development of a progressive restrictive cardiomyopathy in the Zyxko mice in this model. To identify key molecule(s) responsible for the profuse cardiac fibrosis in Zyxko mice, pro-fibrotic gene expression profiles were studied in adult cardiac fibroblasts (ACFs) isolated from Zyxko or WT mice that were exposed in vitro to transforming growth factor-β1. Prominent expression of connective tissue growth factor, collagens I and III as well as lysyl oxidase (LOX) by the Zyxko ACFs supports the notion that CFBs account for the excessive formation of scar tissue in the heart of these animals. Since LOX augments the formation of insoluble collagen deposits in the heart, namely by cross-linking them, it might be together with the enhanced synthesis of collagens I and III primarily responsible for the exaggerated cardiac fibrosis and hence cardiac dysfunction in the Zyxko mice. Whether the age-dependency of this cardiac phenotype is like the vascular phenotype related to a functional compensation between zyxin and LPP in the CFBs or even CMs remains to be determined. Nonetheless, it is likely that the lack of zyxin leads to an altered CM-ECM interaction facilitating CM apoptosis and in turn or in parallel excessive cardiac fibrosis hence contributing to the observed cardiac dysfunction in adult Zxyko mice. In summary, the findings of this study characterize the LIM-domain proteins zyxin and LPP as redundant protective molecules that help to maintain the contractile VSMC phenotype and thus contribute to impeding the development of hypertension-induced arterial and possibly cardiac remodeling. The temporal difference in the appearance of two distinct hypertension-induced phenotypes (vascular and cardiac) might be attributed to the differential abundance of zyxin and LPP in the respective tissue-resident cells contributing to the pathogenesis. Therefore, analysis of old Lppko as well as Lpp/Zyx double-knockout mice has the potential to shed more light on their possible functional compensation in the context of hypertension-induced cardiovascular remodeling processes.
Eine an unterschiedliche Umweltbedingungen angepasste Durchblutung des Körpers ist essentiell für die Aufrechterhaltung der Körperfunktionen. Die Durchblutung wird dabei vor allem durch Änderungen des Tonus von Arterien und Arteriolen gesteuert. Bei Diabetes kann häufig eine dysfunktionale Autoregulation beobachtet werden, was kardiovaskuläre Komplikationen zur Folge hat. Da der BK-Kanal bei der Regulation des Gefäßtonus als negativer Feedbackmechanismus eine entscheidende Rolle spielt, sollte in der vorliegenden Arbeit untersucht werden, inwieweit eine Fehlfunktion des BK-Kanals zur dysfunktionalen Autoregulation bei Diabetes beiträgt. Mittels isometrischer Myographie isolierter Rattenarterien wurde zunächst die Aktivitätssteuerung des BK-Kanals genauer untersucht. Arterien gesunder sowie diabetischer Ratten des STZ-Modells wurden dabei vergleichend analysiert. Die Inhibierung des BK-Kanals mit IBTX führte bei Arterien gesunder Tiere neben einer Verstärkung der Gefäßkontraktion auch zum Auftreten von Oszillationen des Gefäßtonus. Der kontraktile Grundzustand der untersuchten Gefäße ist also durch phasische Kontraktionen gekennzeichnet, welche durch die Aktivität des BK-Kanals in tonische Kontraktionen umgewandelt werden. Es konnte gezeigt werden, dass die SERCA, Ryanodin-Rezeptoren, die Natrium-Kalium-ATPase sowie L- und T-Typ-Calciumkanäle an der Entstehung und Aufrechterhaltung dieser Gefäßoszillationen beteiligt sind und funktionell mit dem BK-Kanal interagieren, ebenso wie Kv-Kanäle. In den Gefäßen diabetischer Tiere waren die IBTX-induzierten Oszillationen schwächer ausgeprägt. Dies lässt darauf schließen, dass bei Diabetes die Funktion der gap junctions gestört und damit die Synchronisation der glatten Muskelzellen nur unzureichend möglich ist oder aber dass der den Oszillationen in einzelnen Zellen zu Grunde liegende Mechanismus durch den Diabetes in seiner Funktion gestört ist. Ein Einfluss des Diabetes auf die Funktionalität des BK-Kanals konnte hingegen nicht festgestellt werden. In der vorliegenden Arbeit konnte also gezeigt werden, dass die Funktionalität des BK-Kanals von verschiedenen Calcium-Transportprozessen sowie Kv-Kanälen mitbestimmt, dies jedoch nicht durch Diabetes beeinflusst wird. Die Entstehung von Oszillationen glatter Muskelzellen oder die Funktionalität der gap junctions wird jedoch durch Diabetes beeinträchtigt.
Malaria affects almost half of the world`s population causing more than 200 million clinical cases each year and thus, remains one of the major infectious diseases of mankind. The protozoan parasite Plasmodium falciparum transmitted by infected Anopheles mosquitoes is responsible for 99 % of deaths, of which most occur in African children. An effective vaccine is urgently required but not yet available. The malaria vaccine candidate Merozoite Surface Protein (MSP)-1 from P. falciparum is an essential and highly abundant protein on the surface of parasite blood stages, which has been associated with protection against malaria in various epidemiological and immunization studies and is currently tested in a clinical Phase I trial. The aim of this work is to characterize the human humoral and cellular immune response against the vaccine candidate PfMSP-1D in African adults with naturally acquired immunity against malaria and after experimental infection of malaria-naïve volunteers with Pf wildtype sporozoites. Antibodies against Plasmodium merozoites are considered as the main actors of naturally acquired immunity against malaria and can target the parasite via different immune mechanisms, such as direct growth inhibition or opsonization and recruitment of effector cells. By using sera from eleven semi-immune adults from Burkina Faso we show that a few sera were capable of directly inhibiting the growth of P. falciparum blood stages in vitro and MSP-1 specific antibodies partly contributed to this effect. Further antigens presumably responsible for the observed growth inhibitory activity were identified via western blot and mass spectrometry analysis. Furthermore, we demonstrate that serum antibodies from all African individuals could opsonize P. falciparum merozoites, recruit neutrophils and elicit antibody-dependent respiratory burst (ADRB). The antibody level against MSP-1 correlated with ADRB activity and MSP-1 specific antibodies, obtained by affinity-purification, induced neutrophil respiratory burst. Opsonizing antibodies effective via ADRB appear to be mainly cross-reactive and – by affinity purification of specific antibodies and antigen-reversal ADRB – we identified MSP-1 and its largest subunit MSP-183 as important targets. Additionally, we show for the first time that opsonizing antibodies can be elicited in non-human primates by immunization with recombinant PfMSP-1D, suggesting that a MSP-1 based vaccine may function via the induction of ADRB-effective antibodies. Since MSP-1 is initially synthesized in Plasmodium infected hepatocytes at late liver stage, it can be a target of the cellular immune response. Cytotoxic CD8+ T-cells secreting IFNγ are supposed to be the key players eliminating infected hepatocytes. By stimulating PBMCs from semi-immune individuals from Burkina Faso and from malaria-naïve European controls with previously identified HLA-A0201 restricted CD8+ T-cell epitopes within MSP-1D, MSP-1 specific IFNγ-secreting CD8+ T-cells were detected in African adults via IFNγ ELISPOT assay. Aiming at a HLA-independent approach, potential CD8+ T-cell epitopes to frequent HLA-types were predicted within MSP-1D and the IFNγ ELISPOT assay was adapted for the use of full-length MSP-1D protein. Furthermore, the immune response of malaria-naïve volunteers experimentally infected once with cryopreserved P. falciparum wildtype sporozoites during the TUECHMI I study was investigated. Antibodies against MSP-1, MSP-6 and MSP-7 were induced by Pf sporozoite infection as well as CD8+ T-cells, which target the cross-stage antigen MSP-1 at comparable levels as the known pre-erythrocytic antigens LSA-1 and CSP. Overall, this work provides a detailed characterization of different immune mechanisms targeting PfMSP-1D in humans with naturally acquired immunity against malaria or after single experimental infection with Pf sporozoites. Moreover, the established immunological assays can be employed to analyze the immune response induced by vaccination with PfMSP-1D during clinical trials.
Mathematiker und Mediziner haben auf den ersten Blick wenig gemeinsam. Doch wenn sie zusammenarbeiten, können völlig neue Ansätze entstehen - z.B. in der Krebsforschung.
Campus Reporter Nils Birschmann hat mit dem Heidelberger Mathematiker Dr. rer. nat. Marcel Mohr gesprochen, der in seiner Doktorarbeit ein mathematisches Modell für die Prognose von Krebserkrankungen entwickelte: das multiple Myelom.
Der Beitrag erschien in der Sendereihe "Campus-Report" - einer Beitragsreihe, in der über aktuelle Themen aus Forschung und Wissenschaft der Universitäten Heidelberg, Mannheim, Karlsruhe und Freiburg berichtet wird. Zu hören ist "Campus-Report" montags bis freitags jeweils um ca. 19.10h im Programm von Radio Regenbogen (Empfang in Nordbaden: UKW 102,8. In Mittelbaden: 100,4 und in Südbaden: 101,1).
TET enzymes are relatively novel players in the epigenetic regulation of mammalian DNA methylation. They participate in DNA demethylation, but their precise roles in different developmental and disease scenarios are not fully understood. The aim of this work was to investigate the biological roles of TET enzymes in lineage-committed normal and cancer cells. To this end, murine primary cells with genetic deletion of TET enzymes and human cancer cells with recurrent mutations in the cofactor providing isocitrate dehydrogenases (IDH), provoking competitive inhibition of TET enzymes, were analyzed. By characterizing mouse embryonic fibroblasts adipogenic differentiation defects, inefficient activation of genes relevant to adipogenesis and widespread gene deregulation upon TET1/2-deficiency were discovered. Examination of the genome-wide DNA methylation landscape demonstrated the hypermethylation of DNA methylation canyons as a main characteristic of the TET1/2-deficient methylome. Canyons were associated with developmentally important genes and canyon collapse due to hypermethylation coincided with developmental gene deregulation, defective induction of adipogenic markers and the hypermethylation of their promoters. Together, these findings uncovered a novel epigenetic regulatory role in the maintenance of DNA methylation canyons for TET1 and TET2 that is essential for epigenetic programming during differentiation. In the second part of this thesis, published array-based DNA methylation profiles of a large acute myeloid leukemia (AML) patient cohort were used to examine mutant IDH- (mIDH) and TET-dependent DNA methylation changes. This confirmed the known association between mIDH and genome-wide hypermethylation. However, similar global methylation changes were not present in TET2 mutant patients and mIDH carrying patients lacked specific canyon hypermethylation. Intriguingly, neither overexpression of mIDH, nor treatment of a leukemia cell line with D-2-hydroxyglutarate, which is a putative TET inhibitor produced by mIDH, recapitulated the mIDH-associated hypermethylation. Instead, comparison with hematopoietic reference methylomes revealed high similarity between mIDH-associated and myeloid progenitor methylation profiles, suggesting the involvement of differentiation state rather than TET inhibition in the hypermethylation phenotype. These findings implicate a previously unnoted factor in the epigenomic changes of AML cells with mIDH, which may be critical to understand and therapeutically target mIDH-dependent pathogenesis.
To fulfill its complex life cycle Plasmodium needs to cross various tissue barriers and invade specific cell types. Its journey inside the mosquito involves active invasion of sporozoites into salivary glands from where these motile forms can be transmitted to the host. To perform active movement inside the mosquito as well as the skin and liver of the host, sporozoites possess an uncommon form of locomotion termed “gliding motility”. Force required for motility is generated by an actin ‐ myosin motor complex and currently thought to be transduced to the sporozoite environment via surface adhesins belonging to the TRAP family. Sporozoites are curved and highly polarized cells capable of active circular movement in vitro. For the first time, our group has observed collective motion of sporozoites within infected salivary glands of Anopheles stephensi mosquitoes following preparation. Most interestingly we observed them to form circling formations, which we termed “vortices” containing up to a hundred sporozoites as well as “swarms” of two to seven sporozoites gliding closely associated to each other. The first part of my thesis was to reach a deeper understanding of these collective migration phenomenons. Here, I show that vortices and swarms emerge from “resting” stacks of sporozoites that redistribute from the central gland cavity to the gland periphery during the preparation process and actively start to migrate individually at the basal membrane surrounding the gland. I further observed vortices to form over several minutes and be stable for hours whereas swarms form in the range of seconds and are stable for up to several minutes. Analysis of basic physical parameters of vortices (e.g. size, speed, angular speed and curvature) helped to broaden our understanding of their characteristics. Most interestingly, we observed vortices to consist of one up to several layers. Investigation of two mutant parasite lines revealed that sporozoites lacking the actin bundling protein coronin are still able to form vortices as well as swarms besides showing aberrant gliding on glass. In contrast, sporozoites lacking the chaperone HSP20 completely fail to form vortices and swarms. In the second part of my thesis I focused on the interplay of the three known sporozoite adhesins (TRAP, S6 and TLP), which have already been characterized independently throughout various studies and are known to play a major role in invasion and gliding motility of sporozoites. As a first step, I used double homologous recombination to create the double knockouts (ΔTRAP/ΔS6, ΔTLP/ΔTRAP and ΔTLP/ΔS6), two independent triple knockout lines (ΔTRAP/ΔTLP/ΔS6) as well as the TRAP complemented ΔTLP/ΔS6 line. Characterization of the generated lines confirmed the dominating TRAP and S6 phenotype blocking and strongly reducing sporozoite salivary gland invasion, respectively. I further demonstrate that once inside the salivary gland, TLP/S6 knockout sporozoites are still capable to undergo natural transmission via mosquito bites. Astonishingly, triple knockout sporozoites in the mosquito hemolymph can still attach and show the so-called patch gliding behavior, a limited form of gliding, indicating the existence of at least one further surface adhesin involved in gliding motility. Taken together, this study provides fundamental insights into the previously undescribed collective motion of Plasmodium sporozoites which might serve as model system for future studies and broadened our understanding of the interplay of sporozoite surface adhesins.
Pericytes are mural cells of the microvascular system of mesenchymal origin, which abluminally ensheath capillary endothelial cells (EC). Pericytes play a pivotal role in the maturation of newly formed vessels and are a hallmark of the quiescent endothelium in most microvascular beds. Pericyte-based research is hampered by the limited availability of robust pan-pericyte markers due to their overlapping marker expression with various cells of mesenchymal origin. In order to identify novel functionally relevant pericyte markers, an expression profiling of five different human primary pericyte populations and other mesenchymal cell populations (EC, adipocytes, fibroblasts, mesenchymal stem cells [MSC]) was performed. Among others, this screen identified Prostaglandin E receptor 2 (PTGER2) and Sphingosine-1-phosphate receptor 3 (S1PR3), two Gprotein-coupled receptors (GPCRs), as novel, highly pericyte-enriched transcripts. Only S1PR3 showed comparable expression levels also in smooth muscle cells (SMC). Subsequent cellular studies demonstrated for the first time that S1PR3 signals via Gαi and Gαq in pericytes and regulates myosin light chain (MLC) phosphorylation. To study the role of pericyteexpressed S1PR3 in a physiological setting, comparative co-culture experiments of EC with S1PR3silenced pericytes were performed and transcriptomic profiles were traced. The expression of S1PR3 by pericytes resulted in subtle, but distinct transcriptomic changes, including changes in cell-cell as well as cell-extracellular matrix interaction molecules. MPRIP, a regulator of RhoA and MLC phosphorylation, was identified as one of the most promising candidate genes. Functional in vitro assays of S1PR3 silenced pericytes resulted in reduced transmigration capacity and increased cell size. Notably, PTGER2 was exclusively expressed by pericytes. Transcriptomic analyses of co-cultured pericytes silenced for PTGER2 revealed expression changes of proliferation-associated genes (downregulation of negative regulators). Correspondingly, functional in vitro assays of PTGER2 silenced pericytes resulted in enhanced proliferation. To enable the validation of pericyte-expressed S1pr3 and Ptger2 and further functional studies in vivo, mouse models for the conditional deletion of these genes are required. Based on the expression profiling and cellular screening experiments and the fact that conditional mice for S1pr3 are not available, experiments were set out with the aim to generate S1pr3 floxed mice by CRISPR/Cas technology. Sequential integration of two LoxP sites using Cas9 wildtype was successfully used to generate conditional S1pr3 mice. Taken together, the experiments identified PTGER2 as novel pericyte marker and S1PR3 as novel mural cell marker within the mesenchymal cell lineage that both control important pericyte functions. Furthermore, CRISPR/Cas technology proved as a suitable approach to generate conditional S1pr3 knockout mice.
The Angiopoietin/Tie system is one of the most important vascular-tissue specific signaling pathways, essential during embryonic vascular development and maturation, and key regulator of adult homeostasis. The Tie receptors, Tie1 and Tie2, and their secreted angiopoietin (Ang) ligands, Ang1 and Ang2, have been identified as the main factors of the system. Ang1 is primarily produced by perivascular cells and acts in a paracrine fashion as strong Tie2 agonist that mediates endothelial cell (EC) survival and maturation signals. Ang2, expressed and stored in EC, functions primarily as an antagonist of Tie2 and promotes vascular destabilization. Nevertheless, it can act as a partial agonist of Tie2 in a context-dependent manner. Tie1 and Tie2 are both expressed by EC and share high similarities in their overall domain structure. Whereas Tie2 acts as the primary signal transducing receptor, Tie1 function as a holoreceptor is not completely understood: It does not activate signaling pathways on its own but rather modulates the Tie2 kinase activity. Tie1 is highly expressed in activated endothelial cells during embryogenesis and during pathological conditions, such as tumor progression and metastasis, but it is downregulated in the adult quiescent endothelium. Thus, Tie1 global deficiency in mice leads to embryonic lethality at late gestation due to perturbed vessel integrity, while its specific deletion in the growing tumor vasculature results in decreased blood vessel density. The combination of high expression in the tumor endothelium and low expression in the adult vasculature makes Tie1 an interesting molecule and a potential therapeutic target. Angiogenesis is one of the most critical steps during tumor growth and metastasis progression. Comparative tumor and metastasis experiments in wildtype and Tie1 endothelial-deficient mice were performed to mechanistically unravel the role of the orphan receptor during individual steps of tumor progression and metastasis. Analysis of the tumor vasculature demonstrated that Tie1 endothelial deletion induces no significant changes in the early steps of tumor growth but rather affects later steps of tumor progression. Endothelial-Tie1 deletion induces progressively normalization of primary tumor blood vessels accompanied by decreased microvessel density, increased mural cell coverage, improved vessel perfusion and reduced tumor cell intravasation into the blood stream as well as extravasation at secondary sites. These effects result in almost complete inhibition of post-surgical spontaneous lung metastasis and improved overall survival of Tie1-endothelial deleted mice. Mechanistically, Tie1 targeting in the primary tumor leads to vascular normalization and stabilization by increasing the proportion of Tie2-positive endothelial cells (stalk cells) and by potentiating the Ang1/Tie2 signaling axis which is primarily essential for the maintenance of endothelial quiescence and survival. The data establish a remarkable contribution of the orphan receptor Tie1 to primary tumor angiogenesis and to individual steps of metastatic cascade. These findings contribute to the mechanism-guided validation of Tie1 as a therapeutic target to sharpen the balance between triggering vascular regression and promoting vascular normalization.
Bei der Strahlentherapie ist die Bestrahlungsdosis limitiert durch Nebenwirkungen im Normalgewebe, v. a. in Zellen des hematopoetischen und gastrointestinalen Systems. Eine Steigerung des Therapieerfolgs könnte durch Tumorsensibilisierung und/oder Radioprotektion von gesundem Gewebe erreicht werden. In der vorliegenden Arbeit wurden die Studien des Labors für zelluläre und molekulare Radioonkologie der Klinik für Strahlentherapie und Radioonkologie der Universitätsmedizin Mannheim zum radioprotektiven Effekt der Überexpression von CAV1 oder MDR1 im TK6-Zellmodell, einer lymphoblastoiden Zelllinie, fortgesetzt, indem untersucht wurde, ob die gleichzeitige Überexpression von CAV1 und MDR1 in der in dieser Arbeit neu etablierten Zelllinie TK6-CAV1-MDR1 im Vergleich zur jeweiligen einzelnen Überexpression eine Verbesserung des Zellüberlebens nach Bestrahlung bewirkt. Zusätzlich wurde die Lokalisation von CAV1 und MDR1 ohne und mit Bestrahlung untersucht und die Bedeutung der Tyr14-Phosphorylierungsstelle von CAV1 im Hinblick auf bestrahlungsinduziertes Zellüberleben analysiert. Für die vergleichenden Untersuchungen wurden TK6-CAV1 erfolgreich mit MDR1 lentiviral transduziert und selektiert. Die Proliferation nach Bestrahlung wurde mittels Langzeit-Assay (10 Tage, 2 Gy bis 4 Gy) und Kurzzeit-Assay (1 Gy bis 6 Gy) untersucht. Im Langzeit-Assay wiesen TK6-MDR1 im Vergleich zu TK6wt, TK6-CAV1 und TK6-CAV1-MDR1 das höchste Überleben auf, während TK6-CAV1 deutlich unter TK6-MDR1 lagen (2 Gy: 16 % niedriger, 4 Gy: 27 % niedriger; p < 0,001). Weder TK6-CAV1 noch TK6-CAV1-MDR1 wiesen eine höhere Proliferation als TK6wt bzw. TK6-MDR1 auf. In Übereinstimmung dazu wurde im Kurzzeit-Assay in TK6-MDR1 ein leichter, nicht signifikanter Proliferationsvorteil im Vergleich zu TK6wt beobachtet, während TK6-CAV1 niedriger lagen (24 h: p < 0,04). Die Kombinationszelllinie TK6-CAV1-MDR1 wies nach 48 h und 72 h die niedrigste Proliferationsrate auf. Die anhand fragmentierter Zellkerne bestimmte Apoptoserate (3- bis 8-fach erhöht, p > 0,35) und die Koloniebildung waren bei den untersuchten Zelllinien vergleichbar. Die Spaltung von Caspase 8 in das Spaltfragment p41/43 als Mechanismus der bestrahlungsinduzierten Apoptose wurde mittels Western Blot untersucht und ergab die höchste Spaltungsrate in TK6-CAV1, gefolgt von TK6-CAV1-MDR1, TK6wt und TK6-MDR1. Interessant war, dass in TK6-CAV1 bereits nach 36 h mehr als 60 % der maximal erreichten Caspase 8 Spaltung erreicht war, während es bei den anderen Zelllinien nur 50 % nach 48 h waren. Insgesamt konnte die radioprotektive Wirkung von MDR1 im Sinne eines Proliferationsvorteils bestätigt werden, die jedoch ohne Auswirkung auf die Apoptoserate und klonogenes Überleben blieb. Entgegen der Erwartung aus Vorarbeiten wurde eine schwache, doch konsistente pro-apoptotische Funktion von CAV1 beobachtet, sodass keine synergistische oder additive Wirkung zur Steigerung der Radioprotektion von CAV1 in Verbindung mit MDR1 gefunden wurde. Veränderte Konditionen (z. B. Zellkulturmedium) oder methodische Ursachen (z. B. schwankungsbehaftete Testmethoden) könnten als Ursache in Frage kommen; die Ergründung der Variabilität bezüglich der Strahlenwirkung auf CAV1-überexprimierende Zellen ist der erforderliche nächste Schritt zur Bewertung der Radioprotektion durch CAV1. Die in der Literatur diskutierte Co-Lokalisation von CAV1 und MDR1 könnte die Grundlage einer direkten Interaktion sein, die u. a. die Transporteraktivität von MDR1 beeinflussen kann. Konfokalmikroskopische Studien bis 24 h nach Bestrahlung (2 Gy) zeigten eine Abnahme der Co-Lokalisationsparameter R (Pearson) um ca. 22 % (p < 0,05) und tM1/tM2 (Manders) um ca. 9 % (nur 1 h: ptM2 < 0,03), während anhand des Intensitätskorrelationskoeffizienten (Li) keine Änderung der Co-Lokalisation ermittelt wurde. Die Verwendung größerer Zellen, eine Kompartiment-spezifische Auswertung und ein objektbasierter Auswerteansatz wären geeignete Maßnahmen zur Präzisierung der Lokalisation von CAV1 und MDR1 nach Bestrahlung. Die Bedeutung der bestrahlungsinduzierten Phosphorylierung von CAV1 an Tyr14 wurde mit der neu etablierten Zelllinie TK6-CAV1Y14F untersucht. Die Phosphorylierung nach oxidativem Stress (H2O2) bzw. Bestrahlung von CAV1 wurde durch Inhibition der Signalkinasen p38 und JNK um ca. 55 % (H2O2) bzw. ca. 25 % gemindert (p ≤ 0,01), während Kinasen der SRC-Familie nur für die durch H2O2 induzierte Phosphorylierung relevant waren (Abnahme: ca. 70 %, p = 0,001). Die Phosphorylierung von CAV1 stieg ca. 5-fach nach 2 Gy bis 4 Gy bzw. nach 1 mM bis 5 mM H2O2 (p < 0,03). Sowohl bis 10 Tage nach Bestrahlung mit 2 Gy (p < 0,005) und bis 72 h nach Bestrahlung mit 1 Gy bis 6 Gy (p < 0,01) war die Proliferationsrate in TK6-CAV1 niedriger als in TK6-CAV1Y14F. Hinsichtlich Kernfragmentierungsrate und Koloniebildung ergaben sich jedoch keine Unterschiede im Zelllinienvergleich. Wahrscheinlich sind unterschiedliche Zellsignalwege relevant für den H2O2- bzw. bestrahlungsinduzierten Zelltod, und möglicherweise werden unterschiedliche Zelltodmechanismen aktiviert, sodass eine multimodale Rolle von CAV1 und der Phosphorylierung an Tyr14 ausgeht.
Local field potentials in the hippocampus are dominated by two different activity patterns - persistent gamma band (30-60Hz) oscillations and intermittent sharp wave-ripple (200Hz) complexes. The former are associated with exploratory behaviours in which information is acquired while the latter are believed to be involved in transfer and consolidation of information to long-term memory. Although there is a great wealth of knowledge about how neuronal networks maintain each oscillatory state, it is not entirely clear what cellular mechanisms mediate the switch between gamma to sharp wave-ripple activity. The goal of this thesis was to characterise this transition at the network and synaptic level. To this end, an \textit{in vitro} model based on acute hippocampal slices was established in which both gamma rhythms and sharp wave-ripples could be evoked. This was achieved with a custom-built holographic illumination system granting targeted and selective optogenetic stimulation of pyramidal cells. Prolonged stimulation of the reduced hippocampal slice network elicited self-synchronisation to a gamma rhythm (50Hz) with a resonance at theta frequency inputs (i.e. 4-5Hz sinusoidal stimulation). This frequency preference was confirmed at the single unit level which revealed most precise coordination of single cell activity within the ongoing gamma rhythm at theta frequency inputs. Additionally, evoked gamma synchronisation emerged suddenly 200ms after stimulus onset indicating a near instantaneous switch of hippocampal network states. This finding was reflected in whole-cell recordings of postsynaptic currents which displayed a sharp increase in both excitatory and inhibitory inputs around 200-300ms after stimulus onset. On the other hand, applying short (5ms) square pulses of low intensity stimulation evoked local field potential signatures closely resembling sharp wave-ripples. This implies that stimulus duration and profile are critical determinants of network output. The results presented here provide experimental evidence for prevailing models of different hippocampal network oscillations and extend the knowledge about how neuronal networks can maintain different oscillatory states and how the transition between these states is brought about.
Cytosolic recognition of viral replication intermediates by RIG-I, the founding member of the RIG-I-like receptor (RLR) family, initiates a signalling cascade which culminates in the activation of latent transcription factors IRF3 and IRF7, inducing the expression of type I and type III interferons (IFNs) and interferon-stimulated genes (ISGs). These work in concert to combat viral infection. Thus, regulation of RIG-I activity is crucial in mounting a balanced antiviral response strong enough to ward off infection, but strictly transient in order to avoid tissue damage. Initially, death-associated protein kinases (DAPKs) were described as initiators of cell death. Since then, they have been found to mediate a variety of cellular processes, such as cell growth and survival, cytoskeletal remodelling and inflammatory responses, but had not been linked to the regulation of innate antiviral signalling. We identified death-associated protein kinase 1 (DAPK1) as a negative feedback-regulator of RIG-I activity. The present study confirms that a minimal DAPK1 construct, DAPK1KCA, comprising only the kinase, calmodulin (CaM)-binding and Ankyrin repeats domains, inhibits RIG-I in a kinase-dependent manner. DAPK1KCA harbours residues targeted by growth factor signalling-related kinases. We could demonstrate, however, that inhibition of RIG-I signalling mediated by DAPK1 happened independently of its role in growth factor signalling. Nevertheless, DAPK1 expression was strongly influenced by cell growth and division. Since the kinase domains of the different DAPKs are homologous and share some substrate specificity, we investigated if, in addition to DAPK1, other DAPK family members would interfere with RIG-I signalling. Upon over-expression, all DAPK family members inhibited RIG-I signalling, except DRAK2 which, in our hands, was kinase-inactive. Reciprocally, siRNA-mediated knockdown of DAPK1 and DAPK3 resulted in increased antiviral signalling activity. Moreover, silencing of DAPK1 and DAPK3 reduced the replication of two different RNA viruses, emphasizing the physiological relevance of these kinases in the regulation of RIG-I signalling. Similar to what was observed for DAPK1, we found DAPK2 to inhibit RIG-I signalling in a kinase activity-dependent fashion. Moreover, the kinase domain of DAPK3 alone inhibited RIG-I signalling. While all DAPK family members phosphorylated RIG-I in vitro, interaction with RIG-I could only be shown for DAPK1 and DAPK2. Although we found DAPKs to physically interact with each other, we observed no interdependence of the kinases regarding their inhibitory function in RIG-I signalling. In order to further study DAPK functions in RIG-I signalling, we created single and combined knockout cell lines for DAPK1, DAPK2, and DAPK3. However, there was no change in the antiviral response in these cells, possibly due to up-regulation of other proteins compensating for loss of the DAPKs. We observed that inhibition of RIG-I signalling by DAPKs happened independently of their well-studied role in apoptotic as well as autophagic cell death induction. In fact, we did not detect any induction of autophagy upon DAPK expression. Ultimately, we discovered that regulation of antiviral signalling by DAPKs is not limited to an inhibition of RIG-I, but that they also inhibited MDA5-mediated signalling. Moreover, we found evidence that DAPKs additionally inhibit antiviral signalling at the level or downstream of IRF3-mediated gene transcription. In summary, the present study identifies a conserved role for DAPKs in the regulation of antiviral RLR signalling independent of known DAPK functions in cell death and survival.
Glioblastoma (GBM) is a highly aggressive tumor that leads to the patient’s deaths within approximately one year. Despite recent considerable advances in our understanding of the pathogenetic alterations present in the tumor cells and the nature of the glioblastoma microenvironment, it has not been yet possible to develop effective therapies. The role of tumor-derived metabolites in the interaction of the tumor cells with cells of the tumor stroma, e.g. immune cells, are a promising aspect of glioblastoma biology for defining new therapeutic targets. This thesis explores different approaches to characterize the branched-chain amino acid (BCAA) metabolism in the tumor-stroma interaction. In the first part I investigated whether and how branched-chain ketoacids (BCKAs), generated in the first step of the BCAA catabolism, are excreted from glioblastoma cells. I found that while the monocarboxylate transporters MCT1 and MCT4 are both capable and sufficient to transport BCKAs across the membranes of living cells, excretion of BCKAs from glioblastoma cells appears to be mediated mostly by MCT1. Additionally, I could show that MCT1 locates in close proximity to the BCKA-generating branched-chain transaminase 1 (BCAT1), suggesting possible functional interaction of the proteins. In the second part I investigated the fate and function of tumor-secreted BCKAs in the tumor-stroma interaction using macrophages as a model of the stroma compartment. Using in vitro isotope tracing analysis, it was demonstrated that BCKAs are taken up by macrophages and catabolized to BCAAs. Additionally, exposure to BCKAs reduced the phagocytic activity of macrophages suggesting that in glioblastoma, tumor cell-secreted BCKAs might be able to modulate the tumor-associated macrophages/microglia, contributing to their role in tumor immune suppression and supporting glioblastoma tumor growth and progression. Furthermore, I was able to provide evidence, that the manipulation of BCAT1 expression in the tumor compartment can impact the immune phenotype of tumor-associated macrophages of the stroma compartment via BCKAs and likely other diffusible factors. Ultimately, this study provides further evidence for the eminent role of BCAA catabolism in glioblastoma by demonstrating that tumor-excreted BCKAs might have a direct role in tumor immune suppression.
Gliomas are tumors of the central nervous system which are classified by the World Health Organization (WHO) from grade I to grade IV, according to the degree of malignancy as defined by histopathological and molecular criteria. Mutations in the isocitrate dehydrogenase (IDHmut) genes are a common cha- racteristic for lower grade gliomas (LGGs, WHO◦II/◦III) and have been shown to be a prognostic marker for a favorable clinical outcome. The metabolic and biologic consequences by IDHmut-induced epigentic alterations have changed our perceptions of gliomas and demonstrated the demand to consider IDHmut LGGs separately. Despite a favourable prognosis, IDHmut LGGs remain deadly since there is still a lack of effective therapies. For that reason, immunotherapeutic approaches are gaining increasing attention. Due to the limited number of known T cell targets in IDHmut gliomas, we aimed to elucidate the reper- toire of spontaneous T cell responses in IDHmut LGGs by performing an unbiased proteomic approach. We systematically analyzed the proteome of IDHmut LGG samples (n = 4) by fractionating tumor tissue lysates and testing resulting fractions by IFN-γ enzyme linked immunospot (ELISpot) assay for recogni- tion by the patients T cell repertoire. Immunogenic tumor protein fractions were subsequently analyzed by quantitative mass spectrometry resulting in 2897 identified proteins. Based on a thorough filter pro- cess 79 proteins have been selected as potential target antigens and were validated in IFN-γ ELISpots by means of synthetic in silico predicted 50-mer peptides. 26 of these were recognized by autologous T cells and were tested in further IDHmut LGG patients as well as in healthy donors. Tumor-specific T cell responses in up to 50 % of IDHmut LGG patients were observed for CRKII, CFL1, CNTN1, NME2, and TKT. Beside the immunogenicity of the antigens, we further characterized their role in oncogenesis, the antigen-specificity as well as the expression levels in the tumor and on glioma stem-like cells (GSCs). By using immunohistochemistry and gene expression analysis we found that four out of five of the most immunogenic tumor-associated antigens (CRKII, CFL1, CNTN1 and NME2) were expressed in astro- cytic and oligodendroglial tumors as well as in IDHmut GSCs, while being almost absent in normal brain tissues. Finally, we identified HLA-A*02-restricted reactive epitopes for CRKII (ALALEVGEL), NME2 (MVWEGLNVV), and TKT (FLAEAELLNL) which are recognized by up to 1.5 % of antigen-specific pe- ripheral cytotoxic T cells in IDHmut LGG patients. By analyzing the repertoire of T cell target antigens in IDHmut LGG patients, we identified four novel immunogenic antigens and even confirmed their expression on IDHmut GSCs, highlighting their potential as T cell targets for the development of new immunotherapeutic approaches.
Light-sheet fluorescence microscopy, also recognised as selective plane illumination microscopy, or SPIM, has paved a new road towards imaging of entire specimens for long periods of time, in vivo. Nevertheless, as in any other microscopy technique, light-sheet fluorescence microscopy also heavily depends on the scattering and absorption properties of the imaged sample in order to generate 3D datasets with high signal to noise even at larger tissue depths. This thesis focuses on the development and implementation of new strategies and methods which target the minimization of scattering and absorption effects stemming from living specimens. Combined, the three methods provide the ability to perform gentle, high contrast deep tissue imaging and photomanipulation. Additionally, it allows easier handling and fusion of 3D multiview light-sheet images.
Adult neurogenesis adds an entirely new level of plasticity to the brain and raises hope to use stem cell therapy to repair damaged nervous tissue. To understand the role of neurogenesis in the adult brain and to harness its potential it is of utmost importance to understand the regulation of the stem cell niches. Our group previously showed that the endozepine DBI is expressed in neuronal progenitors in the SVZ and that it reduces GABA signalling in these cells. Via this mechanism, DBI promotes the proliferation of fast dividing progenitors which leads to a strong increase in neurogenesis. Here I investigated the presence of DBI in other neurogenic niches and its role in regulating postnatal and adult neurogenesis. I found that DBI is strongly expressed in the SGZ and in the walls of the 3rd ventricle both postnatally and in adult mice. Furthermore, I showed that DBI is present in RG cells during embryonic development. I found that DBI is expressed not only in all mouse postnatal and adult neurogenic niches but also across species in the SGZ of the Rhesus monkey and in humans. High expression levels of DBI were detected in all stem cells and in the early population of amplifying progenitors, suggesting that this protein could be considered as an indicator for stemness in the nervous tissue. Focusing on the SGZ, I showed that DBI negatively modulates the activity of the GABAA receptor in stem cells, thereby increasing their proliferation, self-renewal and astrocyte production. In summary, DBI together with GABA regulate the balance between preserving the stem cell pool and neuronal production. External factors such as environmental enrichment and physical exercise strongly enhance neurogenesis. I found in this study that DBI is essential for the pro-proliferative and pro-neurogenic effects of enriched environment and exercise. Therefore, DBI and GABA regulate SGZ neurogenesis in a close partnership enabling multiple levels of control which makes the niche dynamic and capable of reacting promptly to changes in the environment.
In this work, the compressible Navier-Stokes equations describing the dynamics of a dry atmosphere are derived. Based on a scale analysis for Low-Mach number flows, the Low-Mach approximation is derived from the compressible Navier-Stokes equations by neglecting the hydrodynamic part of pressure, which is small compared to the hydrostatic and thermodynamic parts, in the ideal gas law. Both models are discretised by finite elements in space and finite differences in time, where all common parameters of the discretisations are chosen identically in order to minimise influences on the discrete solutions due to differences in the respective discretisation. A solution strategy for both models based on an inexact Newton method is presented, where the linear solvers and preconditioners are adapted to the respective model. In case of the Low-Mach model, a preconditioning technique based on nested Schur complement iterations is proposed. A scenario of two interacting tropical cyclones is presented as benchmark problem in order to compare the solutions of the two models in terms of numerical as well as physical properties. The obtained numerical results show the scalability and robustness of the solution approach. For the considered scenario, the predicted tracks of the cyclones, which are computed by the Low-Mach model, show very good coincidence with those of the Compressible Navier-Stokes model at significantly smaller computational costs, such that the Low-Mach approximation can be regarded as valid in this case.
The notion of positive TFT as coined by Banagl is specified by an axiomatic system based on Atiyah’s original axioms for TFTs. By virtue of a general framework that is based on the concept of Eilenberg completeness of semirings from computer science, a positive TFT can be produced rigorously via quantization of systems of fields and action functionals - a process inspired by Feynman’s path integral from classical quantum field theory.
The purpose of the present dissertation thesis is to investigate a new differential topological invariant for smooth manifolds that arises as the state sum of the fold map TFT, which has been constructed by Banagl as a example of a positive TFT. By eliminating an internal technical assumption on the fields of the fold map TFT, we are able to express the informational content of the state sum in terms of an extension problem for fold maps from cobordisms into the plane. Next, we use the general theory of generic smooth maps into the plane to improve known results about the structure of the state sum in arbitrary dimensions, and to determine it completely in dimension two. The aggregate invariant of a homotopy sphere, which is derived from the state sum, naturally leads us to define a filtration of the group of homotopy spheres in order to understand the role of indefinite fold lines beyond a theorem of Saeki. As an application, we show how Kervaire spheres can be characterized by indefinite fold lines in certain dimensions.
The probability of being affected by osteoarthritis during lifetime is about 20 % in Germany. The disease originates from a degenerative alteration of joints in which the lipid layers covering the cartilage and the cartilage itself are depleted, and the underlying bone is exposed. The treatment mainly includes pain-relieving therapies and physiotherapy for maintaining mobility. A long-term solution is only an artificial joint replacement (endoprosthesis) which, however, has to be inserted by surgery and must be replaced after about 10 to 15 years.
Since the 90s a new treatment called viscosupplementation exists. This non-surgical procedure promises to preserve one's own natural joint while at the same time recovering its functionality. It is based on studies on diseased joints, which showed that the volume of the synovial fluid increases while the concentration and the molecular weight (MW) of the contained hyaluronic acid (HS) decrease. Further investigations indicate that surfaceactive phospholipids (PLs) play an important role in joint lubrication in addition to the HS. Based on these findings, an intra-articular injection of hyaluronic acid (HS) or mixtures of HS and PL is used during viscosupplementation. However, the effectiveness of the method is discussed controversially.
The aim of this work was, therefore, to investigate the interactions of the main components in a joint by means of a simplified model system, and to examine the impact of HS and polymeric substitutes on PL layers covering the cartilage. Simultaneous in-situ neutron reflection (NR) and infrared (IR) measurements at the instrument BioRef at Helmholtz-Zentrum-Berlin (HZB) should provide insights into the internal structure and phase behavior of the lipid layers. The development of a custom-fit shear cell allows to simulate shear in joints and to gain information on the stability of the systems. Further systematic studies on the nature of interactions between PLs and HS or polymeric substitutes were carried out by means of ellipsometric measurements.
For the investigation of the interaction between PLs and polyelectrolyte (PE) or HS solutions, different parameters of the solutions were deliberately changed in the first experiments. In particular, the charge, the molecular weight and the concentration of the PEs or the HS, the concentration and type of added salts and the pH were varied. For this purpose, silicon substrates coated with lipid oligolayers and exposed to water were first characterized and served as an internal reference. After reaching equilibrium, the system was subjected to the above-mentioned changes, measured again and compared with the reference measurement.
For charged PEs, a strong increase in the lamellar layer thickness of the lipids was found at low concentrations, which decreased with increasing PE concentration. Uncharged polymers induced only low swelling, which increased slightly with increasing PE concentration. This indicated that the strong swelling behavior of the lipid layers is based on electrostatic interactions. It was assumed that the selective adsorption of the PEs on the lipid bilayers electrostatically charges the layers. Experiments on charge shielding byexposure to salt solutions and changes in pH as well as the modeling of NR data further confirmed this assumption.
The exact localization of the PE or HS molecules within the lipid layers should be facilitated by NR measurements. Modeling of the reflectivity curves for polyallylamine hydrochloride (PAH) with an average MW of 58 kDa showed an integration into the chain regions. By means of appropriate contrast variation in the systems utilizing deuterated lipids, a direct, model independent detection of the incorporation of PAH into the layer system was feasible so that possible sources of error during modeling could be excluded. Such incorporation suggests bridging of the lipid layers by HS and PEs, which can lead to the formation of a hydrogel-like structure possibly protecting the cartilage.
In line with the concept of the viscosupplementation, a stabilization of the lipid layers against shear could be determined upon addition of HS or the polymeric substitute PAH. However, the expected MW dependency could not be verified. HS showed a stronger stabilizing effect than the PAH, but only after markedly longer incubation times.
In the present work it was shown that HS and polymeric substitutes show a stabilizing effect on the lipid coating of the joints, which can possibly lead to a reconstitution of lowfriction joint movement. The idea of viscosupplementation appears to be promising, and by taking other components such as proteoglycans or cartilage coatings into consideration, an effective non-operative treatment of osteoarthritis should be possible in the future.
In African trypanosomes, the control of transcription initiation by RNA pol II is absent at the level of individual mRNAs. Nevertheless, gene expression changes dramatically during life cycle transitions in response to the changing host environments. In the bloodstream of the mammalian host, Trypanosoma brucei exist as proliferative long slender forms or as a non-dividing stumpy forms; the latter differentiate to procyclic forms in the midgut of the tsetse fly. Differential gene expression between life cycle stages is achieved through regulation of mRNA degradation and translation, and mainly relies on RNA binding proteins. The work focuses on the RNA binding protein RBP10. RBP10 is a bloodstream form specific cytoplasmic protein with a single RRM domain. Depletion of RBP10 in bloodstream forms or forced expression in the procyclic forms is lethal due to mis-regulation of developmentally expressed mRNAs. Bloodstream forms cells depleted of RBP10 differentiate to procyclic forms after transfer into procyclic growth media and incubation at 27oC; within three days, >80% of the cells express GPEET procyclin, and reposition their kinetoplast. Conversely, expression of RBP10 in procyclic cells for two days converts the cells to bloodstream forms. In such cells, eight VSG transcripts, including three with metacyclic promoters, were strongly up regulated, and ~16% of the cells had acquired a VSG surface coat. More importantly, a subset of the cells survived after transfer into bloodstream form growth media and incubation at 37oC, resulting in proliferating cells in about ten days. Tethering of RBP10 to a reporter mRNA inhibits translation and promotes mRNA degradation. RBP10 from bloodstream forms co-precipitated many procyclic specific mRNAs that are normally unstable in bloodstream forms. Indeed, 39% of the mRNAs up regulated after RBP10 depletion were bound by it; these included the transcript encoding procyclic surface coat EP procyclin, several enzymes needed for procyclic energy metabolism, regulatory proteins ZC3H21, ZC3H20, two kinases and a phosphatase. The UA(U)6 motif was found to be highly enriched in the 3' UTR of RBP10 mRNA targets. Binding of RBP10 to EP 3’ UTR was lost when the UA(U)6 motif was deleted from a reporter mRNA, and the motif was also necessary for its regulation by RBP10. In bloodstream forms RBP10 target mRNAs are likely to be blocked from translation hence degraded; this is important for survival. Perturbation of RBP10 expression therefore triggers a regulatory cascade that is sufficient to modulate T. brucei developmental capacity.
Strictosidin ist der gemeinsame Vorläufer vieler medizinisch relevanter, pflanzlicher Monoterpenindolalkaloide, deren Produktion in der Pflanze häufig nicht ausreicht, um den steigenden Marktbedarf zu decken. Die Produktion dieser Alkaloide bzw. ihres Vorläufers Strictosidin in einem mikrobiellen heterologen System könnte eine Alterna-tive zur Synthese/Produktion in der Pflanze bieten. In dieser Arbeit wurde daher ein System zur Co-Expression von drei Schlüsselgenen der Catharanthus roseus Monoterpenindolalkaloid-Biosynthese entwickelt, das die gleichzeitige funktionelle Expression der Tryptophandecarboxylase (TDC), der Stricto-sidinsynthase (STR) und der Strictosidinglucosidase (SGD) in Escherichia coli ermög-licht. Um dies zu erreichen, wurden zunächst zwei unterschiedliche Expressionssysteme auf ihre Eignung hin untersucht. Zum einen wurde mit Pichia pastoris, ein eukaryotes Sys-tem getestet, zum anderen mit Escherichia coli, ein prokaryotes. Für beide Systeme wurden verschiedene Vektorkonstrukte zur Einzelexpression der Catharanthus-Gene hergestellt und deren Funktionalität im jeweiligen System/Organismus überprüft. Die Gene der STR und SGD wurden für die Expression in E. coli Codon-optimiert. Bei der STR, für die bekannt ist, dass sie in E. coli vermehrt in Form von unlöslichen Inclusion Bodies produziert wird, wurde zusätzlich der Einfluss des Vektors (Kopienzahl) und verschiedener Tag-Sequenzen auf die Expression löslicher, enzymatisch aktiver En-zyme untersucht. Die Aktivität der unterschiedlichen Konstrukte wurde über in vivo Enzymassays in E. coli und anschließendem Nachweis des enzymatischen Produkts Strictosidin mittels HPLC verglichen. Da sich die TDC im P. pastoris System trotz vielfältiger Optimierungsversuche nicht ex-primieren ließ, und die STR trotz nachgewiesener funktioneller Expression nicht aus dem Expressionsmedium gereinigt werden konnte, wurden die Arbeiten mit diesem System eingestellt. Alle Arbeiten zur Produktion der drei Catharanthus-Enzyme erfolg-ten von nun an im E. coli System, in dem sich alle drei Enzyme in löslicher, aktiver Form exprimieren und reinigen ließen. Die gereinigten Enzyme wurden hinsichtlich pH- und Temperaturoptima, kinetischer Parameter Km und Vmax und des Einflusses verschie-dener Aktivatoren und Hemmstoffe charakterisiert. Für die Co-Expression aller drei Gene wurden Doppeltransformanten hergestellt, die zum einen den Vektor pET45b(+)_TDC_C-His und zum anderen den Vektor pCDFDuet1_SGD_STR enthielten. Der Erfolg der Expression wurde über SDS-PAGE, die Funktionalität der gebildeten Enzyme über in vivo Enzymassays mit Tryptophan und Secologanin als Substrate und anschließendem Nachweis der Edukte bzw. Produkte über HPLC bzw. LC-MS überprüft. Als kostengünstigere Alternative wurden auch En-zymassays durchgeführt, bei denen anstelle des reinen Secologanin ein Methanol-Ex-trakt von Symphoricarpos albus Beeren verwendet wurde. Zusätzlich zu den in vivo Klonierungs- und Expressionsarbeiten wurde ein säulenba-siertes System zur in vitro Produktion von Strictosidin mittels rekombinanter STR etabliert. Die Ergebnisse dieser Arbeit liefern einen ersten Beitrag zur heterologen Produktion pharmazeutisch wichtiger Monoterpenindolalkaloide in einem E. coli System.
Chromosome capture by microtubules is an early step of cell division essential for alignment and subsequent separation of sister chromatids. Failure to transmit even one chromosome results in aneuploidy, a common cause of infertility, genetic disorders or cancer.
The canonical mechanism of ‘search and capture’ by dynamic astral microtubules has been validated by recent studies, and additionally revealed mechanisms that facilitate microtubule search, ensuring rapid and efficient capture of chromosomes in somatic cells.
However, in specialized cell types such as oocytes with large nucleus chromosomes are located much further from microtubule asters. In these cells, the models that work in small somatic cells are insufficient to explain chromosome capture. Recently, the Lénárt group has shown that in starfish oocytes an actin-driven mechanism facilitates chromosome congression and is required to prevent chromosome loss: a contractile actin meshwork transports chromosomes to within the capture range of microtubule asters of approximately 30 µm. How these actin- and microtubule-driven mechanisms of chromosome capture are coordinated remained an open question.
Here, I investigated the cooperation between the actin meshwork transporting chromosomes and capture by microtubules in meiosis of starfish oocytes using high spatio-temporal resolution tracking of chromosome motion in 3D combined with drug-perturbation experiments. This assay allowed me to characterize chromosome capture kinetics during the two-staged chromosome congression under different conditions.
I find that the actin meshwork, while transporting the distal chromosomes to the vicinity of microtubule asters, also synchronizes their capture. I show that this synchronizing effect is due to an actin-dependent block of chromosome capture active for approx. 5 minutes after NEBD. As a result, chromosomes close to microtubule asters – that in principle could be captured immediately after NEBD – are captured simultaneously with chromosomes transported from distal nuclear locations by the actin meshwork at approx. 5-15 minutes after NEBD and independent of their distance from the asters.
I show that this delay in the capture of the proximally located chromosomes cannot be explained by altered microtubule dynamics when growing through the actin meshwork. The delay is also not the consequence of physical entrapment in the actin network ‘holding back’ chromosomes, because capture is not delayed in slowed or even fully stabilized actin networks.
Together, my results point to an actin-dependent mechanism, which prevents the formation of lateral kinetochore-microtubule attachments. Synchronous disassembly of these F-actin structures exposes kinetochores and thereby synchronizes chromosome capture. This is a first description of a mechanism by which the actin cytoskeleton directly affects spindle assembly, and which actively controls and coordinates chromosome search and capture. I show how this mechanism coordinates chromosome congression in the specialized oocyte nucleus, but it is interesting to speculate whether such mechanisms may have a broader relevance for example to synchronize mitotic events such as cell rounding mediated by the actin cytoskeleton with spindle assembly. The detailed molecular mechanism of how F-actin prevents chromosome-microtubule attachment remains an exciting open question for the future studies.
The replication of the human immunodeficiency virus type 1 (HIV-1) is as yet not fully understood. In particular the knowledge of interactions between viral and host cell proteins and the understanding of complete virus-host protein networks are still imprecise. An integral picture of the hijacked cellular machinery is essential for a better comprehension of the virus. And as a prerequisite, new tools are needed for this purpose. To create such a novel tool, a screening platform for host cell factors was established in this work. The screening assay serves as a powerful method to gain insights into virus-host-interactions. It was specifically tailored to addressing the stage of assembly and release of viral particles during the replication cycle of HIV-1. It was designed to be suitable for both RNAi and chemical compound screening. The first phase of this work comprised the setup and optimization of the assay. It was shown, that it was robust and reliable and delivered reproducible results. As a subsequent step, a siRNA library targeting 724 human kinases and accessory proteins was examined. After the evaluation of the complete siRNA library in a primary screen, all primary hits were validated in a second reconfirmation screen using different siRNAs. The purpose of this two-step approach was to identify and exclude false positives. In the end, 43 genes were reconfirmed to influence the assembly and release of HIV-1. Out of those, 39 were host dependency and 4 host restriction factors. Several of them had already been described in the literature to interact with HIV-1. However, various so far unknown host cell proteins were identified within this work. A subsequent combinatory pathway analysis including hits from other published screens identified several important signaling pathways to be important for HIV-1 assembly and release. The described single key proteins and their underlying protein networks provide a basis for the next steps toward understanding the virus and improving treatment in the future.
Obesity has become a global health problem for both children and adults. White adipose tissue (WAT) displays unique plasticity upon excessive feeding by expanding its mass through proliferation and differentiation of resident adipocyte progenitor cells (AP). Studies have shown that excessive accumulation of WAT is pre-determined during early childhood. However, mechanisms of the body weight programming and effect of WAT expansion during childhood on metabolic disease risk later in life are still elusive. In order to investigate regulation of AP expansion by obesogenic diet, we established a diet-induced obesity model. We observed a rapid gain in body weight and increased accumulation of WAT already one week after high-fat diet (HFD) feeding in young, sexually immature female mice, but not adult mice. Moreover, HFD induced WAT growth via proliferation of APs and partially through mature adipocyte hypertrophy. Employing an isocaloric, pair feeding approach, we found that dietary fat content is able to induce proliferation of APs, while the amount of consumed calories seems to promote inflammation. Moreover we demonstrated that AP proliferation could occur without initial hypertrophy of mature adipocytes. Although we demonstrated that the concentration of insulin-like growth factor 1 (IGF-1) was regulated by calorie load as little as 2 days of HFD, it was not changed upon isocaloric HFD thus could not fully explain the proliferation we observed in APs. Additionally, we found lipids transiently increased after feeding and could be potentially involved in AP activation. In order to identify genes involved in regulating AP responses in the niche upon excessive feeding during childhood, we performed gene expression profiling from whole tissue and specific cellular subpopulations of WAT. A large portion of the differentially expressed genes from WAT profiles were associated with key events in tissue remodeling and cell-to-cell communication, such as inflammation, cytokine production, and extracellular matrix proteins/remodeling enzymes. In order to functionally validate candidate genes, a novel co-culture system with APs and feeder niche-like cells was established to mimic WAT physiological conditions. This system was used for siRNA screening by high-content microscopy to identify alterations in cell proliferation and differentiation. Several gene targets related to increased proliferation of APs in response to HFD feeding have been validated including epithelial membrane protein 1 (EMP1), which known to play a role in cell junctions. EMP1 modulation in feeder cells (siRNA mediated knockdown or overexpression) altered AP-derived colony growth. Our results suggested that high fat feeding induces activation of the network of cell-cycle related genes triggering AP proliferation in early development.
Radius measurements of dust tori around the central engine of luminous active galaxies open up the possibility of probing cosmological models out to redshifts beyond where supernovae can be used. Yet, the value of dust tori as standard candles is constrained by the substantial intrinsic scatter in the size-luminosity relation found for samples of AGNs. Indicated by single objects, a probable cause of this scatter is a non-trivial variation in the dust location with the luminosity of the central engine, due to sublimation events and subsequent long-term reformation of the hot dust surface radiating at near-infrared wavelengths. In this work, I developed a refined dust reverberation model allowing to measure torus sizes and additional observables characterizing the temperature state, stability, and distribution of the innermost, hot dust in AGNs. Optical to near-infrared photometric data were observed for 24 type 1 AGNs. Of these, I have analyzed the Seyfert 1 galaxies NGC 4151, Ark 120, NGC 5548, and NGC 3227. The derived inner torus radii fit very well into the established size-luminosity relation. While three objects are well-described by a standard model without sublimation, the data of one object could be fit satisfactorily only after allowing for dust sublimation events in the model.
In this thesis, I report on the deterministic preparation and the observation of strongly correlated few-fermion systems in single and double-well potentials. In a first experiment, we studied a system of one impurity interacting with a number of majority atoms which we prepared in a single potential well in the one-dimensional limit. With increasing number of majority particles, we observed a decrease in the quasi-particle residue which is in agreement with expectations from the Anderson orthogonality catastrophe. In a second experiment, we prepared two fermions in a double-well potential which represents the fundamental building block of the Fermi-Hubbard model. By increasing the repulsion between the two fermions, we observed the crossover into the antiferromagnetic Mott-insulator regime. Furthermore, I describe a new imaging technique, which allows spin-resolved single-atom detection both in in-situ and in time-of-flight. We use this technique to investigate the emergence of momentum correlations of two repulsive fermions in the ground state of the double well. With the methods developed in this thesis, we have established a framework for quantum simulation of strongly correlated many-body systems in tunable potentials.
Defeating cancer is one of the major challenges that humankind is facing today. To this end, nuclear medicine offers promising approaches including radiometal-based pharmaceuticals. The work presented in this thesis focused on the development of new ligand systems for application as metal-chelating units in radiopharmaceuticals. These novel ligands were evaluated in various non-radioactive and radioactive metal complexation studies concerning their potential application in nuclear medicine.
Human motions result from a complex and well-coordinated interaction between the body segments. Walking and the sit-to-stand transfer are amongst the most challenging human motion in terms of coordination and internal loads, respectively. We propose model-based nonlinear optimal control methods to reconstruct and synthesize these motions while considering the dynamics of the motion over the whole time horizon. The redundant and highly nonlinear character of the computed motions encourages to discretize the optimization problem according to direct multiple-shooting methods. The goal is to identify principles which enable us to describe the patterns of these motions.
We approach human walking from the perspective of unimpaired subjects and subjects walking with unilateral transfemoral prostheses. Their walking motion is reconstructed from motion capture data using subject-specific threedimensional multibody models. The motion of the models is fitted to the recorded data for a whole stride in a least-squares sense in multi-stage optimal control problems. Analyzing the reconstructed motion for the individual foot placement of the subjects suggests that it relates with the Capturability concept: foot locations are chosen by the subjects which enable a balance between the inherently conflicting goals of effortless progression and quick response to perturbations. In addition, the modulation of the ground collision impact forces at heel strike is found to play a major role in the step-by-step stability strategy. Based on these findings, we propose Capturability as a complementary criterion to the established clinical stability assessment methods.
The sit-to-stand motion is particularly demanding for humans with mobility impairments, due to the high joint loads required to lift the body into the standing pose. We synthesize optimal sit-to-stand by solving two-stage optimal control problems. We presume that the sit-to-stand motion is substantially characterized by a preparation phase prior to the actual lift-off. Full body models are established with dynamic model parameters which specifically represent elderly humans from different levels of mobility. For impaired subjects, mobility support is assumed to be provided by generic support actions. The optimization computations result in different patterns which include significant arm motion in both phases. Therefore, the results support our approach to choose a full body representation of the human as well as to consider two stages in the optimal control problem.
The computation of optimal assisted sit-to-stand motions of impaired humans offers the opportunity to optimize design parameters for mobility assistance devices providing adequate support. Based on the support actions for the sit-to-stand motions computed for two different levels of impairment, optimal mechanical design parameters for two different sit-to-stand assistance devices are generated. Our approach to separate the human-device interaction at their interface ensures that the optimal support provided to the human by the device is not compromised by any dynamic coupling between them. Solving large-scale nonlinear optimal control problems with multiple stages, we obtain design parameters for the devices which are optimal in terms of the workspace and the mechanical effort required.
Prevalence of mutations and deregulated gene expression has resulted in a wide variety of tumor antigens which can be targeted for immunotherapy. NY-BR-1 is a breast cancer associated differentiation antigen which is overexpressed in more than 60% of breast cancers. In this study we established the first NY-BR-1 expressing transplantable tumor model using the murine mammary adenocarcinoma cell line EO771 as parental cells for the generation of NY-BR-1-expressing transfectants (EONY cells) which are compatible for transplantation into HLA-DRB1*0401tg mice. Since NY-BR-1 is not endogenously expressed in these mice, transplantation of NY-BR-1 expressing tumors led to the induction of CD4+ and CD8+ T cells specific for HLA-DR*0401- and H2-Db-restcried NY-BR-1 epitopes, respectively, previously identified in our lab. We also generated MHC I knockout cell lines with the aim of using them to study NK cell responses or as parental lines for transection of human MHC I molecules. Since several HLA-restricted NY-BR-1-specific T cell epitopes have been described, these cells could serve as an important tool to study the efficacy of using the epitopes as peptide vaccines and studying the synergistic effect of induced CD8+ and CD4+ T cells in HLA-transgenic mice co-expressing human MHC I and MHC II molecules. Additionally, we also confirmed that previously identified H2-Db-restrcited epitope was indeed naturally processed based on detection of CD8+ T cells which specifically recognized this epitope in mice following transplantation of NY-BR-1 expressing tumors or upon immunization with global NY-BR-1 antigen. We also observed that immunization with Ad.NY-BR-1 was more suitable for generating a NY-BR-1 specific CD8+ T cell line compared to immunization with nonameric peptide. We also obtained evidence suggesting that the H2-Db-restricted CTL epitope might actually be identical to the core sequence of one of the previously identified HLA-DRB1*0301-restricted CD4+ T cell epitopes. Thus binding of the CTL epitope to HLA-DRB1*0301 molecules on feeder cells might have resulted in predominant restimulation of HLA-DR3B1*0401-restricted CD4+ T cells in vitro. Cancer immunotherapy involving T cells have focused primarily on cytotoxic CD8+ T cells. However, a body of evidence has emerged in the recent past that argues for including CD4+ T cells because they not only eliminate tumor cells directly but also greatly enhance the outcome of cancer immunotherapy by various ways. One of the mechanisms by which CD4+ T cells can contribute to successful immunotherapy is by repolarizing tumor associated macrophages (TAMs) into a less immunosuppressive phenotype in an antigen dependent fashion. In this study we could demonstrate that NY-BR-1 specific CD4+ T cells could repolarize M2-like macrophages towards an M1-like phenotype upon antigen specific interaction. However, the majority of the CD4+ T cells infiltrating the EONY tumors appeared to have tumor promoting functions as depletion of CD4+ T cells resulted in delayed tumor growth and was accompanied by switch in the polarization status of TAMs towards an M-1like phenotype. Though Ad.NY-BR-1 immunization resulted in induction of NY-BR-1 specific CD4+ T cells and slight changes in polarization of intra-tumoral macrophages; it did not seem to drastically change the proportion of tumor-promoting CD4+ T cells in the tumor since the protective effect and switch in TAM phenotype was observed in both control virus and Ad.NY-BR-1 immunized mice. Therefore, antigen-specific instruction of TAMs by NY-BR-1-specific CD4+ T cells could not be demonstrated in vivo, possibly due to the prevalence of tumor infiltrating CD4+ T cells with a tumor promoting phenotype occurring in the EO771/NY-BR-1 model established within this thesis.
Glioblastoma multiforme (GBM) (WHO classification IV) is the most common primary malignant brain tumor with a poor prognosis in both adults and children. In the last years several studies have shown that 44% of glioblastoma multiforme tumors are characterized by the same histone mutations manifested in the histone variant H3.3. These studies strongly implicate the K27M amino acid substitution in H3.3 in the pathogenesis of diffuse intrinsic pontine glioma (DIPG). Also, H3.3 has been recently linked to multiple processes in the brain like neuronal specialization, synapses, cognition and contextual fear memory. In this study, two important tools were generated to investigate the role of H3.3 in the brain. Firstly, making use of the RCAS/TVA system, we established a DIPG mouse model overexpressing constitutively active AKT, PDGFB, Luciferase and floxed H3.3K27M in Nestin expressing cells in the brain. This model resembles High Grade Gliomas (HGGs) in histology and shows a key feature observed in the human DIPG tumors that is the H3K27me3 loss. In combination with Bioluminescence Imaging (BLI), the model enables monitoring of tumor growth. The model allowed for H3K27me3 recovery after deletion of H3.3K27M. Secondly, we successfully established an inducible RFP-tagged H3f3b-overexpressing mouse line. By modifying a Bacterial Artificial Chromosome (BAC) carrying the H3f3b gene, the fusion protein H3f3b-RFP-ERT2 was overexpressed. This construct allows for histone tracing in living behaving animals.
Theoretical chemistry has become an important branch of modern chemistry. Theoretical investigations improve our understanding of chemical problems and can predict properties or reaction pathways. Especially in photochemistry, quantum chemical calculations are used along with spectroscopy to analyze the interactions of molecules with light. In recent years, new methods like time-dependent density functional theory (TD-DFT) and the algebraic diagrammatic construction scheme for the polarization propagator (ADC) have been developed allowing calculations of excited states of molecules of chemical relevant size with an accuracy directly comparable with experimental results. These methods allow not only for the calculation of excitation energies, but also of excited state properties, electron densities, absorption strengths and even photoreaction pathways can be calculated. This paves the way for the theoretical investigation of all photochemical processes. Typically, however, chemical reactions and spectroscopic measurements are performed in solution. Unlike in gas phase, molecules in solution are comparatively close together, leading to an interaction between the solvent and solute molecules. In biochemistry, reactions often take place in the active center of a protein and in technical photochemical applications such as organic light emitting diodes (OLEDs) the chromophore is packed in a matrix. Hence, for comparable quantum mechanical calculations, the influence of the environment has to be considered as well. Since a direct treatment of the full environment is generally not feasible due to the computational demand of quantum chemical methods, an approximative treatment of the interaction using specific environment models is made. In my dissertation, I focused on two main topics involving both the application of existing theoretical methods, and the development of new theoretical methods. In the first part, I investigated the photochemical and electrochemical properties of various phosphorus-tetrathia-[7]heterohelicenes. The ground and several excited states of tetrathia-[7]heterohelicene-dialkylphosphane-borane (TTH-DAPB) and tetrathia-[7]heterohelicene-diphenylphosphane-gold(I)-chloride (TTH-DPP-Au(I)) have been analyzed using DFT, TD-DFT and RI-CC2. These molecules belong to the the class of helicenes, which are characterized by multiple annelated aromatic rings forming a helical structure which induces chirality. The optimized ground state equilibrium structures were compared with experimental structures determined by X-ray crystallography and showed generally good agreement. The eight energetically lowest excited singlet states have been calculated. Employing a constant shift accounting for environment effects and intrinsic errors of the applied method, the calculated spectra almost perfectly resemble the experimental absorption and circular dichroism spectra. In both molecules, both the S1 and S2 state contribute to the first absorption band. Therefore, vibrationally resolved absorption spectra have been calculated for these two states for both molecules. It could be shown that only the first excited state determines the absorption band. The second excited state exhibits a very broad band due to many normal modes contributing to the vibronic excitation. In general, the TTH backbone dominates the photochemical properties and the phosphorus and gold atoms exhibit only minor influences. In addition, electrochemical properties of the phosphine-oxide TTH derivatives TTH-(PO(n-Bu)2)2, TTH-(PO(Ph)2)2 and TTH-PO(Ph)2 as well as of the two phosphine-selenide TTH derivatives TTH-(PSe(Ph)2)2 and TTH-PSe(Ph)2 have been calculated. Ionization energies and electron affinities have been computed both in gas phase and solution. In solution, all first electron detachments and attachments are localized on the TTH moiety with only minor influence of the substituents. Each process is qualitatively determined in all molecules by a single frontier orbital, which has been verified by difference density analysis. For the phosphine-oxide TTH derivatives the gas phase results resemble the results in solution. The phosphine-selenides, however, show a different picture. The lone-pairs are shifted higher in energy without stabilization of the environment, leading to an ionization localized at the selenium atom in the gas phase. The second focus of my dissertation was the development, implementation, and testing of a new method for including environment interaction in the excited state of a central molecule. To this end, I combined frozen density embedding thoery (FDET) with the ADC method to develop the new FDE-ADC method. This method is implemented in the quantum chemical program package Q-Chem as the module fdeman, which manages the FDE-ADC calculation. In FDET, the supersystem is divided in two subsystems: the embedded system (A) and the environment (B). The name „embedded system“ comes from the fact that it is embedded in the electron density of the environment. The inuence of the environment is expressed in an embedding potential, which depends on both electron densities of A and B. In fdeman, the whole FDE-ADC calculation is performed in a four step process: a) generation of the electron density of the embedded system _A(~r), b) generation of the electron density of the environment _B(~r), c) calculation of the embedding potential vlin emb(~r) and _nally d) applying vlin emb(~r) in an FDE-ADC calculation by adding it to the Fock matrix during the SCF The second focus of my dissertation was the development, implementation, and testing of a new method for including environment interaction in the excited state of a central molecule. To this end, I combined frozen density embedding theory (FDET) with the ADC method to develop the new FDE-ADC method. This method is implemented in the quantum chemical program package Q-Chem as the module FDEman, which manages the FDE-ADC calculation. In FDET, the supersystem is divided in two subsystems: the embedded system (A) and the environment (B). The name „embedded system“ comes from the fact that it is embedded in the electron density of the environment. The influence of the environment is expressed in an embedding potential, which depends on both electron densities of A and B. In FDEman, the whole FDE-ADC calculation is performed in a four step process: a) generation of the electron density of the embedded system rho_A, b) generation of the electron density of the environment rho_B, c) calculation of the embedding potential v_emb and finally d) applying v_emb in an FDE-ADC calculation by adding it to the Fock matrix during the SCF followed by an ADC calculation using the orbitals influenced by the environment. While the straight-forward implementation of FDE-ADC uses a supermolecular basis to express both density matrices and the embedding potential, an approximate variant named re-assembling of density matrix (RADM) has been introduced in which the density matrix of A is built together from MP(2) and HF based density matrices like a patchwork. The created embedding potential is subsequently cut to the monomer basis which features an FDE-ADC calculation using only the basis functions of the embedded system. This can be done since in the contraction of the density of A with the embedding potential, only the values of the block in the density matrix representing the embedded system contribute. FDE-ADC has been benchmarked up to third order perturbation theory employing three test systems, designed to exhibit an increasing strength of environment interaction. The test systems are 1) benzene with a hydrogen fluoride molecule in plane with the benzene ring, 2) benzaldehyde with a hydrogen-bonded water dimer and 3) uracil surrounded by five hydrogen-bonded water molecules. In the benchmark, the FDE-ADC results have been compared with supermolecular ADC calculations. The deviation from the reference calculation in excitation energies and oscillator strengths determines the accuracy of FDE-ADC. For SE-FDE-ADC(2) and RADM-FDE-ADC(2), mean absolute errors (MAEs) of 0.025 eV and 0.040 eV in excitation energies have been determined, respectively. For RADM-FDE-ADC(3), an MAE of 0.029 eV has been calculated. These errors are well below the intrinsic error of the underlying ADC methods, thus demonstrating the performance of FDE-ADC. This is furthermore demonstrated in three representative applications. First, the excited states of benzoquinone in 42 methanol molecules have been investigated. Next, the vertical photochemical properties of the photoswitch spiropyran in 100 water molecules have been investigated. In the last application, the core-valence excited states of carbon monoxide inside a C60-cage have been calculated. Using a frozen environment neglects the influence of the embedded system on the environment. This is called environment polarization and can be added following two different approaches. In the first variant referred to as pre-polarization, the ground state influence of the embedded system on the environment is treated by an electrostatic potential which is applied during the calculation of the environment density. This way, rho_B is not calculated in the gas phase but instead in the presence of A. In the second variant, referred to as excitation-induced environment polarization, the influence of an electronic excitation of A on the environment is considered. Therefore, the subsystems are interchanged and alternatingly embedded in each other until self-consistency (freeze and thaw). Here, two approximate variants to include excitation-induced environment polarization are introduced. In the first variant, named state-specific iteration (SSI), the alternate embedding is performed once, which prevents changes in the order of the excited states. In the second variant called difference density polarization potential (DDPP), the environment is embedded consecutively in the ground and excited state density of system A. The electron difference density describing the polarization of the environment is used to create a potential which is employed to calculate an energy correction for the excitation energy of the excited state of A. Both SSI and DDPP as well as the pre-polarization are implemented in the module FDEman in Q-Chem. In tests, both the pre-polarization and SSI could increase the accuracy of FDE-ADC. In the case of SSI, up to 35 % increased accuracy is observed. DDPP currently does not improve the results. In total, the FDE-ADC method is a promising approach for considering environmental effects on electronically excited states. The error of this method is lower than the intrinsic error of the employed ADC method. Using the RADM approximation, explicit treatment of extended environments is directly feasible, making FDE-ADC a „black box“ method for the calculation of excited states in complex environments.
Diffusion weighted magnetic resonance imaging (MRI) can be used to gain information on the microstructure of the examined tissue on length scales below the actual image resolution. The large gradient amplitudes required for diffusion measurements can lead to artifacts due to eddy currents and concomitant fields. Another source of image artifacts, is the presence of directed motion such as blood flow or pulsation. In a first MRI sequence, a common approach for eddy current compensation, the twice-refocused spin echo was adjusted for a double diffusion encoding (DDE) sequence. In measurements of healthy volunteers, this approach reduced the falsely elevated microscopic fractional anisotropy (uFA) in the gray matter on average from 0.57 +- 0.19 to 0.50 +- 0.19 and in the ventricles on average from 0.54 +- 0.19 to 0.28 +- 0:27. A second sequence, with a single diffusion encoding, was compensated for any combination of the three artifact sources flow, concomitant fields and eddy currents. For most in vivo measurements, it proved to be sufficient to compensate for flow and concomitant fields. An additional eddy current compensation led only in the brain measurements to a higher reproducibility. The developed sequence was also used to measure the incoherent intravoxel motion (IVIM) effect in the abdomen as well as the prostate of healthy volunteers, where a difference between flow-compensated and non-flow-compensated measurements was observed. This difference could not be seen in patients with prostate carcinoma.
The helium isotope system is an established tool in hydrology for identifying mantle fluids in deep aquifers. This study applies the helium tracer system for the first time in shallow, unconfined aquifers of the Upper Rhine Graben. The Graben is a part of the Cenozoic Rift system of Western and Central Europe, a continental rift zone with unusually high geothermal gradients, making it an ideal region of Germany for geothermal energy development. The aim of this study is to develop a suite of natural groundwater tracers able to achieve a cost and effort reduction in geothermal prospection. The 3He/4He-ratio is therefore applied, as part of a multi-tracer approach including 3H, δ18O, δ2H, δ13C, 14C and 222Rn, to identify and locate fault zones with suitable permeabilities for power plant operation. Three target areas along the graben were studied, each located on one of the main fault lines. A mantle-derived helium signature could be identified and separated from tritiogenic helium in a shallow aquifer in the north-west of the Graben. The mixing component of mantle-derived fluid in the shallow groundwater is calculated to reach up to 5%, based on the analysis of the 3He/4He isotope system. The employed method proves that the local permeability of the fault zone is high. The origin of the locally occurring upwelling of salinated water can be redetermined by the data.
It is well known that the Standard Model is not complete and many of the theories that seek to extend it predict new phenomena that may be accessible in low-energy settings. This thesis deals with some of these, namely, novel spin-dependent interparticle potentials, axion-like particles and Lorentz-symmetry violation. In Part I we discuss the spin-dependent potentials that arise due to the exchange of a topologically massive mediator, and also pursue a comparative study between spin-1/2 and spin-1 sources. In Part II we treat massive axion-like particles that may be copiously produced in core-collapse supernovae, thus leading to a non-standard flux of gamma rays. Using SN 1987A and the fact that after its observation no extra gamma-ray signal was detected, we are able to set robust limits on the parameter space of axion-like particles with masses in the 10 keV - 100MeV range. Finally, in Part III we investigate the effects of Lorentz-breaking backgrounds in QED. We discuss two scenarios: a modification in the Maxwell sector via the Carroll-Field-Jackiw term and a new non-minimal coupling between electrons and photons. We are able to set upper limits on the coefficients of the backgrounds by using laboratory-based measurements.
This thesis is concerned with spatial homology truncation for path connected CW-complexes and the following question: Which continuous maps between two compact pseudomanifolds with isolated singularities induce continuous maps between the corresponding intersection spaces, and when is this assignment functorial? Chapter 1 deals with the construction of a spatial homology truncation functor for path connected CW-complexes, which extends existing results for simply connected CW-complexes. In Chapter 2 we partially use the results of the first chapter to present different approaches to the problem of inducing maps between intersection spaces. Finally, the induced maps between reduced homology groups of intersection spaces and the induced maps between intersection homology groups will be assembled in a morphism of reflective diagrams.
Tumor progression represents an array of complex events that ultimately lead to metastasis, the end-stage of cancer that is responsible for the majority of cancer-related mortalities. Improved ways to target the spread of cancer are thus imperative to treat cancer effectively. Understanding the mechanisms that regulate the dissemination of cancer cells from the primary site to distant places is a pre-requisite for such strategies. Asap1 (Arf-GAP with SH3-domains, Ankyrin-repeats and PH-domains) was identified by our lab in an unbiased screen to identify genes whose expression is associated with the metastatic phenotype. It was shown to be functionally involved in tumor progression in experimental animal models and this expression was correlated with poor metastasisfree survival and prognosis in colorectal cancer patients. To understand the role of Asap1 in normal physiology and in cancer, in my thesis work I studied Asap1 knockout (Asap1GT/GT) mice, generated in our lab by targeted deletion of the gene. I observed that Asap1GT/GT mice can live to maturity, although there is a reduction in the expected number of homozygous knockout offspring at birth. Deletion of Asap1 results in growth retardation, respiratory distress and reduced angiogenesis in the surviving pups. This physiological phenotype in the absence of Asap1 is transient, and adult Asap1GT/GT mice are morphologically undistinguishable from the wild-type mice.I further studied breast tumor development and metastasis in Asap1GT/GT mice using autochthonous models of breast cancer. My results demonstrate that loss of Asap1 in MMTV-PyMT mice leads to an earlier tumor onset, faster tumor growth and increased metastasis to the lungs. I also examined the effect of ASAP1 deficiency on the behaviour of breast cancer cells and fibroblasts taken from Asap1+/+ and Asap1GT/GT mice. Taken together, my results show that Asap1 is a critical regulator of cellular motility and its absence gives rise to developmental defects in newborn mice.Deficiency of ASAP1 also has tumor cell non-autonomous effects, and leads to increased numbers of metastases in the MMTV-PyMT autochthonous mouse model of breast cancer.
In this thesis, we address coupled incompressible flow problems with respect to their efficient numerical solutions. These problems are modeled by the Oseen equations, the Navier-Stokes equations and the Brinkman equations. For numerical approximations of these equations, we discretize these systems by Hdiv-conforming discontinuous Galerkin method which globally satisfy the divergence free velocity constraint on discrete level. The algebraic systems arising from discretizations are large in size and have poor spectral properties which makes it challenging to solve these linear systems efficiently. For efficient solution of these algebraic system, we develop our solvers based on classical iterative solvers preconditioned with multigrid preconditioners employing overlapping Schwarz smoothers of multiplicative type. Multigrid methods are well known for their robustness in context of self-adjoint problems. We present an overview of the convergence analysis of multigrid method for symmetric problems. However, we extend this method to non self-adjoint problems, like the Oseen equations, by incorporating the downwind ordering schemes of Bey and Hackbusch and we show the robustness of this method by empirical results. Furthermore, we extend this approach to non-linear problems, like the Navier-Stokes and the non-linear Brinkman equations, by using a Picard iteration scheme for linearization. We investigate extensively by performing numerical experiment for various examples of incompressible flow problems and show by empirical results that the multigrid method is efficient and robust with respect to the mesh size, the Reynolds number and the polynomial degree. We also observe from our numerical results that in case of highly heterogeneous media, multigrid method is robust with respect to a high contrast in permeability.
Malaria, caused by apicomplexan parasites of the Plasmodium species, is one of the deadliest infectious diseases worldwide. Despite the urgent need to identify new drug targets and vaccine candidates, a large proportion of the Plasmodium genes are uncharacterized, as tools to study gene function are limited. In many eukaryotes, genes can be silenced via RNA interference (RNAi) using artificial short hairpin RNAs (shRNAs). However, Plasmodium parasites lack the machinery required for RNAi. In this study, I therefore engineered a non-canonical RNAi machinery into the rodent parasite Plasmodium berghei (P. berghei). To this end, I exploited a non-canonical RNAi pathway which requires only a single protein, Argonaute 2 (Ago2), and a specifically designed shRNA, a so-called AgoshRNA, for gene silencing. I generated a P. berghei line constitutively expressing Ago2, named PbAgo2, and demonstrated that this parasite can complete its life cycle through the mammalian and insect host, despite exhibiting a reduced growth in blood and mosquito stages. Expression of AgoshRNAs targeting the mRNA of the green fluorescent protein GFP (constitutively expressed by PbAgo2) induced a potent knockdown of GFP both in blood and in non-erythrocytic stages. As different AgoshRNAs mediated gene silencing to various levels, target gene expression could be fine-tuned. AgoshRNA-mediated gene knockdown was also possible for endogenous genes, and the knockdown of a non-essential gene phenocopied the full knockout. Additionally, the expression of a blood-stage-essential gene was reduced using RNAi. The analysis of the transcriptome of PbAgo2 by RNA sequencing suggested a possible interaction between Ago2 and a Plasmodium mRNA storage protein as a putative reason for the growth impairment. To further increase the potential applications of the RNAi-competent parasite, Ago2 expression was restricted to the liver stage using a stage-specific promoter. This transgenic line behavee indistinguishable from wild type and the expression of an AgoshRNA targeting GFP silenced fluorescence exclusively in late liver stages. In summary, PbAgo2 is a potent tool to modulate gene expression without the need to alter the genetic locus. In contrast to existing tools, PbAgo2 provides the option to target genes exclusively in a single life cycle stage, to multiplex different AgoshRNAs enabling the simultaneous knockdown of multiple genes, or to screen for phenotypes using a library of AgoshRNAs. This novel, RNAi-competent parasite line opens a wealth of new options to annotate genes in Plasmodium.
Patients with neck squamous cell carcinoma from unknown primary tumor (NSCCUP) present with lymph node metastases without evidence for a primary tumor. Most patients undergo an aggressive multimodal treatment, which induces severe toxicity. Primary tumors of NSCCUP can be hidden in the oropharynx. Human papillomavirus (HPV) is causally involved in a subgroup of oropharyngeal squamous cell carcinomas (OPSCC) associated with early lymph node metastasis and good prognosis. Detection of markers for HPV transformation in NSCCUP could allow focusing on the oropharynx in primary tumor search and could be of value for choice and extent of treatment. In this retrospective multicenter study analyzing 180 NSCCUP cases from Heidelberg, Treviso and Barcelona, a substantial proportion (16%) was driven by HPV, mainly by HPV16 (89%). The prevalence of HPV-driven NSCCUP varied by geographical region, ranging from 10% in Barcelona to 20% in Heidelberg, and increased with year of diagnosis from 9% during 1988-2004 to 23% during 2005-2014 (p=0.007). Compared to HPV mRNA as gold standard to identify HPV-driven tumors, sensitivity and specificity of HPV DNA, p16INK4a overexpression or the combination of both markers in NSCCUP ranged from 81-100% and 89-100%, respectively, with the lowest concordance for p16INK4a (kappa=0.7) and the highest for the combination (kappa=0.95). HPV seropositivity was a promising diagnostic marker for HPV-driven NSCCUP, since the detection of HPV antibodies in serum from NSCCUP patients had a sensitivity of 91% and specificity of 100% compared to HPV mRNA detected in the metastasis (kappa=0.93). HPV-driven NSCCUP were molecularly different from non-HPV-driven NSCCUP, because they presented with a distinct DNA methylation pattern in five gene promoters and they did not harbor disruptive TP53 mutations, which were common in non-HPV-driven NSCCUP (52% vs. 0%, p=0.0002). Patients with HPV-driven, as well as HPV-seropositive NSCCUP had significantly better overall and progression-free survival rates (p≤0.002). Based on the observed survival benefit, HPV mRNA status assessment should be included in NSCCUP diagnosis. Besides an extended diagnostic work-up of the oropharynx in patients with HPV-driven NSCCUP, de-intensification of radiotherapy concentrating on the oropharynx appears a promising therapeutic strategy, the efficacy of which should be assessed in prospective trials. Analysis of twelve pairs of HPV16-driven OPSCC and corresponding lymph node metastases revealed consistent presence of HPV DNA and mRNA. However, heterogeneity was observed regarding HPV integration status and DNA methylation. Viral-cellular junctions identified in the primary tumor were present in only 43% of corresponding metastases, while new viral-cellular junctions were detected in 14%. Metastases had overall lower methylation levels in the five gene promoters included in the assessed HPV-associated methylation signature compared to primary tumors.
Wie lockt man Kinder vom Smartphone an die frische Luft und weckt in ihnen gleichzeitig Begeisterung für die Forschung? Das schaffen die naturwissenschaftlichen Erlebnistage „Explore Science“ der Klaus Tschira Stiftung. Campusreporter Nils Birschmann hat mit Kim Orzol, Projektleiter von Explore Science, gesprochen.
Der Beitrag erschien in der Sendereihe "Campus-Report" - einer Beitragsreihe, in der über aktuelle Themen aus Forschung und Wissenschaft der Universitäten Heidelberg, Mannheim, Karlsruhe und Freiburg berichtet wird. Zu hören ist "Campus-Report" montags bis freitags jeweils um ca. 19.10h im Programm von Radio Regenbogen (Empfang in Nordbaden: UKW 102,8. In Mittelbaden: 100,4 und in Südbaden: 101,1).
Replicating oncolytic viruses (OVs) that are able to selectively destroy malignant cells are emerging as clinically relevant cancer therapeutics. Along with direct tumor cell lysis, activation of specific anti-tumor immune responses contributes to efficacy of virotherapy, allowing to consider it for a type of cancer immunotherapy. Combinations with different immunomodulation strategies have been shown to enhance the immunostimulatory effects of OVs and contribute to increased therapeutic efficacy. Based on the hypothesis that certain immunomodulation types might more efficiently contribute to efficacy of virotherapy in a given tumor type, this study compared efficacy of oncolytic measles vectors encoding immunomodulators from different classes. Furthermore, to identify immune effector mechanisms associated with successful therapeutic strategies, analysis of the tumor immune environment was performed following treatment with the most promising vectors. Measles Schwarz vaccine strain vectors (MeVac) encoding immunomodulators to target the main phases in establishment of an anti-tumor immune response were developed. Therapeutic efficacy of the novel vectors was compared in a fully immunocompetent murine colon adenocarcinoma model, MC38cea. MeVac vectors encoding an antibody against the negative T cell regulator PD-L1 (anti-PD-L1) and a fusion protein of murine interleukin-12 (FmIL-12), respectively, were identified as the most promising in terms of increased survival of animals. Importantly, MeVac encoding FmIL-12 was the most effective, ensuring complete tumor remissions in 90% of the treated animals. After MeVac therapy, long-term survivors rejected secondary tumor engraftments, indicating establishment of a systemic anti-tumor immune response. Profiling of the tumor environment four days after the last treatment with the anti-PD-L1 encoding vector revealed a slight benefit for cell mediated immune responses, as observed by a slight upregulation of the effector cytokines IFN-γ and TNF-α as well as an increase in the intratumoral T cell population. More pronounced modulation of the tumor immune environment was observed following treatment with the FmIL-12 encoding vector. One day after treatment with MeVac encoding FmIL-12 an increase of effector cytokines IFN-γ and TNF-α was observed, suggesting activation of a cell mediated immune response. Analysis of tumor infiltrating lymphocytes revealed an increase in the T cell population, a massive decrease in the natural killer (NK) cell population and upregulation of an activation marker on NK cells. These results indicated early activation of the immune effector cells following treatment with MeVac encoding FmIL-12, which in case of the NK cells could be associated with activation induced cell death. Furthermore, immune cell depletion experiments revealed that the CD4+ T cells and NK cells do not importantly contribute to the therapeutic efficacy of the MeVac encoding FmIL-12 in this model, but that the cytotoxic CD8+ T cells are essential. This study presents MeVac encoding FmIL-12 as an effective therapeutic for activation of cell mediated anti-tumor immune responses. Furthermore, the MeVac vector is established as a flexible platform for targeted local delivery of immunomodulators. The tumor immune profiling data provide a basis for further rational vector modifications to develop immunomodulation strategies tailored to the individual tumor immune environment.
Embryonic development establishes the body plan, organs, and the shape of the adult animal organism. This process involves cells and tissues that eventually will not be part of the growing embryo, so-called extraembryonic tissues. In insects, extraembryonic tissues contribute to embryonic development by fulfilling important roles during specific morphogenetic movements, such as blastokinesis, germband retraction and dorsal closure, but also in the protection of the embryo against egg desiccation and pathogens. Within insects, extraembryonic tissues differ in number and topology, they may display diverse morphologies even between closely related species, and it is currently not yet clear which specific function each extraembryonic tissue fulfills and how its development is genetically regulated. Most of our current understanding of extraembryonic development and function in insects stems from studies in Tribolium castaneum and Drosophila melanogaster. The two species show several morphological differences, not only at the extraembryonic level but also in the morphology of the embryo. Specifically, T. castaneum has two extraembryonic tissues called amnion and serosa: the serosa separates from the embryo, grows over it, and eventually encloses the embryo, the amnion stays attached to the embryo and covers its ventral side. D. melanogaster, by contrast, develops only one single extraembryonic tissue called amnioserosa that remains in constant contact with the embryo and stays on its dorsal side. The diversity in form, development, and function of extraembryonic tissues in insect species provides an outstanding model to address how form and function of specific epithelia evolved, and how these changes were genetically encoded. In my thesis, I have taken advantage of intermediate characters in extraembryonic development of Megaselia abdita (Diptera, Phoridae), which features a similar embryonic development as D. melanogaster but maintained two extraembryonic tissues and thus part of the ancestral extraembryonic development described in T. castaneum. I have focused my attention on a detailed in vivo analysis of extraembryonic development at a morphogenetic and cellular level by establishing and using light-sheet microscopy. I acquired evidence that links extraembryonic tissues behavior in M. abdita to orthologues of the T-box transcription factor Dorsocross and the tumor necrosis factor Eiger, which in D. melanogaster are key genes that contribute to specification and morphogenesis of the amnioserosa. In vivo observations and functional studies suggest an important interaction of the extraembryonic tissues of M. abdita with the extracellular matrix that seems to be finely regulated. In conclusion, the results of this study increase our knowledge on morphology and development of extraembryonic tissues in M. abdita and provided an in vivo technique for non-model organisms to study in toto dynamics of early development.
Centromeres are the primary constriction sites of mitotic chromosomes and a prereq- uisite for chromosome segregation during mitosis. Centromeres serve as a platform for kinetochore formation and spindle attachment and are epigenetically regulated. Specific proteins and protein modifications discriminate centromeric chromatin from the surrounding pericentromeric heterochromatin. Emerging evidence indicates that RNAs are important factors in centromere identity but the composition and regula- tion of centromeric transcripts are largely unknown. Surprisingly, the role of RNA modifications at centromeres is completely unknown. In this doctoral thesis, a subset of transfer RNAs (tRNAs) was found to localise to mitotic centromeres, as well as a number of different RNA processing enzymes. Among them were the cytosine-5 tRNA methyltransferases Dnmt2 and NSun2. Depletion of these enzymes caused severe chromosome segregation defects, suggesting a role of tRNA methylation in mitosis. Strikingly, analysis of enzymatically inactivated Dnmt2 indicated a direct role of cytosine-5 RNA methylation in the regulation of centromeres. Depletion of Dnmt2 affected (peri-) centromeric chromatin compositions, which presumably lead to the observed mitotic defects. The detection of components of the RNA poly- merase III (RNAPIII) transcription machinery suggested a role of active transcrip- tion at centromeres during mitosis. Indeed, inhibition of RNAPIII-mediated tran- scription caused comparable chromosome segregation defects as observed in tRNA methyltransferase mutant backgrounds. Strikingly, the centromeric localisation of RNAPIII appeared sensitive not only to transcriptional inhibition but also to cen- tromeric levels of Dnmt2. Vice versa, Dnmt2 was dependent on centromeric RNA and RNAPIII transcription, which suggests an interdependent role of RNAPIII tran- scription and tRNA methylation at centromeres. This thesis describes a novel role of RNAPIII transcription and tRNA methylation during mitosis in Drosophila, which functionally connects an epitranscriptomic mechanism and the epigenomic regulation of centromeres. Moreover, the mitotic function of Dnmt2 appeared to be conserved in mammalian cells, which suggests a conserved role of RNA modification in the regulation of centromeric chromatin.
Colorectal cancer (CRC) is among the most frequently diagnosed cancers worldwide. Recent research focused on the association of CRC with an altered microbiome. More specifically, two bacteria, Fusobacterium nucleatum (F. nucleatum) and Streptococcus gallolyticus subspecies gallolyticus (S. gallolyticus) were individually brought in context with CRC. F. nucleatum is predominantly present in oral plaques and was found to be abundant in stool and tumor tissue of CRC patients. S. gallolyticus is a rare commensal in the human intestine and inducer of infective endocarditis that is associated with presence of CRC. The aim of this thesis was to explore potential serological associations of F. nucleatum and S. gallolyticus with CRC using multiplex serology, a high-throughput technology that allows the analysis of large seroepidemiological studies. Multiplex serology was to be developed for F. nucleatum and S. gallolyticus and applied in a retrospective case-control study to analyze potential serological associations with CRC. Prospective studies were to be analyzed to give information on temporality of the association: if serological associations are present prior to diagnosis, these antibodies might serve as early marker for risk of developing CRC. Eleven proteins for each, F. nucleatum and S. gallolyticus, were selected, recombinantly expressed and applied in multiplex serology. Serological validation of the assays was possible only to a limited extent due to a lack of a gold standard assay for comparison. Cut-offs for antibody-positivity to the individual proteins were arbitrarily defined to allow for 10% of controls as positive. Antibody responses to F. nucleatum and S. gallolyticus were analyzed in a retrospective case-control study conducted in Germany and two independent case-control studies nested within multi-center prospective cohorts from Europe and southern United States. Positivity to any of the F. nucleatum proteins was not associated with CRC, neither retro- nor prospectively. In contrast, odds for prevalent and incident CRC in the German case-control study as well as the European prospective study were significantly 2-fold increased with positivity to two or more proteins of a S. gallolyticus 6-marker panel. However, this association was not found in the southern United States study. In conclusion, antibody responses to S. gallolyticus, but not F. nucleatum, were significantly associated with CRC prior to diagnosis and might serve as marker for CRC development. A causal relationship of S. gallolyticus with CRC cannot be inferred from the generated data, however, results of this thesis might stimulate research on the involvement of S. gallolyticus in CRC development as well as risk factors leading to S. gallolyticus colonization.
Miwi2 and Mili are involved in piRNA production and transposon silencing in fetal gonads. It is known that the loss of Miwi2 leads to a complete exhaustion of germline in male mice. Whether the absence of Mili results in the same phenotype, has not been established yet. In this study we show that Mili is also required for germ cell maintenance, but presents a much weaker phenotype than Miwi2 with only a partial loss of the germ line observed in aged mice. The difference between the two mutant phenotypes could be explained by the fact that in Mili deficient fetal testes a small amount of piRNAs is produced that can mount a residual defense against transposable elements as determined by bisulfite sequencing data. We have determined that piRNAs made in the absence of Mili make up a small pool of piRNAs that are also present in the wild type mice. Mili mutant piRNAs are mainly primary in nature and are most likely bound by Miwi2 since Miwi2 partially retains its nuclear localization in the absence of Mili in mouse fetal gonadocytes. In summary, a primary piRNA biogenesis pathways exist in mouse in the absence of Mili that contribute to both germ line reprogramming and maintenance. Miwi2 and Dnmt3L are involved in de novo DNA methylation in fetal gonads. Miwi2 and Dnmt3L mutant mice exhibit a depletion of germline with age. Dnmt3L mutant mice completely lose germ cells within a two-month period, while Miwi2 mutant mice exhibit Sertoli-only phenotype at nine-month of age. Nonetheless, the phenotype of both reprogramming mutants indicates Miwi2 and Dnmt3L’s possible role in germline maintenance. In this study we have shown that the main role of Miwi2 and Dnmt3L in establishing and maintaining the population of undifferentiated spermatogonia precursor cell population in the adult is executed through their role in reprogramming fetal gonadocytes. We have observed that even though Miwi2- and Dnmt3L-deficient spermatogonial precursor cells exhibit transposon de-repression, no apparent loss these cells can be associated with DNA damage. We hypothesize that actively transcribed TE containing locus generates hybrid transcripts triggering abnormal gene expression program in spermatogonia stem cells, which leads to a great reduction in the numbers of actual GFRa1pos stem cells observed n Miwi2 and Dnmt3L mutant testis. Overall, this study shows the importance of germ cell reprograming in establishing a healthy, functional stem cell population in the adult tissue.
The Epstein-Barr-Virus is an oncogenic herpesvirus that establishes a lifelong infection in humans. It is ubiquitous in the population and is responsible for the development of multiple diseases including cancer. Analysis of the EBV DNA and RNA sequences has predicted that the viral genome encodes approximately 100 protein-coding genes. However, the existence of many putative proteins has not been confirmed by biochemical methods yet. Furthermore, the identification of EBV open reading frames (ORFs) is difficult as viral genes are encoded on both strands of the double-stranded DNA genome and often overlap. Moreover, EBV encodes different types of non-coding RNAs. In order to identify the full scope of EBV’s coding capacity, ribosome profiling of replicating and non-replicating EBV strains was performed. Ribosome profiling combines classical ribosome footprinting experiments with current deep sequencing technology to map translating ribosomes on mRNA at single nucleotide resolution. This approach confirmed the majority of previously identified ORFs and has enabled the identification of 28 novel small open reading frames and of 8 alternative translation initiation sites. 25 of the 28 small ORFs were localized in the 5’leaders of several mRNA transcripts and are classified as upstream open reading frames (uORFs). Several of these uORFs were found to repress the translation of the downstream encoded main ORF. In summary, ribosome profiling of EBV-infected cells has allowed a comprehensive identification and annotation of the EBV ORFs and has revealed a novel mode of viral gene expression regulation at the translational level.
Studies as early as in the 70s showed that the gut and its intrinsic gut microbiota is a possible site of drug modification and later studies confirmed that human microbiota metabolism with its diverse set of genes can be a cause for drug side effects. Yet, our knowledge of the biochemical capabilities of gut bacteria to interact with or metabolize therapeutic drugs remains largely incomplete. To our knowledge, there has not been any systematic screen of xenobiotic-microbial interactions elucidating how wide-spread bacterial drug modification is across therapeutic drugs or the gut microbiota. In my PhD work, I tested, under anaerobic conditions, 450 bacteria-drug interactions covering 25 metabolically diverse gut bacteria and 18 structurally diverse FDA-approved drugs. This revealed almost 50 novel bioaccumulation or biotransformation links between 19 bacterial species and 10 drugs. The implicated bacteria are phylogenetically diverse, including commensals, probiotics and bacteria associated with diseases. The affected drugs span diverse indication areas, from asthma (montelukast) to depression (duloxetine and aripiprazole). As a case in point, the results from this bacteria-drug interaction study are followed upon in more details through investigation of interactions involving duloxetine – a widely used antidepressant. I found that duloxetine induces higher diversity in synthetic bacterial communities, and its bioaccumulation by community members affects the community dynamics. Following, I found that duloxetine affects the native metabolism of B. uniformis and C. saccharolyticum, in particular the purine metabolism. These interactions might in turn influence bacterial behavior in a community. To find the direct protein targets of duloxetine in C. saccharolyticum, I used click chemistry-based methods and proteomics. Two of the five strongly enriched binding proteins are part of a NADH:quinone dehydrogenase complex. Two potential underlying mechanisms for duloxetine interactions are suggested: i) Duloxetine inhibits NADH:quinone dehydrogenase by binding to its quinone binding site. The resulting NADH excess leads to a change in downstream pathways like purine metabolism. ii) Duloxetine binds competitively on the NADH binding site of NADH:quinone dehydrogenase and other proteins. In addition to discovering new xenobiotic interactions, the study highlights a new dimension to gut microbiota-drug interactions, namely bioaccumulation, which so far has been largely overlooked. My results suggest that bioaccumulation of drug compounds might be a common feature to many gut bacteria and thus have broad and far-reaching implications for drug dosage decisions and personalized medicine.
Novel genes are being discovered at constantly increasing rates by sequencing bacterial genomes and bacterial communities. Gene function discovery has been lagging behind, but recent technological advances allow us to apply reverse genetics approaches on a genome wide scale. In this study I profile the growth of more than 3800 gene deletion mutants of the pathogen Salmonella Typhimurium in more than 550 perturbations including physical stresses, nutrient limitation, antibiotics and host defense molecules. Analysis of gene-drug interaction scores reveal significant phenotypes for 75% of the tested mutants. The data set provides a number of novel biological inferences, linking genes of unknown function to known pathways and providing insights into drug mode-of-action, uptake and efflux. Using similar high-throughput data available for E. coli., I provide the first comprehensive cross-species comparison of genetic networks in bacteria. Correlation analysis and detection of functional modules reveals broad conservation of cellular pathways and drug responses between Salmonella and E. coli. However, I also find intriguing cases of network rewiring and investigate how species-specific genes connect to conserved modules. Lastly, I investigate the highly different resistance levels of Salmonella and E. coli to the type 2 diabetes drug metformin and determine the Salmonella-specific efflux pump SmvA as the major component conferring drug resistance. Furthermore, I identify more transporters capable of exporting metformin and examine their wiring into the cellular networks of E. coli and Salmonella. This analysis reveals that many enterobacteria may have the potential to develop resistance against metformin leading to important implications for diabetic patients.
One outcome of compartmentalization in eukaryotic cells is that each organelle has the ability to establish a unique pH environment. Homeostasis of the luminal pH within these organelles is essential for survival of the cell because important processes such as secondary active transport and enzymatic reactions depend on optimal pH conditions. Luminal pH is a product of the cooperative effort between H+ pumps and ion transporters located on the limiting membrane of the organelle. The vacuolar H+-ATPases (V-ATPases) are one class of proton pumps that are well conserved within eukaryotic cells. V-ATPases are multisubunit complexes that hydrolyze ATP and transport protons into the lumen of organelles. In plant cells, the V-ATPase is important for pH homeostasis at the vacuole and early endosome but most of the information available about the eukaryotic V-ATPase complex comes from studies in yeast and mammals. In plants, nothing is known about the mechanism of targeting in the cell, the regulation of its activity and whether the pump has secondary functions aside from pH homeostasis. Our research focus was on subunit a (VHA-a) of the VO subcomplex of plants. The localization of the V-ATPase in the cell is determined by the isoforms of VHA-a. The sorting signal of any VHA-a subunit was not known. In the first chapter, we report the discovery of an acidic cluster and a critical leucine (L159) residue in the N-terminus of VHA-a1 that serves as both an ER export signal and as a TGN retention motif. This motif is reminiscent of mammalian endosomal targeting acidic clusters but the specific sequence of amino acids is unique to the plant kingdom and the motif originates in the gymnosperm sequences. In the second chapter we investigated whether the TGN localized V-ATPase not only fulfils its primary function of acidification, but whether it is also a component of the molecular machinery that senses the level of luminal acidification and its recruits cytosolic proteins involved in vesicle trafficking to the membrane. In the third chapter, we explore a new form of modification of the tonoplast localized isoform (VHA-a3). We show that Sacylation is not involved in targeting of VHA-a3 to the tonoplast and that VATPase activity is not compromised by a lack of S-acylation in the presence of high concentrations of zinc.
Theories of spin-2 fields take on a particular role in modern physics. They do not only describe the mediation of gravity, the only theory of fundamental interactions of which no quantum field theoretical description exists, it furthermore was thought that they necessarily predict massless gauge bosons. Just recently, a consistent theory of a massive graviton was constructed and, subsequently, generalized to a bimetric theory of two interacting spin-2 fields. This thesis studies both the viability and consequences at cosmological scales in massive gravity as well as bimetric theories. We show that all consistent models that are free of gradient and ghost instabilities behave like the cosmological standard model, LCDM. In addition, we construct a new theory of massive gravity which is stable at both classical background and quantum level, even though it suffers from the Boulware-Deser ghost.
We build robots that are meant to look and work like humans, with humans, inspired by humans. But many are the human characteristics that we have not yet understood, as humans are highly complex systems. One fundamental characteristic is compliance, which characterizes human movements. If our body was completely rigid, we would not be able to climb up trees or walk on mountainous paths as easily as we do. But despite being inspired to be a copy of human beings, humanoid robots had rigid links connected with rigid joints since their first appearance. It is only recently that they started to be more “human-like”, with the development of compliant actuators.
In this thesis the objective is to analyze of the role of compliance in human walking and in humanoid robots motions. We model both the human body and humanoid robots as rigid multi-body systems. Both systems are highly redundant, reason for which optimization represents an essential tool to achieve our goals. In particular, we adopt optimal control approaches.
In many state of the art compliant walking mechanisms, compliance is introduced at joint level by means of elastic components with constant stiffness, due to the difficulty of varying stiffness and the considerable dimensions of currently available variable stiffness actuators. This is the reason for which many studies focused on finding constant joint stiffness during human walking. However, biomechanics studies have shown that stiffness changes in human joints during movements. The questions we want to address are therefore: how does stiffness modulate during human walking and what is the influence of such modulations on the gait? To answer these questions, we used walking motions from motion capture data and a 2D dynamic model of the human body, where the actuation of the leg joints are modeled with torsional springs and bi-articular coupling springs with variable stiffness. We computed the stiffness profiles of these springs, which showed how stiffness changes over the walking cycle and can also assume big values, contrasting with many state of the art walking mechanisms. We proceeded by analyzing how walking gaits are modified if the stiffness modulation is reduced. This further step showed that the original walking gait could be approximated in unconstrained walking scenarios such as level ground and slopes but not in constraint ones as stairs. This result demonstrated the importance of stiffness modulation during walking and can serve for future compliant actuators design.
There are several existing humanoid robots with compliant actuators. Among these, the iCub is a widely spreaded advanced research humanoid that has recently acquired legs with Series Elastic Actuators (SEA). The reduced version of it, HeiCub, was delivered to Heidelberg University by the end of 2014 and is the robot used in this thesis. We first analyzed the motion of squatting. The problem is formulated as an optimal control problem where only the three pitch joints of the legs are considered active and the whole-body dynamics of the robot is used. Squat motions for different objective functions are generated for the robot with and without the use of SEA. A step further is taken in using all the actuated degrees of freedom of the robot to generate push recovery motions with the same approach, also considering the SEA. As there is a lack of literature and experiments of iCub walking, for this complex task we aimed at exploiting the capabilities of HeiCub by measuring its walking performances. We used the table cart model to generate walking trajectories on level ground, slope and stairs, which have never been achieved before by other iCub robots. In this way we could gain details of the platform that were unknown beforehand that are fundamental to be used in future optimal control formulations. Thanks to this study, future developments of walking control frameworks for the iCub family robots have now a point of reference.
Technological advances of recent years have changed the way research is done. When describing complex phenomena, it is now possible to measure and model a myriad of different aspects pertaining to them. This increasing number of variables, however, poses significant challenges for the visual analysis and interpretation of such multivariate data. Yet, the effective visualization of structures in multivariate data is of paramount importance for building models, forming hypotheses, and understanding intrinsic properties of the underlying phenomena. This thesis provides novel visualization techniques that advance the field of multivariate visual data analysis by helping represent and comprehend the structure of high-dimensional data. In contrast to approaches that focus on visualizing multivariate data directly or by means of their geometrical features, the methods developed in this thesis focus on their topological properties. More precisely, these methods provide structural descriptions that are driven by persistent homology, a technique from the emerging field of computational topology.
Such descriptions are developed in two separate parts of this thesis. The first part deals with the qualitative visualization of topological features in multivariate data. It presents novel visualization methods that directly depict topological information, thus permitting the comparison of structural features in a qualitative manner. The techniques described in this part serve as low-dimensional representations that make the otherwise high-dimensional topological features accessible. We show how to integrate them into data analysis workflows based on clustering in order to obtain more information about the underlying data. The efficacy of such combined workflows is demonstrated by analysing complex multivariate data sets from cultural heritage and political science, for example, whose structures are hidden to common visualization techniques.
The second part of this thesis is concerned with the quantitative visualization of topological features. It describes novel methods that measure different aspects of multivariate data in order to provide quantifiable information about them. Here, the topological characteristics serve as a feature descriptor. Using these descriptors, the visualization techniques in this part focus on augmenting and improving existing data analysis processes. Among others, they deal with the visualization of high-dimensional regression models, the visualization of errors in embeddings of multivariate data, as well as the assessment and visualization of the results of different clustering algorithms.
All the methods presented in this thesis are evaluated and analysed on different data sets in order to show their robustness. This thesis demonstrates that the combination of geometrical and topological methods may support, complement, and surpass existing approaches for multivariate visual data analysis.
The antibiotic roseoflavin (RoF) is the only known natural riboflavin (vitamin B2) analogue and is active against gram-positive bacteria. RoF is produced by Streptomyces cinnabarinus (S. cinnabarinus) and Streptomyces davawensis (S. davawensis) and can be considered to be an “antivitamin”. In RoF biosynthesis one of the methyl groups of the predicted precursor riboflavin undergoes a site-specific replacement by a dimethyl amino group whereby 8-demethyl-8-amino-riboflavin (AF) was postulated to be an intermediate. The first discovered enzyme of roseoflavin biosynthesis was the S-adenosyl methionine (SAM) dependent dimethyltransferase RosA which converts AF to RoF. Subsequent systematic gene deletion experiments carried out in the RoF-producer S. davawensis suggested that a single enzyme (RosB) is responsible for the formation of AF. However, when recombinant RosB was tested in an assay mixture containing riboflavin-5’-phosphate (RP) the formation of the predicted final reaction product AF was not observed. Instead the compound 8-demethyl-8-formyl-riboflavin-5’-phosphate (HOC-RP) was detected, probably an intermediate of the RosB reaction. How the formyl-group of HOC-RP was replaced by an amino group to give AF or 8-demethyl-8-amino-riboflavin-5’-phosphate (AFP) remained unclear. The present work was initiated to investigate the predicted oxidation of HOC-RP to 8-demethyl-8-carboxyl-riboflavin-5’-phosphate (HOOC-RP), to identify the amino group donor of the RosB reaction and to shed light on the reaction mechanism of the multi-step enzyme RosB. It was found that RosB accepts only RP as a substrate and not riboflavin (RF). RosB activity depends on the presence of O2, thiamine and the amino group donor glutamate. HOOC-RP was found to be an (additional) intermediate of the RosB reaction. The crystal structure of RosB was solved with bound AFP (1.7 Å) and HOC-RP (2.0 Å). RosB is composed of four flavodoxin-like subunits which have been upgraded with specific extensions and a unique C-terminal arm. Structure-based active site analysis was complemented by mutational and isotope-based mass-spectrometric data to propose an enzymatic mechanism. The present work also shows that the RoF biosynthetic pathway still has not been completely resolved. RosB releases AFP, yet the substrate for the subsequent RosA reaction is AF. Consequently, a phosphatase must be present which has not yet been identified.
Natural Killer (NK) cells are important effector cells in Hepatitis C Virus (HCV) infection, a virus that chronically infects around 2.5% of the world population and is a major cause of liver disease and hepatocellular carcinoma. The exact mechanisms, however, through which NK cells are activated in response to HCV remain elusive. Using the well-established HCV replicon cell-culture model we show that after co- culture of HCV replicon-carrying hepatocytes with peripheral blood mononuclear cells (PBMCs), NK cells increase expression of the high-affinity IL-2 receptor chain CD25, proliferate rapidly and produce IFN-gamma. Activation of NK cells was dependent on IL-2, most likely produced by T cells and on cell-cell contact mediated signals from monocytes. Monocytes from replicon-carrying co-cultures showed increased expression of OX40L, a member of the tumor necrosis factor family and concurrently its receptor OX40 was increased on NK cells. Blocking of OX40L in those co-cultures, as well as depletion of CD14+ monocytes abrogated the virus-induced activation and effector functions of NK cells. Together, our data reveals a novel mechanism of monocyte mediated NK cell activation against virus-infected cells involving the OX40/OX40L axis with potential relevance for therapeutic intervention by e.g. agonistic antibodies against OX40, which are already tested in cancer therapy.
The work presented in this thesis aimed at the development of antibody immunoconjugates for the delivery in tumor cells of highly immunogenic T cell epitopes that mediate the antigen-specific recognition by tumoricidal T cells. For this purpose, antibody-targeted pathogen-derived peptides (ATPPs) were generated by conjugating immunodominant, cysteine-containing MHC class I peptides from Epstein-Barr or Influenza A virus to tumor antigen-specific antibodies via a disulfide bond. The integral membrane protein CUB domain-containing protein 1 (CDCP1) was chosen as proof of concept target, as it is upregulated on various cancer types and known to efficiently internalize after antibody binding. After binding to the target and subsequent internalization of ATPPs, fluorescence resonance energy transfer (FRET) imaging revealed that delivered peptides are released upon disulfide reduction in an early endosomal compartment, where they can be loaded into recycling MHC class I complexes. Transport of these MHC-peptide complexes to the cell surface triggers activation of human peptide-specific cytotoxic CD8+ T cells as revealed by interferon-γ ELISA and ELISPOT. Moreover, peptide-specific CD8+ T cells from human donors efficiently lysed ATPP-treated tumor cell lines of various cancer types in a target-dependent manner in vitro. Importantly, targeting of different tumor antigens (e.g. CD138) was equally efficient. The possibility to utilize various peptides with differing HLA-restrictions further highlights the broad applicability of the ATPP approach for T cell mediated targeting of cancer. The usage of a non-cleavable construct or an extended peptide that can not bind to MHC class I molecules additionally revealed the importance of disulfide-dependent peptide release and epitope delivery independent of the classical MHC class I antigen processing pathway. In vivo, ATPPs mediated approximately 60% tumor growth inhibition of established, PD-L1 expressing MDA-MB231 xenografts after 3 weeks of treatment in combination with αPD1-mAb therapy and adoptive transfer of human, peptide-specific CD8+ T cells in NOG mice. These data indicate the potential of ATPPs as novel immunotherapeutic agents, which can be employed to redirect pre-existing virus-specific memory T cells against cancer. Since the immune response will be directed against an exogenous, viral antigen, ATPP therapy reduces the risk for autoimmune side effects as observed with other immunotherapies. Furthermore, the use of highly immunogenic target epitopes circumvents the limitations of the T cell repertoire directed against tumor-associated auto-antigens. The flexible design of ATPPs allows development of an off-the-shelf repertoire of immunoconjugates comprising immunogenic T cell epitopes encoded by highly prevalent pathogens and presented by various high frequency HLA allotypes, thereby providing a means for T cell mediated tumor targeting in a broad patient population.
Designing an exoskeleton to reduce the risk of low-back injury during lifting is challenging. Computational models of the human-robot system coupled with predictive movement simulations can help to simplify this design process. Here, we present a study that models the interaction between a human model actuated by muscles and a lower-back exoskeleton. We provide a computational framework for identifying the spring parameters of the exoskeleton using an optimal control approach and forward-dynamics simulations. This is applied to generate dynamically consistent bending and lifting movements in the sagittal plane. Our computations are able to predict motions and forces of the human and exoskeleton that are within the torque limits of a subject. The identified exoskeleton could also yield a considerable reduction of the peak lower-back torques as well as the cumulative lower-back load during the movements. This work is relevant to the research communities working on human-robot interaction, and can be used as a basis for a better human-centered design process.
Systems to regulate gene expression from an Adeno-associated viral (AAV) vector are widely used. In most cases, the transgene expression has to be switched on by applying a drug. In terms of safety of gene therapy, a shut-off system for AAV vectors would be beneficial to silence gene expression in case of side-effects, ideally by destruction of the vector genome. Therefore, the aim of the present study was to develop a system for elimination of gene expression from an AAV vector after systemic injection using an inducible Cre recombinase. In presence of tamoxifen, the inducible Cre recombinase is activated which should result in excision of DNA fragments flanked by loxP sites within the vector. Before evaluating the final shut-off system, several experiments were performed to analyze the background activity of the induced CreERT2 recombinase, the best suited positions of the loxP sites within the AAV genome and the influence of the loxP sites on the transgene expression. Moreover, a co-transduction of an AAV vector encoding the CreERT2 and a second AAV vector expressing the reporter gene flanked by loxP sites was tested. AAV vectors of serotype 9 were used for packaging the final shut-off system consisting of the inducible CreERT2 recombinase, a luciferase reporter gene, and different positions of loxP sites to investigate the effect of loxP localization within the vector genome. To drive reporter gene and CreERT2 expression, the CMV promoter was used. All vectors were first tested in vitro. Afterwards, the vectors which showed a significant down-regulation of transgene expression after tamoxifen administration were also analyzed in vivo. Here, a significant reduction in reporter gene activity could be detected in animals receiving AAV vectors containing loxP sites one week upon tamoxifen administration. Another finding was that the insertion of loxP sites has a negative influence on the expression levels of the transgene. Thereby, the vector expressing the reporter gene flanked by loxP sites showed the lowest expression but also the highest extent of down-regulation after tamoxifen treatment. Finally, the shut-off system used was improved in terms of coding capacity of the AAV genome used. Therefore, the “self-cleaving” peptide P2A of the Porcine Teschovirus-1 was used to replace the promoter driving CreERT2 expression to increase the limiting coding capacity for the gene of interest. Again, these AAV9 vectors were tested in vitro and in vivo, also showing a significant reduction in reporter gene expression after tamoxifen administration. Taken together, expression of an inducible Cre recombinase allows efficient inactivation of AAV-mediated gene expression on the expense of reduced overall expression efficiency due to insertion of loxP sites. These results contribute to the generation of a novel shut-off system for AAV-mediated gene transfer applicable for the use in combination with various promoters and AAV serotypes to target cell types or tissues of choice.
This thesis investigates robust strategies of optimal experimental design for discrimination between several nonlinear regression models. It develops novel theory, efficient algorithms, and implementations of such strategies, and provides a framework for assessing and comparing their practical performance. The framework is employed to perform extensive case studies. Their results demonstrate the success of the novel strategies.
The thesis contributes advances over existing theory and techniques in various fields as follows:
The thesis proposes novel “misspecification-robust” data-based approximation formulas for the covariances of maximum-likelihood estimators and of Bayesian posterior distributions of parameters in nonlinear incorrect models. The formulas adequately quantify parameter uncertainty even if the model is both nonlinear and systematically incorrect.
The thesis develops a framework of novel statistical measures and tailored efficient algorithms for the simulation-based assessment of covariance approximations for maximum-likelihood estimator for parameters. Fully parallelized variants of the algorithms are implemented in the software package DoeSim.
Using DoeSim, the misspecification-robust covariance formula for maximum-likelihood estimators (MLEs) and its “classic” alternative are compared in an extensive numerical case study. The results demonstrate the superiority of the misspecification-robust formula.
Two novel sequential design criteria for model discrimination are proposed. They take into account parameter uncertainty with the new misspecification-robust posterior covariance formula. It is shown that both design criteria constitute an improvement over a popular approximation of the Box-Hill-Hunter-criterion. In contrast to the latter, they avoid to overestimate the expected amount of information provided by an experiment.
The thesis clarifies that the popular Gauss-Newton method is generally not appropriate for finding least-squares parameter estimates in the context of model discrimination. Furthermore, it demonstrates that a large class of optimal experimental design optimization problems for model discrimination is intrinsically non-convex even under strong simplifying assumptions. Such problems are NP-hard and particularly difficult to solve numerically.
A framework is developed for the quantitative assessment and comparison of sequential optimal experimental design strategies for model discrimination. It consists of new statistical measures of their practical performance and problem-adapted algorithms to compute these measures. A state-of-the-art modular and parallelized implementation is provided in the software package DoeSim. The framework permits quantitative analyses of the broad range of behaviour that a design strategy shows under fluctuating data.
The practical performance of four established and three novel sequential design criteria for model discrimination is examined in an extensive simulation study. The study is performed with DoeSim and comprises a large number of model discrimination problems. The behaviour of the design criteria is examined under different magnitudes of measurement error and for different number of rival models.
Central results from the study are that a popular approximation of the Box-Hill-Hunter-criterion is surprisingly inefficient, particularly in problems with three or more models, that all parameter-robust design criteria in fact outperform the basic Hunter-Reiner-strategy, and that the newly proposed novel design criteria are among the most efficient ones. The latter show particularly strong advantages over their alternatives when facing demanding model discrimination problems with many rival model and large measurement errors.
The Hypothalamic-Pituitary-Adrenal (HPA) axis and its final effector, glucocorticoids (GCs), are important players in maintaining homeostasis of an organism upon stress exposure. However, overexposure to GCs during early life is involved in developmental programming of the HPA axis and is linked to detrimental effects in health. The hypothalamus is a key target for developmental programming due to its pivotal role as an integrator of input signals coming from sensory systems and other brain regions and as a translator of neuronal signals into endocrine signals. Yet, little is known about the molecular mechanisms involved in hypothalamic programming mediated by stressful experiences during early life. Elucidation of these mechanisms is essential for understanding the link between early life stress, dysregulation of the stress response, and detrimental health in later stages. Here, I used the zebrafish model to elucidate the molecular correlates of early adverse experience in hypothalamic cells. First, I developed a stimulation protocol using vortex flows to activate the hypothalamic-pituitary-interrenal (HPI) axis, the homolog of the HPA axis in teleost, and characterized the stress response at early stages by measuring cortisol (the main GC in zebrafish) and behavioral correlates. I then identified a critical time window in which HPI axis activity matures. Subsequently, I established an early life stress protocol to induce hypercortisolic states and alter stress response maturation. Endocrinological, behavioral, and cellular characterization of the early life stress paradigm showed an overall downregulation of the stress response with attenuated locomotor and cortisol response to subsequent stressful events as well as reduced calcium activity and expression of stress related peptides (AVP, CRH, and OXT) in hypothalamic cells. To dissect the molecular correlates of early adverse experience, I then performed transcriptomic analysis of hypothalamus-specific cell populations after exposure to the early life stress paradigm. Candidate molecules involved in the adaptive process occurring in hypothalamic cells were identified. Moreover, gene ontology and pathway analysis showed that lipid metabolism and molecular transport pathways were downregulated after zebrafish larvae were subjected to the early life stress protocol. In contrast, cellular movement and inflammatory response pathways were upregulated. Finally, I characterized the cortisol profiles of optogenetic and targeted transgenic tools which have been generated to manipulate the HPI axis activity in freely swimming larvae. Here, I show evidence of altered levels of endogenous cortisol in larvae that were manipulated at any of the three levels of the HPI axis. Altogether, the main contributions of this thesis are: 1) establishment of a novel stress protocol to activate the HPI axis in zebrafish larvae in a highly controlled and strength-dependent manner; 2) characterization of the cortisol response of developing zebrafish and identification of a critical time window of stress response maturation; 3) development of an early life stress paradigm and elucidation of the effects of early adverse experience at the cellular, behavioral, and endocrinological level; 4) identification of candidate molecules and metabolic pathways in hypothalamic cells involved in adaptive processes after early adverse experience, and 5) characterization of the cortisol profiles of optogenetic and genetic tools to manipulate the HPI axis activity at any of its three levels (hypothalamus, pituitary, and interrenal gland).
The novel HALO mini-DOAS instrument was developed for measurements of UV/vis/near-IR spectra of scattered skylight in limb and nadir geometry aboard the new research aircraft HALO. The absorptions of a suite of trace gases (O3, O4, NO2, CH2O, BrO, OClO, and others) are identified in the measured spectra using the DOAS-technique. Previously employed methods to infer absolute concentrations from DOAS measurements rely on a priori knowledge of aerosols and cloud cover. The recently developed scaling method promises to enable the retrieval of target gas concentrations under all sky conditions. Effective light path lengths are estimated by employing a scaling gas, whose concentration at flight level is known, in conjunction with modelled profile shapes, radiative transfer calculations, and using the measured absorptions of the targeted species relative to those of the scaling gas. The present thesis describes the development and characterises the measurement properties of the HALO mini-DOAS instrument. For the first time, random and systematic errors of the scaling method are thoroughly investigated. It is argued that random errors are 10 – 20% for most measurement conditions and that the scaling method is practically unperturbed by changing cloud cover if applied appropriately. It is however shown that biases may occur if the assumed profile shapes are significantly different from actual profile shapes. Retrieved mixing ratios of BrO and NO2 from measurements obtained during the science mission TACTS/ESMVal in August/September 2012 indicate that (a) no enhanced tropospheric BrO was detected in the mid-troposphere (3.5 – 9 km altitude) near the Antarctic continent (65° S) in spring (Sept. 13, 2012), (b) LMS and bottom polar vortex [BrO] agree with previous measurements, (c) other oxidants beside O3 influence NO oxidation in the UT/LS where [N2O] < 310 ppb, and (d) the same finding was confirmed for very low-NOx conditions, although the latter measurements are uncertain.
During central nervous system (CNS) development, extensive reciprocal interactions exist between different cell types, including neural progenitors, neurons, endothelial cells, microglia and other glial cells that are crucial in driving proper development. Moreover, neurons and vessels share striking anatomical and molecular features and are presumably orchestrated by an overlapping repertoire of signaling systems. Here, we identify an important neuron/vessel/glia-interaction essential for the correct formation of the neurovascular system in the CNS that involves the CD95 receptor and ligand system. Deletion of CD95 receptor in neurons, as shown before, and in endothelial cells result in aberrant branching and growth of both cell types in the postnatal CNS. Furthermore, we identify microglia as the main source of CD95L and microglial-specific deletion thereof impairs proper neurovascular development. CD95 promotes endothelial cell branching and proliferation by acting as a receptor tyrosine kinase that upon activation leads to recruitment of SFK and p85 which further elicit Akt and Erk signaling, two effectors that are crucial in regulating endothelial cell growth. These data highlight a coordinated neurovascular development instructed by microglial-derived CD95L and impact the importance of microglia for the establishment of the neurovascular network during CNS development. In tumorigenesis, angiogenesis is reactivated and takes place extensively to support tumor cell growth. Glioblastoma multiforme (GBM), the most aggressive and deadly type of tumor in the brain, is characterized by a high level of angiogenesis. The CD95 receptor and ligand system also fosters tumor angiogenesis. However, this does not occur by direct activation of CD95 on endothelial cells, but rather results from CD95 activation on GBM cells. Activation of CD95 on GBM cells by CD95L leads to an increase in the release of pro-angiogenic factors that promote vessel growth in the tumor. Together, our data demonstrate a novel role of CD95 in angiogenesis both during development and tumorigenesis and show a potential to target CD95 in neurovascular diseases or tumor angiogenesis.
The phenomena of photo-induced chemical reactions can exhibit kinetics as short as a few femtoseconds. The development of femtosecond UV pulses enables the investigation of such ultrafast phenomena in more experimentally challenging, UV absorbing systems. This thesis describes, chronologically, development of a UV transient absorption (TA) system and its application to various coumarin-based molecular systems. The experimental section details the TA system comprising of three major components: two tunable UV excitation sources giving a total excitation range of 250-350 nm, supercontinuum generation enabling 240-700 nm probe range, and multichannel detection with greater than 5 ×10-5 OD sensitivity. The potential of the TA setup was tested on the study of ultrafast relaxation in 7-hydroxy coumarin. Following successful tests, the photo-induced cleavage reaction of coumarin dimer was studied in order to understand the ultrafast dynamics. The experiments utilized 280 nm excitation and broadband (300-650 nm) probing. The results revealed fast cleavage occurring through short-lived nonradiative (<200 fs) singlet states. Two branched kinetic models were developed to describe the monomer formation and dimer relaxation dynamics, identify intermediate states, and determine the quantum yields. The anti-hh displayed the highest cleavage efficiency of ~20 %. Finally, variation in the quantum yields between isomers was rationalized using a sequential bond cleavage mechanism over a concerted, “pericyclic-style” ring cleavage.
Umgangsprachlich heißt es: "Ein guter wissenschaftlicher Text besteht aus drei Teilen: Den ersten Teil versteht jeder, den zweiten Teil verstehen Fachleute und den dritten Teil versteht niemand." Leider ist das häufig wirklich so. Dabei nutzt Forschung vor allem dann auch der Allgemeinheit, wenn man sie verstehen kann. Darum vergibt die Klaus Tschira Stiftung jedes Jahr den Klaus Tschira Preis für verständliche Wissenschaft, kurz "KlarText!". Campus-Reporter Nils Birschmann hat mit der Preisträgerin Christiane Heinicke und der Geschäftsführerin Beate Spiegel geredet.
Der Beitrag erschien in der Sendereihe "Campus-Report" - einer Beitragsreihe, in der über aktuelle Themen aus Forschung und Wissenschaft der Universitäten Heidelberg, Mannheim, Karlsruhe und Freiburg berichtet wird. Zu hören ist "Campus-Report" montags bis freitags jeweils um ca. 19.10h im Programm von Radio Regenbogen. (Empfang in Nordbaden: UKW 102,8. In Mittelbaden: 100,4 und in Südbaden: 101,1)
Anti-apoptotic Bcl-2 (B-cell lymphoma 2) proteins such as Bcl-2 itself, Bcl-xL and Mcl-1, prevent mitochondrial activation and thereby the induction of the intrinsic apoptotic signaling pathway. Since the avoidance of cell death is a prerequisite for malignant transformation, anti-apoptotic proteins are frequently overexpressed in tumor cells. Additionally, there is growing evidence that Bcl-2, Bcl-xL and Mcl-1 also play a role in other cellular processes, such as cell cycle regulation, DNA repair and autophagy induction, which can also be important for the onset and progression of cancer. One of the most frequently diagnosed malignancies worldwide is colorectal cancer. Even though improvements in treatment and population screenings have led to a decreased mortality in many countries, especially patients with metastasized colorectal cancer still face a poor prognosis. Until now, it has been an open question whether and to which extend anti-apoptotic Bcl-2 proteins influence colorectal cancer initiation, progression and metastasation, with a few studies showing contradictory results. Hence, the purpose of this work was to shed more light on the role Bcl-2, Bcl-xL and Mcl-1 play for the maintenance of intestinal tissue homeostasis and for colorectal cancer development and outgrowth. First, their expression levels in the human intestinal mucosa as well as in adenoma and adenocarcinoma tissue were determined. This revealed a significant increase of Bcl-xL in the malignant state and an unaltered expression of Bcl-2. By contrast, Mcl-1 has been found to be significantly downregulated in colorectal cancer specimens. Results obtained in subsequent in vitro experiments clearly showed that Mcl-1 has an anti-proliferative effect which cancer cells preclude by downregulation of the protein. Neither in intestinal epithelial cells nor in colorectal cancer cells has a cell cycle inhibiting mode of action been described so far for Mcl-1. Additionally, further in vitro experiments have shown for the first time that the siRNA mediated silencing of anti-apoptotic Bcl-2 proteins significantly decreased the migratory capacity and invasiveness of human colorectal cancer cells. In a second step, protein functions were studied in further detail in vivo. Since previous publications showed that the constitutive deletion of both Bcl-xL and Mcl-1 induces embryonic lethality, two intestine-specific knockout mouse models were generated during this work. For the first time, they allowed to study functioning of Bcl-xL and Mcl-1 in the murine intestine. The mouse models revealed a strong discrepancy between Bcl-xL and Mcl-1, regarding their influence on tissue maintenance and tumorigenesis. While Bcl-xL turned out to be dispensable for normal tissue homeostasis, it has been found to be a crucial factor for colorectal cancer cell survival in a chemically induced tumor model. This confers Bcl-xL tumor-promoting properties and explains its overexpression in human adenomas and adenocarcinomas. The loss of Mcl-1, by contrast, caused a severe intestinal phenotype, comprising high levels of cell death, an accompanying increase of proliferation and chronic inflammation. From an age of about six months, spontaneous tumorigenesis was observed in intestine-specific Mcl-1 knockout mice which was promoted by the loss of the anti-proliferative effect Mcl-1 exerts on intestinal epithelial cells and the inflammatory environment. Therefore, it has been proven that Mcl-1 possesses tumor-suppressing properties in the intestine, what explains its downregulation in human colorectal cancer specimens. The presented results highly recommend the utilization of Mcl-1 sparing inhibitors in the context of colorectal cancer treatment. A first step towards clinical application was done in this work by treating viable human colorectal cancer tissue ex vivo with the Bcl-xL/Bcl-2-specific inhibitor ABT-737. Subsequent analyses revealed a significantly decreased viability of human colorectal cells in presence of the inhibitor. Since proliferation turned out to be unaltered under ABT-737 treatment, inhibition of Bcl-xL in combination with classical chemotherapy could be an interesting approach for further studies with a focus on clinical applicability.
Motivation. Patient-specific, knowledge-based, holistic surgical treatment planning is of utmost importance when dealing with complex surgery. Surgeons need to account for all available medical patient data, keep track of technical developments, and stay on top of current surgical expert knowledge to define a suitable surgical treatment strategy. There is a large potential for computer assistance, also, and in particular, regarding surgery simulation which gives surgeons the opportunity not only to plan but to simulate, too, some steps of an intervention and to forecast relevant surgical situations.
Purpose. In this work, we particularly look at mitral valve reconstruction (MVR) surgery, which is to re-establish the functionality of an incompetent mitral valve (MV) through implantation of an artificial ring that reshapes the valvular morphology. We aim at supporting MVR by providing surgeons with biomechanical FEM-based MVR surgery simulations that enable them to assess the simulated behavior of the MV after an MVR. However, according to the above requirements, such surgery simulation is really beneficial to surgeons only if it is patient-specific, surgical expert knowledge-based, comprehensive in terms of the underlying model and the patient’s data, and if its setup and execution is fully automated and integrated into the surgical treatment workflow.
Methods. This PhD work conducts research on simulation-enhanced, cognition-guided, patient-specific cardiac surgery assistance. First, we derive a biomechanical MV/MVR model and develop an FEM-based MVR surgery simulation using the FEM software toolkit HiFlow3. Following, we outline the functionality and features of the Medical Simulation Markup Language (MSML) and how it simplifies the biomechanical modeling workflow. It is then detailed, how, by means of the MSML and a set of dedicated MVR simulation reprocessing operators, patient-individual medical data can comprehensively be analyzed and processed in order for the fully automated setup of MVR simulation scenarios. Finally, the presented work is integrated into the cognitive system architecture of the joint research project Cognition-Guided Surgery. We particularly look at its semantic knowledge and data infrastructure as well as at the setup of its cognitive software components, which eventually facilitate cognition-guidance and patient-specifity for the overall simulation-enhanced MVR assistance pipeline.
Results and Discussion. We have proposed and implemented, for the first time, a prototypic system for simulation-enhanced, cognition-guided, patient-specific cardiac surgery assistance. The overall system was evaluated in terms of functionality and performance. Through its cognitive, data-driven pipeline setup, medical patient data and surgical information is analyzed and processed comprehensively, efficiently and fully automatically, and the hence set-up simulation scenarios yield reliable, patient-specific MVR surgery simulation results. This indicates the system’s usability and applicability. The proposed work thus presents an important step towards a simulation-enhanced, cognition-guided, patient-specific cardiac surgery assistance, and can – once operative – be expected to significantly enhance MVR surgery. Concluding, we discuss possible further research contents and promising applications to build upon the presented work.
In Eukaryotes vesicular transport mechanisms collectively ensure the transport and distribution of proteins and lipids between cellular compartments to maintain their unique composition and their specialized functions. COPI vesicles mediate retrograde transport from the ERGIC/Golgi to the ER as well as intra-Golgi transport. Formation as well as consumption of COPI-coated transport vesicles is directly controlled by small GTPases of the Arf family, which in turn are regulated via specific Guanine nucleotide exchange factors (GEFs) and GTPase Activating Proteins (GAPs). COPI vesicle formation is initiated by recruitment of Arf1 to membranes, which subsequently recruits the heptameric coat complex coatomer. GTP hydrolysis within Arf1 is a prerequisite for COPI vesicle uncoating. Three ArfGAPs are associated with COPI vesicle formation in mammalian cells: ArfGAP1, ArfGAP2 and ArfGAP3. During the course of this work mechanistic aspects of COPI vesicle biogenesis were investigated: i) the interaction of ArfGAPs with coatomer isoforms, ii) the regulation of ArfGAP activity by p24 family proteins, and iii) the molecular mechanism of COPI vesicle uncoating. We have determined the dissociation constants of the ArfGAPs for each of the four individual coatomer isoforms and found that all three ArfGAPs displayed a higher affinity for the γ1ζ1 isoform than for the other isoforms. This result is in accordance with the localization of both ArfGAP2/3 and γ1ζ1 to the cis-Golgi, whereas ArfGAP1 is equally distributed throughout the Golgi apparatus. Furthermore, we have investigated an effect on the GAP activity of ArfGAP1 and ArfGAP2 of the cytoplasmic tail of the transmembrane protein p23, a COPI vesicle machinery protein. p23 was reported to induce a conformational change in coatomer, resulting in a structure that resembles coatomer conformation within the polymerized COPI coat. Interestingly, p23 influenced the activity of ArfGAP1 and ArfGAP2 in an opposite fashion: whereas ArfGAP2 displayed a higher rate of Arf1 GTP hydrolysis in the presence of p23, ArfGAP1 displayed a lower rate. Thus, ArfGAP2/3 might preferentially interact with polymerized coatomer as found on a completed COPI vesicle. Although GTP hydrolysis in Arf1 is commonly considered necessary for coat disassembly, it remains obscure whether it is sufficient to complete this process. To investigate this pivotal mechanistic question, we have established a real-time assay to follow the fate of the COPI coat components of purified vesicles upon addition of ArfGAPs, and discovered an unanticipated essential role of the non-catalytic domains of ArfGAPs. While GTP-hydrolysis within Arf1, induced by the isolated catalytic domain of the ArfGAP, released the small GTPase from the coat, the network of coatomer remained associated with vesicle membranes. Only in the presence of full-length ArfGAP1, including the non-catalytic part, the coat network was completely disassembled. We propose that the energy released upon GTP-hydrolysis in Arf1 is coupled by GAP-coatomer interactions to mediate conformational changes in coatomer that are required for COPI coat disassembly.
To date, despite significant advances in early detection and treatment of breast cancer, metastatic progression remains a major cause of morbidity and mortality in breast cancer patients. Importantly, patient stratification according to biomarker expression has led to continuous improvement of therapeutic strategies and better understanding of the diseases. However, the aggressive triple-negative subtype of breast cancer is still lacking effective therapeutic options.
The important role of the tumor microenvironment in the formation of metastasis is well recognized. The extracellular matrix glycoprotein tenascin-C (TNC) has been previously described as a functional player of the metastatic niche, supporting the growth of breast cancer cells at the distant site. In this study, we investigated the clinical prognostic value of TNC in breast cancer patient cohorts. We found that TNC predicts poor clinical outcome only in the triple-negative subtype and that these tumors are enriched for TNC expression. Interestingly, while other subtypes rely on the stromal compartment as a source of TNC, triple-negative tumors express TNC in an autocrine manner. In addition, we confirmed that TNC promotes the growth of triple-negative breast cancer cells in the lungs in vivo. Therefore, we suggest that triple-negative breast tumors benefit from high, autocrine TNC expression to promote metastasis.
Several cell surface receptors have been suggested to interact with TNC. However, the receptor(s) mediating the pro-metastatic signaling downstream of TNC remained unclear. We identified two integrin receptor subunits, integrin beta 1 (ITGB1) and integrin beta 3 (ITGB3), as TNC receptors. We demonstrated the binding of these molecules to TNC in an endogenous setting and showed that ITGB1 and ITGB3 support the growth of breast cancer cells in the lungs in vivo. Furthermore, we observed that the expression of these receptors, similar to TNC, is enriched in the triple-negative subtype. Using a large patient cohort, we showed that the prognostic value of TNC depends on the expression of the identified receptors, underscoring the clinical relevance of our findings. Importantly, ITGB1 and ITGB3 are supporting stem cell properties in triple-negative breast cancer cells.
We found that the TNC knockout phenotype was associated with a decrease in stem cell properties of the normal mammary gland epithelium. In addition, we showed that TNC signaling members are upregulated during stages of the mammary gland development and maturation associated with expansion of the stem cell compartment. More importantly, TNC knockout mice showed an impairment in the formation of alveolar structures during pregnancy. All in all, our data strongly suggest a functional role of the TNC signaling in mammary stem cell biology.
In this study, we identified two integrin receptors of the mammary gland (ITGB1 and ITGB3) as the receptors mediating the TNC pro-metastatic signaling in triple-negative breast cancer. Furthermore, we showed that TNC supports stem cell properties in the mammary gland. Therefore, we propose that the TNC signaling might play an important role in the mammary stem cell to support its activity and that triple-negative breast cancer cells benefit from high expression of TNC and its receptors to promote metastatic growth in this subtype. Deeper understanding of the mammary stem cell biology might support the development of targeted therapy for triple-negative breast cancer patients.
The goal of this work was two-fold: 1) To apply serial block-face electron microscopy (SBEM) to the spinal cord of a larval zebrafish, in order to gain a mechanistic understanding of motoneuron (MN) recruitment, based on a reconstruction of the wiring between spinal interneurons and MNs and 2) to implement technological improvements to SBEM that would allow datasets to be acquired at much higher speed, in order to acquire a dataset of a complete larval zebrafish brain. The spinal cord of vertebrates contains a neural circuit known as a central pattern generator (CPG), which can generate the rhythmic muscle contractions underlying locomotion independently of the brain. In fish, the rhythm consists of muscle contractions that alternate between the left and right side of the tail and that travel down the length of the fish, from head to tail. When swimming fast, such as during escapes, the rhythm has a high frequency and muscles contract vigorously. During slow, routine swimming, the rhythm has a low frequency and muscles contract with less strength. The MNs in the spinal cord, which elicit the contractions of the tail musculature, are recruited to different degrees during these different behaviors. With increasing contraction strength, more and larger MNs are activated. This phenomenon is called orderly recruitment. The rhythmic excitation that recruits MNs is provided by Circumferential Descending (CiD) interneurons located in the spinal cord. These interneurons also follow a specific recruitment pattern: During weak swimming, ventral cells are active exclusively and dorsal cells are silent. As swims increase in vigor, the activity in these cells shifts towards more dorsal cells, with more ventral cells becoming inactive. The aim of the first part of this thesis was to reconstruct the MNs along with the CiDs that excite them, using a high resolution SBEM dataset of the spinal cord, to identify the pattern of connectivity between these types of neurons and distinguish between competing hypotheses of orderly MN recruitment. Conceptually, orderly recruitment could either be implemented with unspecific connectivity, in which case it would be a consequence of the interplay of size-dependent biophysical properties (in particular the input resistance) with the strengths of the synapses driving them. Alternatively, the wiring pattern could be specific and the CiDs could select the subset of MNs to activate by making synapses with just those cells. MNs in the larval zebrafish spinal cord clustered into distinct subtypes, depending on their size: Small, intermediate and large. The small MNs received almost no synaptic inputs and appeared to be immature. CiDs differentially innervated the intermediate and large MNs: Ventrally located CiDs did not differentiate between the two subtypes, but the dorsal CiDs made synapses onto large MNs with high specificity. Since dorsal CiDs are active only during the fastest swims, this finding can be interpreted as a labeled line specifically recruiting the strongest MNs during the most vigorous behaviors. During weaker behaviors, when the dorsal CiDs are inactive and the more ventral ones are active exclusively, differences in MN excitability due to size would encode the recruitment order. The second objective was to improve SBEM technology to acquire a whole larval zebrafish brain in a relatively short period of time. Due to the very high resolution required to trace small neurites and to identify synapses, even very small brains, such as the brain of a larval zebrafish, would take many months to acquire using a typical SBEM setup. Two main techniques were used to increase net speed. First, line-scanning of individual image tiles was implemented, where the electron beam scans the image in one axis only and the other axis is scanned by moving the stage. This allows larger individual images to be taken, greatly reducing the number of motor moves between images. Second, dynamic adaptation of the image tile mosaic to the shape of the sample was used to avoid scanning the blank plastic regions surrounding an irregularly shaped sample. These improvements allowed the complete brain of a 5 day old larval zebrafish to be imaged in less than 30% of the time than would have been required previously. In a collaborative project with Dr. Fumi Kubo, two-photon calcium imaging was performed prior to EM imaging, revealing pretectal cells active during optokinetic stimulation. The two-photon dataset was successfully registered to the EM data and a functionally identified pretectal cell could be traced. This dataset will be used to reconstruct the complete neural networks that compute the optokinetic response.
Camera motion estimation and dense scene reconstruction are essential for modern computer vision applications such as autonomous driving, robot navigation and virtual reality. State-of-the-art methods are usually based on stereo camera systems that use the information about the distance between the two cameras to uniquely estimate the depth map. However, these systems need to be calibrated and are too expensive for some special industrial applications. Thus, we focus in this work on monocular camera systems that consist of a single moving camera. To increase the robustness of the method we use temporal information in terms of filters. These use temporal consistency to improve the accuracy of the estimation of the current state of a system, e.g. the unknown camera motion or the depth map. Instead of using established stochastic filters such as extended Kalman filters, unscented Kalman filters or particle filters we use novel minimum energy filters that do not base on a stochastic model but on the minimization of an energy function. In a first step we derive the minimum energy filter and provide differential equations for the optimal state and the corresponding second-order operator. We demonstrate that this filter is as exact as state-of-the-art stochastic filters for most problems and, in addition to it, superior in more involved scenarios. Then we consider a simple geometric setting for the reconstruction of the camera motion within a static scene based on stereo image data. There we formulate a non-linear filtering problem on the special Euclidean group based on non-linear observations of optical flow and depth map. In experiments we show that the underlying camera motion can be reconstructed with minimum energy filters as accurate as in other state-of-the-art stereo methods. Finally, we present an approach for the joint reconstruction of camera motion and disparity map (inverse of depth map) in a monocular approach by means of minimum energy filters. By introducing a novel disparity group we can derive the filter without additional constraints or barrier functions. Further we generalize the used energy function to a Charbonnier penalty function which is robuster against outliers in the optical flow. We also demonstrate that additional regularizers can be easily integrated within the overall filtering problem providing a rich basis for many applications. From the mathematical point of view we solve by means of minimum energy filters a non-linear filtering problem on a Lie group for a high dimensional problem -- thus a problem which is infeasible for most stochastic filter.
Non-local electronic decay mechanisms constitute important pathways for the relaxation of cations produced by the action of ionizing radiation in van-der-Waals or hydrogen bonded chemical environment. Electronically excited cations may undergo the ultrafast Interatomic Coulombic Decay or ICD process, whereby the excess electronic energy is transferred to the environment and used to ionize it. It has been extensively studied by computational and experimental techniques during the last two decades and shown to operate in a variety of systems from rare gas dimers to large biomolecules. In this thesis we investigate using ab initio methods the Electron Transfer Mediated Decay or ETMD process which is responsible for the charge redistribution in environment, whenever atomic cations with a low excess energy and high electron affinity are produced. In ETMD electron transfer to the cation leads to the emission of an electron from the neighboring species. The net result is partial neutralization of the cation and the increase of the charge of the environment by two.
The light rare gas atoms He and Ne have a high ionization potential and, in the presence of a suitable neighbor are likely to undergo ETMD when they are singly ionized, e.g. by photoionization. In particular, we showed that a HeMg cluster efficiently decays by ETMD whenever He is photoionized and a ground state He+ ion is produced. The joint process of photoionization and ETMD corresponds to a one-photon double ionization of Mg. Remarkably, we found that the cross section of this process is three orders of magnitude higher than the cross section of the atomic one-photon double ionization, which demonstrates the prominent role of the neighboring He species in the double ionization. This mechanism of the ETMD driven one-photon double ionization was recently demonstrated experimentally in doped He nanodroplets and is proposed as a method for the experimental production of cold molecular dications.
Multiply charged rare gas cations have higher electron affinities and undergo ETMD with a larger variety of neighboring atoms or molecules. Such cations are naturally produced by the Auger decay following core ionization of rare gases in the X-Ray absorption. The ETMD process reduces their positive charge by one, i.e. leads to their partial neutralization and serves as a purely electronic alternative to neutralization mechanisms driven by the movement of the nuclei. Our calculations show that in small Ne2+Xe and Ne2+Kr2 clusters the ETMD process takes place on a picosecond timescale. The ETMD in these systems is accompanied by nuclear dynamics which in turn enhance the rate of the electronic decay. We show that for such systems ETMD is an important mechanism responsible for the fast redistribution of the localized charge produced in the Auger decay process.
We also demonstrated that multiply charged hydrated metal cations are likely to decay via complicated cascades comprising both ETMD and ICD steps. Our calculations in the Mg2+(H2O)6 microsolvated cluster showed that such a cascade proceeds on a timescale of few hundreds of femtoseconds and leads to a massive degradation of the imetal’s solvation shell through its multiple ionization and emission of slow electrons. Repulsive nuclear dynamics at later stages of the cascade, which were not taken into account explicitly, are expected to considerably reduce its duration. We expect that studying interatomic decay cascades of metal cations is important for understanding mechanisms of the damage caused by X-Rays to metal containing biomolecules such as DNA, metalloproteins etc. For the latter of particular importance is the knowledge of the duration of different interatomic decay steps, since it determines the timescale at which proteins become damaged by X-Rays and beyond which their structure becomes compromised.
These considerations led us to investigate the dependence of ICD lifetimes on atomic charge in excited microhydrated Na2+ and Mg3+ cations. Our ab initio results reproduce within the numerical error the experimental ICD lifetimes of the respective ions in aqueous solutions. We show that the microsolvated Mg3+ cations decay faster than the Na2+ ones, in accordance with experiments on aqueous solutions. The detailed analysis reveals that at characteristic metal-water separations the polarization of the water neighbor enhances ICD the stronger the higher the charge of the metal is. This, together with the shorter Mg-water equilibrium distances, leads to the observed ordering of the ICD rates. We also showed that polarizing the neighbors causes sub-linear growth of ICD rates with the number of water molecules in the first solvation shell. This investigation of ICD in microsolvated metal cations demonstrated the prominent role the cation’s charge and the consequent polarization of the medium have on the decay rate. It also leads to a reasonable expectation that even faster, sub-femtosecond decay lifetimes might be achieved for highly charged solvated metals ions.
The general topic of this dissertation is the analysis of impact ionization time-of-flight mass spectra of ice grains in Saturn’s E ring sampled in-situ by the Cosmic Dust Analyzer (CDA) onboard the Cassini-Huygens spacecraft. The source of these E ring ice grains is the subsurface ocean of Saturn’s cryo-volcanically active icy moon Enceladus. The Chemical Analyzer subsystem of CDA generated mass spectra of cations that form when the ice grains impinge onto the instrument’s target plate with high speed. The first aim of this work is a detailed compositional analysis of the organic material in the ice grains ejected from subsurface Enceladus’ into the E ring. Many of these ice grains carry diverse organic material that is characterized in this work. A laser-based analogue laboratory experiment is used to simulate the impact ionization CDA spectra of ice grains enriched in organic material. This experiment allowed to understand the varying cationic fragmentation patterns from organic material in a water ice matrix. Despite the relatively low mass resolution of the CDA, results of the analogue experiment allow to identify characteristic finger prints of certain classes of organic compounds in many CDA mass spectra. Three main categories are classified: (i) Amine-, (ii) Carbonyl-, and (iii) Aromatic-type mass spectra. Furthermore, some aromatic-type CDA spectra show features that correspond to breakup-products of larger complex aromatic species with masses above 200u. On the whole, the analysis of E ring ice grains in this work gives first insights into the largely varying and complex organic chemistry inside the ocean of Enceladus. The second aim of this thesis is to infer the compositional profile of ice particles in the E ring in the vicinity of Saturn’s moon Rhea, from a series of spectra recorded on Cassini’s Rhea flyby (R4) in 2013. No striking change in the frequency of different compositional types is observed along the spacecraft trajectory. However, a varying size distribution of different compositional populations of ice grains is observed and discussed. Sodium salts and organic compounds are more frequent in relatively large ice grains, whereas pure water ice particles become more abundant in smaller E ring grains. A generally higher number density of ice grains is observed in the close vicinity of Rhea, which might indicate either the presence of an ejecta cloud from Rhea’s surface or a general confinement of particles near the equatorial plane of the E ring.
The primary subject of this thesis is the two-dimensional Hubbard model. Using the functional renormalization Group in static approximation we will present the phase diagram of the 2D-Hubbard model and refine it by including frequency dependencies and full self-energy effects. The focus will fall on the vicinity of the quantum critical point, where the system shows a non-Fermi-liquid-like behavior. In mean-field calculations, we show that the frequency dependence of the self-energy has a substantial impact on the ground state. Furthermore, we investigate the relation between the density-density interaction and the quasiparticle lifetime. During the course of our study, we will develop a saddle-point formulation of Schwinger-Dyson equations and present a new renormalization group scheme which is valid for all fermionic systems.
In dieser Arbeit wurden Kernspinresonanz (NMR)-Eigenschaften von braunem Fettgewebe (BAT) charakterisiert. Mittels Magnetresonanzspektroskopie (MRS)-Messungen wurde zunächst der Wasser- und Fettanteil im thermoneutralen Zustand bestimmt und anschließend die Auswirkungen einer Kälteexposition auf beide Anteile analysiert. Ein Messprotokoll für eine Magnetresonanztomographie (MRT) zur Detektion von BAT im interskapulären Bereich wurde entwickelt. Das Protokoll ermöglicht, neben der Bestimmung des BAT-Volumens, die Quantifizierung des Fettverhältnisses (FF) in BAT. Dazu wurde eine Machbarkeitsstudie mit zehn gesunden Probanden bei 3T durchgeführt und die Reproduzierbarkeit evaluiert. Mit einer Weste wurde die Kälte- und Wärmeexposition des interskapulären Bereichs durchgeführt. Zur FF-Quantifizierung wurden die Wasser- und Fettanteile mit einer 2-Punkt-Dixon (2PD)-Sequenz bestimmt und deren Änderungen für drei verschiedene Temperaturphasen evaluiert. Magnetfeldunabhängige Temperaturmessungen wurden parallel durchgeführt. Mit dem entwickelten Messprotokoll konnte gezeigt werden, dass unter Kälteexposition in einem mittleren Volumen von (1,31 ± 1,43)ml eine mittlere, signifikante (p < 0,001) Abnahme des FF in potentiellem BAT von (−2,9 ± 2,0)%/h stattfand. Ebenso konnte eine Korrelation (r > 0,5) dieser FF-Abnahme mit der mittleren Abnahme der Rückentemperatur von (3,8 ± 1,0)°C der Studienteilnehmer beobachtet werden. Die Körpertemperatur blieb konstant bei (37,2 ± 0,9)°C. Signifikante Änderungen (p = 0,008) der T1-Zeit konnten zwischen kälteexponiertem (T1,4C = (411 ± 69)ms) und thermoneutralem (T1,RT = (373 ± 45)ms) in vitro BAT beobachtet werden. Bei einer ersten 7T Vergleichsmessung konnten vergleichbare Auswirkungen einer Kälteexposition auf BAT beobachtet werden.
The primary aim of this thesis is to shed a quantitative light on the mechanics of dynamic biological interfaces with different levels of structural complexities, ranging from lung surfactant models to regenerating tissues. In chapter 3, the correlation between biophysical properties and function of the native extracellular matrix (ECM), mesoglea, of the freshwater polyp Hydra was studied. In the body design of Hydra, mesoglea acts as an interlayer between external (ectodermal) and internal (endodermal) cell layers, sustaining the mechanical integrity of polyps. In this study, nano-focused grazing incidence small angle X-ray scattering on isolated mesoglea revealed that the packing order of Hydra collagen type I was comparable to its vertebrate homologue. The structure was anisotropic with respect to the oral-aboral axis, supporting the extensive extension and contractions of the body along this axis. In the next step, the spatio-temporal evolution of mesoglea mechanics was tracked ex vivo by nano-indentation using an atomic force microscope. The experimental data demonstrated that freshly detached polyps initially had a uniformly soft mesoglea, but mesoglea changed the characteristic "elasticity patterns" during the asexual reproduction. This change could be explained by a quantitative proteome analysis, implying that the mechanical remodeling of Hydra was highly correlated with protease expression activity. When the body column tissue was transformed into head tissue either by a drug or by the over-expression of β-catenin, mesoglea had low elastic moduli over the whole body. This result suggests that the spatio-temporal patterns in mesoglea mechanics is strongly correlated with the stem cell activity. In chapter 4 a highly sensitive two-fingered micro-robotic hand was used to determine the viscoelastic properties of Hydra tissue fragments (regenerates) during early stages of regeneration. Owing to the dexterous grasping motion of microobjects realized by the micro-robot, the bulk elastic modulus of Hydra regenerates could be determined by linearly compressing the tissue by keeping the strain level low. Under a constant strain, the stress relaxation behavior could be interpreted by applying the Maxwell model of viscoelastic materials, yielding the Stokes frictional coefficient and viscous modulus. Furthermore, the forces actively generated by the regenerate were measured and shown to correlate well with shape fluctuations of a freely regenerating sample. In chapter 5, lung surfactant inactivation by serum proteins during the acute respiratory distress syndrome (ARDS) was simulated. As the model of dynamic, oscillating interfaces in lung, the competitive adsorption of dipalmitoylphosphatidylcholine (DPPC) and bovine serum albumin (BSA) to the air/water interface was monitored by periodically changing the surface area. The model was used to investigate the impact of perfluorohexane (PFH) as a potential therapeutics. The lipid-protein composite films at the air/water interface in the presence and absence of PFH gas could be visualized by fluorescence microscopy, indicating an accelerated displacement of a pre-adsorbed BSA by DPPC in saturated PFH atmosphere. The acceleration of BSA-DPPC replacement under PFH atmosphere was accompanied by significant changes in viscoelasticity of the interface, suggesting the incorporation of PFH to the protein layer.
This thesis investigates how a single highly excited atom, called Rydberg atom, can be optically imaged. Direct detection methods based on the scattering of light are hardly applicable due to the small scattering rate of the ground to Rydberg state transition. Instead, a cloud of ground state atoms, normally absorptive, is rendered transparent using electromagnetically induced transparency (EIT), involving an auxiliary probe Rydberg state. The cloud acts as a contrast medium, whose optical response is locally perturbed by the strong Rydberg-Rydberg interaction between the probe and the Rydberg impurity which we want to detect. This perturbation restores absorption within a small volume around the impurity, readily detected and spatially resolved on a camera. We call this technique Interaction Enhanced Imaging (IEI). To implement IEI we characterize the optical response of the EIT contrast medium in absence of interactions. By combining measurements of the spatially resolved optical spectrum and of the total Rydberg atom number, we can reconstruct the full one-body density matrix of the three-level system. Next, we excite |nS> or |nP> states and, using IEI, we demonstrate spatially resolved imaging, enabling us to study dipolar energy transport. To reach single impurity sensitivity we investigate our current detection fidelity and characterize the signal and noise contributions in IEI. We model our interacting system, finding good agreement with experimental data. Based on this model, we predict combinations of Rydberg states for which single-shot single impurity sensitivity should be possible in future experiments.
Since their introduction in the 1980s, locally stationary time series play an important role in time series analysis. As a generalization of stationary processes, they allow the observations to change their distribution properties over observation time. In many locally stationary time series models this change over time is characterized by parameter curves, whose estimation is of essential interest. In this work we develop methods and prove theoretical results for bandwidth selection for nonparametric estimators of these curves. We focus on local maximum likelihood estimators. Their strong connection to martingale difference sequences is fundamental in many of our proofs.
In the first part of this dissertation we define a global bandwidth selector for linear locally stationary processes which is motivated by the cross validation method that was first introduced in the nonparametric regression model. We prove that the selector is asymptotically optimal in the sense that the Kullback Leibler distance of the model connected with this selector to the true model converges to the minimal possible Kullback Leibler distance as the observation time increases to infinity. In simulations we analyze the quality of the method. The proofs are based on bias-variance decompositions of the estimators. The formal discussion of these decompositions gets harder in the case of nonlinear locally stationary time series models.
In the second part of this dissertation we develop general techniques to approximate locally stationary processes by stationary processes. These techniques allow us to obtain the decompositions mentioned above. We introduce so called derivative processes and give conditions under which existence and uniqueness can be guaranteed. An important result is a Taylor-like expansion of locally stationary processes. These findings are of independent interest for further research. We emphasize this point by using the approximation techniques to obtain new versions of standard theorems like a law of large numbers and a central limit theorem for locally stationary processes under minimal moment assumptions.
In the last part of this thesis we define a local bandwidth selector for a large class of locally stationary processes which is based on a contrast minimization approach which was first applied to nonparametric regression models. We show that our selector is minimax optimal up to a logarithmic factor (which is typical for local model selection procedures) with respect to the Euclidean distance and the Kullback-Leibler distance. For the proofs we use the approximation techniques which were discussed before. In a simulation we analyze the behavior of the selection routine for different time series models.
The findings of this thesis regarding bandwidth selection routines can be interpreted as a generalization of the original methods in the nonparametric regression model, because this model is included as a special case. Due to the more general formulation this thesis makes a contribution to understand these methods more deeply.
Protein quality control is a well-organized cellular process in which potentially toxic misfolded proteins are either refolded back to their native state, degraded or deposited into special deposition sites. Sequestration of misfolded protein species into specific deposition sites occurs in all kingdoms of life and serves as a second line of defense when refolding or degradation machineries that normally deal with these misfolded proteins are overwhelmed. In Saccharomyces cerevisae, three major subcellular sequestration sites have been described for deposition of different protein aggregates: INQ (intranuclear quality control compartment)/JUNQ (juxtanuclear quality control compartment), IPOD (insoluble protein deposit) and CytoQ. Amorphously aggregating proteins are targeted either to the INQ/JUNQ by the nuclear sorting factor Btn2, or they are targeted to a peripheral deposition site termed as Cyto Q with the aid of the cytosolic small heat shock protein Hsp42.
Amyloidogenic aggregates including yeast prions are predominantly sequestered at the IPOD, a perivacuolar deposition site. The perivacuolar IPOD is located in close proximity to the PAS (Phagophore Assembly Site) where the cells initiate formation of autophagosomes and CVT (Cytoplasm-to-Vacuole Targeting) vesicles. The cellular machinery, however, by which amyloid aggregates are recognized and deposited at the IPOD is still unknown.
Using a fishing approach with immobilized prion fibers formed by the prion domain of Sup35 (PrD), I identified components of actin cable-based and SNARE-mediated vesicular transport machinery to bind to PrD fibers. Using an auxin-based depletion system, I show that proper recruitment of the model prion amyloid PrD-GFP to the IPOD and the CVT substrate preApe1 to the PAS is disrupted upon depletion of essential components of the actin-based transport machinery, Myo2, Cmd1 and Tpm1/2, as well as Sec18-mediated SNARE function. Interestingly, the IPOD substrate PrD-GFP and the two PAS markers preApe1 and Atg8 accumulate reversibly in the cytosol in these mutants. Using fluorescence microscopy, I observed that PrD-GFP aggregates are associated with Atg9 transport vesicles similar to preApe1 and are targeted to the IPOD through these vesicles along actin cables. In addition, these PrD-GFP aggregates are shown to interact with Myo2 in vivo upon disruption of Sec18 SNARE function.
In a next approach, I investigated the possible fate of PrD-GFP aggregates deposited at the IPOD, that is located adjacent to the site in the cell where autophagosomes/CVT vesicles are formed. I demonstrate that PrD-GFP aggregates are not turned over in bulk via autophagy, but can be degraded by proteasomal and other unknown cellular degradation pathway(s) only after their slow and progressive extraction by the disaggregase Hsp104 from the IPOD. Thus accumulation of PrDGFP amyloids at the IPOD might serve a temporary storage function when downstream cellular degradation systems are overwhelmed. Based on the above findings, a model was proposed where PrD-GFP and preApe1 use an Atg9-vesicular transport machinery and Myo2 as a linking factor to be deposited at their recruitment sites IPOD and PAS, respectively, along tropomyosin-coated actin cables.
The co-receptor CD8 plays an important part in the proper functioning of cytotoxic T lymphocytes. In order to sense stimuli, the T cell surface recep- tor (TCR) engages peptide-specifically with its ligand pMHC, while CD8 makes peptide-unspecific contact to the MHC subunit. In this work, the entirety of interactions between TCR, CD8, and pMHC are elucidated by confronting of a family of mathematical pMHC-TCR-CD8 interaction mod- els with accurately measured dose response data. The interaction model being in best agreement with the data, termed CBM, consists of a TCR- CD8 complex having the striking property that its CD8 subunit exhibits increased affinity to pMHC compared to CD8 alone. A T cell triggering model, founded on multivalent binding, is con- structed that enables affinity-based ligand discrimination. In combination with CBM, the TCR triggering model is capable to correctly predict key aspects of dose response T cell activation data, and introduces a novel mechanism for the contribution of CD8 in ligand discrimination and T cell activation. The high affinity CD8 binding site of the TCR-CD8 complex prevents low affinity (self) ligands to establish pMHC-TCR contacts and thereby reducing the intracellular signal intensity in response to self pep- tides. High affinity (foreign) ligands, on the other hand, can counteract by forming pMHC-TCR contacts with TCR-CD8 complexes. Because CD8 also binds the kinase Lck, this leads to enhanced intracellular signal in- tensity in response to foreign antigen. Thus, CD8 amplifies affinity-based ligand discrimination and the proposed mechanism leads to improved self tolerance as well as sensitivity towards foreign antigen of T cells allocating CD8 a significant contribution to T cell immunity.
Cancer is largely a disease of the genome. Cancer development is thought to involve the gradual acquisition of mutations that can activate oncogenes and/or inactivate tumor suppressor genes, resulting in a series of genetic changes that stimulate growth, attenuate cell death, destroy checkpoint controls, promote further genetic instability, and enable metastasis.
In the first part of my thesis, I focused on deciphering how activation of oncogenes by structural copy number alterations (SCNAs) that relocate enhancers in close proximity to oncogenes can be achieved, rather than activation by mutation or amplification. This mechanism was recently described as enhancer hijacking (EHJ). I contributed to the understanding of the EHJ mechanism at two genomic loci, IGF2, a known oncogenic locus in colorectal cancer and IRS4, a gene identified as a top pan-cancer EHJ candidate. To achieve this, I recapitulated the rearrangements associated with EHJ in colorectal and lung cell lines using the CRISPR/Cas9 genomic engineering system and tested for IGF2 and IRS4 overexpression, respectively. The rearrangements were successfully reconstructed; however an increase in gene expression was not achieved, suggesting a more complex mechanism of activation or context-dependency than initially anticipated. Investigation of the tumor promoting role of IRS4 was supported using mouse xenografts, where constitutive overexpression of IRS4 leads to formation of larger tumors in comparison to control tumors.
In the second part of this thesis I emphasized on the identification of genes, which, when disrupted, lead to sustained cell growth and can be potential tumor suppressors. To achieve this, I employed systematic screens on cells with different genetic backgrounds using a combination of CRISPR/Cas9-based whole genome knockout libraries and the powerful anchorage independent growth assay. I was able to verify known tumor suppressor genes, which include components of the Hippo and mTOR pathways, as well as to identify novel candidates including FRYL and AHR. Furthermore, a growth screen under non-selecting conditions was performed and identified numerous candidates found in the initial anchorage independent growth screen, which further supports the growth promoting roles of the candidate genes. In conclusion, in my study I identified potential tumor suppressors that lead untransformed cells to enhanced as well as anchorage independent growth.
Platelets are an important component of blood that help maintain haemostasis. They are derived from large bone marrow residents, called megakaryocytes, which release tubular processes into the blood stream that fragment and eventually form 2 to 3 μm sized enucleate discoid platelets. Activation of platelets is caused by factors released into the bloodstream upon endothelial damage, to which they respond by undergoing a distinct order of shape transition, from discoid, to spheroid, to dendritic, and finally an extended morphology, required to form a clot. Despite the simple architecture, platelets are able to drastically alter their morphology owing to the repertoire of cytoskeletal proteins they express. Microtubules, which form a bundle running along the platelet periphery, are known to be important for maintaining the resting discoid morphology of a platelet, while actin is heavily implicated in the later stages that require adhesion. During the first step of platelet activation, the microtubule marginal band undergoes coiling, while the platelet changes from a disc to a sphere shape. Both actin and microtubules are implicated in this process but the mechanics of the process are not clearly understood.
The following project has been carried out to explore the role of cytoskeletal mechanics in triggering activation of a platelet. A combination of experimental and analysis techniques was used to quantitatively assess mechanical properties of the system in a resting state, as well as during activation, by direct measurement of the marginal band morphology. The structure and composition of the marginal band was analyzed using electron tomography, which provided detailed information on individual microtubules. Super-resolution microscopy was also used to visualize the overall morphology and composition of the marginal band. With this data we could infer the mechanical properties of the resting marginal band in fixed platelets. To analyze the dynamics of coiling process, live cell fluorescence microscopy was used in combination with a microfluidic system. With this setup, changes in the marginal band shape could be followed in response to treatment with agonists or inhibitors that affect the cytoskeleton, that is, a process analogous to mechanical perturbations of the platelet. A large population of platelets was also analyzed to infer the intrinsic variations mechanical properties of the marginal band. The Cytosim software was used to set up simulation of marginal band coiling.
By using a multifaceted approach, we were able to get novel insight into the mechanics of marginal band coiling. Firstly, we showed that length distribution of microtubules in a set of resting platelet marginal bands follows an exponential distribution. The sum of all polymerized microtubule length was found to be 101.84µm ±12.63 per platelet. The typical distance between two microtubules was found to be 30nm in a tightly packed marginal band. Secondly, by measuring the dynamics of coiling, we could infer that the marginal band behaves like a visco-elastic ring upon activation with ADP. This response was found to be dependent on actin, while thrombin activation elicited a response that manifested in an actin independent manner. Analysis of large population of platelets showed that the tubulin intensity scales as a power of five to the platelet radius, indicating a possible enrichment of tubulin in platelets. Finally, our data suggests that platelets with longer marginal bands have a higher propensity to coil.
Although some of our results need to be followed up with further investigations, this study provides an experimental and analysis framework that allows us to quantitatively analyze platelet cytoskeleton morphology with an aim to understand the mechanics of platelet activation in healthy and disease states.
In this thesis the dynamics of a trapped ion immersed in a spatially localized buffer gas is investigated. For a homogeneous buffer gas, the ion’s energy distribution reaches a stable equilibrium only if the mass of the buffer gas atoms is below a critical value. This limitation can be overcome by using multipole traps in combination and/or a spatially confined buffer gas. Using a generalized model for elastic collisions of the ion with the buffer gas atoms, the ion’s energy distribution is numerically determined for arbitrary buffer gas distributions and trap parameters. Three regimes characterized by the respective analytic form of the ion’s equilibrium energy distribution are found. One of these is a novel regime at large atom-to-ion mass ratios where the final ion temperature can tuned by adiabatically decreasing the spatial extension of the buffer gas and the effective ion trap depth (forced sympathetic cooling). The second part of the thesis presents a hybrid atom ion trap designed for sympathetic cooling of hydroxide anions. In this hybrid trap the anions are immersed in a cloud of laser cooled rubidium atoms. The translational and rovibrational temperatures of the anions is probed by photodetachment tomography and spectroscopy which shows the first ever indication of sympathetic cooling of anions by laser cooled atoms.
Die vorliegende Arbeit befasst sich mit der Synthese und Strukturuntersuchung neuartiger Indiumamidinate von der Typ R2InX (R = R”NCR’NR”; R’ = Ph, R” = SiMe3, iPr, dipp; X = Br, Cl) mit der Koordinationsnummer 5 sowie R3In (R = Me3SiNCPhNSiMe3) mit der Koordinationsnummer 6. Durch die bereits verwendeten Synthesemethoden sollen Carbodiimiden mit Organo-Lithium-Verbindungen die entsprechenden Lithiumamidinate erhalten und mittels Transmetallierung mit InBr3 and InCl3 in Indiumamidinate überführt werden. Alle erfassten Strukturen wurden durch Kristallstrukturanalyse, NMR-Spektroskopie, sowie Elementaranalyse geprüft.
T cells are one of the key players in cell-mediated immunity. The peripheral blood consists of an intricate balance between various T cell subpopulations, which vary in their differentiation state and memory potential. Many of these T cell subpopulations are associated with diseases, and numerous studies highlight the importance of monitoring the frequencies of T cell subsets in peripheral blood for determining the immune competence status of a person. Particularly elderly persons show a wide spectrum of immunosenescence stages that need to be taken into account in targeted medicine. Methods that are used up to date to identify T cell subsets are based on the expression of an enormous and increasing number of surface markers that depend on cells’ differentiation and activation state. This is often insufficient since T cells display a great variety of differentiation states. In this thesis, a novel marker-free, fast and low-cost approach for T cell subpopulation identification is presented that is based on changes in adhesion- and migration-related parameters during T cell differentiation. We hypothesized that these fine phenotype shifts can be visualized with Reflection Interference Contrast Microscopy (RICM) imaging and suffice to allow clear classification of T cell subpopulations. To first prove the sensitivity of our approach we imaged CD4+ T cells isolated from human peripheral blood at different time points during in vitro differentiation. Several morphological parameters correlated with memory T cell marker expression and were distinct from less differentiated states, e.g. an increased fractal dimension (Df) parameter that describes the cell´s membrane topology or increased projected and adhesion cell area. In a next step we determined adhesion parameters and fractal dimensions from RICM images of T cell subsets that were differentiated in vivo. We separated naïve, central memory and effector memory CD4+ T cells from human CD4+ populations. For all determined parameters, naïve T cells showed significant lower values compared to memory cells. Using principal component analysis, the parameters that described the data variance of subsets best were identified to be area, adhesive area, excess perimeter and fractal dimension of contour/topology. These parameters were then used for generating cluster centres for each subpopulation with k-mean clustering. T cell subsets isolated from three different donors were then designated to these clustering points. With this approach, 80 % of T cells were assigned to their correct subtype. In accordance to in vitro differentiation experiments, we found an increase in the complexity of membrane topology and an increase in cell area from naïve to central memory and to effector memory cells. These results suggest that all differentiation states of T cells may be continuously represented in adhesion maps. This visualisation approach will allow including T cell subsets that cannot be considered by conventional surface marker sets but are highly representative for distinct immune competence states. To apply our concepts of a marker-free adhesion-based assay to discriminate between a healthy CD4+ T cell “fingerprint” and one in disease state, we used an in vitro Graftversus- Host-Disease (GVHD) murine model. In this approach we did not evaluate adhesion properties on static RICM images but tracked adhesion-dependent intestine T cell homing on migration chips. For this, the chemokine CCL25 was anchored to 2D gold nanoparticle distance gradients and ICAM-1 was covalently linked to functional PEG molecules in between. CD4+ T cells with a GVHD phenotype were able to respond to decreasing CCL25 distances by increasing cell velocity, which was not observed for control CD4+ T cells from healthy mice. Furthermore, GVHD phenotype CD4+ T cells displayed a lower degree of complexity of cell trajectories than control cells. Our approaches of imaging T cell adhesion and/or in vitro homing is very promising for future marker-free immunodiagnostics that read out the immune competence status of a patient within short time, which can be applied in therapy monitoring and advising primary personalised treatment.
Plasma cell dyscrasias are characterised by accumulation of malignant plasma cells in the bone marrow. Asymptomatic multiple myeloma (AMM) evolves from monoclonal gammopathy of unknown significance (MGUS) and progresses to symptomatic myeloma involving end organ damage. Three main questions are addressed by mathematical modelling. Firstly, how is growth of malignant plasma cells characterised? Secondly, how fast does progression from early asymptomatic stages (MGUS, AMM) to symptomatic myeloma happen? Thirdly, how many malignant plasma cells initially arrive at the bone marrow?
New mathematical models consisting of piecewise-smooth ordinary differential equations are formulated describing the dynamics of healthy and malignant plasma cells in the bone marrow and its niche. Model analysis refers to existence and uniqueness of solutions, characterisation of solutions within invariant sets, and existence and stability properties of equilibria. Partial equilibria are identified extending the classical notion of equilibria. The models are validated using clinical data consisting of serum and urine samples (n = 8398) of patients with AMM and MGUS (n = 322 and n = 196, respectively).
Model analysis and parameter estimation imply that accumulation of malignant plasma cells can be quantified by the doubling time. A faster doubling time relates to a higher probability of progression to symptomatic myeloma and correlates with a small initial number of malignant plasma cells. Instead of one single initial malignant plasma cell, initiation of myeloma can rather be explained by a „malignant wave“ comprised of a population of malignant plasma cells arriving at the bone marrow and perturbing healthy homoeostasis.
This thesis is the result of an interdisciplinary doctorate and the joint work with Prof. Dr. Anna Marciniak-Czochra (Institute of Applied Mathematics, Heidelberg University) as well as with PD Dr. Dr. Dirk Hose and Dr. Anja Seckinger (Multiple Myeloma Research Laboratory, Heidelberg University Hospital).
Image sequences of dynamic scenes recorded using various depth imaging devices and handling the artifacts arising within are the main scope of this work. First, a framework for range flow estimation from Microsoft’s multi-modal imaging device Kinect is presented. All essential stages of the flow computation pipeline, starting from camera calibration, followed by the alignment of the range and color channels and finally the introduction of a novel multi-modal range flow algorithm which is robust against typical (technology dependent) range estimation artifacts are discussed. Second, regarding Time-of-Flight data, motion artifacts arise in recordings of dynamic scenes, caused by the sequential nature of the raw image acquisition process. While many methods for compensation of such errors have been proposed so far, there is still a lack of proper comparison. This gap is bridged here by not only evaluating all proposed methods, but also by providing additional insight in the technical properties and depth correction of the recorded data as base-line for future research. Exchanging the tap calibration model necessary for these methods by a model closer to reality improves the results of all related methods without any loss of performance.
Expression of accessory proteins during human immunodefiency virus-1 (HIV-1) life cycle is essential to ensure efficient viral replication and spread. Despite of being dispensable for HIV-1 infection of cell lines in vitro, use of primary target cells and in vivo experiments proved them to be critical key pathogenicity factors. Along with counteraction of restriction factors to facilitate immune response escape, they play an important role as manipulators of host cell fundamental biological processes to favor replication. This work focuses on the study of the accessory proteins. Both these proteins are known to strongly alter intracellular vesicular transport. As a consequence, the composition of the plasma membrane is modified upon their expression, a phenomenon studied in the first part of this thesis. Here, it has been demonstrated the specificity of the Nef- and Vpu-mediated downregulation of tetraspanins, the most drastically affected host receptors, and identification of molecular determinants of both proteins necessary for this activity was pursued. It was also studied the ability of Vpu to retarget the T lymphocyte master regulator Lck, a well described phenomenon induced by Nef. Another yet unstudied field for Vpu examined herein was the induction of extracellular vesicle release. This work revealed that Vpu, like Nef, enhances extracellular vesicle release when expressed by T lymphocytes with comparable or even higher efficiency. The second part of this thesis addressed the effect of these proteins on T cell polarity disruption in the context of T cell migration. Unlike Vpu, Nef has been reported to inhibit F-actin cytoskeleton rearrangements. This requires the interaction of Nef with PAK2 which facilitates a specificity switch of the host kinase resulting in hyperphosphorylation and thus inactivation of the actin severing factor cofilin. As a consequence, the motility of HIV-infected T lymphocytes is hampered. A previous report showed that in murine CD4+ T cells, the central Nef-mediated block in vivo and ex vivo is at the level of transendothelial and subendothelial migration. Whereas the latter is dependent on Nef-PAK2 on actin dynamics, the effect on transmigration involves other, yet uncharacterized protein interactions and is remarkably paralleled by a pronounced reduction of cell polarization. This thesis shows that Nef also hinders polarization of HIV-1 infected primary human CD4+ T lymphocytes. Vpu was entirely unrequired for this effect. Nef-expression in human T cell lines caused a marked polarization defect conserved among Nef proteins from HIV-1, -2 and simian immunodeficiency virus. The interaction surface of Nef with the Nef-associated kinase complex (NAKC) was found to be the essential molecular determinant for polarization disruption by Nef, both when overexpressed or in HIV-1 infection. Pharmacological inhibition of NAKC components evidenced the involvement of protein kinase C (PKC) in this Nef activity. This work also details setting up of experimental conditions to study the possible implication of this Nef amino acid stretch on transendothelial migration, that is on diapedesis ex vivo. Altogether, the results presented here underline the importance of the studied redundant functions between Nef and Vpu in HIV-1 infection and the relevance of these accessory proteins as pathogenicity factors. Also, they highlight the role of Nef as the main HIV-1 negative regulator of T lymphocyte migration.
The aim of this thesis is to analyze several aspects of dependence structures for stochastic processes. To this end, new dependence measures for spatial stochastic processes will be introduced. Further, different analytical properties of correlation functions and their relation to properties of the realizations of the corresponding stochastic processes will be studied. The newly introduced dependence measures are based on the distance correlation. Thus, they are defined for larger classes of processes than the well-known Pearson correlation function. In addition, the new dependence measures allow to quantify non-linear dependencies, as well as dependencies in multivariate stochastic processes. On the one hand, the investigation of analytical properties of correlation functions considers isotropic positive definite functions on spheres of different dimensions. These functions are characterized by Gegenbauer expansions involving so-called Schoenberg coefficients. Relationships between those Schoenberg coefficients will be given. On the other hand, characterizations of correlation functions of random vector fields with almost surely divergence-free and irrotational realizations will be considered. Further, the dimple property of spatio-temporal random fields will be analyzed. This dimple corresponds to a non-monotonic temporal behaviour of the correlation function, which can be generated by so-called transport fields. These transport fields and their relation to the dimple property will be investigated.
The nervous system, built by the powerful capability of neurons to intercommunicate, is the most fascinating biological achievement, which through evolution has given rise to highly complex structures responsible for remarkable animal behaviours. It is therefore surprising, and at the same time highly motivating for a scientist, to realise how little we know about this evolutionary process. Traditional approaches like palaeontology and phylogenomics lack the resolution to confidently solve the history of neuronal circuits. Nevertheless, thanks to modern comprehensive techniques and integrative approaches, it is possible to molecularly, morphologically and functionally describe entire nervous systems by their constituent components, the cell types. The comparison of cell types and circuits across animals will help elucidate the different evolutionary steps that led to extant nervous systems. Within this thesis, the reader will find a description of the research I have conducted on the implementation of system-level approaches to characterize cell types, with the goal of achieving an integrative understanding of the nervous system of Platynereis dumerilii, an animal suited for these system-level evolutionary studies. A special emphasis has been given to the generation of a new automatic pipeline to build gene expression atlases for complex body plans, the design of image analysis routines to monitor and quantify animal behaviour, and the implementation of the Crispr/Cas9 technique in this organism. I also describe pioneer work to reconstruct the full connectome of the larvae at six days post fertilization, the combination of light-sheet microscopy and calcium indicators to monitor neuronal activity in thousands of neurons, and the proof-of-principle of using optogenetics to manipulate neuronal activity in Platynereis. Because of the relevance of the control of muscle contraction for the evolution of nervous systems, and the vast amount of information collected for various animals with regards to locomotion (Goulding, 2009), I focus my analysis on the post-mitotic Platynereis ventral nerve cord. I show that this structure contains the circuits responsible for the crawling behaviour, and describe in detail the kinematics of these movements, which suggest a similar organization of circuits than vertebrates and segmental protostomes. Using the expression atlas, I unbiasedly unravel the molecular substructure of the ventral nerve cord, which consists of cell types grouped into general medio-lateral domains, remarkably similar to those in vertebrates, as well as different territories unique to protostomes. I further characterize in detail a commissural cell population, showing strong similarities with both vertebrates and Drosophila in terms of position, molecular profile, morphology and function. These findings support the idea of an ancestral cell type, specified by a newly acquired gene, which controlled the coordination between the two sides of the body during locomotion in the bilaterian ancestor.
In this thesis we address supervised algorithms and semi-manual working steps which are used for scenarios where automatic computer vision approaches cannot achieve desired results. In the first part we present a semi-automatic method to acquire depth maps for 2D-3D film conversions. Companies that deal with film conversions often rely on fully-manual working steps to ensure maximum control. As an alternative we discuss an approach which uses computer vision methods to reduce processing time but still provides opportunities to interactively control the outcome. As result we receive detailed, smooth and dense depth maps with sharp edges at discontinuities. Part II, which presents the major contribution of this work, deals with human annotations used to assist ground truth acquisition for computer vision applications. To optimize this labour-intensive method, we analyse whether annotations created by different online crowds are an adequate alternative to running such projects with experts. For this purpose we propose different methods for improving acquired annotations. We show that appropriate annotation protocols run with laymen can achieve results comparable to those of experts. Since online crowds have much more users than typical expert groups used to run according projects, the presented approach is a viable alternative for large data acquisition projects.
Eukaryontische Zellen weisen neben einer hochgradig organisierten Genregulation auch eine besonders ausgefeilte Kompaktierung der DNA auf, deren Basiseinheit Nukleosomen sind. Diese bestehen aus einem Histonkern mit je zwei Kopien der einzelnen Histonproteine – H2A, H2B, H3 und H4 – und einer 147 bp langen DNA, die in 1,7 Windungen um den Histonkern gewickelt ist. Im Chromatin sind die einzelnen Nukleosomen über DNA-Linker (10 – 90 bp) miteinander verbunden. Die Konformation dieses Komplexes kann durch posttranslationale Modifikation der Proteine und der DNA verändert werden. Von besonderer Bedeutung sind hierbei die aus dem Histonkern herausragenden H3-Histonschwänze, deren Deletion zur Destabilisierung des Komplexes führt. Aus Simulationen lässt sich schließen, dass dies mit Veränderungen der elektrostatischen Umgebung im inneren des Histonkerns, der α3-Region des H2A-Histonproteins, einhergeht. Da bis heute unklar ist, ob die elektrostatische Umgebung dieser Region einen direkten Einfluss auf die Stabilität hat, wurde in dieser Arbeit der Einfluss von Mutationen der Positionen R81 und R88 des H2A-Histonproteins auf die Nukleosomenarchitektur und –Stabilität analysiert. Hierfür wurde ein Aminosäureaustausch zu Alanin bzw. zu Glutaminsäure durchgeführt und dessen Einfluss mit Hilfe von Förster-Resonanz-Energie-Transfer (FRET) charakterisiert. FRET bezeichnet einen distanzabhängigen, strahlungsfreien Energie-Transfer zwischen zwei Fluorophoren und kann als Maß für Distanzveränderungen im Bereich von 2 – 10 nm verwendet werden. Durch den Vergleich von mutierten und Wildtyp-Nukleosomen unter Verwendung verschiedener Markierungsstrategien konnte eine durch die Mutationen hervorgerufene Destabilisierung detektiert werden. Deren jeweiliges Ausmaß lässt Rückschlüsse auf die Relevanz der Position (SWt > SR88 > SR81 > SR81R88), sowie der Ladung (SRA > SRE) der eingebrachten Mutation für die Stabilität der Nukleosomen zu. Eine Einzelmolekülanalyse der salzinduzierten Öffnung der Nukleosomen weist auf eine Schwächung der Dimer:Tetramer-Interaktion als Grund für die Destabilisierung hin. Im zweiten Teil der Arbeit wurden die Dynamik und die intranukleosomalen Wechselwirkungen des H3-Histonschwanzes näher betrachtet. Hierfür wurde unter Verwendung von zwei mutierten H3-Histonproteinen (H3K4C und H3K9C) eine neue Markierungsstrategie etabliert. Die Ergebnisse weisen daraufhin, dass Interaktionen im Bereich der Dyadenachse auftreten und diese einen dynamischen Prozess darstellen. Es kann daher davon ausgegangen werden, dass die intranukleosomalen Interaktionen des H3-Histonschwanzes das Nukleosoms stabilisieren.
In this thesis, motivated by the simulation of fuel cells and batteries, we develop an adaptive discretization algorithm to reduce the computational cost for solving the coupled parabolic/elliptic system. This system is the model for the electrochemical processes within the cathode of a solid oxide fuel cell (SOFC). First, the coupled system is discretized in time and in space by the Finite Element Method. Then, it is split into parabolic and elliptic sub-problems through an operator splitting method. These two equations are solved sequentially by the multirate iterative solving method that allows for different time step sizes for the temporal discretizations.
The main focus of this work is to derive goal-oriented, a posteriori error estimators based on the Dual Weighted Residual method that are computable and separately assess the temporal discretization error, the spatial discretization error and the splitting error for each sub-problem. Instead of natural norms, the errors are measured in an arbitrary quantity of interest, as is often used in practical applications.
The sub-problems are solved in temporal discretizations with different step lengths. If the ratio between the two step lengths is too large, this can result in the divergence of the coupling iteration within the multirate scheme. In this case, the algorithm uses the information from the splitting error estimator to control the convergence behavior. The error contributions of both discretizations and splitting method are balanced at the end of the refinement cycle that halts when the error estimators reach a desired accuracy.
The described methods are validated on a simplified model that simulates the cathode of a SOFC. In this application, the parabolic part consists of a reaction-diffusion equation describing the concentration distribution of ions, and the elliptic part describes electrical potential. For a given accuracy, the adaptive algorithm finds the least required number of degrees of freedom of the parabolic and the elliptic parts of the system. Since the electrical potential equation has the faster time scale, we use the multirate method and see that the elliptic problem requires a smaller number of degrees of freedom to attain the same desired accuracy within the system. This significantly saves the total computational cost, since the elliptic equation in the coupled system is more expensive to solve. Therefore, this combination of the degrees of freedoms is optimal, in that it gives the least computational cost and the convergence within the algorithm.
To date, there are no specific treatments available that efficiently target the loss of neural connectivity after a spinal cord injury (SCI). Thus patients usually suffer from life-long motor, sensory and autonomic dysfunction. Neuron-intrinsic growth programs are activated after a lesion in the peripheral nervous system (PNS) and can contribute to enhanced regeneration in a subsequent central lesion. Yet this so-called conditioning lesion (CL) holds little translational potential for SCI. Electrical stimulation (ES) can influence various cellular functions, including neuronal growth and could provide a practical approach to enhance regeneration after SCI. However, the mechanisms and a practical means for applying ES as a therapy after SCI are insufficiently understood. I hypothesized that evoked neuronal activity by direct ES of the peripheral nerve can enhance the growth potential of dorsal root ganglia (DRG) neurons in a similar way to CL, supporting the regeneration of the injured central branch ascending in the dorsal column. ES (20Hz, 2*MT, 0.2ms, 1h) was applied in vivo to the sciatic nerve of adult Fischer 344 rats, followed by ex-vivo assessment of the growth potential, showing about 2-fold enhanced neurite growth compared to sham animals. ES increased the percentage of neurons with neurites >100um, but there was no change in the percentage of neurite bearing neurons, indicating that the effect on growth is due to enhanced elongation and not initiation. Longer duration stimulation (7h) also enhances growth by 67 ± 25%, as well as repeated stimulation for 7 days (55 ± 24%). The pattern of growth and timeline is similar to a CL, suggesting a similar or a partial overlap in the mechanism. Growth effects of 1h ES were also assessed in vivo in a model of spinal cord injury, together with cell transplantation of BMSCs (bone marrow stromal cells) at 4 weeks post-injury. Stimulated fibers were labeled by sciatic nerve injection of the transganglionic tracer Cholera toxin B (CTB). Animals with ES for 1h showed significantly increased axonal regeneration into the spinal cell graft within the lesion compared to sham animals. Repeated stimulation with chronic electrodes showed a similar effect, but also a slight influence from chronic electrode implantation in chronic sham animals. Dieback of axons was not modified in any of the conditions. To evaluate possible side effects that may interfere with clinical applicability, I also tested pain-like behavior, showing a lack of allodynia or thermal hyperalgesia after ES. This further highlights the translational potential of this strategy in combinatorial approaches such as cell transplantation. In parallel, I investigated the mechanisms underlying the observed neuronal activity-mediated increases in neurite growth. Using in vitro depolarization of DRG neurons as a model, my data show that neurite growth is influenced depending on the duration of the depolarization and the delay between stimulation and measurement. Since depolarization induces calcium influx, I examined in a separate set of experiments calcium signaling, showing that blocking nuclear calcium signaling with recombinant calmodulin-binding proteins reduces growth in DRG cultures at 72h by 50 ± 10%. However, a cytoplasmic block enhances growth by 35 ± 11%, and has similar effects in vivo after adeno-associated virus gene transfer into lumbar DRGs. This differential effect of nuclear and cytoplasmic calcium signaling provides an explanation for previous reports, which have shown stimulation or reduction of growth following neuronal activity. Furthermore, I investigated HDAC5 (histone deacetylase 5), showing export from the nucleus in DRGs (92 ± 5% nuclear before and 14 ± 1% after depolarization). These in vitro experiments suggest that neuronal activity-mediated effects on axon growth could involve epigenetic mechanisms, dependent on calcium/calmodulin signaling. To follow up on these experiments, RNA sequencing was performed to investigate differential gene expression at 1 day and 7 days after ES, compared to sham animals, naive animals and animals that underwent a peripheral lesion, collecting 30M SE reads/sample on a HiSeq2000. As expected CL induces and represses an extensive number of genes compared to naïve animals. ES induced/reduced expression of a much lower number of genes relative to sham animals with smaller changes in gene expression. Several genes and pathways could be identified that are known to play a role in regeneration, suggesting that ES-mediated effects on axon regeneration are likely a summation of several activated pathways that overlap only partially with CL. Taken together, my results reveal the capacity of neurons to modulate their growth response depending on their activity in vivo. Electrical stimulation is shown to be an effective means to increase axonal regeneration in a central lesion, and could provide a feasible therapeutic approach either alone or in combination with other strategies such as cell transplantation.
Inflammation is an organism’s concerted response to damage and infection. For an inflammatory signal to spread it relies on specific signaling pathways to activate proinflammatory genes. One is set off by receptors that, after detecting an appropriate stimulus, nucleate the assembly of a large multimolecular signaling platform called the inflammasome. Currently, thanks to the advances in live microscopy, questions in immunology that can only be solved by live imaging are beginning to be addressed. In this work, we have established zebrafish and medaka as vertebrate model systems for the visualization of inflammasome signaling by three approaches. Since inflammasome assembly is driven by the aggregation of the adaptor molecule ASC, one approach was to study the dynamics of this molecule’s switch from a cellular cytoplasmic localization to a single aggregate, called a speck, using zebrafish. We saw that speck formation leads to pyroptosis, a proinflammatory type of cell death, in vivo. This is the first time this process is visualized in a live organism. Furthermore, we generated a zebrafish transgenic line with endogenously tagged ASC that can be used to study the role of inflammasome activation live in zebrafish infection models. Second, we used medaka to study the proinflammatory cytokine interleukin-1 (il1), whose activation is downstream of inflammasome assembly. We generated a transgenic line to track the transcriptional activation of the gene and the protein’s cleavage. Based on our results, we propose that il1 genes in teleost fish correspond genetically and functionally to both il1 paralogues in mammals, instead of only for il1β. Lastly, we generated a zebrafish reporter line to visualize and quantify NF-κB activity, a master regulator of proinflammatory genes. We show that the line has potential to be used in high-throughput screens. Overall, this work reveals unknown features of the functional role of the inflammasome signaling cascade in fish and its evolution.
Umweltschutz wird immer mehr zum Top-Thema. Nicht nur für die Weltpolitik, wie beim Klimagipfel in Marrakesch. Überall auf der Welt gibt es besondere Schutzgebiete, die Biosphären-Reservate der UNESCO. Bei uns zum Beispiel im Pfälzerwald und auf der Schwäbischen Alb. Mit modernster Satelliten-Technik können solche Gebiete jetzt viel besser geschützt werden. Dazu wurde in Heidelberg der erste UNESO Lehrstuhl Baden-Württembergs eingerichtet.
Der Beitrag erschien in der Sendereihe "Campus-Report" - einer Beitragsreihe, in der über aktuelle Themen aus Forschung und Wissenschaft der Universitäten Heidelberg, Mannheim, Karlsruhe und Freiburg berichtet wird. Zu hören ist "Campus-Report" montags bis freitags jeweils um ca. 19.10h im Programm von Radio Regenbogen. (Empfang in Nordbaden: UKW 102,8. In Mittelbaden: 100,4 und in Südbaden: 101,1)
Introduction: Colorectal cancer (CRC) is the most common type of gastrointestinal cancer and a major cause of morbidity and mortality throughout the world. Today we know that exposure to exogenous chemicals (xenobiotics) combined with a modified ability to detoxify carcinogens increases the risk of developing cancer. Xenobiotic metabolizing enzymes (XMEs) play a major role in the activation and detoxification of several carcinogens but their expression and activity in human colorectal tissue as well as their role in the development of CRC has not been sufficiently explored. In order to shed light on this question, this study aimed to link XME metabolism, lifestyle and risk factors with the etiology of CRC investigating two different aims. Therefore, the thesis is split into two parts:
1) Evaluation of mRNA, protein and enzyme activities of relevant phase I and phase II xenobiotic metabolizing enzymes in normal colorectal tissue of colorectal cancer patients: In this study, three different layers of XME abundance (mRNA, protein, and enzyme activity) were evaluated in cytochromes P450 (CYPs), glutathione S-transferases (GSTs), and UDP-glucuronosyltransferases (UGTs). Gene expression was assessed by quantitative real-time PCR (qRT-PCR), protein expression evaluated by immunoassay detection, and enzymatic activities measured by biochemical assays, in the normal tissue of 97 patients with CRC. The mean relative expression levels in normal colorectal tissue were highest for GSTP1 [mean (± standard deviation): 7.70 (0.60)] and lowest for GSTM1 [mean: 4.08 (1.80)]. Associations of xenobiotic metabolism-related gene expression, protein level and enzyme activities with clinical parameters in patients with CRC, were evaluated by the Mann-Whitney U-test and the Kruskal-Wallis test. Results of the univariate analysis revealed a 1.2-fold lower UGT1A8 expression and 1.7-fold lower UGT activity in normal tissue of rectal compared to colon cancer patients (p=0.008; pFDR=0.34 and p=0.002; pFDR=0.17, respectively). Furthermore, lower GSTP1 expression levels among recent nonsteroidal anti-inflammatory drug (NSAIDs) users compared to non-users (p=0.04; pFDR=0.58) were detected. Associations with lifestyle and dietary factors were evaluated using linear regression models. Results of the multivariable models adjusted for relevant covariables, showed that regular consumption (>1x/week) of cooked vegetables was associated with higher CYP3A4 protein levels (β=5.62, pn=0.009; pFDR=0.65) and regular consumption of raw vegetables was associated with lower UGT activities (β=-0.56; pn=0.03; pFDR=0.72) compared to non-regular consumption of raw and cooked vegetables (≤ 1x/week) in the normal mucosa of CRC patients.
Relation of mRNA expression, protein levels and enzyme activities were assessed using Spearman correlation coefficients. No statistically significant associations were found between mRNA expression and neither protein levels, nor enzymatic activities for the CYPs and GSTs. For the UGTs, statistically significant, albeit weak, positive associations between UGT1A8 protein and mRNA abundance (r=0.20, p≤ 0.05) and UGT1A10 mRNA levels and UGT activity (r=0.28, p ≤0.01) were observed. However, between UGT1A10 protein level and UGT activity, a statistically significant negative correlation (r= -0.27, p ≤0.01) was detected.
2) Evaluation of differentially expressed XMEs in normal and tumor colorectal tissue: The expression of eight selected XMEs (GSTP1, GSTA1, GSTM1, UGT1A10, UGT1A8, CYP2W1, CYP2C9, and CYP3A4) in colorectal carcinomas and adjacent normal mucosa (n=71) was compared and associations of sociodemographic, lifestyle and dietary factors with the expression of these genes were investigated. Differences between XMEs’ gene expression (IlluminaHT-12 Expression BeadChips) in tumor and normal mucosa were tested by the paired Wilcoxon-Rank-sum test. Among the genes analyzed, GSTM1, GSTA1, UGT1A8, UGT1A10, CYP3A4, and CYP2C9 were down-regulated in tumor tissue as compared to normal tissue, while GSTP1 and CYP2W1 were up-regulated. Linear regression models were used to evaluate potential associations between sociodemographic, lifestyle and dietary factors and the relative gene expression in tumor and normal mucosa tissue. Although none of these relationships remained statistically significant after false discovery rate (FDR)-adjustment for multiple testing, a trend toward significance (β=-0.21; pn=0.0005; pFDR=0.05) with lower CYP2C9 expression in normal tissue of rectal compared to colon cancer patients was detected. CYP2C9 plays a key role in the metabolic activation of many environmental and dietary mutagens and interactions of its expression with these factors should be considered in larger studies.
Overall conclusions: In summary, it can be concluded that in normal tissue of CRC patients: - Correlations between XMEs’ mRNA, protein and enzyme activities are moderate to poor - Colon and rectum showed considerable differences regarding expression and activities of several XMEs - Regular consumption of cooked vegetables was associated with CYP3A4 gene expression and protein levels Taken together, the results of this thesis suggest some interesting differences, which should be considered in larger studies to elucidate its potential contribution to CRC etiology.
Although the fight against malaria has achieved a remarkable progress during the last 15 years, there were still 214 million new cases and 438000 estimated deaths caused by malaria worldwide in 2015. Transporters play a crucial role in Plasmodium biology but they can also be considered as double edge swords: on the one hand, they are potential new antimalarial drug targets but on the other hand, they are the main players in the development of drug resistance. PFE0825w is a putative organic cation transporter that has been proposed as the target of the candidate antimalarial drug albitiazolium and the locus where it is localized has been linked to chloroquine transport. In this study, different PFE0825w isoforms were identified and studied using the X. laevis oocyte system. Two of the isoforms were expressed at the oocyte oolemma but no significant transport of putative organic cation substrates was detected, restricting further characterization of this transporter. A better characterized transporter that plays a significant role in resistance against chloroquine (CQ) and quinine (QN) is the chloroquine resistance transporter PfCRT. It is known that this transporter has at least three different phosphorylation sites and that the phosphorylation of one of these sites -T416- is essential for the correct trafficking of PfCRT to the food vacuolar membrane. In this study the role of phosphorylation in the drug-resistance-mediating function of PfCRT was investigated. CQ-resistant parasites treated with the kinase inhibitor ML-7 accumulated more CQ than untreated parasites and showed CQ and QN IC50 values comparable to those of sensitive strains. Along the same line, the mutagenesis of the phosphorylation site S33 to alanine in PfCRT led to reduced CQ and QN IC50 values although no increase in drug accumulation was observed. Furthermore, PfCRTS33A conferred a fitness advantage to the parasites in the absence of CQ and a fitness cost in the presence of the drug. Two protein kinases were analyzed regarding their roles in PfCRT phosphorylation, PfCK2 and PF11_0488, the latter being identified in a Y2H assay. The downregulation of PfCK2 did not have an effect on CQ accumulation, but the overexpression of the C-terminal part of PF11_0488 resulted in reduced levels of CQ accumulation. However, the same fragment did not show any catalytic activity when recombinantly expressed and used in in vitro phosphorylation assays. Downregulation of this kinase was not achievable, most likely due to its essential function. Altogether, these results point to the fact that the parasite susceptibility towards CQ and QN is regulated by phosphorylation, although the exact molecular mechanism needs to be further examined.
Photosynthesis is a very important process in plants which occurs in chloro- plasts. Plants use photon energy to oxidize water molecule, release oxygen, and convert carbon dioxide to sugar molecule. The process of photosynthe- sis contains two main parts: light dependent reactions and light independent reactions.
A mathematical model, which describes the diffusion-transport and related chemical reactions in a multi-component flow in a single C3 plant leaf cell, is constructed. A sub-domain of a leaf cell is considered containing multiple organelles: chloroplast, mitochondria, vacuole, cytoplasm, and peroxisome. A typical distribution of a finite number of these organelles inside a cell is considered. The cell domain is decomposed in 5 sub-domains, separated by fixed interfaces. The interacting chemical reactions induced by light, of the Calvin cycle, the starch synthesis, sugar synthesis, respiration and photores- piration are investigated. A sensitivity analysis was performed, the results allows a reduction of the complex system. A system of partial differential equations, which describes the diffusion-transport and also related chemical reactions is formulated and simulated using the software Gascoigne. For the reduced system, the resulting flow of substances is analyzed.
Geoengineering oder Climate Engineering bezeichnet technologische Konzepte zur Manipulation des Klimasystems durch Eingriffe in den globalen Kohlenstoffkreislauf oder die Reduktion der einfallenden Sonnenstrahlung. Das Heidelberg Geoengineering Forum (HGF) verbindet Wissenschaftler aus den Regionen Rhein-Neckar und Karlsruhe, die zu Potential, Grenzen und Risiken des Climate Engineering forschen.
Projekte im Rahmen des Wassernetzwerks Baden-Württemberg Zwei Forschungsverbünde zum Thema Wasser, an denen Wissenschaftler des Heidelberg Center for the Environment (HCE) der Ruperto Carola beteiligt sind, erhalten eine Förderung des Landes Baden-Württemberg. Die beiden inter- und transdisziplinären Konsortien gehören zu drei Forschernetzwerken im Rahmen des Programms Wasserforschung Baden-Württemberg. Forschernetzwerk "Eff-Net“ ("Effect Network in Water Research": im Projekt "Eff-Net" werden Wirkungszusammenhänge für die Risikobewertung von Chemikalien in Gewässerökosystemen untersucht.
Unter dem Thema "Umwelt und Gesundheit in ariden Regionen: Neue Herausforderungen im Kontext von Urbanisierung" befasst sich die interdisziplinäre Nachwuchsgruppe mit den Dynamiken von gekoppelten sozial-ökologischen Systemen. Das Projekt wird in Kooperation zwischen dem Südasien-Institut, dem Geographischen Institut und dem Institut für Public Health innerhalb des HCE umgesetzt.
Das interdisziplinäre Projekt "Land unter?" soll für den Oberrhein zwischen Straßburg/Kehl und Mannheim/Ludwigshafen eine regionale Geschichte der Hochwasser und Hochwasserschutzmaßnahmen auf Grundlage archäologischer und geomorphologischer Untersuchungen erarbeiten.
Die Forschernachwuchsgruppe untersucht die Verflechtung und das Ineinandergreifen von Umwelt und Gesellschaft. Im Zentrum stehen dabei die frühneuzeitlichen Nahrungskrisen. Die Gruppe ist interdisziplinär ausgerichtet und verknüpft Ansätze der Umweltgeschichte, der Paläoklimatologie, der Sozialen Ökologie und der postcolonial studies. Nachwuchsgruppe: Umwelt und Gesellschaft. Handeln in Hungerkrisen der Frühen Neuzeit
Das Heidelberg Center for the Environment wirkt als Netzwerk der Umweltwissenschaften an der Universität Heidelberg. Im Film wird die Struktur des HCE vorgestellt und ein Einblick in aktuelle Aktivitäten gegeben. Institut/Arbeitsgruppe: Heidelberg Center for the Environment
Axons of the adult mammalian central nervous system (CNS) are unable to regenerate following axonal injury. Thus, spinal cord injury (SCI) leads to devastating and permanent functional impairments, the extent of which depends on the position of the lesion. At present, our understanding of the response to axonal injury and what underlies the failure of CNS axons to regenerate is far from complete. Therefore, despite various strategies proposed and tried, a robust method to improve axon regeneration after SCI is yet to be found. Due to growing evidence highlighting the role of post-transcriptional control towards protein expression and the importance of localised protein synthesis in axon physiology, we decided to investigate the post-transcriptional regulation mechanisms that could govern the regeneration of CNS neurons.
At a very early time window following SCI, axon regrowth still occurs, but is however limited in duration and extent. This however offers a rare opportunity of learning how nature initiates a regenerative response in the CNS. In order to study the difference between the total level of RNA and the subset that is actually translated, we profiled and compared total and polysome-bound RNAs from spinal cords early after injury and naïve ones, and revealed substantial uncoupling between mRNA abundance and ribosome loading. mRNAs of genes related to nervous system development were highly reduced following injury, while still being stably loaded onto ribosomes. By analysing motifs recognised by RNA-binding proteins, it was discovered that mRNAs harbouring the cytoplasmic polyadenylation element (CPE) exhibit increased transcript abundance upon SCI relative to those that do not, and were highly enriched in nervous system development genes in both mouse and Drosophila genomes. By manipulating the expression of Cpeb1, the binding protein of CPE, we found that Cpeb1 is a positive regulator of regeneration in both mouse and Drosophila CNS neurons. In an attempt to identify the targets of Cpeb1 mediated injury response, we analysed the transcriptome of naïve and injured processes from wild-type and Cpeb1 knockout cortical neurons with RNAseq. It was found that Cpeb1 knockout processes have a much attenuated transcriptional activation-response towards injury. In particular, the Jun and Fos family of transcription factors, which are highly up-regulated upon injury in wild-type processes, failed to do so in their Cpeb1 knockout counterparts. In addition, Cpeb1 knockout was found to have an effect on alternative polyadenylation. However, the precise molecular mechanisms underlying observed changes remain a subject of future studies.
In conclusion, this study demonstrates widespread uncoupling between mRNA abundance and ribosome-loading in the injury response of CNS neurons, and identifies Cpeb1 as a conserved positive regulator of regeneration, as well as a mediator of this uncoupling effect.
Cerebral malaria (CM) is one of the most severe manifestations of Plasmodium falciparum, characterised by seizures, coma and death within a short time period. The aetiology of the disease remains poorly understood and is limited by ethical constraints in dealing with human patients. A large body of research dedicated to CM has therefore focussed on delineating the mechanisms involved using the rodent model of malaria. Infection of mice with sporozoites or infected red blood cells (iRBCs) of PbANKA parasites recapitulates several features of CM including haemorrhaging, oedema and blood-brain barrier breakdown and is termed experimental CM (ECM).
The development of ECM relies on a complex series of interactions between the parasite and host. Although regarded as an immune-mediated pathology, sequestration of iRBCs is considered a central event for ECM to ensue, thus supporting the notion that ECM is purely the outcome of host-parasite interactions at the pathological blood stage. However, previous studies have shown that both surface antigens of iRBCs and the host’s immune response differ between naturally transmitted (sporozoites) and blood-passaged parasites. This thesis is therefore aimed at describing the role of a novel Plasmodium antigen in the development of ECM and outlining differences in ECM progression between naturally transmitted and blood-passaged parasites.
The antigen, PbmaLS_05 is expressed in both liver-stage- and blood-stage schizonts and localises to the apicoplast of individual merozoites. Deletion of the endogenous PbmaLS_05 had no effect on parasite viability, but abrogated the development of ECM in mice, after both sporozoite and iRBC infections. PbmaLS_05 (-) parasites displayed retarded growth rates in the blood and enhanced clearance by the spleen, both of which were more pronounced on the days when PbANKA wild type infected mice showed signs of ECM. The absence of ECM in PbmaLS_05 (-) infected mice was accompanied by reduced infiltration of activated CD8+ T cells within the brain and reduced cross-presentation of a known parasite antigen (GAP50) by the brain endothelium, after iRBC but not sporozoite infection. Further investigations into sporozoite infections revealed an important role for PbmaLS_05 in the priming of CD8+ T cells responsible for causing ECM. These data thus highlight the existence of multiple mechanisms leading up to the development of ECM relevant to sporozoite or iRBC infections, with potential implications for vaccines or therapeutics designed to alleviate CM.
Die Mikrostrukturierung auf biokompatiblen Oberflächen ist eine wichtige und hilfreiche Methode, um die Funktion bei Anordnungen kleiner Präparate zu erforschen und verstehen zu lernen. In der vorliegenden Bachelorarbeit war es mit Hilfe dieses Werkzeugs möglich das Wachstum von Zellen zu beeinflussen und damit kontrollierbar zu machen. Dabei wird der Aufbau eines Biochips beschrieben, mit dem es nach erfolgreicher Zellanordnung in einem Linienmuster möglich ist, das Verhalten der sich darauf befindlichen Muskelzellen zu untersuchen. Beim Anlegen einer definierten elektrischen Spannung über Elektroden wird das Ruhemembranpotential der zu Tuben differenzierten Zellen verändert und es erfolgt eine Umwandlung dieses Signals in eine mechanische Reaktion. Die Zelle kontrahiert. Auf diese Weise ist es mit dem Biochip bzw. Elektrodenarray möglich, Myotuben zu analysieren, wenn sie mit einem Spannungssprung beaufschlagt werden oder mit chemischen Reizen in Berührung kommen. Es ist also realisierbar, die Funktion und eventuelle Funktionsstörungen von Zellen anhand ihrer Signalweiterleitung zu untersuchen. Bei der Konstruktion des Elektrodenarrays wurden die bestmöglichen Konturen ermittelt und es kamen unterschiedliche Verfahren der Photolitographie zum Einsatz. Unter Anwendung verschiedener chemischer Stoffe wurde die optimale Strukturierung ermittelt, womit der Zellenträger reproduzierbar hergestellt werden konnte.
Parallel information processing in distinct channels is a common functional principle of nervous systems to facilitate rapid and precise extraction of specific features. A hallmark of such parallel processing is that the originally acquired information is initially segregated into individual processing channels that are tuned to extract distinct features of the input before re-converging them to guide appropriate responses. Parallel processing also applies to aversive olfactory memories in Drosophila where the metabolically costly and more enduring memory channel is sensitive to cold anesthesia (ASM) whereas the parallel anesthesia resistant memory channel (ARM) is only transient. The molecular basis and functional significance of this segregation of aversive olfactory memories in parallel channels is currently unclear. Here, we show that an aversive unconditioned stimulus (US) used in classical olfactory conditioning experiments is responsible for synaptic activity-driven neuronal nuclear calcium transients in distinct areas of the fly brain. These areas include the fly's association center, the mushroom bodies (MBs), as well as the fly's master regulator of its neuropeptidergic system, the pars intercerebralis (PI). Blockade of nuclear calcium signaling allowed us to functionally and morphologically separate the role of cAMP, a classical signaling pathway in learning and memory, and nuclear calcium signaling in the establishment of consolidated long-term memories (LTM) (Weislogel et al., 2013). In addition, we show that the US activates the fly’s widespread neuropeptidergic system and, in particular, the PI which results in multiple local signaling events or even systemic responses. Furthermore, we show that the acquisition and formation of all ASM phases requires additional release of mature neuropeptides from a single pair of dorsal paired medial (DPM) neurons. DPM neurons form a recurrent network with mushroom body neurons that has been shown to be involved in the formation of serotonin-dependent ARM, consolidation of memory and linking these consolidation processes to sleep. Our results reveal that DPMs define a qualitatively distinct parallel memory channel that strictly depends on mature neuropeptides and that is, within the first hours after training, behaviorally additive to the neuropeptide-independent ARM channel. Afterwards, in its subsequent consolidated phase, the ASM channel becomes exclusive towards the ARM channel. Thus, we propose that DPM neurons are capable of gating the simultaneous formation of two parallel memory channels by means of using two distinct signaling systems. Finally, given that neuropeptide signaling appears to be more widely involved in the processing of the US, it could represent a general mean of defining parallel processing channels.
Unlike many other species, where the body plan is already pre-patterned in the oocyte or upon fertilization, in the early mouse embryo there is no asymmetry up to 8-cell stage when all cells in the embryo have the same morphology and developmental potential. As development proceeds initially identical cells of the embryo segregate into two distinct cell lineages: trophectoderm (TE) and the inner cell mass (ICM) (Wennekamp et al., 2013; Rossant and Tam, 2009; Yamanaka et al., 2006). While both apical-basal cell polarity (Hirate et al., 2013; Alarcon, 2010) and cell-cell adhesion (Stephenson, Yamanaka and Rossant, 2010) are required for this differentiation, the decisive cue that breaks symmetry between the cells and is sufficient for specifying the first cell fate remains to be identified (Wennekamp et al., 2013). To understand the mechanism underlying the symmetry breaking in the mouse embryo, in this study I have established a new experimental system in which a blastomere isolated at the 8-cell stage (1/8th blastomere) recapitulates the first lineage segregation between TE and ICM during its development into 4/32th mini-blastocyst. Using live-imaging and quantitative image analysis, I identified that inheritance of the apical domain during 1/8th-to-2/16th-cell stage division allows for predicting the process leading to TE fate specification. The majority of 8-cell blastomeres undergo asymmetric division defined by the differential segregation of the apical domain among daughter cells. In the 8-cell stage embryo, the apical domain, emerging at the center of the contact-free surface of the blastomere, recruits microtubule organizing centers to the sub-apical region, thereby forming one of the acentrosomal spindle poles and inducing the asymmetric division. After asymmetric 8-to-16-cell stage division, all cells that inherit the apical domain express a TE marker, Cdx2. In contrast, apolar cells can either acquire ICM fate, as previously described, or, if positioned on the embryo surface, form a new apical domain and turn on Cdx2. Thus, contrary to the previous model (Johnson and Ziomek, 1981b), cell fate is determined by its position within the embryo, but not by the division pattern. Finally, using 1/8th blastomere, I showed that cell contact, not mediated by Cdh1, facilitates cellular symmetry breaking and directs the apical domain formation in the center of the contact-free surface, and that the inheritance of this apical domain predicts the acquisition of TE fate.
Programmed cellular execution is one of the hallmarks of cellular homeostasis as well as the development of multicellular organisms. To date, two major signaling pathways for programmed cell death have been elucidated, namely apoptosis and necroptosis. The induction of these two regulated cell death programs can be initiated by a variety of extrinsic as well as intrinsic stimuli, like genotoxic stress, death receptor activation, pathogen infection or the depletion cIAPs. Apoptosis thereby relies on the activation of caspase-8, which induces a downstream caspase cascade, finally leading to apoptotic execution. In contrast, caspase-independent necroptosis relies on the activation of the kinases RIP1 and RIP3, resulting in the phosphorylation of the pseudokinase MLKL. This finally leads to necroptotic plasma membrane permeabilization. Additionally, recent studies highlighted that certain cellular conditions can induce the formation of an intracellular 2 MDa multi-protein complex, called Ripoptosome, which can lead to apoptosis as well as necroptosis based on the stoichiometric composition. This protein complex consists of FADD, caspase-8, cFLIP and the name giving RIP1. However, it is still unclear, if all components or interacting proteins have been identified. In this study purified Ripoptosome complexes were analyzed for the identification of novel components by mass spectrometry. Interestingly, the non-canonical IKKs; TBK1 and IKK-ε as well as TRAF2 could be identified as integral elements of the complex. The association of these molecules with the Ripoptosome was observed upon TNF treatment or TLR3 stimulation or the overexpression of RIP1 as the initial stimulus. Moreover, the non-canonical IKKs were highly phosphorylated in the complex, arguing for substantial activation. The cell death regulating function of the Ripoptosome was not affected by the loss of TBK1 and IKK-ε, indicating that these molecules could play a pivotal role in non-cell death signaling pathways. In contrast, decrease of TRAF2 resulted in a sensitization against apoptotic cells death. This was true for the expression of RIP1 as well as the combined treatment of poly (I:C) or TNF with IAP antagonists. Additionally, RIP1-induced Ripoptosome formation could be linked to the activation of NF-κB and the induction of inflammatory cytokines. The reduction in TBK1 and IKK-ε protein levels was leading to a decrease in the RIP1-regulated gene induction. Additionally, the formation of the Ripoptosome was inducing the phosphorylation of autophagy receptors optineurin and sequestosome-1. This activation was highly depending on the kinases TBK1 and IKK-ε, indicating a possible Ripoptosome-dependent activation of the autophagy machinery. The novel identified components highlighted the Ripoptosome as a key complex not only for cell death regulation, but also for cell death-independent signaling pathways such as inflammatory responses and autophagy.
The organisation of chromatin is non-random and shows a broad diversity across cell types, developmental stages, and cell cycle stages. During G0 and G1 phase of interphase, chromatin displays a bivalent status. The condensed chromatin (heterochromatin) at the nuclear periphery is mostly associated with low levels of gene expression, while the loosened chromatin (euchromatin) towards the interior of the nucleus is associated with higher gene expression. This quiescent picture of interphase radically changes when the cell cycle progresses toward cell division. Firstly, during S phase, DNA is replicated, and chromatin progressively condenses. This is followed by the G2 phase that shows a compact heterochromatin recruited towards the centre of the nucleus. At the beginning of mitosis, the chromosomes condense with a significant topological change in their organisation and are segregated during the next stages of the cell division. Meiotic chromosomes are also highly condensed as mitotic chromosomes but show a particular functional structure, which prepares germ cells to exchange DNA sequences between their homologous chromosomes to generate diversity. To summarise, chromatin experiences dramatic organisational changes during mitosis and meiosis. These changes in chromatin organisation during the lifetime of a cell show that chromatin is not a static entity but highly dynamic in nature.
For a variety of reasons, conventional light and electron microscopy have not been able to fully capture the finer details of chromatin organisation and dynamics. For a long time, description of the interphase nucleus was limited to delineate the euchromatin-heterochromatin dichotomy or describe some specific nuclear elements such as the nucleolus. Advancements in molecular biology during the last thirty years have brought an immense amount of information about how chromatin is organised and genes are regulated. As a classical example, the globin gene has been shown to display a highly constrained shape forced by chromatin looping that brings the regulatory regions to the promoter of the gene. Nowadays, genomic studies can acquire an immense amount of information regarding chromatin organisation and gene regulation, leaving one with the expectation that structure of individual genes could potentially be described visually if sufficient specificity and resolution were reached. With the advent of various super-resolution methods, in particular, single molecule localisation microscopy (SMLM) based methods and recently developed strategies for labelling DNA, it is now possible to study chromatin organisation and underlying gene regulatory mechanisms at the nanoscale.
During my PhD, I have analysed a broad range of nuclear phenotypes using SMLM. My analyses contribute to the description of a periodic and dynamic structure of chromatin. Moreover, I have described several elementary chromatin structures that I call chromatin domains, both in interphase and meiosis, that are potentially associated with a local function such as gene activation or silencing.
Firstly with colleagues, I established an experimental setup to study chromatin organisation with single molecule localisation microscopy. I investigated how UV-induced photo-conversion of conventional DNA dyes allows increasing sufficiently the labelling density such that it is possible to study various organisational aspects of chromatin in basal interphase. An adequate imaging protocol has been established to bring DNA minor groove binding dyes such as Hoechst 33258, Hoechst 33342 and DAPI (4’,6-diamidino-2-phenylindole) into an efficient blinking state necessary to record single molecule locations with high precision. This method was applied to several cell types to investigate the chromatin organisation during different stages of the cell cycle at the highest resolution currently achievable with light microscopy.
The results show that the method can capture several hierarchical levels of chromatin organisation. In reverse hierarchical order, I could describe previously known chromatin territories of 1000 nm, subchromosomal domains of 500 nm, chromatin domains of 100 to 400 nm (and further sub-categories of active or repressed domains) and chromatin fibres below 100 nm, mostly between 30 to 60 nm. Individual nucleosomal domains are also described, which tend to cluster in batches of 10-15 nucleosomes, a number close to one found in genomic studies upstream to promoter regions. Next, with colleagues, I studied the dynamics of chromatin using stress as a model system. It was found that short-term oxygen and nutrient deprivation provokes chromatin to shrink to a hollow, condensed ring and rod-like configuration, which reverses back to the initial structure when the stress conditions cease. The condensed network of rods and rings interspersed with large, chromatin-sparse nuclear voids were 40-700 nm in dimension, capturing another level of chromatin organisation not described before.
Finally, I explored the unique properties of chromatin during meiosis, which has escaped analysis at the single-molecule level until now. Single molecule analysis revealed unexpected highly recognisable periodic patterns of chromatin. Firstly, I observed that meiotic chromatin show unique clusters of 250 nm diameter along the synaptonemal complex, extended laterally by chromatin fibres forming loops. These clusters show a remarkable periodicity of 500 nm, a pattern possible to spot because of the highly deterministic nature of pachytene chromosomes and the resolution of the experimental setup. Furthermore, guided by genomic data, I selected histone modifications associated with different chromatin states to dissect the morphology of meiotic chromosomes. I could examine the morphology of these chromosomes into three spatially distinct nanoscale sub-compartments. Histone mark H3K4me3 associated with active chromatin was found in a lateral position, potentially located at the places of \textit{de novo} double-strand breaks. Repressive histone mark H3K27me3 was shown to display a surprising medial symmetrical and periodic pattern, putatively associated with recombination. Finally, centromeric histone mark H3K9me3 locates at one of meiotic chromosome ends and is potentially associated with repression of repeated regions and pairing of homologous chromosomes at early stages. I summarise these findings in a comprehensive final model.
Overall, I have used new information brought by super-resolution technologies to show the dynamics of chromatin in various processes and novel orders of chromatin compaction, which were not reported previously. Among these new levels of chromatin compaction are the interphase hierarchical chromatin domains, the stress pattern of cells upon oxygen and nutrients deprivation and the novel epigenetic domains found at pachytene stage of meiosis. These architectures show that the organisation of chromatin is more complex than thought before, dynamic in nature and shows a high order of periodicity. Further investigation is, therefore, necessary to understand how chromatin transits from a ’beads-on-string’ model to the intermediary chromatin domains and finally to the commonly observed X-shaped chromosomes.
The efficient and reliable estimation of model parameters is important for the simulation and optimization of physical processes. Most models contain variables that have to be adjusted, e.g. in the form of material properties, and the uncertainty of state estimates and predictions is directly linked to the uncertainty of these parameters. Therefore, efficient methods for parameter estimation and uncertainty quantification are required. If the physical system is spatially highly heterogeneous, then the number of model parameters can be very large. At the same time, imaging techniques and time series can provide a large number of measurements for model calibration. Many of the available methods become inefficient or outright unfeasible if both the number of model parameters and the number of state observations are large.
This thesis is concerned with the development of methods that remain efficient when a large number of measurements is used to estimate an even larger number of model parameters. The main result is a special preconditioned Conjugate Gradients method that can achieve both quasilinear complexity in the number of parameters and pseudo-constant complexity in the number of measurements. The thesis also provides randomized methods that allow linearized uncertainty quantification for large systems, taking redundancy in the measurements into account if applicable.
Oncogenic activation of MYC drives cell proliferation in Burkitt lymphoma (BL), but also evokes stress signals that have to be counterbalanced to escape apoptosis. Pro-survival signals from tonic B cell receptor and PI3K signaling are essential, but additional mutations are required for malignant transformation. TP53 mutations were identified in 43% of BL patients. Mutations in the conserved MYC box I that abrogate activation of pro-apoptotic Bcl2-family member Bim were present in 17% of BL patients. Notably, MYC box I and TP53 mutations occurred independently and accounted for 54% of cases, suggesting that alternative failsafe mechanisms may be inactivated in BL. To study alternative mechanisms of transformation in the absence of TP53 mutations, we analyzed the pattern of recurrent genetic aberrations in BL for associations with TP53 status. We observed an overrepresentation of chromosome 1q gains in TP53 wild-type BL patients, which was not observed in diffuse large B cell lymphoma (DLBCL) patients. Minimally gained regions comprised 1q21-q23 and 1q32 and amplified regions displayed a gene dosage effect as shown by gene expression analysis. To identify genes essential for p53 wild-type BL cells we performed a RNAi loss-of-function screen in a panel of genetically defined cell lines. We used a pooled shRNA library targeting 5,000 genes in key signaling pathways across the genome. Our data was probed against published RNAi screens across cancer entities and showed a high overlap of common essential and non-essential genes. p53 wild-type BL cell lines showed a strong and specific dependence on the p53 inhibitor MDM4. Depletion of MDM4 resulted in an upregulation of p53 target genes and induced cell cycle arrest. In a mouse xenograft model, MDM4 knock-down significantly reduced tumor growth. These effects were p53 dependent as confirmed in an isogenic p53 knock-out cell line. MDM4 is located within the minimally gained region 1q32 associated with p53 wild-type BL patients and might therefore contribute to BL pathogenesis by inactivation of the p53 pathway. Our data suggest that reactivation of p53 in patients lacking TP53 mutation, e.g. by specific MDM4 inhibition, is a promising therapeutic approach. Re-analysis of published RNAi screening data revealed p53-specific sensitivity of MDM4 knock-down across cancer cell lines, suggesting a broader application for MDM4 inhibitors. Our data set on essential genes in Burkitt lymphoma proved to be a valuable resource for identification of genotype-specific vulnerabilities. This analysis could be extended by integration of published RNAi screening data in non-lymphoid cell lines to identify interesting potential entity-specific vulnerabilities.
Blood flow-generated shear stress (FSS) is the major determinant of endothelial nitric oxide synthase (NOS-3) expression. In humans, a promoter variant of the NOS3 gene, the C-variant of the T-786C single nucleotide polymorphism, renders the gene insensitive to shear stress, resulting in a reduced endothelial cell (EC) capacity to generate nitric oxide (NO). Endothelial dysfunction, commonly associated with decreased NO bioavailability, may facilitate vascular inflammation. Consequently, individuals homozygous for the C-variant have an increased risk of developing cardiovascular (e.g., coronary heart disease (CHD)) and rheumatic diseases (e.g., rheumatoid arthritis (RA)).
However, there are at least two mechanisms by which insuffcient NO production can be counterbalanced in CC-genotype endothelial cells (ECs), one of which involves a multicomponent pathway leading to the increased release of the anti-inflammatory prostanoid 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2). Exposure of human ECs to physiological levels of FSS effectively reduced monocyte migration, not only through monolayers of TT- but most notably also of NO-deficient CC-genotype ECs. FSS up-regulated the expression of COX-2 and L-PGDS, the rate-limiting enzymes for 15d-PGJ2 synthesis, solely in CC-genotype ECs, and only these cells revealed an increased release of 15d-PGJ2 in response to FSS. Exogenously added 15d-PGJ2 significantly reduced the transmigration of monocytes through EC monolayers. In addition, pre-treatment with 15d-PGJ2 or exposure to FSS-pretreated CC-genotype ECs exerted a pronounced anti-inflammatory effect on the (transmigrated) monocytes, as demonstrated e.g. by an inhibitory effect on interleukin-1 beta (IL-1 beta) expression, a marker for monocyte pro-inflammatory activation. This inhibition occurs at the transcriptional level, as 15d-PGJ2 repressed tumor necrosis factor-alpha-induced IL-1 beta promoter activity in transiently transfected HEK293 cells.
The anti-inflammatory activity of 15d-PGJ2 in monocytes involves the Nrf2-antioxidant response element (ARE) pathway. Similar to 15d-PGJ2, constitutive activation of Nrf2 reduced the expression of IL-1 beta. Bioinformatic analysis revealed three putative Nrf2-responsive elements (i.e., AREs) in the human IL-1B promoter, suggesting that Nrf2 may act through an as yet unknown mechanism to repress transcription of the IL-1B gene. Chromatin immunoprecipitation showed a 15d-PGJ2-induced binding of Nrf2 to the promoter of the IL-1B gene. Deletion of two of the identified ARE motifs attenuated the inhibitory potency of 15d-PGJ2 toward IL-1B promoter activity, thereby corroborating Nrf2 as a downstream effector of this prostanoid’s transcriptional effects.
Given its powerful immunomodulatory properties, 15d-PGJ2 has been proposed to have anti-atherogenic potential. To evaluate its prognostic relevance, the relationship between plasma 15d-PGJ2 levels and disease severity and outcome in patients suffering from CHD, RA or both was investigated. The levels of 15d-PGJ2 were found to be significantly increased in the CHD group compared to age-matched controls, suggesting that 15d-PGJ2 may constitute a general defense mechanism to counteract the ongoing chronic inflammatory process in affected individuals. Moreover, pre-treatment with 15d-PGJ2 potently inhibited the in vitro transendothelial migration of interferon-gamma-producing human T helper type 1 cells, major players in atherosclerosis as well as various other chronic inflammatory disorders.
Despite an inadequate capacity to form NO, CC-genotype ECs maintain a robust anti-inflammatory phenotype by enhancing the shear stress-dependent synthesis of 15d-PGJ2. Its anti-inflammatory activity on human monocytes may ascribe a novel role to Nrf2 as a direct repressor of pro-inflammatory gene expression.
Only with the evolution from circular to linear genomes, allowing easy exchange of genetic information by sexual reproduction, complex organisms with large genomes could evolve. But the linearity of a chromosome inherits a major problem, namely two chromosome ends that have to be protected. Telomeres at the very end of the chromosomes ensure cell survival. These nonprotein coding DNA repeats are essential features of chromosomes, as their loss leads to irreversible cellular senescence and chromosome loss. Paradoxically, telomeres resemble DNA double-strand breaks (DSBs), however, unlike DSBs, they are refractory to repair events. This socalled “end protection” function carried out by telomeres ensures that chromosomes do not fuse together in an end-to-end manner and avoids the DNA damage response machinery from being activated, leading to cell cycle arrest. Although end protection has largely been attributed to the major telomere binding complexes such as shelterin in mammals and the CST complex in yeast, it has also been proposed that a three-dimensional structure at the telomere may contribute to safeguarding telomeres. These so called telomere loops (t-loops) have been demonstrated via electron- and super-resolution-microscopy in mammalian cells, however, the short length and base composition of yeast telomeres prevent such approaches. By using a combination of Chromatin Immunoprecipitation (ChIP) and transcriptional readouts it has been demonstrated that yeast telomeres loop back onto their respective subtelomeres; however, both methods are indirect and unsatisfactory in terms of analyzing the dynamic regulation of loop structures. In this study we have established a new assay based on Chromosome Conformation Capturing (3C) to directly detect and quantify interactions between a telomere and its subtelomeric region in S. cerevisiae, as a measure of telomere looping. In this manner we could exploit the genetic advantages of the yeast system to understand the mechanistic details of telomere loop formation and maintenance. Since telomere shortening leads to an unprotected telomere, we wondered whether telomere length may have an impact on looping. We were able to show a significant looping defect in cells that lack telomerase as well as in other mutants that harbor short telomeres. On the contrary, elongated telomeres were able to maintain the looped structure. This suggests that a critical telomere length is essential to maintain the telomere loop and that telomeres in senescent cells are likely in an open conformation, rendering them susceptible to nucleolytic end resection and unscheduled DNA repair events. Gene looping is another kind of looped chromatin that brings promoter and terminator together. Gene loops depend on transcription, a functional transcription initiation complex and several components of the mRNA processing machinery. Indeed, we could detect a telomere looping defect upon loss of RNA polymerase II and in a mutant of the transcription preinitiation complex (sua7-1). It has been shown that certain chromatin loops that bring promoter and enhancer regions in close proximity depend on a non-coding RNA species that interacts with components of the promoter associated mediator complex. Depletion of total RNA levels or mutation of mediator resulted in a telomere looping defect, indicating a similar regulation at the telomere.
Cancer is a complex genomic disease characterized by accumulation of somatic mutations over the lifetime of a patient. Identification of somatic driver mutations that contribute to tumorigenesis is a major goal of cancer genomics. With the recent advances in the sequencing technologies it became possible to study somatic mutations on the whole-genome scale in multiple cancers. While most of the cancer genomics studies were previously focused on identification of driver mutations affecting exons, several examples of driver events within the non-protein-coding regions of the genome were identified, including the recurrent TERT promoter mutations. Such findings have spurred searches for similar examples of recurrent non-coding mutations using computational cancer genomics. In my PhD thesis, I present several computational approaches aimedto identify somatic driver mutations with a specific focus on intergenic regions of the genome. The first part of this thesis focuses on the somatic mutational patterns along the cancer genome and addresses a fundamental problem of computational identification of recurrently mutated regions – regional mutational heterogeneity. Here I studied the correlation of specific genomic features with background somatic mutation rates and devised a background model that accounts for regional mutational heterogeneity. The second part of this thesis describes three different computational approaches designed to identify somatic driver events of functional relevance in cancer. The first approach integrates somatic mutation calls with gene expression data to identify variants associated with altered mRNA levels. The second approach is designed to predict changes in transcription factor binding sites in presence of recurrent somatic mutations. The third approach uses cross-validation scheme to enable parameter tuning in screens for recurrently somatically mutated regions in cancer genomes in an unbiased genome-wide manner. Using this approach, we identify several known cancer-relevant targets, both exonic (e.g., the TP53, MYC, and SMARCA4 genes) as well as non-coding regulatory regions (e.g., the TERT promoter) and uncover novel candidate regulatory driver regions. Among those, a cluster of recurrent intergenic mutations, occurring in an enhancer element near the FADS2 gene, which encodes a critical enzyme in the biosynthesis of long chain polyunsaturated fatty acids and has been previously implicated in cancer. Collectively, the computational approaches presented here helped in uncovering novel somatic candidate events of relevance in cancer and can be further used for various applications in cancer genomics.
Therapy failure with subsequent tumor progress is a common problem in radiotherapy of high grade glioma. Definition of treatment volumes with CT and MRI is limited due to uncertainties concerning tumor outlines. The goal of the presented work was to enable assessment of tumor physiology and prediction of progression patterns using multi-modal image analysis and thus, improve target delineation. Physiological imaging modalities, such as 18F-FET PET, diffusion and perfusion MRI were used to predict recurrence patterns. The Medical Imaging Interaction ToolKit together with own software implementation enabled side-by-side evaluation of all image modalities. These included tools for PET analysis and a module for voxel wise fitting of dynamic data with pharmacokinetic models. Robustness and accuracy of parameter estimates were studied on synthetic perfusion data. Parameter feasibility for progression prediction was investigated on DCE MRI and 18F-FET PET data. Using the developed software tools, a pipeline for prediction of tumor progression patterns based on multi-modal image classification with a random forest machine learning algorithm was established. Exemplary prediction analysis was applied on a small patient set for illustration of workflow functionality and classification results.
In dieser Arbeit wurde ein Verfahren zur Darstellung der quantitativen Wasserkonzentration im Wadenmuskel bei 3 Tesla entwickelt. Außerdem wurden quantitative Messungen der Natriumkonzentration bei 7 Tesla durchgeführt, um mit diesen beiden Konzentrationsgrößen das zu untersuchende Gewebe beschreiben zu können. Zur Quantifizierung des Wassergehaltes wurden die Methoden mit einem Phantom mit schwerem Wasser getestet und Korrekturmessungen zu den Relaxationszeiten T1 und T2*, dem Sende- und Empfangsfeld B1+ und B1- der Ganzkörperspule und dem Sensitivitätsprofil der lokalen Empfangselemente durchgeführt. Um quantitative Messungen zu ermöglichen wurde ein Referenzröhrchen bei der Messung der Wade hinzugefügt. Bei der Gehirnmessung erfolgte eine Normierung auf den Wert der Zerebrospinalflüssigkeit. Im Gehirn wurde bei der B1- -Korrektur die Methode mit dem Reziprozitätsprinzip, die Linearer-Fit-Methode und die Minimaler-Kontrast-Methode verglichen. Bei den Messungen der Unterschenkelmuskulatur konnten die üblichen B1- -Korrekturverfahren nicht angewendet werden. Es stellte sich heraus, dass das Reziprozitätsprinzip bei den Wadenmessungen nicht gilt und B1- sich stark von B1+ unterscheidet. Deshalb wurde eine veränderte Form der Minimaler-Kontrast-Methode entwickelt. Für das Verhältnis aus Natrium- und Wasserkonzentration im Gehirn ergaben sich für graue Substanz 0,42 ± 0,11 ‰, weiße Substanz 0,44 ± 0,10 ‰ und Zerebrospinalflüssigkeit 0,83 ± 0,13 ‰. Für den Wadenmuskel wurde eine Wasserkonzentration von 92 ± 10 mol/l bestimmt und das Verhältnis 0,21 ± 0,04 ‰.
Quantum many-body systems far from equilibrium can approach a nonthermal fixed point during their real-time evolution. One example is scalar field theory, which occurs in models of cosmological inflation, and similar examples are found for non-Abelian plasmas relevant for heavy-ion collisions and for ultracold Bose gases. Investigating nonthermal fixed points of different microscopic theories, we present two novel universality classes that provide links between these systems.
One of them involves nonrelativistic, N-component relativistic and expanding scalar systems. It occurs in the deep infrared regime of very high occupancies and is governed by a self-similar evolution. Its nonequilibrium dynamics leads to the formation of a Bose-Einstein condensate. The scaling properties of this region can be described by a vertex-resummed kinetic theory that is based on a systematic large-N expansion at next-to-leading order.
The other novel universality class encompasses scalar field theories and non-Abelian plasmas in a longitudinally expanding background and corresponds to an early dynamical stage of heavy-ion collisions in the high-energy limit. We show that these systems share the same self-similar scaling properties for a wide range of momenta in a limit where particles are weakly coupled but their occupancy is high.
Both universality classes are found in separate momentum regions in a longitudinally expanding N-component scalar field theory. We argue that the important role of the infrared dynamics ensures that key features of our results for scalar and gauge theories cannot be reproduced consistently in conventional kinetic theory frameworks. Moreover, the observed universality connects different physics disciplines from heavy-ion collisions to ultracold atoms, making a remarkable link between the world's hottest and coldest matter.
This work addresses homogeneous and heterogeneous nucleation phenomena in atmospheric aerosol particles. First, the heterogeneous freezing of supercooled droplets containing feldspar particles has been investigated in a novel droplet freezing assay instrument. Second, the efflorescence of NaCl-dihydrate in supercooled NaCl and sea salt solute droplets has been studied with a technique combining electrodynamic balance (EDB) and Raman microscopy. The study was aimed to clarify the fundamental stochastic nature of homogeneous and heterogeneous nucleation in metastable aerosol systems. The ice nucleation efficiency of feldspar particles has been measured by depositing several hundred suspension droplets onto continuously cooled silicon substrate. Three types of experiments have been conducted: cooling ramp, isothermal freezing at constant temperature and freeze-thaw cycles. The observed freezing behavior have been interpreted in a framework of classical nucleation theory (Soccer Ball Model, SBM). The SBM was shown to adequately describe the shift of the freezing curves towards lower temperature with dilution, the cooling rate dependence and the ice nucleating active sites (INAS) surface density n_s (T) in a wide temperature range. Moreover, the SBM was capable of reproducing the variation of INAS surface density n_s (T) with concentration of IN in the suspension droplets and correctly predicting the leveling-off of the n_s (T) at low temperature. The freeze-thaw experiments have clearly shown that the heterogeneous freezing induced even by very active ice nucleating species still possesses a stochastic nature, with the degree of randomness increasing towards homogeneous nucleation. The reliability of the experimental approach has been also demonstrated during the Fifth Ice Nucleating intercomparison campaign (FIN02). Single solution droplets consisting of pure NaCl or simulated sea salt were suspended into the EDB maintained at constant temperature and relative humidity. A statistical analysis of several hundreds of individual efflorescence events allowed to describe the probability of NaCl dihydrate crystallization as a function of temperature, droplet volume, and solute concentration, and to derive the homogeneous and heterogeneous nucleation rate coefficients of NaCl dihydrate. Based on these measurements, the discrepancies between temperature dependent efflorescence of NaCl dihydrate observed in the previous experiments could be quantitatively explained for the first time. Furthermore, the experiments with the sea salt solution droplets showed an enhanced formation of NaCl dihydrate due to the faster crystallization of secondary sea salt components triggering the heterogeneous nucleation of NaCl dihydrate.
The development of pixelated photon counting semiconductor detectors enables to resolve the spectral composition of the incident photons in X-ray imaging, provided that the detector is operated in the absence of pulse pile-up and sensor polarisation. The purpose of this study is to examine the imaging properties of Medipix2 MXR detectors under high photon fluxes, which do not meet this requirement. At first, it is shown that the critical photon flux, at which the detector’s linearity breaks down, can be shifted towards higher values by increasing the IKrum current in the charge sensitive preamplifier, which corresponds to reducing the pulse shaping time. The negative impact of this procedure on the detector’s spectroscopic performance seems only moderate. Furthermore, the deviation from an ideal linear response is determined as a function of the photon flux. The deviations are used to correct single projections and eventually acquire spectral CT images at high photon fluxes which contain corrected absorption coefficients. Thus, K-edge imaging with the contrast agents iodine and gadolinium can be performed at high photon fluxes. It is shown that the spectroscopic information vanishes for energy ranges, which correspond to energies higher than approximately the mean photon energy of the incident spectrum.
The cortex is a contractile cross-linked network of actin filaments and myosin motors lining the plasma membrane. It defines the shape of animal cells, and regulated changes in cortex mechanics drive many cellular processes, including cell migration and division. The molecular mechanisms controlling cortical contractility in space and time are therefore essential for cell physiology, but are still not well understood. During cell division, in cytokinesis, tightly controlled changes to cortical contractility separate the two daughter cells. When very large cells undergo cell divisions, they exhibit highly stereotypical patterns of cortical contractility, termed surface contraction waves (SCWs). These waves occur in cells of a wide variety of species and move across the cells immediately prior to the division. The molecular mechanisms underlying this striking phenomenon are not known. I set out to investigate SCWs in starfish oocytes, which display a prominent contraction wave during meiotic division that can be imaged live using fluorescence microscopy. Combined with quantitative image analysis, this allowed me to correlate cell shape changes with the localization of key cortical and cell cycle proteins in untreated oocytes and following biochemical and physical manipulations. I find that morphologically the contraction wave is a band of flattening that forms at the vegetal pole and moves across the cell to the animal pole. The flattening is driven by increased cortical contractility induced by localisation of myosin II to the cortex. Myosin II recruitment is controlled by RhoA kinase and RhoA, which in turn is activated by release of its inhibition by the cell cycle kinase, cdk1-cyclin B. Importantly, I could show that cdk1-cyclin B activity forms a gradient along the animal-vegetal axis originating from accumulation of cdk1-cyclin B in the nucleus which is located at the animal pole. Therefore, as cyclin B is degraded, the bottom threshold of cdk1 activity will be reached first opposite of the animal pole, marking the starting point of the contraction wave. The gradient of cdk1-cyclin B activity furthermore controls the progression of the contraction wave across the cell. Additionally, I show that feedback internal to the downstream signalling network contributes to defining the speed of the wave and determines the width of the band of activity. Overall, this data for the first time establishes the molecular mechanisms underlying SCWs, a phenomenon observed in oocytes of many species. I show that the contraction wave is driven by the highly conserved RhoA-Rok-Myosin II pathway, and is patterned in space and time by an activity gradient of cdk1 as well as feedbacks internal to the signalling pathway. My work thereby reveals how this biochemical signalling network can define a spatially and temporally complex cellular behaviour.
Stars form within molecular clouds in galaxies. Therefore, understanding the formation, evolution and collapse of molecular clouds is critical for understanding galactic evolution. We present a systematic series of numerical simulations of a kiloparsec-scale size, elongated box, with sub-parsec resolution, developed to study the dynamics of molecular clouds in a galactic environment. We explore the origin of empirically observed relations such as the velocity dispersion-size relation in molecular clouds (Chapter 4), where we find that supernova explosions appear to be ineffcient at driving strong turbulent motions inside the clouds, where instead gravity appears to be the dominant process driving the observed fast motions. However, supernova explosions do play an important secondary role in the mass accretion histories of molecular clouds, simultaneously enhancing and suppressing inflow of gas onto the clouds by compressing and disrupting their mass reservoirs, (Chapter 5). We complete our analysis by studying the relative importance of magnetic fields in the evolution of molecular clouds and their envelopes. We find that, although we recover magnetic field strengths comparable to the observed values, they appear unable to prevent clouds from collapsing but capable of maintaining the diffuse envelopes supported, while restricting the gas flows in the diffuse ISM along field lines (Chapter 6). Together these results strongly support a picture of molecular clouds as highly dynamical objects that collapse quickly, and shortly after begin forming stars. However the subsequent evolution of these clouds must be strongly influenced by the newborn stars to avoid star formation effciencies higher than those observed.
Alcohol consumption is a broadly accepted part of many cultures around the world. While for some people it is not a problem to control their intake and use alcohol recreationally, others escalate their drinking until it becomes compulsive. Through cycles of excessive drinking and abstinence, alcohol dependence develops. This process is accompanied and supported by adaptations in the brain, including neurotransmitter and hormone systems as well as ion channels. Many of these systems are also altered in nicotine dependence, schizophrenia and depression, in part explaining the high comorbidity between these disorders and alcohol dependence. For the development of new drug therapies, an endeavor necessitated by the lack of efficient medications, it is imperative to understand the underlying mechanisms of each of these disorders. One possible target are the L-type calcium channels (LTCCs), which are influenced by both alcohol and nicotine, and have also been implicated in the risk to develop schizophrenia and depression. However, the two central LTCC subtypes CaV1.2 (Cacna1c) and CaV1.3 (Cacna1d) may play different roles, which have not yet been defined. This thesis aims to identify the individual involvement of CaV1.2 and CaV1.3 in alcohol dependence, and determine whether similar contributions of these subtypes can be found in comorbid disorders.
In Study I, Cacna1c mRNA levels are found to be dynamically regulated during intoxication, withdrawal, and protracted abstinence, with a strong increase in the amygdala and hippocampus after 21 days of abstinence. While Cacna1d mRNA remains unchanged at this time, CaV1.2 protein levels and currents are also increased. Furthermore, antagonism of central LTCCs prevents cue-induced reinstatement of alcohol seeking. Other genetic and functional models of alcohol dependence do not show a clear distinction between Cacna1c and Cacna1d mRNA expression (Study II). Transgenic mice with a CaV1.2 knockout (KO) in Ca2+/calmodulin-dependent protein kinase II (CaMKII)-positive neurons did not show a dependence-induced increase of alcohol intake, which their control littermates displayed clearly (Study III). Similarly to alcohol dependence, Study IV shows increased Cacna1c mRNA after chronic administration of nicotine and subsequent abstinence, while Cacna1d mRNA is increased one day after a single nicotine injection. An augmented nicotine sensitization after abstinence from chronic nicotine was prevented by nifedipine administration during abstinence. Additionally, CaV1.2 KO in CaMKII-positive neurons prevented nicotine self-administration behavior. Study V investigated CACNA1C and CACNA1D mRNA expression in postmortem samples of schizophrenia patients, alcoholics, and suicide completers. In schizophrenia, both subtypes were decreased in the prefrontal and temporal cortex. No changes caused by alcohol dependence or depression were found.
In conclusion, this thesis provides evidence for a crucial role of CaV1.2 in prolonged abstinence from chronic alcohol and nicotine, with effects on drug seeking and craving. It further underlines the importance of central LTCCs in some aspects of schizophrenia. Altogether, it highlights CaV1.2 as promising target for the development of new therapies for drug dependence and comorbid mental disorders.
Gut-derived bacteria enter the liver via the portal vein where they induce an innate immune response leading to inflammation. Lipopolysaccharide, a part the bacterial cell wall component endotoxin, functions as stimulus for toll-like receptors in non-parenchymal liver cells leading to secretion of di- verse cytokines. Among these cytokines tumor necrosis factor (TNF) is one of the first to be produced. It binds to the TNF receptors of hepatocytes and activates NF-κB signalling. The transcription factor NF-κB enhances gene expressions of acute phase proteins. Its signalling primes hepatocytes for cell proliferation. In this work, I have studied NF-κB signalling in hepatocytes in various ways, using computational models trained and validated with experimental data from primary murine cells. First, I extended an ODE model of canonical NF- κB signalling to include the experimentally validated influence of p38 MAPK signalling on this signalling pathway. Additionally, by including the receptor level to the model, I ensured an accurate description of dose response mea- surements for the main pathway components. This was especially important for the second part of this work, where I used the ODE intra-hepatocellular model to investigate the influence of different non-parenchymal cells on hepatocytes in the liver. By combining information on cell abundance and cell size, and experimental TNF secretion profiles in response to LPS with this ODE model, I was able to establish for the first time a computational model combing all liver cell types relevant to LPS-induced TNF secretion and intra-hepatocellular NF-κB signalling. I could show that liver resident macrophages and liver sinusoidal endothelial cells produce the most TNF in response to LPS. Furthermore, my simulations showed that not the final lev- els of TNF regulate the in vivo response, rather the initial cytokine increase defines how strongly NF-κB signalling is activated in hepatocytes. As a third part I converted the ODE model into a PDE model, which describes possible temporal and spatial aspects of single cell microscopy measurements. I was able to show that the relevant reaction parameters of the ODE model could be used for PDE simulations to describe experimental data on the localisa- tion of fluorescently labeled NF-κB molecules after TNF stimulation. Further- more, I could show that the dynamics observed on a population-based level were comparable to those observable on a single cell level. These new insights into NF-κB signalling in the liver may change experi- mental procedures with respect to cytokine administration when analysing inflammation. Furthermore, the new multi-cellular model can serve as a basis for simulating the influence of non-parenchymal cells on hepatocytes under various experimental conditions.
Death ligands such as CD95L and TRAIL initiate the extrinsic apoptotic signalling cascade by activation of the death-inducing signalling complex (DISC). Upon ligand binding to the receptor, the adaptor protein FADD is subsequently recruited to the receptor, thereby allowing for further recruitment of caspase-8 and its negative regulator cFLIP. In addition to the well-known caspase-8-mediated cell death induction, DISC-induced gene expression via the activation of NF-κB ruled out to be an important signalling pathway. Even if the major key players in DISC signalling have been described, the molecular mechanisms in DISC formation remain to be elucidated. In addition, the function of some DISC-associated proteins is unknown to date. To this day, the role of caspase-10, a homologue of caspase-8, in DISC signalling remains unknown and is controversially discussed. In this study, we elucidated the function of caspase 10 in DISC-induced cell death signalling and unexpectedly observed anti-apoptotic features under endogenous protein conditions. In contrast to previous thoughts, our data reveal that caspase-10 negatively regulates caspase 8-mediated cell death signalling in the DISC by blocking the recruitment to the complex and thereby the activation of caspase-8. Furthermore, we demonstrate that caspase 10 functions independent of cFLIP for inhibition of caspase-8 activation in the DISC. In addition, we show that caspase-8 does not compete with other tandem DED proteins such as cFLIP or caspase-10 in binding via FADD to the receptor as current models suggest. By utilising CRISPR-Cas9 mediated homologous recombination, we generated caspase-8 knockout cell lines and were able to demonstrate that caspase-8 has to be placed upstream of both cFLIP and caspase-10 in the DISC. We found that even FADD association with the DISC was drastically reduced in the absence of caspase-8. Interestingly, reconstitution of wild type caspase-8 and its active site mutant rescued the phenotype, indicating that caspase-8 is indispensable for the formation and/or stability of the DISC independent from its enzymatic activity. Moreover, we identified caspase 10 to promote DISC-mediated NF-κB activation. Caspase-10 favours at least the degradation of IκBα upon DISC stimulation resulting in enhanced NF-κB activation and inflammatory gene expression. Therefore, our data are consistent with a model in which caspase-10 rewires DISC signalling to NF-κB activation and cell survival. As a consequence, caspase-10 and cFLIP co-ordinately regulate caspase-8-mediated cell death signalling whereas both proteins contrast in their ability to induce gene expression upon death receptor activation.
Budding yeast cells deficient of the ubiquitin E3 ligase Rtt101 are highly sensitive to the genotoxic agents MMS and CPT, which cause DNA damage that leads to replication fork stalling in the ensuing S-phase. In a genetic screen we identified the replisome component Mrc1 as a key suppressor of rtt101 drug sensitivity. The rescue of rtt101 cells by MRC1 deletion depended on Rad52. The loss of Mrc's checkoint function was not sufficient to alleviate rtt101 drug sensitivity. Instead, the replicative function of Mrc1 seemed tobe toxic for rtt101 cells. We propose that the ubiquitination of Mrc1 (or an unknown factor regulating Mrc1) by Rtt101 modulates the replisome at the stalled fork, possibly inducing the uncoupling of the helicase from the polymerase. This could allow the production of ssDNA that might trigger replication fork repair or restart by a homologous recombination-based pathway. Telomere shortening is a natural process during replicative senescence. We could show that Rtt101 prevents premature senescence without affecting telomere length. Our results indicate that Rtt101 and Mrc1 protect shortening telomeres through a common mechanism. We speculate that Rtt101 and Mrc1 act in concert to prevent precocious senescence signalling by delaying the creation of subtelomeric ssDNA. Thus this study provides insights into how the Rtt101 ubiquitin E3 ligase functions to promote genome stability in the face of replication stress and replicative senescence.
For irreducible admissible representations of the group of symplectic similitudes GSp(4,F) of genus two over a p-adic number field F, we obtain the parahoric restriction with respect to an arbitrary parahoric subgroup. That means we determine the action of the Levi quotient on the invariants under the pro-unipotent radical in terms of explicit character values. Especially, we get the parahoric restriction of local endoscopic L-packets in terms of lifting data.
The inner cohomology of the Siegel modular variety of genus two with an arbitrary l-adic local system admits an endoscopic and a Saito-Kurokawa part under spectral decomposition. For principal congruence subgroups of squarefree level N they define simultaneous representations of the absolute Galois group and the Hecke action of GSp(4;Z/NZ). We decompose them into irreducible constituents and give explicit character values. As an application, we prove the conjectures of Bergström, Faber and van der Geer on level two.
Linear acenes are a widely studied class of materials in the field of Organic Electronics. Their aromatic system and the strong interaction of the π-electrons of neighbouring molecules in the solid state allow an efficient charge transport in these materials. The defined molecular structure of these small molecules and the possibility to tune their optical and electronic properties as well as the solid state packing through careful chemical design and synthesis have resulted in numerous applications of acenes in organic transistors and optoelectronic devices. This work focuses on the application of N-Heteroacenes and non-conjugated pentacene-based polymers as semiconductors in solution-processed organic field-effect transistors. These devices are used to evaluate the charge transport properties of the materials and derive structure-function relationships for the various compounds. To draw structure-function relationships from the studies described in this thesis, a myriad of characterisation techniques was employed to obtain an insight into the optical, electronic, electrical and morphological properties of each material. The effects of order, energetics and processing of the semiconductor on the transistor performance are all investigated. While non-conjugated pentacene-based polymers offer an ease of processability, their amorphous nature inhibits efficient hole transport, resulting in a relatively poor transistor performance. The fashion in which the pentacene systems are connected to the polymer backbone changes their flexibility and therefore affecting the injection behaviour and charge transport properties. The nitrogen substitution in N-Heteroacenes results in an energetic stabilisation of the frontier molecular orbitals, allowing for an enhanced electron injection into these materials. For the symmetrical tetraazapentacene and two halogenated phenazine derivatives relatively high electron mobilities were achieved demonstrating their potential for future application as n-type semiconductors in organic field-effect transistors. The use of N-heteroacenes is not limited to electron transport only. Their N,N’-dihydro forms are electron rich compounds that exhibit good hole transport. This is demonstrated for differently substituted tetraazapentacenes as well as for a N,N’-dihydro diazahexacene and -heptacene. For these materials it is shown that the processing conditions not only affect the macroscopic transistor performance, but also influence the formation of polymorphs in thin films. The solid state packing of functionalised acenes is typically determined by their solubilising side chains. A norbornadienyl substitution at the side chain of the well-known 6,13-bis(triisopropylsiliylethynyl)pentacene and its tetraaza derivative was shown to result in an enhancement of the charge transport properties for the p-type derivatives and deterioration of the performance of the n-type transistors. These observations are related to changes in the charge transfer integrals, the film microstructure and the solid state packing. In conclusion, this work contributes to the development of guidelines for the design and synthesis of next generation N-heteroacenes to be applied in the future in state of the art organic electronic devices.
Oligodendrocytes are the myelin-forming cells of the white matter of the central nervous system (CNS). Oligodendrocytes are derived from Oligodendrocyte Progenitor Cells (OPC) or from Neural Stem Cells (NSC) at different development stages. Although numerous factors involved in oligodendrocyte production have been identified, the transcriptional control of oligodendrocytogenesis remains largely unknown. However, the treatment of demyelination diseases like multiple sclerosis (MS) and periventricular leukomalacia (PVL) could greatly benefit from this knowledge.
The forkhead (Fox) gene family encodes transcription factors characterized by a DNA binding domain with a variant of the helix-turn-helix configuration. Foxb1 encodes a forkhead transcription factor expressed in the mouse neural plate and early mesoderm in the primitive streak stage. In midgestation, Foxb1 is expressed in restricted areas of the neuroepithelium (ventricular zone) as well as the brain parenchyma of midbrain, thalamus, hypothalamus, superior and inferior colliculi, pons, medulla oblongata and spinal cord. Preliminary work in our lab showed that the number of CNS cells belonging to the Foxb1 lineage (i.e. born from Foxb1-expressing ventricular zone) is much larger than the number of CNS cells actually express Foxb1 in the adult mouse. For my PhD work I wanted to know, first, which CNS cells are generated by Foxb1-expressing NSC; second, I wanted to learn about the specific function of Foxb1 in those cells.
To approach those questions I analyzed the phenotype of heterozygous (Foxb1Cre/+) and homozygous (Foxb1Cre/Cre) mice of the knock in-knock out Foxb1-Cre-EGFP mouse line (generated previously in our lab). I found that the Foxb1-expressing neuroepithelium generates large numbers of oligodendrocytes (as well as some astrocytes and neurons); in mice deficient in Foxb1, immature oligodendrocytes as well as OPC are abnormally abundant but can differentiate into oligodendrocytes able to produce normal myelin sheaths. I concluded that transcription factor Foxb1 is a novel player in the regulation of OPC generation, on which it exerts a potent inhibitory function.
Bei der klinischen Magnetresonanztomographie (MRT) wird das Signal von Wasserstoff (1H) detektiert. In der vorliegenden Arbeit wurden verschiedene Bildgebungsverfahren für die Phosphor (31P)-MRT mit hoher räumlicher und spektraler Auflösung entwickelt, um nichtinvasiv Informationen über den menschlichen Energiestoffwechsel zu gewinnen. Dabei wurde insbesondere auf die geringe In-vivo-Sensitivität und die große spektrale Bandbreite der 31P-Metaboliten-Resonanzen eingegangen. Zur Separation der einzelnen 31P-Metaboliten wurden die frequenzselektive Anregung (FreqSel) und das Multipunkt-Dixon-Verfahren (MP-Dixon) analysiert. FreqSel sollte bei 31P-Messungen eingesetzt werden, bei denen nur ein Metabolit von Interesse ist. Bei der Untersuchung einer größeren spektralen Breite lieferte dagegen MP-Dixon ein höheres Signal-Rausch-Verhältnis (SNR). Die Signalintensität konnte mithilfe von bSSFP-Sequenzen in Kombination mit dem Kern-Overhauser-Effekt um bis zu (82 ± 13)% bei einer Magnetfeldstärke von B0 = 3T und (37 ± 9)% bei 7T gesteigert werden. Dabei ist zu berücksichtigen, dass aufgrund der höheren Magnetfeldstärke das SNR bereits um einen Faktor von 2,5 erhöht ist. Weiterhin wurde die iterative Rekonstruktion von 31P-Daten unter Anwendung von A-priori-Informationen charakterisiert. Mithilfe einer aus morphologischen 1H-Bildern erstellten Trägerregion konnten Partialvolumeneffekte und Gibbs-Ringing-Artefakte in 31P-Bildern reduziert werden. Iterativ rekonstruierte Bilder repräsentieren somit die Verteilung der 31P-Metaboliten realistischer als konventionell rekonstruierte Bilder. Mit diesen Techniken ist die 31P-MRT bei einer isotropen Auflösung von (1cm)^3 in klinisch realisierbaren Messzeiten von 10min in vivo bei 7T möglich.
Highly integrated and increasingly complex video-based driver assistance systems are rapidly developing nowadays. Following the trend towards autonomous driving, they have to operate not only under advantageous but also under adverse conditions. This includes sight impairments caused by atmospheric aerosols such as fog or smog. It is an important part of environmental understanding to thoroughly analyze the optical properties of these aerosols.
The aim of this thesis is to develop models and algorithms in order to estimate meteorological visibility in homogeneous daytime fog. The models for light transport through fog are carefully derived from the theory of radiative transfer. In addition to Koschmieder's well-established model for horizontal vision, a recursively-defined sequence of higher-order models is introduced which yields arbitrarily good approximations to the solutions of the radiative boundary problem.
Based on the radiative transfer models, visibility estimation algorithms are proposed which are applicable to data captured by a driver assistance front camera. For any one of these algorithms, the recording of luminances from objects observed at distinct distances is required. This data can be acquired from moving objects being tracked as well as from depth-extended homogeneous objects such as the road. The resulting algorithms supplement each other with respect to different road traffic scenarios and environmental conditions. All given algorithms are extensively discussed and optimized regarding their run-time performance in order to make them applicable for real-time purposes. The analysis shows that the proposed algorithms are a useful addition to modern driver assistance cameras.
Global climate change has significantly impacted the terrestrial ecosystems and water cycles over the past century. This dissertation aims to further improve our knowledge of the linkages and interactions between vegetation, climate, streamflow, and drought. First, the current study investigated long-term variations in vegetation and climatic variables and their scale-dependent relationships by using Rhineland-Palatinate (Southwest Germany) as a case study area. Based upon the monthly normalized difference vegetation index (NDVI), precipitation and temperature data for six different vegetation types in two precipitation regimes (low and high precipitation regimes) of Rhineland-Palatinate, the temporal trends in the original time series of these variables and their relationships were examined. In addition, the further objectives were to evaluate which time-scale is dominantly responsible for the trend production found in the original data and find out the certain time-scales that represent the strongest correlation between NDVI and climatic variables (i.e., precipitation and temperature). A combined approach using the discrete wavelet transform (DWT), Mann-Kendall (MK) trend test and correlation analysis was implemented to achieve these goals. The trend assessment in the original data shows that the monthly NDVI time series for all vegetation types in both precipitation regimes have upward trends, most of which are significant. The precipitation and temperature data for six vegetation types in two precipitation regimes present weak downward trends and significant increasing trends, respectively. The most important time-scales contributing to the trend production in the original NDVI data are the 2-month and 8-month events. For precipitation, the most influential ones are 2-month and 4-month scales. The 4-month periodic mode predominantly affects the trends in the original temperature data. The results indicate temperature is the primary driver influencing the vegetation variability over this study area, while there is a negative correlation between NDVI and precipitation for all vegetation types and precipitation regimes. For the scale-dependent relationships between NDVI and precipitation, the 2-month and 8-month scales generally present the strongest negative correlation. The most significant positive correlation between NDVI and temperature is obtained at the 8- and 16-month scales for most vegetation types. The results might be valuable for water resources management as well as agricultural and ecological development planning in Rhineland-Palatinate, and also offer a helpful reference for other regions with similar climate condition. Then, this study presented a detailed regional investigation of the probabilistic and multi-scale relationships between streamflow and hydroclimatic variables (precipitation, temperature and soil moisture) and the potential links to large-scale atmospheric circulations over Baden-Württemberg, Southwest Germany. First, the joint dependence structure between seasonal streamflow and hydroclimatic variables was established using copulas. On the basis of the joint dependence structure, this study estimated the probability (risk) of hydrological droughts and floods conditioned upon two different scenarios of hydroclimatic variables for different seasons over the study area. Then, it was evaluated how the relationships between hydroclimatic forcings and streamflow vary among different temporal scales using wavelet coherence. The results reveal that the strong positive coupling between streamflow and both precipitation and soil moisture occurs at most temporal scales, particularly at decadal scales, while the multi-scale relationships between temperature and streamflow are significantly weak compared to precipitation and soil moisture. The connections between streamflow variability and large-scale atmospheric circulations were explored by using composite analysis. Although the atmospheric circulation patterns vary in different seasons, it can be found that the high streamflow anomalies for most seasons over Baden-Württemberg are related to strong westerly atmospheric circulations that play an important role in favoring the warm and moist air from the North Atlantic Ocean towards the study area and thus enhancing the precipitation. Moreover, the low streamflow anomalies are generally linked to the northerly circulations that induce the movement of cold air from northern Europe towards this study area and thus result in the reduced precipitation. Finally, a general probabilistic prediction network was developed in this dissertation for hydrological drought examination and environmental flow assessment. This methodology is divided into three major components. First, the joint streamflow drought indicator (JSDI) was proposed to describe the hydrological dryness/wetness conditions based on the monthly streamflow data. The JSDI relies on a high-dimensional (12-d) multivariate probabilistic model to establish a joint distribution model. In the second part, the drought-based environmental flow assessment method was introduced, which provides dynamic risk-based information about how much flow (the environmental flow target) is required for drought recovery and its likelihood under different hydrological drought initial situations. The final part involves estimating the conditional probability of achieving the required environmental flow under different precipitation scenarios according to the joint dependence structure between streamflow and precipitation. Two catchments in Germany were used to examine the usefulness of this network. The results show that the JSDI can provide an overall assessment of hydrological dryness/wetness conditions and does well in identifying both drought onset and persistence. The method also allows quantitative prediction of targeted environmental flow that is required for hydrological drought recovery and evaluates the corresponding risk. In addition, the results confirm that the general network can estimate the conditional probability associated with the required flow under different precipitation scenarios. The presented methodology offers a promising tool for water supply planning and management and for environmental flow assessment. The network has no restrictions that would prevent it from being applied to other basins worldwide.
Bluthochdruck gilt als Risikofaktor für kardiovaskuläre Erkrankungen wie Schlaganfall oder Herzinfarkt. Der erhöhte transmurale Druck, der auf die arteriellen Gefäße wirkt, resultiert nach dem Gesetz von Laplace in einer erhöhten tangentialen Wandspannung und führt zu pathologischen Veränderungen in der arteriellen Gefaßwand. Infolgedessen erhöht sich die Gefäßsteifigkeit, die - über einen Anstieg des peripheren Widerstands und somit einer Nachlasterhöhung für das Herz - die Progression der Hypertonie weiter fördert. Durch einen Anstieg der Wandspannung verlieren glatte Gefäßmuskelzellen ihren kontraktilen, ruhenden Phänotyp, der durch die Expression von Genprodukten des kontraktilen Apparates (z.B. SM-MHC, SMalphaA) gekennzeichnet ist. Gleichzeitig erhöht sich die Proliferation und Migrationsaktivität der glatten Gefäßmuskelzellen einhergehend mit einer gesteigerten Synthese von Matrixmolelekülen (z.B. Kollagen I) und Matrixmetalloproteinasen wie MMP-2, was sich auf die Architektur und Komposition der Extrazellulärmatrix auswirkt. In diesem Zusammenhang wurde beschrieben, dass der Transkriptionsfaktor NFAT5 (nuclear factor of activated T-cells 5) diesen aktivierten, synthetischen glattmuskulären Phänotyp fördert, indem er beispielsweise eine verstärkte Genexpression des Matrixmoleküls Tenascin C (TNC) stimuliert, welches die Proliferation der Zellen fördert. Basierend auf diesen Beobachtungen diente diese Arbeit der Aufklärung von biomechanisch induzierten Mechanismen, die zur Aktivierung von NFAT5 führen. Darüber hinaus sollte die funktionelle Rolle von NFAT5 während Bluthochdruck-induzierter Remodellierungsprozesse in vitro und in vivo untersucht werden. Die biomechanische Dehnung glatter Gefäßmuskelzellen aus Arterien in vitro resultierte sowohl in einer erhöhten Proteinsynthese als auch in einem verstärkten Kernimport von NFAT5, das nach Bindung an die DNA die Genexpression von Actin-beta-like-protein 2 (ACTBL2) induzierte. ACTBL2 kodiert dabei für die Aktin-Isoform Kappa-Aktin, die funktionell einen kritischen Beitrag zur gerichteten glattmuskulären Migration lieferte. Ferner konnte erstmalig gezeigt werden, dass eine divergente Regulation der NFAT5-Isoformen A und C nach Dehnung auftritt. Die Überexpression der entsprechenden Isoformen führte dazu, dass die nukleäre Akkumulation von NFAT5c, das in ruhenden Zellen sowohl im Zytoplasma als auch im Zellkern vorliegt, durch biomechanische Dehnung verstärkt wird. Im Gegensatz dazu war NFAT5a sowohl in ruhenden als auch in gedehnten Zellen ausschließlich im Zytoplasma lokalisiert. Der Einfluss von posttranslationalen Proteinmodifkationen auf die Kerntranslokation von NFAT5 wurde mithilfe der zielgerichteten Mutation von spezifischen Aminosäuren im NFAT5c-Protein bzw. durch die Inhibition bestimmter Signalwege untersucht. So war eine u.a. durch die Kinase c-Abl vermittelte Phosphorylierung von Tyrosin-143 ebenso wie eine Palmitoylierung durch Palmitoyltransferasen der CPT1-Familie (Carnitin-Palmitoyltransferasen) essentiell für den dehnungsinduzierten Kernimport von NFAT5. Im Gegensatz dazu schien die Kerntranslokation durch eine Phosphorylierung an Serin-1197 inhibiert und somit kontrolliert zu werden. Durch die Verwendung von induzierbaren, glattmuskulär NFAT5-defizienten Mäusen war es möglich, die funktionelle Rolle von NFAT5 in vivo zu untersuchen. Bei der Hypoxie-induzierten pulmonalen Hypertonie resultierte ein Verlust von glattmuskulärem NFAT5 in einer verminderten Rechtsherzhypertrophie, einer geringeren medialen Verdickung der kleinen peripheren Lungenarterien sowie einer verminderten Expression der NFAT5-abhängigen Zielgene TNC und Kappa-Aktin. Außerdem führte der Verlust von glattmuskulärem NFAT5 im Rahmen einer experimentell induzierten arteriellen Hypertonie (DOCA/Salz-Modell) zu einer verminderten Proliferation der glatten Gefäßmuskelzellen in den Femoralarterien. Im Rahmen dieser Arbeit konnte somit gezeigt werden, dass der Transkriptionsfaktor NFAT5 – möglicherweise durch die Regulation der Genexpression von Kappa-Aktin und TNC – eine bisher unbekannte, wichtige Rolle in der Pathogenese von Bluthochdruck-induzierten Gefäßumbauprozessen spielt. Die der dehnungsinduzierten Translokation von NFAT5 in den Zellkern zugrunde liegenden Mechanismen könnten zukünftig als Angriffspunkt zur Entwicklung einer neuartigen Therapie Hypertonie-induzierter arterieller Umbauprozesse genutzt werden.
In recent years, various highly charged ions (HCI) with optical transitions have been proposed for metrology and searches of a possible variation of the fine-structure constant α. Optical transitions in HCI are uncommon due to the scaling of energy levels with atomic number Z^2 . At the 4f–5s level crossing, three configurations are nearly degenerate, and thus many optical transitions can exist. The complex many-electron couplings reduce the accuracy of current calculations. Moreover, experimental data to benchmark the predictions is lacking. Spectra in the optical and extreme-ultraviolet (EUV) range of several ion species near the 4f–5s level crossing were measured at the Heidelberg electron beam ion trap. A collisional-radiative model was employed for the interpretation of the EUV data, resulting in the first identification of the long sought-after 5s–5p transitions in Pm-like Re^14+ , Os^15+ , Ir^16+ , and Pt^17+. The characteristic line shapes of optical transitions in Ir17+ were studied, with the aim of identifying transitions with a high sensitivity to α-variation. Previously suggested candidates could be excluded and new candidates were proposed. This data provides a stringent benchmark for state-of-the-art precision atomic theory.
Epidemiological studies indicate a reduced risk and a lower incidence of hormone-dependent cancer types in populations following a traditional Asian diet rich in phytoestrogens such as isoflavones (IF). Lifestyle and timing of exposure to IF seems to be a critical factor. In rodent models, especially multi-generational or prepubertal exposure was shown to modulate mammary gland (MG) morphology, resulting in anti-tumorigenic activity. Many processes during early development are regulated by epigenetic mechanisms, therefore IF might alter the epigenetic reprogramming of the MG affecting normal cell growth and susceptibility to breast cancer. Several investigations provide evidence for the impact of IF on DNA methylation but most of the work focused on selected candidate genes. Investigations of genome-wide changes of DNA methylation in the MG upon IF treatment are scarce. The present thesis was aimed to analyzed DNA methylation profiles in MGs of healthy rats and during estrogen-induced mammary carcinogenesis on a genome-wide scale. Our focus was to clarify the impact of different IF doses and identify critical developmental windows of exposure in Wistar rats, and to investigate the chemopreventive efficacy of IF during estrogen-induced MG tumor development in ACI rats. Genome-wide methylation analyses were initially performed with Wistar rats bred on a diet with medium IF levels that were switched to either IF-free or high dose IF diet for two weeks after ovariectomy (OVX). We observed only few IF-related DNA methylation changes (measured by MCIp-Seq technology). This weak epigenetic effect was attributed to the fact that all rats had been exposed to the phytoestrogens during all critical developmental phases until adult life. Most methylation changes were observed outside of classical regulatory regions (promoters, CpG islands or 5’ UTRs) but interestingly were enriched for binding sites for ETS-domain and basic leucine zipper-domain containing transcription factors. For validation of the genome-wide results, selected candidate genes were analyzed quantitatively by EpiTYPER MassARRAY technology. Two groups of rats with lifelong exposure (from conception to post OVX) to low and high IF doses were included to consider dose effects. We observed a U-shaped dose-response pattern, with the low IF concentration reducing methylation levels, whereas medium and high doses increased methylation compared to an IF-unexposed control group. The relevance of these changes needs to be further investigated. However, lifelong exposure to high IF levels significantly reduced estrogenic and proliferative response of the MG, whereas lower IF concentrations were not sufficient to induce the beneficial health effects and even provoked opposing effects, i.e., significantly induced PCNA and PR protein expression. Different from the lifelong exposure to high IF levels, IF intervention exclusively during puberty exhibited only minor effects on DNA methylation and mRNA expression levels of candidate genes. Also, expression of proliferation markers were not affected. An exception was an increase in Vdr and Gata3 mRNA expression which might sensitize the MG towards enhanced differentiation. In contrast, short term IF exposure only after OVX affected DNA methylation patterns of candidate regions mostly in an opposed direction when compared to the lifelong IF exposure. Post OVX exposure to IF also induced pro-proliferative as well as pro-estrogenic properties and sensitized the animals towards the estrogen treatment. These results contradict the use of high IF concentrations, e.g., for hormone replacement therapy after menopause. In order to investigate lifelong IF-mediated effects on estrogen-induced rat mammary carcinogenesis, ACI were exposed lifelong to the highest IF dose. IF intervention reduced incidence and multiplicity (56 %, p=0.018) of MG tumors but shortened tumor latency by 5 weeks (p<0.0001) and enhanced tumor growth (>2 fold), if tumors escaped the preventive effect. IF intake increased the estrogenic and proliferative response of the MG during puberty when carcinogenesis was induced by exogenous estrogen exposure, and epigenetic modifying enzymes such as DNMT3a and 3b were significantly downregulated. Genome-wide methylation profiling analyzed by quantitative reduced representation bisulfite sequencing (RRBS) again indicated hypo- and hypermethylation mainly outside of classical regulatory regions. Genes with differential promoter methylation induced during carcinogenesis which could be modulated by IF exposure were enriched for biological processes and signaling pathways involved in reproductive tissue homeostasis and endocrine system. After applying stringent selection criteria, we were able to validate selected methylation changes from the RRBS analysis by MassARRAY. In all regions investigated, the carcinogenic process significantly modified methylation levels up to 30 %, inducing both, hypo- and hypermethylation. Interestingly, lifelong intervention with the highest IF dose prevented this carcinogenesis-mediated loss and/or gain in methylation. For half of the selected candidate genes methylation changes significantly correlated with mRNA expression. Interestingly, lifelong high dose IF exposure significantly reduced mRNA levels of DNMT1 in healthy MGs and prevented the estrogen-induced upregulation of DNMT1 during tumor formation. In conclusion, we identified genome wide DNA methylation changes induced by dietary IF. The impact of IF on DNA methylation is highly dependent on exposure time and IF dose. Lifelong exposure to IF reduced estrogen-induced MG tumor incidence but shorted tumor latency. This phenomenon might be partly explained by the downregulation of epigenetic modifiers such as DNMTs during early MG development as well as during breast carcinogenesis. Additional investigations are required to gain a comprehensive insight on IF-induced epigenetic regulation unraveling the functional mechanisms of biological effects exerted by phytoestrogenic soy IF.
The amplification of the MYCN oncogene, occurring in 20% of neuroblastomas, a tumor of early childhood, is associated with drug-resistant relapse and poor prognosis. High MYCN expression has paradoxical effects in most cells: it promotes cell cycle progression and sensitizes to cell death. This work aims to characterize, at population and single-cell level, the mechanisms through which amplified MYCN allows tumor regrowth after chemotherapy in neuroblastoma. In MYCN-regulatable neuroblastoma cell line models, MYCN shortens the lengths of cell cycle phases, preferentially in G1, and increases the proportion of cycling cells under exponential growth. Upon DNA-damage induced by the chemotherapeutic agent, doxorubicin (DOX), MYCN delays activation of cell cycle checkpoints and boosts the proportion of transiting cells. During and after chemotherapy MYCN favors cell death and suppresses cellular senescence, shifting p53 downstream effects from cell cycle arrest to apoptosis. However, MYCN also drives clonal regrowth of a small fraction of surviving resister cells after DNA-damage. These resister cells exhibit nearly identical molecular and phenotypic profiles as cells before treatment. Live-cell imaging reveals that resister cells arise exclusively from the G1-phase-arrested subpopulation and rapidly repair DNA-damage-induced double-strand breaks (DSBs). The suppression of DNA repair via ATM inhibition during chemotherapy results in reduction of G1 phase arrest and prevents DNA DSB repair, completely eradicating resister cells. Taken together, these data show that non-genetic tumor heterogeneity and a key oncogenic lesion, MYCN, synergize to resume cellular proliferation after DNA damage and probably cause chemotherapy resistance. This work indicates that improved first-line therapies could specifically target resister cells and help avoid cellular regrowth.
Campus-Reporter Nils Birschmann hat an der Universität Heidelberg Prof. Werner Aeschbach getroffen. Seit Jahren erforschen er und weitere WissenschaftlerInnen die Ursachen von Flucht und Vertreibung.
Der Beitrag erschien in der Sendereihe "Campus-Report" - einer Beitragsreihe, in der über aktuelle Themen aus Forschung und Wissenschaft der Universitäten Heidelberg, Mannheim, Karlsruhe und Freiburg berichtet wird. Zu hören ist "Campus-Report" montags bis freitags jeweils um ca. 19.10h im Programm von Radio Regenbogen. (Empfang in Nordbaden: UKW 102,8. In Mittelbaden: 100,4 und in Südbaden: 101,1)
Nowadays, X-ray absorption spectroscopy (XAS) techniques are important tools to investigate the electronic structure of molecules. Mostly, these methods are applied in the field of organic electronics to study unoccupied molecular levels, which provide information about charge generation and transport properties. With the help of modern synchrotron soft beam sources, molecules can absorb high-energy X-ray photons, thereby promoting an electron from the core level, e.g. K-shell 1s orbitals, to the unoccupied molecular level. As a result, meta-stable bound core-excited states are generated. Since core orbitals are energetically well-separated from the remaining occupied and virtual orbital space, they are strongly contracted and the corresponding core-excited states are very localized. As a consequence, the generated core-hole interaction induces a rearrangement of the valence electrons, because the effective shielding of the nucleus is reduced. This effect leads to a lowering of the core-excitation energy of the final state. This rearrangement of the electrons can be understood as an orbital relaxation effect. To fully understand and interpret experimental spectra, an accurate knowledge about core-excitation energies, transition moments, the character of the core-excited states as well as their corresponding properties is necessary. Such information can be obtained with quantum chemical (QC) methods. They help to analyze and interpret experimental spectra, thereby providing a deep insight into the nature of core-excited states. Generally, a plethora of methods is available to calculate excited states and simulate absorption spectra. The larger the system, the more expensive are the computations. Hence, certain levels of approximation have to be introduced to lower the computational cost. This leads to a loss of reliability and accuracy of the results. The time-dependent density functional theory (TD-DFT), for example, currently is the prevalently used excited-state method for the calculation of large molecules up to 300 atoms. However, TD-DFT has several disadvantages like the self-interaction error (SIE), which leads to wrong descriptions of certain kinds of excited states, e.g. charge-transfer states or core-excited states. The excitation energies of these types of states are strongly underestimated, but if these issues are kept in mind, TD-DFT is a useful tool, providing proper spectral features. The algebraic diagrammatic construction scheme (ADC) is a prominent QC method for the calculation of excited states, which is known to provide accurate valence-excited states of small- and medium-sized molecules in an adequate computational time. The ADC approach is based on a Green’s function formalism in combination with partitioning the Hamiltonian using perturbation theory. Due to its size-consistency and Hermitian ADC secular matrix structure, the level of approximation can be improved systematically and properties can be computed straightforwardly. It is possible to calculate one-particle state properties in combination with the intermediate state representation (ISR) approach, e.g. static dipole moments and state densities, which altogether provide enhanced information about absorption spectra. A further advantage of ADC is the indirect inclusion of orbital relaxation effects via couplings to higher-excited configurations, which are important to describe core-excited states properly. However, the calculation of core excitations is tedious using the unmodified ADC approach, because, in order to solve the ADC eigenvalue problem, numerical iterative eigenvalue solvers are employed usually only providing the energetically lowest eigenstates. Core-excited states, however, are located in the high energy X-ray region of the optical spectrum and in order to calculate them directly, one has to compute all energetically underlying valence excitations as well. This is computationally very expensive and not feasible for medium-sized systems. The direct calculation of the core excitations is prevented by couplings between the valence and core-excited states. A solution to this issue is the application of the core-valence separation (CVS) approximation to the ADC approach, which results in the CVS-ADC method. This approximation is based on the fact that core orbitals are energetically well-separated from the remaining orbital spaces and as a consequence, the couplings between core- and valence-excited states are small and can be neglected. In other words, the CVS approximation decouples the core and valence excitation spaces from each other and allows for a direct computation of core-excited states. In former work, it was proven that a very good agreement with experiments can be obtained at the extended second order level CVS-ADC(2)-x.
My PhD project mainly consists of two important parts. One was to enhance and develop variants of the CVS-ADC method and implement all approaches efficiently in the adcman program, which is part of the Q-chem program package. Secondly, I benchmarked these implementations and simulated X-ray absorption spectra of small- and medium-sized molecules from different fields. In this thesis, I present my implementations, as well as the results and applications obtained with the CVS-ADC methods and give a general introduction into quantum chemical methods. At first, I implemented the CVS-ADC approach up to the extended second in an efficient way. The program is able to deal with systems up to 500 basis functions in an adequate computational time, which allows for accurate calculations of medium-sized closed-shell molecules, e.g. acenaphthenequinone (ANQ). Afterwards, the CVS-ADC implementation was extended for the first time to deal with open-shell systems, i.e. ions and radicals, which implies a treatment of unrestricted wave functions and spin-orbitals. The resulting method is denoted as CVS-UADC(2)-x. For the first time, I applied the CVS approximation to the the third order ADC scheme, derived the working equations, and implemented the CVS-ADC(3) method in adcman. As the last step, I applied the CVS formalism for the first time to the ISR approach to enable calculations of core-excited state properties and densities. This provides the basis for subsequent evaluations of transition- and density matrices, which give access to exciton sizes, e.g. hole sizes or distances between hole and electron densities. All implementations are presented and discussed in the scope of my thesis.
To benchmark all restricted and unrestricted CVS-ADC/CVS-ISR methods up to third order in perturbation theory, I chose a set of small molecules, e.g. carbon monoxide (CO). The calculated values of core-excitation energies, transition moments and static dipole moments are compared with experimental data or other approaches, thereby estimating complete basis set (CBS) limits. Furthermore, a comprehensive study of different basis sets is performed. As it turns out, the CVS-ADC(2)-x method provides the best agreement with experiments, while CVS-ADC(3) overestimates the core excitation energies. In combination with the CBS limit of the aug-cc-series, a mean error of -0.23%±0.12% for core-excitation energies can be identified at the CVS-ADC(2)-x level for carbon, nitrogen and oxygen K-edge excitations, whereas CVS-ADC(3) exhibits errors of 0.61%±0.32%. This is due to fortuitous error compensation of basis set truncation, electron correlation, orbital relaxation and neglect of relativistic effects at the CVS-ADC(2)-x level. I show that this error compensation is broken at the third order level, because the ratio between terms describing relaxation and polarization effects is shifted in a way that the excitation energy increases. However, transition moments and spectral features, as well as static dipole moments, are excellently described with both CVS-ADC(2)-x and CVS-ADC(3). Overall, considering the detailed investigation of the basis set influence on the results, I conclude that the use of restricted or unrestricted CVS-ADC(2)-x in combination with a diffuse triple-ζ basis set in its Cartesian version can be seen as a black-box method for the calculation of core-excited states of organic molecules. Especially the 6-311++G** basis set provides an excellent ratio of accuracy to computational time. Another important topic is the description of orbital relaxation effects. In the scope of this thesis, I show, how these effects are included indirectly within the CVS-ADC approaches. For this purpose, two different descriptors are used, i.e. electron promotion numbers and the amount of doubly excited amplitudes. Furthermore, with the help of detachment/attachment (D/A) densities, which can be constructed via the CVS-ISR approach, relaxation effects can be visualized. For this purpose, the (D/A) densities are compared with hole/electron (h/e) densities based on the transition density matrix. With this knowledge, the X-ray absorption spectra of medium-sized molecules and radicals from the fields of organic electronics and biology are investigated and analyzed. On the basis of these studies, the restricted and unrestricted versions of CVS-ADC(2)-x in combination with the 6-311++G** basis set exhibit mean errors of core-excitation energies around 0.1%, compared to experimental values. Additionally, core-excited state characters are analyzed with the help of state densities obtained via the CVS-ISR approach or the transition density matrix. To demonstrate that the CVS-ADC(2)-x approach can be employed as a benchmark black-box method, TD-DFT results are compared directly with the ones at the CVSADC(2)-x level. As expected, TD-DFT underestimates core-excitation energies up to 4% due to the SIE, which is about 10 eV in the case of carbon 1s excitations. Since the CVS approximation leads to both a simplification of the ADC working equations, as well as a restriction of the excitation space to correspond only to core excitations, the computational cost is reduced compared to the general ADC approach. To demonstrate the computational savings as a function of the size of the core space, several systems are investigated. CVS-ADC(3) calculations take about 8 – 10 times longer than CVS-ADC(2)-x calculations and since the results are generally more accurate with the latter method, the use of CVS ADC(3) is not justified. Compared to general ADC(2)-x, the speed-up at the CVS-ADC(2)-x level is about a factor of 4.0, but this factor strongly depends on the size of the system and the size of the core space. Next, I present applications from the field of organic electronics. The remarkable agreement with experimental data at the CVS-ADC(2)-x level justifies the prediction of yet non-recorded experimental X-ray absorption spectra. Therefore, I chose the anthracene cation, which can be seen as a model system of pentacene and its derivatives, which are commonly used as hole conductors (p-type). X-ray absorption spectra of the pentacene cation could provide deeper insight into its charge carrier properties, but measurements of experimental spectra of ionized species are usually very challenging. With the help of CVS-UADC(2)-x calculations, I show that the anthracene cation exhibits additional peaks due to the half-filled single-occupied molecular orbital. They are located approximately 3.5 eV – 1.5 eV below the first peak of neutral anthracene, which may help to distinguish a cation from the neutral species. Furthermore, the cationic spectrum exhibits peak broadening, compared to the two first peaks of neutral anthracene. Other applications concentrate on the trends of core-excited state properties along important potential energy surfaces (PES) of ANQ, phenol and bithiophene. Therefore, static dipole moments, energies, and exciton sizes are analyzed as a function of the C–O distances of ANQ and phenol, as well as the torsion around the central dihedral angle of bithiophene. Finally, another aspect of the CVS-ISR method is the accessibility of transition moments between two states, which can be used to calculate oscillator strengths for core-excited state absorption (CESA) spectra. To the best of my knowledge, no experimental data of CESA processes between two core-excited states have been recorded yet. However, such spectroscopic data could exhibit new insights and the calculation of CESA transition moments using the CVS-ADC/CVS-ISR approach is straightforward. Hence, first results of CESA processes were calculated and are presented in this thesis. In the case of ANQ, particularly bright transitions can be identified from the lowest oxygen 1s excited-state to higher ones.
Hepatitis C Virus (HCV) is a positive stranded RNA virus, grouped into the family of Flaviviridae. The HCV genome encodes a single polyprotein, which is co- and posttranslationally cleaved into ten structural and non-structural (NS) proteins by cellular and viral proteases. The coding sequence is flanked by 5’ and 3’ untranslated regions (UTRs), which contain essential cis-acting elements, regulating translation and RNA synthesis, e.g. an internal ribosome entry site (IRES) for cap-independent translation. HCV RNA replication involves the synthesis of a negative strand replication intermediate, serving as a template for the generation of multiple strands of genomic RNA. This process requires a concerted action of several viral nonstructural proteins, cis-acting replication elements and host factors, and is poorly understood at the molecular level. The first part of this study aimed to characterize the viral non-structural proteins comprising the replicase complex in vitro and their mode of action during (-)-strand RNA synthesis. Since the natural 3’(+)-end is a poor template for the viral polymerase NS5B, supporting roles of the viral protease/helicase NS3 and the phosphoprotein NS5A were hypothesized. Optimal conditions for NS3 activity were established by an in vitro helicase assay. By combining the individual proteins with different RNA templates, it was observed that initiation and processivity of NS5B were stimulated by active NS3, but not by inactive mutants. Inhibition of NS3 helicase activity did not impair the stimulatory effect on NS5B, but led to an altered mode of initiation. Addition of purified NS5A further augmented the effect of NS3. In conclusion, this work demonstrates that NS3 and NS5A can improve RNA dependent RNA polymerase activity on a natural template, thereby providing an experimental model to study the molecular mechanisms governing initiation of RNA synthesis. Liver -specific microRNA (miR)-122 is an important host factor of HCV replication, and recognizes two conserved target sites within the first 45nt of the HCV 5’ UTR, close to the IRES. Previous studies suggested a role of miR-122 in RNA stability, translation, and RNA synthesis. The mechanisms, by which miR-122 exerts these functions, remain enigmatic. Insertion of a heterologous IRES element, allowing for miRindependent translation of the non-structural proteins, was sufficient to enable replication in miR122deficient Hep3B cells, suggesting a substantial role of miR-122 in IRES-dependent translation. The miR122 binding region is engaged in a strong secondary in the complementary negative strand. Additionally, we found that a similar structure was predicted inthe positive strand, which would interfere with IRES formation. We therefore hypothesized that miR-122 binding in this region might prevent such alternative structures, thereby facilitating IRES-mediated translation. Indeed, mutations in the miR-122 binding region, but not the IRES sequence, which were designed to stabilize or destabilize the IRES, enhanced or decreased initial translation, respectively, independent of miR-122. Translation enhancement was independent from RNA stability, but short-lived, suggesting additional roles of miR-122, e.g. recruitment of host proteins facilitating steady state translation. Moreover, structural analysis suggested that the HCV IRES folds into a number of conformers in solution, which can be modified by miR-122 under certain conditions. Apart from the 5’ UTR, HCV also contains several seed-matches for miR-122 in the coding sequence of NS5B, and the 3’ UTR, with unknown functional significance. Two novel sites were identified to be conserved over a number of genotypes. The functional characterization of these miR-122 binding sites was evaluated by insertion of point mutations, abrogating miR-122 binding to single and multiple sites, revealing a previously unappreciated role in virion assembly or release. However, assembly of the mutants could not be rescued by a corresponding mutant miR, suggesting a specific need for wild type miR-122. Conclusively, this study provides evidence for miR-122 involvement in almost every intracellular stage of HCV infection, and defines translation enhancement by suppression of RNA structures interfering with IRES activity as a key function of miR-122.
The primary aim of this thesis is to clarify how the structures and functions of biological membranes are influenced by the oxidative damage mediated by free radicals. As a precisely defined model systems, artificially reconstituted lipid membranes (Langmuir monolayers, vesicles, supported membranes, multilamellar membranes) incorporating two oxidized phospholipids bearing aldehyde or carboxyl groups at the end of truncated sn-2 acyl chains were fabricated. By the combination of various experimental methods, the generic impact of chain oxidization on physical characteristics of membranes (e.g. lateral cooperativity, fine-structures perpendicular to membrane planes, electrostatics) and the specific interactions of oxidized phospholipids with EO6 peptides and acute immune response proteins was investigated. In the first step, the influence of oxidized phospholipids (OxPL) on the thermodynamics and electrostatics were investigated using Langmuir film balance at the air-water interface. The pressure-area (π-A) isotherms and surface potential (Δψ-A) measurements implied that both OxPLs lead to a decrease in the isothermal compression modulus. In fact, surface potential measurements suggest changes in the orientation of oxidized moieties that decrease the lateral cooperativity. Further increase in the fraction of oxidized lipids resulted in the loss of molecules into bulk water, which seems consistent with the destabilization of cell membranes under oxidative stresses. In the second step, the impact of lipid oxidization on the electrostatics of membranes was examined by the combination of high-energy specular X-ray reflectivity (XRR) and grazing-incidence X-ray fluorescence (GIXF). The scattering length density profiles reconstructed from XRR results suggested that both OxPL leads to membrane thinning, which seems plausible from the decrease in the lateral cooperativity suggested by Langmuir isotherms. GIXF offers an unique possibility to localize specific target elements within Å accuracy, suggesting that the binding affinity (Ca2+ > Cs+ > K+) could be interpreted in terms of the solvation entropy (Hofmeister series). Further, the impact of oxidization on the vertical structural ordering of vertically stacked membrane models was investigated by off-specular neutron scattering. A decreased lamellar periodicity d indicated that incorporation of OxPL into the membrane displace water molecules from the inter-membrane region due to the reorientation of oxidized moieties. In the third step, the combination of experimental techniques was utilized to shed light on specific interactions of OxPLs with peptides and proteins; C-reactive protein that is characteristic for the acute immune responses and monoclonal antibody EO6 to oxidized lipids. Following the fundamental characterization of membrane-protein interactions using isothermal titration calorimetry (ITC) and dynamic light scattering (DLS) of vesicle suspensions, in addition to XRR, GIXF, off-specular neutron scattering, dual waveguide polarization interferometry (DPI) was used to monitor the changes in thickness, refractive index, and the optical anisotropy (birefringence) of lipid membranes simultaneously. Furthermore, the specific binding of EO6 was verified from the fluorescence imaging of glioblastoma multiforme cells undergoing apoptosis, where a clear accumulation of OxPLs could be identified in apoptotic blebs. The obtained results demonstrated that the combination of well defined membrane models and unique physical techniques is a powerful tool to shed a new quantitative light on the generic and specific impacts of lipid oxidization on the lateral cooperativity, vertical fine-structures, electrostatics, and specific interactions in inflammation and apoptosis.
The model of experimental autoimmune encephalomyelitis (EAE) is the most common model for studying pathological processes in multiple sclerosis (MS). In a variant of this model, induced by immunisation of Brown Norway rats with myelin oligodendrocyte glycoprotein (MOG), pathological mechanisms reflects many aspects of MS, observed by the activation of adaptive, innate and humoral immune response (Fairless et al., 2012). In addition to “the classical EAE pathology” characterised by inflammatory demyelination of the spinal cord, these animals also develop autoimmune optic neuritis (AON). The dynamics of the pathological events in AON are similar to those observed in acute optic neuritis in patients, a common early manifestation of MS. Taking this into consideration, the aim of this study was to provide a detailed characterisation of the optic nerve pathology during the course of AON in order to gain further insight into mechanisms of axonal injury and visual dysfunction associated with optic neuritis. Testing of visual functions by recording of visual evoked potentials (VEPs) revealed a decrease in visual acuity already during the induction phase of AON. During this phase, major pathological changes (such as inflammatory demyelination and axonal loss) in the optic nerve were still absent. However detailed examination of the optic nerve revealed that the observed visual impairment is timed with disruptions in axonal domains involved in saltatory conduction of action potentials (nodes of Ranvier and paranodes). Further evidence of stress was detected in both compartments of paranodal axon-glia junctions observed by alternation in the axonal neurofilament cytoskeleton and increased production of alpha B-crystallin (cryαB), a heat shock protein involved in oligodendrocyte stress response. Observed signs of stress were more prominent in the optic nerve head (ONH), the area which was also characterised by increased numbers of activated microglia and the deposition of autoantibodies. Following the onset of the clinical disease phase animals were characterised with greater impairments in visual functions and with the presence of inflammatory demyelination and increasing signs of axonal injury in optic nerves. In addition to increased expression of cryαB, a portion of oligodendrocytes associated with inflammatory lesions underwent the process of apoptosis. In order to investigate the origins of observed axonal and oligodendroglial stress and their contribution to disrupted axon-glia paranodal junction in the late induction phase, two different approaches were made. Firstly, the oligodendrocyte compartment was targeted by auto-antibodies through the transfer of sera from MOG-immunised into naïve animals. This led to deposition of auto-antibodies and the presence of oligodendrocyte stress in the ONH, in a similar manner to that observed in the late induction phase. Secondly, a model of primary retinal injury by intravitreal injection of glutamate was performed and successfully mimicked aspects of the retinal pathology characteristic of the early induction phase. This led to the presence of axonal stress in optic nerves in the similar extent to one observed in the late induction phase of AON. In both instances, the observed signs of stress did not translate across the paranodal axon-glia junction to the other compartment. This suggests that axons and oligodendrocytes could be targeted independently during the induction phase of AON by mechanisms involving a primary retinal insult and the actions of the humoral immune response. Collectively, the pathological image observed in the induction phase of AON shares great similarities with the pathology of normal-appearing white matter in MS, in terms of both axonal and oligodendroglial stress, further suggesting that the model of AON is useful for studying early degenerative processes in MS. Observed disruptions of axonal domains in the induction phase of AON could serves as a structural correlate of impairment of visual functions. These findings could be relevant for human studies, particularly in a sub-set of MS patients which are characterised by impaired visual functions in the absence of clinically-defined optic neuritis.
Eukaryotic chromosomal ends are formed by special structural and functional units, termed telomeres, protecting them from fusion, degradation, and unwanted DNA damage repair events. In this way, telomeres preserve genome stability and integrity that are vital to every cell and organism, whereas genomic instability is a hallmark of cancer and ageing. Telomere length is maintained mainly by telomerase which is expressed in 85–90 % of human malignancies, although most human cancer types arise from somatic tissue that is telomerase negative. Thus, telomerase can serve as a nearly universal marker for cancer, as well as a drug target against tumour cells. Genome-wide studies in Saccharomyces cerevisiae identified approximately 7 % of the genome (roughly 400 genes) to be associated with telomere length maintenance (TLM genes).
Based on these studies, a strong enrichment of genes encoding endosomal sorting complex required for transport (ESCRT) factors has been found within the TLM genes that led to telomere shortening when deleted. ESCRT factors define a system of five multi-protein complexes which is involved in deforming and scissioning cytosol filled membrane stalks. Although they can be linked to genomic stability and integrity, only little literature exists on the relationship of telomere homoeostasis and the ESCRT machinery. The data presented here shows that the whole ESCRT system is required to safeguard proper telomere length maintenance. More in-depth experimental investigations of the ESCRT-0 factor Vps27 suggested a model of impaired Exo1-mediated resection resulting in too little telomeric overhang such that Cdc13 binding is suppressed, preventing either telomerase recruitment or telomeric overhang protection. A protein-protein-interaction network analysis indicated a potential feedback mechanism to regulate telomere homoeostasis via the ESCRT system.
The results from the present thesis uncovered the ESCRT system, especially Vps27, as promising drug candidate directed against malignant growth of cancer or diseases caused by dysfunctional telomeres.
BACKGROUND Cancer is a direct consequence of genomic aberrations, such as somatic copy number alterations that frequently occur in the cancer genome affecting not only oncogenic genes, but also multiple passenger and potential co-driver genes. An intrinsic feature resulting from such a disruption of the genome is deregulation of the tumor metabolic landscape, as a result of which, multiple metabolic genes have been identified as oncogenes, tumor suppressor genes or targets of oncogenic signaling.
RESULTS Here we elucidate that linear proximity of metabolic and cancer-causing genes in the genome can lead to metabolic remodeling through copy number co-alterations. We observed that cancer-metabolic gene pairs are unexpectedly often proximally positioned in the chromosomes and share loci with altered copy number, thus being either co-deleted or co-amplified across all cancers analyzed (19 cancer types from The Cancer Genome Atlas). We have developed an analysis pipeline - Identification of Metabolic Cancer Genes (iMetCG), to infer the functional impact on oncogenic metabolism from such co-alteration events and delineate genes truly driving cancer metabolism from those that are neutral. Using this approach, we have identified novel and well known metabolic genes that target crucial pathways relevant for tumors. Moreover, using these identified metabolic genes we were able to classify tumors based on its tissue and developmental origins. We further observed that these putative metabolic cancer genes (identified across cancers) had higher network connectivity, were indicators for patient survival, had significant overlap with known cancer metabolic genes and shared similar features with known cancer genes in terms of their isoform diversity, evolutionary rate and selection pressure.
CONCLUSIONS This thesis provides novel insights into the functional mechanism of metabolic regulation and rewiring of the metabolic landscape in cancer cells. Our pan-cancer, genomic data driven approach revealed a hitherto unknown generic mechanism for large scale metabolic reprogramming in cancer cells based on linear gene proximities and identified 119 new metabolic cancer genes likely to be involved in remodeling tumor cell metabolism. Furthermore, our newly identified metabolic cancer genes will serve as a vital resource to the experimental community engaged in tumor metabolism and genomics research to further expand the scope of this field.
Neurodegenerative diseases are a growing burden in the modern ageing societies. Especially Alzheimer’s disease (AD)—the most common form of dementia—has gained lot of attention lately. Although several drugs are available to enhance the life-quality of people with AD, none of them can stop the progression or cure this disease. Therefore new medications for treatment and prevention are needed. Medicinal plants are a rich source for drug leads and active compounds. Furthermore, plant extracts are potential multitarget drugs that can be particularly useful for diseases with complex pathology like AD. Therefore, in the present work plants from Traditional Chinese Medicine (TCM) were tested for their efficacy against two prominent pathological markers in AD: beta-amyloid (Aβ) aggregates and oxidative damage. For the present study the model organism Caenorhabditis elegans was deployed. In a screening of 55 TCM plant extracts on a C. elegans strain expressing human Aβ peptide in muscles, several extracts that could reduce Aβ aggregation were identified. From those the three most active ones were chosen for further evaluation. The methanol extract of Glycyrrhiza uralensis proved to have the best characteristics for therapeutic use. Additionally to the reduction of Aβ aggregates by 30 %, this extract could also counteract Aβ toxicity in a paralysis assay by increasing the mean time of paralysis (PT50) by 1.8 h and showed antioxidant activity in the heat shock protein (HSP) expression assay. The major compounds in the G. uralensis extract were identified via LC-MS/MS. Four substances—glycyrrhizic acid (GA), glycyrrhetinic acid (GRA), liquiritigenin (LG), and isoliquiritigenin (ILG)—were chosen as possible active compounds. From those ILG showed the strongest activity by reducing Aβ aggregation by 26 % and counteracting Aβ toxicity in paralysis assay (1.2 h delay in PT50). It also affected serotonergic neurotransmission in C. elegans with neuronal Aβ expression. Furthermore, significant antioxidant activity was shown in the HSP expression assay, and the survival of worms under oxidative stress was increased by 82 % after treatment with ILG. This compound could induce nuclear translocation of the transcription factor DAF-16 that is responsible for stress resistance and longevity in C. elegans. The mechanism of action of ILG in counteracting Aβ toxicity could therefore involve hormesis and modulation of serotonergic neurotransmission. The present work also reports for the first time the effect of Carlina acaulis against Aβ toxicity and its antioxidant activity in vivo. The dichloromethane extract of this plant delayed the Aβ-induced paralysis by 1.6 h. GLC-MS analysis identified Carlina oxide as the main compound in this extract. Carlina oxide alone was not as active as the extract in paralysis assay, but it was responsible for the antioxidant activity. Both the extract and Carlina oxide were active in the HSP expression assay and could induce DAF-16 delocalisation. The mechanism of action for C. acaulis against Aβ toxicity still needs further study, although hormetic effects and the antioxidant activity may contribute to this effect. The plants and compounds identified in this study should be considered for further investigation in vertebrate models. Especially their bioavailability and drug safety need broader attention. The initial results reported here suggest G. uralensis, C. acaulis, and ILG as possible candidates for prevention or treatment of AD. Their positive effects counteracting protein aggregation and oxidative stress might also be useful against other neurodegenerative diseases and for healthy ageing in general.
In recent years, simulations have steadily replaced real world experiments in science and industry. Instead of performing numerous arduous experiments in order to develop new products or test a hypothesis, the system to be examinded is described by a set of equations which are subsequently solved within the simulation. The produced vector fields describe the system's behavior under the conditions of the experiment. While simulations steadily increase in terms of complexity and precision, processing and analysis are still approached by the same long-standing visual techniques. However, these are limited by the capability of the human visual system and its abilities to depict large, multi-dimensional data sets.
In this thesis, we replace the visual processing of data in the traditional workflow with an automated, statistical method. Cluster algorithms are able to process large, multi-dimensional data sets efficiently and therefore resolve the limitations we faced so far. For their application to vector fields we define a special feature vector that describes the data comprehensively. After choosing an appropriate clustering method, the vector field is split into its features.
Based on these features the novel flow graph is constructed. It serves as an abstract representation of the vector field and gives a detailed description of its parts as well as their relations. This new representation enables a quantitative analysis and describes the input data. Additionally, the flow graphs are comparable to each other through a uniform description, since techniques of graph theory may be applied. In the traditional workflow, visualization is the bottleneck, because it is built manually by the user for a specific data set. In consequence the output is diminished and the results are likely to be biased by the user. Both issues are solved by our approach, because both the feature extraction and the construction of the flow graph are executed in an un-supervised manner.
We will compare our newly developed workflow with visualization techniques based on different data sets and discuss the results. The concluding chapter on the similarity and comparison of graphs applies techniques of graph theory and demonstrates the advantages of the developed representation and its use for the analysis of vector fields using flow graphs.
An angular analysis of the decay B0→ K*0mu+mu- is performed on a data set from LHCb Run 1 corresponding to a total integrated luminosity of 3 fb-1. A complete set of CP-averaged angular observables and their correlations are determined simultaneously in intervals of the dimuon invariant mass squared, q². Special care is taken to separate the two contributions in which the kaon and the pion either originate from a spin 1 or a spin 0 resonance.
The measurement reveals a good agreement with Standard Model predictions. However, observables S5 and S6s show small discrepancies with respect to the predictions in the q² region 1-6 GeV². The measurement is also performed in a second angular basis, confirming a previously observed local deviation in one of its observables, P'5. The agreement between the measurement of P'5 and predictions in the q² region 0.1-8 GeV² corresponds to a p-value of 8.2 10^-4 only. The observations are compatible with theoretical models which predict additional physics contributions at the TeV scale.
Reactive halogen species (RHS) have a significant impact on the chemical composition of the atmosphere. With its high halite abundance and unique topography the Dead Sea Valley (DSV) is predestined for the investigation of RHS. In the frame work of this thesis, two extensive measurement campaigns were carried out at the DSV in May 2012 and November 2014. The comparison of MAX-DOAS and LP-DOAS data with meteorological measurements indicates strong impact of transport process on the observed trace gas dynamics. The strong depletion of NO2 mixing ratios during daytime coincides with increased RHS abundance and suggests the formation of halogenated nitrate compounds. These are assumed to enhance the release of RHS from aerosol surfaces by heterogeneous processes. Elevated, confined layers of BrO of up to 50 pptv suggest the release of reactive bromine compounds from aerosol surfaces. The correlation of IO with surface waves indicates abiotic iodine release as a result of increased gas exchange at the water surface. For the first time, gaseous molecular iodine (up to 70 pptv) was detected at the DSV serving as a precursor for reactive iodine. Further, first direct evidence for reactive chlorine chemistry at the DSV was found by the detection of OClO at mixing ratios of up to 6 pptv.
The aim of this thesis is to study the role of interstellar turbulence in the process of star formation. We demonstrate that supersonic turbulent motions significantly affect various properties of the interstellar medium (ISM). Therefore, we run numerical simulations of molecular clouds in different environments. In particular, we study typical clouds located in the Milky Way disk as well as clouds which can be found in more extreme regions in our Galaxy, e.g. in the Central Molecular Zone (CMZ) near the Galactic Center. In addition, we perform radiative transfer calculations of numerous diagnostic fine structure lines and compare our results with observational measurements. Furthermore, we analyze the influence of the turbulence on different observables, e.g. on the structure functions, the ∆-variance, the power spectra as well as the star formation efficiencies. We also study the impact of turbulent motions on the chemistry and the different phases of the ISM. Our studies about Milky Way disk clouds show that the statistical properties of the turbulence are significantly influenced by the individual gas tracers. Moreover, our investigations about CMZ-like clouds show that high levels of turbulence can significantly suppress, but never inhibit star formation, owing to local compression of gas by turbulent shocks. Finally, we show that various atomic tracers accurately reflect most of the physical properties of both the H2 and the total gas of the cloud and that they provide a very useful alternative to common molecular lines when we study the ISM in the CMZ.
Die Entwicklung prädiktiver in silico Modelle mit unmittelbarer medizinischer Relevanz ist eines der wesentlichen Ziele der Systembiologie. Solche Modelle umspannen bei hinreichend komplexer Fragestellung mehrere biologische Organisationsebenen, um den meist vorhandenen Vorwärtsschleifen und Rückkopplungen der betrachteten funktionellen Prozesse des Organismus über diese Ebenen hinweg Rechnung zu tragen. Mit einem solchen, methodisch betrachtet, multiskalierten Modell, lässt sich eine semantische Brücke von der Subzell- zur Zell- bis hin zur Gewebe- oder sogar Organebene schlagen. Um die i.d.R. vielfältigen zu berücksichtigenden biologischen Prozessen auf unterschiedlichen räumlichen wie zeitlichen Skalen adäquat abzubilden, werden in multiskalierten Modellen eine Reihe unterschiedlicher Modellierungs- und Simulationsansätze kombiniert und semantisch miteinander verwoben. Dies erfordert nicht nur einschlägige Kenntnisse aus den Domänen Biologie und Medizin, sondern Erfahrung mit Methoden aus den Bereichen Biophysik, Biochemie, Mathematik und nicht zuletzt eine hohe technische Kompetenz in Sachen Programmierung sowie, bei größeren Projekten, Software Engineering. Diese vielschichtige Komplexität multiskalierter Modelle motiviert, wie auch wiederholt in der einschlägigen Literatur gefordert, die Bereitstellung von computergestützten Systemen zur Erstellung und Simulation derselben. Um diese an Bedeutung zunehmende Form der in silico Modellierung einem Personenkreis mit einem, fachlich gesehen, biologisch-medizinischen Schwerpunkt zugänglich zu machen, müssen solche Systeme die Komplexität der Modellerstellung insbesondere mit Blick auf die technische Umsetzung der Modellsimulation reduzieren. Das übergeordnete Ziel der vorliegenden Arbeit war daher der Entwurf und die Realisierung eines computergestützten Systems zur (multiskalierten) in silico Modellierung und Simulation (CMS) von Epithelgeweben. Die Fokussierung auf Epithelgewebe ist wegen deren Relevanz im Bereich der Onkologie und der Einbettung dieser Arbeit in einen Forschungskontext, der in der systembiologischen Erforschung der Epidermis seinen Schwerpunkt hat, vorgenommen worden. Neben der Gebrauchstauglichkeit, Flexibilität sowie Erweiterbarkeit soll insbesondere die Benutzerfreundlichkeit ein wesentliches Merkmal dieses Systems sein und eine Anwenderzielgruppe mit keinen oder nur geringfügigen Programmierkenntnissen im Fokus haben. Zu diesem Zweck sind neben verschiedenen Konzepten zur konkreten technischen Realisierung des Systems eine modulare multiskalierte (Gewebe-)Modellarchitektur und ein mehrstufiges modellgetriebenes Systementwicklungs¬konzept entworfen worden. Den Kern der Modellarchitektur bildet die grafische Modellierung des Verhaltens von räumlich diskret repräsentierten Zellen in einem multizellulären Kontext. Hierfür ist eine grafische Modellierungssprache entwickelt worden, mit der Zellverhalten in Form von Prozessdiagrammen deterministisch, stochastisch oder hybrid modelliert werden kann. Ein grafisches Zellverhaltensmodell (CBM) ist dynamisch an ein seitens des CMS bereitgestelltes, wählbar zwei- oder dreidimensionales, biomechanisches Modell (BM) gekoppelt. Die dynamische Kopplung ermöglicht potentiell die Wiederverwendung von CBMs in unterschiedlichem räumlichem Kontext. Ein BM bildet die räumlichen und biophysikalischen Zelleigenschaften auf Basis eines der hierfür entwickelten diskreten Zellmodelle ab. Das BM verbindet das zellbasierte CBM mit der Gewebeebene, da über dieses ein (biomechanischer) semantischer Bezug zu den benachbarten Zellen, bspw. durch Abbildung von Kontaktinhibition bei proliferierenden Zellen, und damit zur zellulären Mikroumgebung hergestellt wird. Ergänzt wird dieser wechselseitige Bezug von Zell- und Gewebemodellebene durch extrazelluläre Diffusionsfelder (DF), die ebenfalls modularer Bestandteil der Modellarchitektur sind. Mittels eines DF kann die Sekretion, Absorption, der Zerfall sowie die gewebeübergreifende Ausbreitung eines Stoffs abgebildet und simuliert werden, der im Modell einem Zytokin oder Chemokin entsprechen kann. Schließlich können quantitative subzelluläre Modelle (QSMs), die im weit verbreiteten Modellstandard Systems Biology Markup Language (SBML) vorliegen, semantisch in ein CBM integriert werden. Somit umfasst die modulare multiskalierte (Gewebe-)Modellarchitektur die Subzell-, die Zell- und die Gewebemodellebene. Die zuvor beschriebenen Teilmodelle CBM, BM, DF und QSM werden durch das entwickelte und realisierte Konzept der automatisch vom CMS erzeugten Modell-Konnektor-Komponenten (MKK) semantisch miteinander verbunden. Die MKKs übernehmen in diesem Kontext auch die Abbildung der unterschiedlichen räumlichen und zeitlichen Skalen der Teilmodelle. Das mehrstufige modellgetriebene Systementwicklungskonzept ermöglicht auf der ersten Stufe die Formalisierung der grafischen Zellverhaltensmodellierungssprache als, ebenfalls grafisches, Meta-Modell derselben. Aus diesem Meta-Modell können Softwarekomponenten, die die grafische Zellverhaltensmodellierungssprache technisch realisieren, automatisch erzeugt werden, wodurch eine flexible und effiziente Anpassung an neue Anforderungen möglich wird. Auf der zweiten Stufe wird das Zellverhalten mit dieser grafischen Sprache unter semantischer Einbindung der zuvor beschriebenen modularen Bestandteile eines multiskalierten (Gewebe-)Modells abgebildet. Aus dem grafischen CBM wird mittels eines hierfür entwickelten Code-Generators lauffähiger, bezüglich Rechenzeit optimierter Code erzeugt, der dynamisch in eine multiagentenbasierte Simulation dieses Modells integriert wird. Als Realisierung des zuvor skizzierten Entwurfs des CMS sind die beiden Endanwender-Softwaresysteme EPISIM Modeller als grafisches Modellierungssystem (GMS) und EPISIM Simulator als Simulationsumgebung (SE) entstanden. Mit dem GMS werden die (multiskalierten) grafischen CBMs erstellt, validiert, in lauffähigen optimierten Code übersetzt und in einer Modellarchiv-Datei gespeichert. Auf der Basis dieser Datei führt die SE eine multiagentenbasierte Simulation des Modells durch und bietet in diesem Zusammenhang die Möglichkeit, in Echtzeit das zwei- bzw. dreidimensionale Gewebe zu visualisieren und die Simulationsdaten zielgerichtet vorzuverarbeiten bzw. auszuwerten. Für das CMS ist eine Modellinfrastruktur geschaffen worden, die sich in Basismodelle für die SE und in BMs gliedert. Für die SE sind Basismodelle für Stoffaustausch zwischen Zellen und damit Zell-Zell-Kommunikation, ein Reaktions-Diffusions-Modell für die extrazellulären DF und in diesem Zusammenhang ein Sekretions- bzw. Absorptionsmodell entwickelt worden. Diese Modelle können über entsprechende Funktionen im GMS in einem CBM verwendet werden. Ferner sind ein gitterfreies sowie ein gitterbasiertes BM entwickelt und jeweils eine zwei- wie dreidimensionale Variante realisiert worden. Beim gitterfreien BM handelt es sich um ein Zell-Zentrumsmodell, das die Annahme einer elliptischen bzw. ellipsoiden Zellmorphologie und die Berücksichtigung bzw. Parametrisierung der Kräfte Zelladhäsion und Zellabstoßung erlaubt. Das gitterbasierte BM ermöglicht hingegen die Abbildung einer gerichteten Zellmigration in einem extrazellulären Stoffkonzentrations¬gradienten (Chemotaxis). Das CMS ist anhand einer Reihe von Anwendungsfällen der methodischen Ansätze und der bereitgestellten Modellinfrastruktur evaluiert worden. Diese Evaluation umfasste sowohl die Demonstration der Möglichkeiten des CMS, die Erprobung von deren technischer Umsetzung als auch die Anwendung des CMS im Kontext von forschungsrelevanten Fragestellungen. Die methodische Eignung zur multiskalierten zellbasierten Modellierung von Geweben, insbesondere die diesbezügliche Eignung der grafischen Zellverhaltensmodellierungssprache, ist anhand des von Grabe et al. 2005 veröffentlichten, zweidimensionalen in silico Modells der humanen epidermalen Homöostase erfolgt. Das Modell sowie die Simulationsergebnisse konnten mit dem CMS in vollem Umfang reproduziert werden. Darüber hinaus ist das Modell auf die dritte Dimension erweitert worden, indem das CBM an das dreidimensionale gitterfreie BM gekoppelt wurde. Damit wurde demonstriert, dass CBMs potentiell in verschiedenen räumlichen Kontexten simuliert werden können, ohne dass hierfür, abgesehen von Anpassungen an Modellparametern, strukturelle Anpassungen am Modell als solchem notwendig sind. Die Unterschiede zwischen zwei- und dreidimensionaler Simulation des Modells in Abhängigkeit von der Form der Basalmembran sind unter Nutzung der entsprechenden Möglichkeiten der SE ausführlich charakterisiert und dargestellt worden. Ein wesentliches Ergebnis hiervon war die Feststellung, dass die Homöostase der in silico Epidermis bei einer Basalmembran mit Reteleisten stabiler ist als bei einer flachen Basalmembran, wie man sie bspw. bei in vitro Vollhautkulturen vorfindet. Anhand verschiedener realisierter Zellzyklusmodelle wurde die semantische Integration von quantitativen subzellulären Modellen in diskrete CBMs demonstriert. Tysons Zellzyklusmodell bildete in diesem Zusammenhang einen Anwendungsfall, mit dem erfolgreich die automatische Abbildung verschiedener zeitlicher Modellskalen durch das CMS gezeigt werden konnte. Mit einen qualitativen in silico Modell der Reepithelialisierung akuter epidermaler Wunden konnte das CMS direkt in den systembiologischen Forschungskontext eingebracht werden. In einem neu etablierten in vitro Wundmodell, basierend auf Vollhautkulturen, ist die Reepithelialisierung im zeitlichen Verlauf anhand von histologischen Schnitten der kompletten Wunde untersucht worden. Auf Basis der so gewonnenen experimentellen Ergebnisse ist ein neuer Reepithelialisierungsmechanismus abgeleitet und postuliert worden. Mit dem in silico Modell wurde dieser Reepithelialisierungsmechanismus in einer zwei- und dreidimensionalen Simulation übereinstimmend reproduziert, was die theoretische Plausibilität des Mechanismus als solchem im Kontext der Publikation der Ergebnisse untermauerte. Neben verschiedenen Anwendungsfällen mit Test- und Veranschaulichungscharakter konnte mit dem gitterbasierten BM unter kooperativem Einsatz des entwickelten CMS im klinischen Forschungskontext ein T-Zell-Migrationsmodell realisiert und simuliert werden. Abgebildet wurde die gerichtete Migration von T-Zellen in einem chemotaktischen Stoffkonzentrationsgradienten in der Grenzregion von gesunder Leber und Lebermetastase eines kolorektalen Primärtumors. Von Interesse war die sich ergebende räumliche Verteilung der T-Zellen in Abhängigkeit von der räumlichen Dichte sekretorischer Zellen in der Grenzregion, die den Stoffkonzentrations-gradienten erzeugen. Die Simulationsergebnisse zeigten eine quantitativ signifikante Übereinstimmung der räumlichen T-Zell-Verteilung in Gegenüberstellung mit entsprechenden histologischen Schnitten von Biopsien aus der zuvor beschriebenen Grenzregion. Neben den zuvor geschilderten Anwendungsfällen wird die Gebrauchstauglichkeit des CMS auch durch den erfolgreichen Einsatz desselben durch Dritte u.a. im Kontext der Anfertigung von Masterarbeiten im Bereich der Systembiologie und einer medizinischen Dissertation demonstriert. Auf der Grundlage der Anwenderrückmeldungen ist das CMS kontinuierlich verbessert worden. Verschiedene im weiteren Sinne vergleichbare CMS sind zeitlich parallel in anderen Institutionen entstanden. Betrachtet man die Chronologie der Systempublizierung, dann kann festgestellt werden, dass das im Rahmen dieser Arbeit entwickelte CMS zu den ersten veröffentlichten Systemen einer einschlägigen wissenschaftlichen Gemeinschaft gehört, die sich kontinuierlich erweitert. Stellt man die Systeme vergleichend gegenüber, dann ergibt sich eine Zweiteilung in Frameworks, die sich an Anwender mit vergleichsweise soliden Programmierkenntnissen richten, und Endanwender-Softwaresysteme, wie das hier vorgestellte CMS. Unter den Endanwender-Softwaresystemen bietet dieses einen vergleich-baren Funktionsumfang mit Blick auf die bereitgestellte Modellinfrastruktur. Es ist jedoch das einzige mit einer grafischen Modellierungssprache, automatischer Codeerzeugung und weitreichenden Datenauswertungsmöglichkeiten in der Simulationsumgebung. Auch ist es das einzige, das eine automatische semantische Integration von SBML-basierten Modellen ohne Modellkonvertierung oder die Erfordernis manueller Codierung einer MKK zur Abbildung der zeitlichen Skalen bietet. Schlussfolgernd kann festgehalten werden, dass mit dem im Rahmen dieser Arbeit entwickelten CMS ein relevanter Beitrag zur Vereinfachung der Erstellung multiskalierter multizellulärer (Gewebe-)Modelle geleistet werden konnte. Das CMS als solches, dessen methodische Infrastruktur sowie die durch es bereitgestellten Modelle konnten in forschungsrelevanten Fragestellungen erfolgreich zur Erstellung und Simulation von in silico Modellen eingebracht und publiziert werden. Ausblickend ist ein erweiterter Einsatz des CMS in Forschungsprojekten abzusehen. Beispielhaft sei hier die kooperative Weiterentwicklung des in silico Modells der Epidermis genannt, das um das Stratum corneum als strukturelles Kompartiment und die explizite Abbildung eines Wasser- sowie pH-Gradienten erweitert wird.
Adipose tissues in mammals can be broadly classified into two main types: white and brown adipose tissue. Although both are defined as adipose tissues, they differ drastically in their function. The main function of white adipose tissues (WAT) is the storage of fat. Unlike its white counterpart, brown adipose tissue (BAT) specializes in burning fat via thermogenesis and is known to play an important role in non-shivering thermogenesis especially in hibernating animals and newborn babies. Recent evidence of functional BAT in adult humans and its ameliorating effect on metabolic disorders has brought BAT under the spotlight for treatment of metabolic diseases like obesity and type 2 diabetes mellitus. WAT also acts as an endocrine organ by secreting signaling molecules called adipokines such as leptin and adiponectin. Adipokines constitute the secretome of WAT and not only play an important role in WAT function but also affect whole-body energy homeostasis. Various studies have investigated the role of adipose tissue secretome in metabolic disorders like obesity and insulin resistance. The WAT secretome has also been extensively characterized in various settings such as in whole WAT, mature white adipocyte etc. However, the BAT secretome and its adipokines (‘batokines’) have not yet been investigated. Thus, the main aim of this dissertation work was a comparative study of the white and brown adipocyte secretomes using a combination of Click-iT® AHA labeling and pulsed-SILAC (stable isotope labeling by amino-acid in cell culture). In total 1013 proteins were detected and a subset of these proteins was selected based on their secretion with norepinephrine stimulation. An in vitro assay was developed and optimized to test their putative effect on insulin secretion. In addition, one of the secretome candidates, inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4) was investigated as a potential batokine and BAT activity marker. Although, the serum levels of ITIH4 did not correlate with BAT activity under cold stimulation, its expression was found to increase with adipogenesis and browning of white adipocytes. Using in vitro knockdown studies, a reduction in differentiation was observed which was characterized by reduction in mature adipocyte functions such as lipolysis, lipid and intracellular triglyceride storage, glucose uptake and lipogenesis. Therefore, rather than being a batokine, ITIH4 was shown to be important for adipogenesis and adipocyte biology. In summary, this dissertation sheds light on BAT secreted proteins and also introduces a new player in field of adipogenesis, both of which might have a significant impact in BAT biology and in the treatment of metabolic disorders like obesity.
Die Vergrößerung des Öffnungswinkels entlang des optischen Achse eines Mikroskopes durch die kohärente Überlagerung der Foki zweier sich gegenüberstehender Objektive (4Pi-Anordnung) ermöglicht die Erzeugung der lichteffizientesten und schärftesten Punktbildfunktionen, um die dreidimensionale Auflösung in der Fernfeldmikrokopie weit unter die Beugungsgrenze zu bringen. Um bei diesem Verfahren jedoch möglichst eindeutige Aufnahmen zu erhalten, müssen die detektierten Signale, von Hintergrundsignalen über- und unterhalb der Fokalebene getrennt werden. Bisher wurden deshalb hauptsächlich nur fixierte Proben untersucht, da deren Aufnahmebedingungen sehr gut kontrolliert werden können. In dieser Arbeit stelle ich ein hochauflösendes 4Pi-Verfahren vor, das Aufnahmen von lebenden Zellen mit vernachlässigbarem Hintergrundsignal und Lichtschäden ermöglicht. Basierend auf RESOLFT Mikroskopie mit schaltbaren fluoreszierenden Proteinen und Zweiphotonenaktivierung und in Verbindung mit modifizierten Probenprotokollen und optimierter Optik zur Vorbeugung von Aberrationen, ermöglicht es einen robusten Zugang zu dicht gepackten, entlang der optischen Achse ausgedehnten, zellulären Regionen, die bislang weitestgehend vermieden wurden. Die Auflösung und die Signalqualität der Aufnahmen konnten zudem weiter durch die zeitliche Auswertung des Auslesesignals, auf Grund der hohen molekularen Sensitivität der entwickelten Methode auf lokale Schaltraten der Proteine, verbessert werden.
Live cell imaging is a powerful tool for studying the distribution and dynamics of proteins. However, due to the difficulties in absolute quantification and standardization of data obtained from individual cells, it has not been used to map large sets of proteins that carry out dynamic cellular functions. Cell division is a good example of this challenge for an essential cellular function, as rapid changes in protein localization and protein interactions result in dramatic changes to subcellular structures and cellular morphology, which in turn influence the behavior of the enclosed proteins.
Here, I report an integrated experimental and computational pipeline to map the dynamic protein network of dividing human cells in space and time. Using 3D live confocal microscopy, I imaged human cell lines that stably expressed fluorescently tagged mitotic proteins throughout mitosis. To obtain the absolute quantities of protein abundance with high subcellular resolution over time, the microscopy pipeline was calibrated by fluorescence correlation spectroscopy (FCS). Cell and chromosome volumes were segmented as references of cellular context for temporal and spatial alignment based on fluorescent landmarks. Together with my colleague Julius Hossain, we computationally generated a canonical model of mitotic progression for both kinetics (“mitotic standard time”) and morphology (“mitotic standard space”) by averaging and kinetically and geometrically parametrizing many registered dividing cells. The resulting model enabled us to subdivide the mitotic process into 20 characteristic kinetic steps and integrate our complete proof of concept dataset of 13 mitotic proteins imaged in over 300 dividing cells, represented as the 3D protein localization probability of each protein over time.
To measure localization similarities between different proteins and make predictions about their dynamic interactions, the integrated data was then mined using supervised as well as unsupervised machine learning. The power of this approach was demonstrated by our ability to automatically identify the major subcellular localizations of all proteins in the dataset and quantify protein fluxes between subcellular compartments and structures. Due to the quantitative nature of our imaging data, we were able to estimate the abundance of each protein in mitotic structures and complexes such as kinetochores, centrosomes, and the midbody, and determine the order and kinetics of their formation and disassembly.
The integrated computational and experimental method I present in my thesis is generic and scalable and makes many dynamic cellular processes amenable to dynamic protein network analysis even for large numbers of components. The pipeline provides a powerful instrument for analyzing large sets of quantitative live imaging data of fluorescently tagged proteins. It allows the systematic mapping and prediction of dynamic protein networks that drive complex cellular processes such as mitosis, thus promoting our understanding of the mechanisms by which many molecules together achieve spatio-temporal regulation.
This work investigates the potential of an in-time parallelization of atmospheric chemical ki- netics. Its numerical calculation is one time-consuming step within the numerical prediction of the air quality. The widely used parallelization strategies only allow a limited potential level of parallelism. A higher level of parallelism within the codes will be necessary to enable benefits from future exa-scale computing architectures. In air quality prediction codes, chem- ical kinetics is typically considered to react in isolated boxes over short splitting intervals. This allows their trivial parallelization in space, which however is limited by the number of grid entities. This work pursues a parallelization beyond this trivial potential and investigates a parallelization across time using the so called “parareal algorithm”. The latter is an iterative prediction-correction scheme, whose efficiency strongly depends on the choice of the predictor. For that purpose, different options are being investigate and compared: Time-stepping schemes with fixed step size, adaptive time-stepping schemes and repro-models, functional representations, that map a given state to a later state in time. Only the choice of repromodels leads to a speed-up through parallelism, compared to the sequential reference for the scenarios considered here.
The African lithospheric continent has an extended history over 3.8 Ga and is tectonically active since more than 2.9 Ga. Ever since the topography of that continent was changing under influences of a series of endogenic (tectonic) and exogenic (surface) processes. Generally the earth’s topography has major influences on the planet, examples include but are not limited to species distribution, forest succession, erosion, sedimentation, fluvial systems and climate. The topographic changes are accompanied by rock exhumations in either way of endogenic forces or as response to exogenic processes. These exhumation events could be traced by low temperature thermochronology (LTT) techniques. The LTT techniques date the rock passing through a certain isotherm (closure temperature) and are used to quantify the cooling rates. The closure temperature is function of the applied LTT technique and mineral type. Combining these cooling ages and LTT data with the time-temperature (t-T) modelling enables visualizing and quantifying the rock movement through the upper crust. Therefore, these combinations were used to compare and reconstruct the topographic changes in key areas dominated by various geologic environments as response to different magnitudes from multiple landscaping processes in the African continent. Furthermore, the ability of LTT to answer difficult questions related to landscaping processes (e.g., landslide detection and quantifying and the endogenic-exogenic processes relationship) was also tested. Comparing and reconstructing rift flanks uplifted areas (the Albertine Rift; the Rwenzori Mountains and the Gulf of Suez; the Samra Mountain area) on an old craton revealed a relatively long cycle of life. The non-uniform uplift through fault-bounded blocks was the dominant mechanism of response for all the induced far-field continental scale tectonics and surface processes. Only a uniform uplift was demonstrative during the rifting event. The thermochronological record of the Samra area has started earlier with the East African Orogeny (EAO) plutonism and accretion. Afterwards, both areas (the Rwenzoris and the Samra) were affected by the post orogenic erosional event. Shortly after, each area of them was affected differentially by a series of far-field tectonic events. Then, the rift started to activate affecting the whole areas with corresponding uplift. While the Gulf of Suez was nearly deactivated by the movement along the Dead Sea transform fault at mid-Miocene. The movement along the footwall of Bwamba fault caused additional uplift to the Rwenzoris at the Pliocene. On the other hand, comparing and reconstructing volcanic islands (Fuerteventura and La Gomera; Canary Islands) on passive margin revealed a relatively short cycle of life. That cycle started by emerging, followed by formation of the shield stage with adding a huge amount of magmatic materials forming a highly topographic island (Fuerteventura; ~20 Ma, La Gomera; ~10 Ma). Afterwards, the topography destruction starts with landsliding (Fuerteventura; ≤20 Ma, La Gomera; ~7 Ma) when suitable topographic and climatic conditions, among others, were dominated. Then the volcanic island experience other cycle, starting with constructing high topography by feeding with new magmatic materials till the hot spot related magmatic activities transfer to other regions. That activity shift was recorded by a lateral movement of the Canary plume materials beneath northwest Africa to west the Mediterranean Sea produced a track of intraplate volcanism through its course. Furthermore, LTT techniques were able to detect, differentiate, and quantify different landscaping events (including landslides) with various magnitudes in different geologic environments. - In rifted regions; the Rwenzori Mountains have experienced 4 rapid cooling/exhumation events. 1) the Silurian-Devonian (420- 390 Ma) event associated with ~3.5 (1.5) km of rock uplift as response to the post Pan-African orogeny deep erosional event. 2) The Triassic (240-220 Ma) event that caused ~3.0 km of rock uplift associated with rapid cooling and a major erosional event at the end of the Karoo sedimentary regime. 3) The Eocene- Miocene (52-10 Ma) event resulted in an average rock uplift of ~3.0 (0.2) km, the Early Eocene tectonic events were associated with India drifting afterwards the Eastern Rift activity was started. 4) The Pliocene-Pleistocene event (3-2.5 Ma) caused ~≤2.0 km of rock uplift along the footwall of Bwamba fault. The last two exhumation events with ~5 km of corresponding rock uplift produced the exceptionally high Rwenzori Mountains in the EARS extensional regime as a rift flank within two stages. The latter movement caused the tilt uplifting in the western flank of the mountains. While, the Samra Mountain area has experienced 5 rapid cooling/exhumation events. 1) The Neoproterozoic (775-640 Ma) event caused ~5.8 (0.1) km of rock uplift as a response to the accretion and plutonism during the EAO. 2) The Cambrian-Devonian (507-457 Ma) event causing ~5.6 (0.2) km of rock uplift as response to the post-EAO erosional event. 3) The Carboniferous-Permian (390-230 Ma) event resulted in ~4.2 (1.6) km of rock uplift as response to the Hercynian tectonic event. 4) The Jurassic-Cretaceous (170-70 Ma) event resulted in ~2.9 (0.5) km of rock uplift as a response to the Gondwana breakup. 5) The Oligocene-Miocene (27-22 Ma) event causing rock uplift of ~1.3 (0.3) km as response to the rift initiation. Additional reheating event was reported in the time span extending between the uplift associated with the Gulf of Suez and the prior cooling event causing an average subsidence of ~0.6 (0.3) km. - The Albertine rift flanks uplift is double the Gulf of Suez related flanks uplift which suggests an additional heat component during the Albertine rift formation. That heat component resulted from being the corresponding mantle plume directly beneath the EARS and more than 2000 Km away from the Gulf of Suez (Afar plume). - In volcanic islands; Fuerteventura Island has experienced two rapid cooling/exhumation events; one has started ~20 Ma with ~2.7 (0.5) km of corresponding rock uplift that caused the onset of the Fuerteventura landslide. The other has been initiated ~7 Ma with ~2.3 (0.2) km of corresponding rock uplift forming the doming stage on the western part of Fuerteventura ~5 Ma. Finally, these domes were eroded to nowadays surfaces. La Gomera Island also has experienced two rapid cooling/exhumation events; the first event has started between ~10 and 7 Ma with corresponding ~2.7 (0.2) km of rock uplift causing the onset of the La Gomera landslide. The second rapid cooling event occurred by ~4 Ma resulting in ~2 km of rock uplift. Finally, this topography was eroded to reduce elevation to nowadays surfaces
Orientalist und Astronom, Dozent in Heidelberg. Jakob Christmann regte bereit 1590 die Einrichtung eines Lehrstuhls für arabische Sprache an, den er 1608 in Heidelberg erhielt. Er befasste sich neben seinen Sprachstudien mit Geometrie und Astronomie.
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Max Born studierte im Sommersemester 1902 in Heidelberg. Hier hörte er eine Vorlesung über Differentialgeometrie von Leo Koenigsberger.
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The present thesis reports on the world's first measurements of the second most important ozone-depleting halogen bromine at the entrance to the stratosphere (14 - 18.5 km, theta = 330 - 400 K) over the East and Central Pacific in late winter 2013. The measurements were performed within the NASA-ATTREX project from aboard the unmanned aerial vehicle Global Hawk. For the interpretation of the remote-sensing DOAS measurements of O3, NO2 and BrO, use of complementary measurements of brominated source gases (SGs) and dynamical tracers (e.g. CH3Br, halons, very short-lived species (VSLS), CH4) and model simulations of the chemical transport model (CTM) SLIMCAT/TOMCAT, is made. The agreement of measured and modelled CH4, O3, and NO2 shows that the major dynamicaland photochemical processes are represented well in the CTM. Considering surface concentrations of the brominated organic SGs of in total 20.5 ppt, the measured BrO mixing ratios (0.5 - 9.0 ppt) are well explained. An exception are regions where the contribution of the short-lived CH2Br2 or the partitioning of BrONO2 plays an important role. The present observations confirm previous findings on the formation of BrONO2 of our workgroup. Depending on the flight, a total bromine budget (Bry) in the tropical tropopause layer (TTL) of 20.3 ppt to 22.3 ppt is inferred. For each fligh the contribution to total bromine of the long-lived brominated SGs stays constant (CH3Br + halons = 14.6 ppt), while the amount of VSLS and inorganic bromine varies between 5.7 ppt and 7.7 ppt. Thus, the present observations set a tighter constraint on the role of bromine for ozone depletion in the TTL than previous studies.
Ludwig Boltzmann betrieb im Sommersemester 1870 postgraduale Studien in Heidelberg.
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The rational design of organic semiconductors for optoelectronic devices relies on a detailed understanding of how their molecular and morphological structure condition the energetics and dynamics of charged and excitonic states. Investigating the role of molecular architecture, conformation, orientation and packing, this work reveals mechanisms that shape the spatially resolved densities of states in organic, small-molecular and polymeric heterostructures and mesophases. The underlying computational framework combines multiscale simulations of the material morphology at atomistic and coarse-grained resolution with a long-range-polarized embedding technique to resolve the electronic structure of the molecular solid. We show that long-range electrostatic interactions tie the energetics of microscopic states to the mesoscopic structure, with a qualitative and quantitative impact on charge-carrier level profiles across organic interfaces. The computational approach provides quantitative access to the charge-density-dependent open-circuit voltage of planar heterojunctions. The derived and experimentally verified relationships between molecular orientation, architecture, level profiles and open-circuit voltage rationalize the acceptor-donor-acceptor pattern for donor materials in high-performing solar cells. Proposing a pathway for barrier-less dissociation of charge transfer states, we highlight how mesoscale fields generate charge splitting and detrapping forces in systems with finite interface roughness. The associated design rules reflect the dominant role played by lowest-energy configurations at the interface.
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Der Mathematiker und Physiker Daniel Bernoulli (1700-1782) aus der bekannten Schweizer Gelehrtenfamilie studierte 1718 in Heidelberg Medizin.
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The maintenance of pluripotency in embryonic stem cells (ESC) is regulated by a network of chromatin-associated proteins coordinated by three master transcription factors Oct4, Sox2 and Nanog. To understand how different states of pluripotency are established, I developed three methods for studying chromatin-associated proteins globally, protein-targeted and locus-targeted in mouse ESC. Firstly, to study chromatin protein composition in a global manner, for the first time I developed in-vitro enzymatic labeling of chromatin by biotinylated nucleotides using Terminal deoxynucleotidyltransferase (TdT). As a result, more than 5000 proteins were significantly enriched in mouse ESCs in comparison to the negative control omitting the biotinylation step. In addition to the canonical chromatin-binding proteins, SICAP suggests chromatin association of some unexpected proteins such as Fgf4, which is a growth factor. This observation was further verified by immuno-staining. Secondly, I combined SICAP with chromatin immuno-precipitation (ChIP-SICAP) to identify proteins that interact with a target protein specifically on chromatin. Using endogenous Oct4, Sox2 and Nanog (OSN) as the targets of ChIP-SICAP, I identified about 400 proteins, as the overlap of the three assays. These 400 proteins include a large number of established interaction partners of the target proteins known to participate in the core pluripotency network (e.g. Rex1, Prdm14, Tcf3, Sall4, Esrrb, Tbx3, Stat3 etc). To reveal the co-localization sites with OSN, I selected Trim24. Interestingly, using ChIP-seq it turned out that Trim24 co-localizes with OSN on many super-enhancers of pluripotency. Thirdly, I developed a method to identify proteins bound to the Nanog promoter using biotinylated oligonucleotides. The specificity of the method, called targeted isolation of genomic regions (TIGR), was validated using qPCR and high-throughput sequencing. Hence, several proteins have been identified that are known to bind to, and regulate transcriptional activity of Nanog. Comparing the meta-stable and the ground-state of pluripotency, TIGR identified several nucleoporins that associate with the Nanog promoter preferentially in the ground-state of pluripotency. Using ChIP-qPCR I could validate the association of Nup98 to the Nanog promoter. Taken together, the data generated by the aforementioned methods expand the circuitry of pluripotency, and shed a new light on the differences between the ground-state and meta-stable state of pluripotency. Additionally, the newly developed methods are highly generalizable and independent of cell culture or genetic engineering so that they can be used for studying diverse biological systems.
Das Anthrazyklin Doxorubizin ist eines der am häufigsten verwendeten Chemotherapeutika. Es wird allerdings in seiner Anwendung durch seine Kardiotoxizität, mit der Gefahr der Entwicklung einer ernsten Herzinsuffizienz, eingeschränkt. Da bislang nur eine symptomatische Therapie der Doxorubizinkardiomyopathie möglich ist, liegt der Fokus auf der Vermeidung hoher Doxorubizindosen. Die einzige präventive Therapie ist für ein großes Patientenkollektiv, Kinder und Jugendliche, nicht geeignet. Daher müssen neue Strategien für eine Protektion des Herzens gegenüber Doxorubizin gefunden werden. Einen interessanten Ansatz bietet hier die Übertragung natürlicher Resistenzmechanismen von Tumoren in eine kardiale Therapie. Das Chaperon "Glukose Reguliertes Protein 78" (GRP78) ist in vielen Tumoren hochreguliert und wird dort mit der Resistenz gegen Anthrazykline in Verbindung gebracht. Ziel der vorgelegten Arbeit war daher, das protektive Potenzial einer spezifischen, durch Adeno-assoziierte Viren vermittelten Überexpression von GRP78 gegenüber Doxorubizin im Herzmuskel zu untersuchen. Dafür wurden klinisch relevante in vivo und in vitro Modelle der Doxorubizinkardiotoxizität etabliert. Im in vitro Modell zeigte sich die Doxorubizinkardiotoxizität durch eine erhöhte Aktivierung von Apoptose. Im Zusammenhang mit der Toxizität war eine Akkumulation von p53 und die Aktivierung der "Ca2+/Calmodulin-abhängigen Proteinkinase II" (CaMKII) zu beobachten, was auf die durch Doxorubizin induzierte DNA-Schädigung und Störungen der Ca2+-Homöostase zurückzuführen ist. Analog konnten im Mausmodell nach der Doxorubizinbehandlung erhöhte Werte des kardialen Zelltodmarkers TroponinT (hsTnT) sowie eine beginnende kontraktile Dysfunktion festgestellt werden. Durch die Überexpression von GRP78 sank in vitro sowohl die Apoptose, als auch die p53 Akkumulation und die Aktivierung der CaMKII. Im in vivo Modell war die GRP78 Überexpression mit niedrigeren hsTnT-Leveln und einer signifikanten Verbesserung der Relaxation des Ventrikels verbunden. Darüber hinaus war eine deutliche Verlängerung des Gesamtüberlebens zu beobachten. In dieser Arbeit konnte somit erstmals gezeigt werden, dass eine kardiale Überexpression von GRP78 die Doxorubizinkardiotoxizität in vitro und in vivo verringert. Der protektive Effekt konnte über p53 und CaMKII mit der Regulation der Antwort auf DNA-Schädigung bzw. der Ca2+-Homöostase in Verbindung gebracht werden. Damit stellt diese Arbeit auch erstmals Hinweise auf einen Einfluss von GRP78 auf CaMKII-Aktivität vor und eröffnet zudem Möglichkeiten für neue präventive Maßnahmen und ein besseres Verständnis der Doxorubizinkardiotoxizität.
Microorganisms in nature live in interconnected communities, where the language of biochemistry creates means for communicating, fighting, and cooperating with each other. This work investigates one of the ways for microbial interactions - nutrient exchange. It is focus ed primarily on co - metabolism of Saccharomyces cerevisiae and lactic acid bacteria - Lactococcus la ctis and Lactobacillus plantarum - community whose composition was inspired by co - occurrence of yeast and LAB in a multitude of naturally fermented foods. Specifically, I w as interested in detecting metabolic interactions between budding yeast and lactic a cid bacteria, identifying transferred molecules, exploring metabolic mechanisms of their biosynthesis and excretion, and understanding possible causes behind them. A c ombination of experimental and computational methods was used to understand how nutritio nal dependencies shape communit ies of microorganisms. First step involved compos ing a synthetic community of common laboratory strains of yeast and lactic acid bacteria . Following a series of experiments with chemically defined media, LAB revealed their me tabolic dependency on yeast for growth and survival. This m ixed species community appears to be stable and is sustained through the flow of small nitrogenous molecule s from yeast to bacteria. Nutrient cross feeding was found to be a result of overflow meta bolis m in yeast, which release excess catabolit es under particular combinations of available nitrogen sources. The o bserved nutrient excretion involve s a set of genes that regulate yeast nitrogen metabolism when depleted of preferred nitrogen sources. We h ave recreated co - metabolism of yeast - LAB community, as well as multiple natural bacterial communities, with multi - species genome - scale metabolic modeling. Simulation results demonstrated a link between metabolic cross - feeding and species co - occurrence, and proved its high potential of the method for predicting metabolite exchange in microbial communities. In this project, the inter - kingdom model community of wild type microorganisms has been established and characterized. Peculiarities of yeast regulatory n etwork, in certain media compositions, cause “wasteful” excretion of amino acids and other metabolites. This in turn cr e ates a stable niche for growth of lactic acid bacteria , which are auxotro phic for multiple amino acids. D escribed scenario of metabolic dependency between yeast and lactic acid bacteria demonstrates how survival of one species can be driven by metabo lic idiosyncrasy of the other. The y east - LAB interaction is established instantly, and thus can serve as a first step in evolution of cooperat ion.
Sexual reproduction is a key evolutionary innovation which sets the ground for sexual selection. Sexual selection exhibits a strong dependence on the degree of competition in a mating population. The tie between active perception of competition and sexual behavior is a crucial process for intra and intersexual selection, however, its mechanisms remain largely unknown due to experimental intractability. Unicellular mating occurs under the same constraints but population and environmental parameters can be experimentally controlled and dynamic measurements of molecular and behavioral outputs can be performed. In this work, we propose that on the prototypical chemosensory mating system from Saccharomyces cerevisiae, the response magnitude generated by the presence of the complementary sex equals the probability of forming a sexual pair by chance. In chemosensory (pheromonal) mating systems, perception of competition as an indicator of mating likelihood is constrained by the following fact. Given that the most reasonable measure of the degree of competition/mating-likelihood in the population is the operational sex ratio, i.e. the fraction of individuals of a particular sex in the sexually active population (OSR in animals or theta in this work), sensory systems would need information about the abundance of individuals of both sexes, whereas the sexual response is induced by pheromones produced only by the opposite sex. Therefore, the OSR seems sensorially indistinguishable from the absolute number of potential mates, which would make mating likelihood imperceptible. By using experiments where the emitted pheromone concentration is isotropic and therefore does not depend on the distance separating mates, we manipulated population parameters and measured quantitative mating-pheromone pathway outputs to show that yeast is able to effectively sense the population sex ratio (theta) and the absolute mate number as separate cues by using a sensory disentangling mechanism. The mechanism is based on sensory input attenuation, i.e. the enzymatic degradation of the sexual pheromone produced by the opposite sex. As revealed by a simple physical model, the population displays specific sensitivities to sex ratio and cell density by modifying the time profile of pheromone concentration, with its maxima depending linearly on emitter cell density, and scaled by the inverse square root of receiver cell density. We show that in a random collision scenario the sex-ratio of the population indeed determines the likelihood of successful sexual pairing, matching the gene-expression response to sex ratio. Sensing mating likelihood allows control of mating investments, minimizing growth arrest and pathway overstimulation. Pheromone-based mate-sensing constitutes an example of a population-level fractional sensing mechanism, aided by the coupling of population-dependent signal attenuation and internal non-adaptive signal transduction. The study can be framed within the context of quantitative biology in its experimental methodology, and within (cellular) sensory systems, cell-cell communication and sexual selection theory because of its implications.
The human papillomavirus (HPV) belongs to the family of Papillomaviridae with more than 200 members, including HPVs but also papillomaviruses (PV) infecting for example cattle or rodents. Due to the causative association of HPV infection with the development of cervical cancer intensive investigation on HPV has been conducted over the last decades. Therefore, many aspects on the viral structure, infection as well as the transforming properties especially of the high risk HPV types have already been deciphered. In the course of these investigations, the HPV minor capsid protein L2 has been identified as an important player in the establishment of viral infection. Even though, the protein is dispensable for capsid formation, it has been demonstrated to have several functions crucial for e.g. DNA encapsidation, viral entry and the delivery of the viral genome to the host cell nucleus. However, the exact function of L2 during some of these processes is still unknown and under continuous investigation. In this context, many functional domains of the L2 protein have been identified especially in the highly conserved N-terminus of the protein however the function of the remaining parts is still unrevealed. Regarding the importance of the L2 protein for viral infection further investigation on potential functions still represents a promising field of research. The objective of this thesis was the identification of novel cellular interaction partners of L2. To this end, three independent experimental approaches were established, allowing the co-purification of interacting proteins. A) Tandem affinity purification (TAP) a two-step purification method based on the overexpression of HPV16 L2 as fusion protein with the TAP tag. B) Immunoprecipitation (IP) of HPV16 L2 from pseudovirus (PsV) infected cells, mimicking the natural infection pathway of HPV16. C) Peptide pull down of cellular proteins using immobilized HPV16 L2 epitopes which are targets for neutralizing antibodies. For identification of the co-purified interaction candidates the samples derived from the different experimental approaches were analyzed by mass spectrometry (MS).
The outer atmosphere of the first generation of low-mass stars retain to a great extent the original composition of the interstellar medium at the time and place of their birth. Hence the earliest phases of Galactical chemical evolution and nucleosynthesis can be investigated by means of studying the old, metal-poor stars. A minority of these stars exhibit dramatic enhancements in their abundances of heavy neutron-capture elements and/or of carbon. The key question for Galactic chemical evolution models is whether these peculiarities reflect the composition of the natal clouds, or if they are due to later (post-birth) mass transfer of chemically processed material from a binary companion. If the latter is the case, these stars should all be members of binary systems. This thesis presents high-resolution elemental-abundance analysis for a sample of 23 very metal-poor (VMP; [Fe=H] < -2.0) stars, 12 of which are extremely metal-poor (EMP; [Fe/H] < -3.0), and 4 of which are ultra metal-poor (UMP; [Fe/H] < -4.0). The results of radial velocity monitoring of 17 r-process enhanced stars (r-I and r-II stars), 24 CEMP-no stars, 18 CEMP-s and four CEMP-r/s stars, are also presented. The stars, for which the abundance analysis were performed, were targeted to explore differences in the abundance ratios of Li, C, N, O, the alpha-elements, the iron-peak elements, and a number of neutron-capture elements. These are elements that constrain the possible astrophysical sites of element production. This sample has substantially increased the number of known carbon-enhanced metal-poor (CEMP) and nitrogen-enhanced metal-poor (NEMP) stars. The sample of stars include eight that are considered "normal" metal-poor stars, six CEMP-no stars, �ve CEMP-s stars, two CEMP-r stars, and two CEMP-r/s stars. One of the CEMP-r stars and one of the CEMP-r/s stars are possible NEMP stars. Lithium is detected for three of the six CEMP-no stars, all of which are Li-depleted with respect to the Li plateau for metal-poor dwarfs found by Spite & Spite. This suggests that whatever site(s) produced C either do not completely destroy lithium, or that Li has been astrated by early-generation stars and mixed with primordial Li in the gas that formed the stars observed at present. Carbon and nitrogen abundances for the CEMP stars reveal, for the majority, that a small degree of mixing has happened in their progenitor stars ([C/N] > 0). However, signs of a larger degree of mixing ([C/N] < 0) is found in some CEMP-no stars, but these stars are only found at the lowest metalicities ([Fe/H] < -3:4). CEMP-no stars with large enhancements in Na, Mg, and Al are also only found below this metallicity. This sample confirms the existence of two separate bands in the absolute carbon abundances of CEMP stars, as suggested by Spite et al. The derived abundances for the alpha-elements and iron-peak elements of the stars are similar to those found in previous large samples of metal-poor stars. Finally evidence for a 'floor' in the absolute Ba abundances of CEMP-no stars at A(Ba) ~ -2.0 is also presented. Binary frequencies of 18%, 17%, and 75% are found for the r-process enhanced, CEMPno and CEMP-s stars, respectively. These results show that the nucleosynthetic processes, responsible for the strong carbon excess in the CEMP-no stars, and the r-process element enhancement in the r-I and r-II stars, are unrelated to their binary population. Instead, the element excess was imprinted on the natal molecular clouds of these stars by an external, distant source. The high frequency of binary stars found for the CEMP-s stars however, demonstrate that the peculiar abundance pattern of these stars is coupled to the binary nature of the stars.
Since both healthy human hematopoietic stem and progenitor cells (HSPC) and leukemia initiating cells (LIC) are sustained in a dormant state in bone marrow niche, they are protected against cytotoxic effects of chemotherapy. Thus, quantitative identification of differential adhesion of HSPC vs. LIC to bone marrow niche would help for the development of an effective clinical therapy of leukemia. The main aim of the present thesis was the fabrication and application of self-assembled, planar phospholipid membranes on solid support as in vitro model of bone marrow niche. A special focus was put on the influences of relevant ligand-receptor pairs and acute myeloid leukemia (AML) on the adhesion and morphological dynamics of HSPC. As the model of bone marrow niche, supported lipid membranes functionalized with N-cadherin and SDF1alpha were utilized to study their relative significance. In Chapter 2, the deposition of supported membranes and their quantitative functionalization with N-cadherin and SDF1alpha were confirmed by high energy specular X-ray reflectivity (XRR) and quartz crystal microbalance with dissipation monitoring (QCM-D). The fine structures perpendicular to the membrane surface and the lateral density of membrane-anchored proteins were determined by XRR with sub-Ångström resolution. Real-time monitoring by QCM-D of membrane deposition and functionalization demonstrated the quantitative variability of the average intermolecular distance <d> of proteins and elucidated their viscoelastic properties such as the shear elastic modulus and shear viscosity. In Chapter 3, the strength of HSPC adhesion was quantitatively evaluated by the determination of (a) the fraction of adherent cells, (b) the area of tight adhesion and (c) the critical force of cell detachment as a function of the average intermolecular distance <d> of N-cadherin nd SDF1alpha. The results clearly demonstrated that the binding of HSPC to the in vitro niche model was a positively cooperative process, and the adhesion mediated by the SDF1alpha/CXCR4 axis was stronger compared to adhesion mediated by the homophilic N-cadherin axis. The statistical image analysis of stochastic morphological dynamics unraveled that HSPC on in vitro niche models displaying SDF1a dissipated energy by undergoing oscillatory deformation, whereas cell locomotion mediated by the homophilic binding of N-cadherin was hardly impaired with morphological deformations. In order to verify the clinical relevance, the adhesion of leukemic blasts (LB) from AML patients was investigated in a systematic manner. In comparison to HSPC, LB exhibited a significantly higher affinity to the in vitro niche model reflecting the partial ineffectiveness of chemotherapy and the difficulties of replacing them by allogenic transplanted HSPC. The obtained results demonstrated that the combination of precisely defined cell surface models, a novel non-invasive assay for evaluating the cell adhesion strength, and statistical analysis of live cell images in Fourier space is a powerful tool to quantitatively analyze different functions of ligand-receptor pairs in bone marrow niche, which cannot be assessed by phenomenological observation.
The goal of this thesis is the development of a novel and efficient algorithm to determine the global optimum of an optimal control problem. In contrast to previous methods, the approach presented here is based on the direct multiple shooting method for discretizing the optimal control problem, which results in a significant increase of efficiency. To relax the discretized optimal control problems, the so-called alpha-branch-and-bound method in combination with validated integration is used.
For the direct comparison of the direct single-shooting-based relaxations with the direct multipleshooting-based algorithm, several theoretical results are proven that build the basis for the efficiency increase of the novel method. A specialized branching strategy takes care that the additionally introduced variables due to the multiple shooting approach do not increase the size of the resulting branch-and-bound tree. An adaptive scaling technique of the commonly used Gershgorin method to estimate the eigenvalues of interval matrices leads to optimal relaxations and therefore leads to a general improvement of the alpha-branch-and-bound relaxations in a single shooting and a multiple shooting framework, as well as for the corresponding relaxations of non-dynamic nonlinear problems. To further improve the computational time, suggestions regarding the necessary second-order interval sensitivities are presented in this thesis, as well as a heuristic that relaxes on a subspace only.
The novel algorithm, as well as the single-shooting-based alternative for a direct comparison, are implemented in a newly developed software package called GloOptCon. The new method is used to globally solve both a number of benchmark problems from the literature, and so far in the context of global optimal control still little considered applications. The additional problems pose new challenges either due to their size or due to having its origin in mixed integer optimal control with an integer-valued time-dependent control variable. The theoretically proven increase of efficiency is validated by the numerical results. Compared to the previous approach from the literature, the number of iterations for typical problems is more than halved, meanwhile the computation time is reduced by almost an order of magnitude. This in turn allows the global solution of significantly larger optimal control problems.
Primary cilia are whip-like structures, usually 1-3μm long, protruding from the cell membrane of almost all mammalian cells. During the last decades, it has been uncovered that primary cilium is crucial for Hedgehog (Hh) signalling. Its involvement in the appropriate function of Hh signalling pathway indicated by extension its importance in developmental processes. Midbrain dopaminergic neurons are responsible for the regulation of voluntary movements, as well as for cognitive functions such as emotion and reward. Studies have revealed that midbrain dopaminergic (mDA) neurons are dysfunctional in a number of neuropsychiatric or neurodegenerative diseases. Nowadays it is also widely known that Hh signalling is necessary for the generation and specification of dopaminergic (DA) neurons in the ventral midbrain. This thesis, through a collaboration with PD Dr. Sandra Blaess and her colleagues at the University of Bonn, investigates the role of primary cilia in the development of mDA neurons by using two mouse mutants defective in intraflagellar transport protein 88kDA (Ift88) or kinesin family member 3a (Kif3a), two genes important for the genesis and maintenance of the primary cilium. This is the first time that the role of primary cilia in the generation of mDA is examined. Study of conditional inactivation of Ift88 after embryonic day (E) 9.0 resulted in a progressive loss of primary cilia that was completed by E10.5 and a significant reduction of both mDA progenitors and mDA neurons at later stages, as observed by immunostainings against tyrosine hydroxylase (TH) – a typical marker for dopaminergic neurons and forkhead box A2 (FoxA2), a transcription factor induced by Sonic hedgehog (Shh) and commonly used as a floor plate marker. These observations show that functional primary cilia are necessary for the patterning of the ventral midbrain. Additionally, in situ hybridizations for glioma-associated oncogene 1 and -3 (Gli1 and Gli3) revealed that in the ventral midbrain of Ift88 knock-down (cko) mutants Shh signalling was inactivated. Shh pathway inactivation was attributed to the loss of primary cilia. Moreover, employment of a mouse model in which Smoothened (Smo) was constitutively active demonstrated that the constitutive activation of Shh signalling in these mice led to the expansion of the mDA precursor domain. However, this was not the case in the absence of Ift88; hence in the absence of primary cilia. This indicates that Smo acts downstream of Ift88 and by extension of primary cilia in Shh signalling. Surprisingly, conditional inactivation of Kif3a did not produce a similar phenotype of the mDA neurons. However, this could be attributed to the slightly delayed loss of primary cilia and the delayed inactivation of Shh signalling in Kif3a cko in comparison to Ift88 cko. Taken together, these results uncover the importance of the relationship between primary cilia and Shh signalling and identify for the first time a crucial window at which the two are critical for the induction of mDA neurons.
The study of elemental abundance ratios from spectroscopy of stars has for a long time been used to investigate the structure and the chemical evolution history of the Milky Way. However, even with the ever-increasing number of stars with detailed abundances, many details about the Milky Way evolution are still not understood. While elemental abundance measurements already provide a lot of information, nucleosynthesis models predict not only bulk abundances of an element, but also its isotopic composition. When these can be measured, additional details about the nucleosynthesis can be obtained. The isotopic composition of elements in stars has only been measured for the lightest elements and even for these, observations of the highest quality are needed. In addition, detailed modeling of the line-formation in the stellar atmospheres is needed to correctly interpret the data.
The purpose of this thesis is to: - Investigate the chemical evolution history of the massive, high metallicity globular cluster 47 Tucanae, by performing an extensive study of a range of elements in cool giants. - Perform the first study of Mg isotopes in this cluster, to further constrain its chemical evolution history. In addition, this work represents the first study ever of the effects of using 3D stellar atmospheric models to derive the Mg isotopic mixture. - Perform the first study of Mg isotopes in stars in the inner disk of the Milky Way and the Milky Way bulge, including stars in the globular cluster NGC 6522. - Demonstrate that this type of study is feasible for stars in the bulge, and show how the Mg isotopic ratios can be used to constrain chemical evolution models for this part of the Galaxy.
The Mg isotopic ratios were successfully measured in all 21 observed stars and provided additional constraints on the chemical evolution history of 47 Tucanae. In addition, the first results for Mg isotopes with 3D stellar atmospheres gave improved fits to the MgH molecular features, compared to 1D. This also resulted in an increase of the measured fraction of 25Mg, improving the agreement with chemical evolution models.
For the inner disk and the bulge, we reached a level of accuracy on the Mg isotopic ratios that will allow us to distinguish between different chemical evolution models. For the one field star in the bulge, we see an indication of more efficient star formation compared to the disk, but a larger sample of stars is needed before firm claims can be made.
This thesis presents the measurement of the CP-violating asymmetry in B0-B0bar mixing using data corresponding to an integrated luminosity of 3.0 fb^{−1} collected at the LHCb experiment in proton-proton collisions at the center-of-mass energies of 7 TeV and 8 TeV. This analysis uses untagged, semileptonic B0 → D−μ+ν and B0 → D∗−μ+ν decays, where the D− decays into K+π−π−, and the D∗− decays into D0(→ K+π−)π−. The neutrino in the semileptonic B decays is not reconstructed. A decay time dependent fit allows to disentangle the CP asymmetry from the possible B0-B0bar production asymmetry. Detection and reconstruction asymmetries are calibrated using promptly produced Cabibbo-favored D+ decays, and inclusive secondary J/ψ decays. The CP-violating asymmetry is measured to be a^{d}_{sl} = (−0.02 ± 0.19 (stat) ± 0.30 (syst))% . This result is consistent with the Standard Model prediction, and it is the most precise measurement from a single experiment to date. This measurement is published in Physical Review Letters
Tools from materials science are increasingly used to decrease the variability that often limits the quantitative analysis of cell experiments. Most prominently, micropatterned substrates can be used to normalize cell shape and internal organization. For long-term experiments, however, cells must be able to divide and migrate. This creates the need to design networks of micropatterns that ensure normalization to be maintained even in a dynamic context. The design of such networks requires an efficient model predicting the degree of normalization for dividing and migrating cells. Here we extend earlier formulations of the two-dimensional cellular Potts model with the aim to achieve good agreement with existing cell experiments on cell shape and forces on micropatterns. We show that our model correctly predicts cell spreading, division and migration on micropatterns, both for single cells and cell communities. The inverse problem of network optimization is then addressed with genetic algorithms.
The p53 protein is one of the most well-known tumor suppressor proteins, and it plays a variety of functions in somatic cells. Once activated, p53 induces cell cycle arrest and inhibits cell proliferation. Since it was found that p53 is highly expressed in murine embryonic stem cells, a cell type that proliferates very fast because of a shortened G1 phase, it remained a mystery whether p53 is active in this cell type. I observed that a significant part of p53 is localized in the nucleus of murine embryonic stem cells and that the majority of this nuclear p53 is bound to DNA. In addition, the anti-proliferative activity of p53 is compromised in stem cells, and this control is due, at least in part, to the high amount of MDMX that is present in embryonic stem cells. This high amount of MDMX is most likely due to exclusion of exon 7 of the MDMX RNA during retinoic acid induced differentiation. MDMX co-eluted with p53 from sucrose gradient assays and downregulation of MDMX in mESCs increased MDM2 abundance, a transcriptional target of p53, indicating that MDMX controls p53’s transcriptional activity in stem cells. P53 is posttranslationally modified in mESCs and these modifications endow a neutral isoelectric point (pI) of a fraction of the p53 protein that is only present in stem cells. Moreover, according to its nuclear localization in mESCs, p53 influences the transcriptome of mESCs. However, in contrast to the anti-proliferative activity that p53 has in differentiated cells, p53 controls transcription of pro-proliferative genes in embryonic stem cells including c-myc and c-jun. Chromatin-Immunoprecipitation showed that p53 binds to the responsive element of these proto-oncogenes. The impeded anti-proliferative activity of p53 and the induction of certain proto-oncogenes by p53 in murine embryonic stem cells can explain why stem cells proliferate efficiently despite having high levels of p53.
Apomixis refers specifically to asexual reproduction through seed in plants. Like other modes of asexual reproduction it has received much attention from evolutionary biologists and has been subject of many studies throughout the last decades. At the same time, it attracts interest from an economic point of view, as the long-term goal to technologically induce apomixis in major crop plants offers the prospect of a potential agricultural revolution. Hence, interests have been growing in the scientific community in order to elucidate the evolution and underlying molecular genetic mechanisms of apomixis. Here I present a multifaceted approach to the problem by (1) the development of biotechnological tools in order to (2) apply molecular evolutionary methods to narrow down the possible causes and consequences of asexual reproduction in plants. In this work, representatives of two genera (Ranunculus L. and Boechera Á. LÖVE & D. LÖVE) were studied in order to advance current apomixis knowledge from different perspectives. In the framework of a microarray based transcriptomic analysis of ovules extracted from sexual and apomictic Boechera, a list of housekeeing genes (HKGs) was selected based on a stability of expression analysis subsequently conducted in different vegetative and reproductive tissues of apomictic and sexual species. Using a GeNorm algorithm, different combinations of HKGs were identified, including Ribosomal Subunit 18 (BoechRPS18), Elongation Factor Alpha 1 (BoechEfα1), Actine 2 (BoechACT2) and Ubiquitine (BoechUBQ), based on their pairwise stability in ovules, anthers, and vegetative tissue in apomictic and sexual species. These genes, specifically chosen to be reproduction mode- and tissue-specific, have subsequently been used for normalization in the experimental validation of two candidates genes related to apomixis in Boechera Next, molecular evolutionary causes and consequences of apomixis were investigated by analyzing the transcriptomic effects of asexual reproduction and its correlated traits (i.e. hybridization, polyploidy and mutation accumulation). Flower-specific RNA from sexual and apomictic species of the wild apomictic Ranunculus auricomus complex was used for high throughput transcriptome sequencing (RNAseq). The first de novo assembled transcriptome for these species was used as a reference sequence for mining Single Nucleotide Polymorphism (SNP) and insertion-deletion xii (indel) polymorphisms. The data were further used to design and manufacture a custom 3 x 1.4 million spot expression microarray. Comparative SNP analysis between apomictic and sexual individuals (specifically, two apomicts from two populations and three sexuals from three populations) corroborated the hybrid origin of apomictic Ranunculus, as proposed by Paun et al. (2006b), and could furthermore elucidate the Pleistocene origin and subsequent divergence of the apomictic individuals. In addition, sites of divergent selection were detected with the analysis of non-synonymous (dN) to synonymous (dS) substitution rates, strengthening the idea of rapid divergence in the hybrids. Finally, the custom microarray was used for the hybridization of RNA from live-microdissected ovules (four developmental stages) from the three apomictic and four sexual individuals used in the SNP analysis. The comparative stage specific transcriptome analysis was used to detect stage specific differentially expressed genes in ovules, in order to identify signatures of apomixis and to produce a list of potential candidates underlying the reproductive switch. 555 stage specific genes were found to be differentially expressed throughout ovule development, and eight genes showed a significant shift in expression pattern throughout ovule development in apomicts compared to sexuals. A further classification was conducted following the predictions made from Nogler’s extensive work in Ranunculus in which different genetic factors were proposed for the induction and penetrance of apomixis. In that light, differentially expressed homoeologous genes were classified into three categories based on their relative expression in apomicts compared to their phylogenetic sexual parent, with the final aim of classifying the number of genes potentially responsible for apomixis. In doing so, we have provided a solid base for future studies in wild (i.e with little or no genetic information available) Ranunculus species. By developing biotechnical tools for their study, identifying genes potentially involved in the establishment of apomixis, and analyzing their evolutionary history, this work presents an important step towards a more comprehensive understanding of the processes and patterns connected to apomixis in model and non-model plants, and has far-reaching potential for agricultural use.
Einer Anregung Ernst Hoffmanns folgend, gab die Heidelberger Akademie der Wissenschaften ab 1927 die Werke Nikolaus Cusanus', des vielseitigen Gelehrten des 15. Jahrhunderts in einer kritischen Gesamtausgabe heraus.
Der Präsident bzw. geschäftsführende Sekretar der Akademie referierte (mit wenigen Ausnahmen) jedes Jahr in seinem Jahresbericht über den Fortschritt der Cusanus-Edition der Heidelberger Akadmie der Wissenschaften. In den ersten Jahren waren diese Berichte sehr ausführlich; ab 1933 hatten sie einen Umfang von 1/3 bis 1/2 Seite.
Aus Briefen und unveröffentlichten Dokumenten können wir die langen und schwierigen Verhandlungen rekonstruieren, die zu Helmholtz' Berufung an die Universität Heidelberg und später zu seiner Berufung nach Berlin führten.
Bereits 1851 hoffte Helmholtz auf eine Heidelberger Berufung. Jedoch wurde aus diesen Plänen nichts, als sein Fürsprecher J. Henle 1852 Heidelberg verließ. Erst 1857 nahm die Regierung des Großherzogs von Baden wieder Berufungsverhandlungen mit Helmholtz auf. Es war hauptsächlich R. W. Bunsen, der Chemiker und Freund Helmholtz', dessen private und amtliche Fürbitte für die Ernennung von Helmholtz entscheidend war. Es folgten ermüdende taktische Manöver von Helmholtz und der preußischen Regierung. Helmholtz wollte Bonn (Preußen) verlassen, Prinz Wilhelm und Königs Friedrich Wilhelm IV. von Preußen wollten ihn behalten. Im September 1858 war Helmholtz endlich in der Lage den neu eingerichteten Lehrstuhl für Physiologie in Heidelberg zu ungewöhnlich günstigen Bedingungen zu übernehmen: er erhielt ein großzügiges Gehalt, ein neu erbautes Institut ("Friedrichsbau") und eine beträchtliche Summe für dessen Ausstattung/Unterhalt.
Nach wiederholten Versuchen der preußischen Regierung und auf ausdrücklichen Wunsch des Deutschen Kaisers Wilhelm I., verließ Helmholtz im Jahr 1871 Heidelberg und übernahm zu besonders günstigen Bedingungen den Lehrstuhl für Physik in Berlin.
H. Weber betont die Rolle der Wahrscheinlichkeitsrechnung bei der Auffindung von Naturgesetzen und führt als Beispiel die Planetenbewegung an.
The prevalence of cutaneous squamous cell carcinoma (cSCC) is constantly increasing worldwide, however, the mechanisms driving tumorigenesis are not yet understood. The aim of this study was to understand the contribution of the Wnt/Beta-catenin and the Ras-Raf-MEK-ERK mitogen-activated protein kinase (MAPK) signaling pathways in cSCC development and progression using 2D keratinocyte monolayer cultures, 3D organotypic cultures of the skin (OTCs) as well as primary human cSCC samples.
Deregulated canonical Wnt signaling in tumor cells, as detected by nuclear translocation of Beta- catenin, is well described for a number of human cSCCs. Here, we found that aberrant Wnt/Beta- catenin activation is not restricted to tumor cells, but was additionally or exclusively detected in stromal fibroblasts. This suggested a second role of aberrant Wnt signaling in the stroma of developing cSCCs. Gene expression analysis of fibroblasts identified Interleukin-8 (IL-8), C-C motif chemokine 2 (CCL-2) and Matrix metalloproteinase-1 (MMP-1) as targets of Wnt/Beta-catenin. In agreement, IL-8 and CCL-2 were secreted by Wnt-3a-stimulated fibroblasts in OTCs and could also be detected in human cSCCs in situ. As consequence, IL-8 and CCL-2 caused hyper-proliferation and early invasion of non-tumorigenic HaCaT keratinocytes. Importantly, neutralizing antibodies against IL-8 and CCL-2 abolished these Wnt- dependent effects in OTCs. MMP-1 was also highly expressed in Wnt-3a-stimulated OTCs as well as in human cSCCs and was shown to be active. Furthermore, degradation of the basement membrane correlated with invasive growth of benign-tumorigenic H-Ras G12V -transfected HaCaT-RAS A-5 cells, demonstrating the importance of the Wnt-dependent fibroblast-keratinocyte cross-talk for skin cancer progression.
Ras-Raf-MEK-ERK MAPK signaling is constitutively active in most melanomas due to a B-Raf V600E - mutation leading to increased proliferation and survival. Clinical trials targeting oncogenic B-Raf in melanoma by mutation-specific inhibitors like Vemurafenib displayed high response rates, but caused at the same time development of cSCCs. This suggested a direct involvement of Vemurafenib in the progression of pre-existing lesions. Vemurafenib stimulation of B-Raf V600E -mutant A375 melanoma cells confirmed a block in MEK-ERK signaling, while all B-Raf wild-type cells, such as dermal fibroblasts and different keratinocytes, revealed MEK-ERK hyper-activation. Despite MAPK-activation, neither proliferation nor survival was altered in the keratinocytes. Analyses of continuously Vemurafenib treated HaCaT cells did not show genomic instability, but rather led to a selective growth advantage of genetic subpopulations. To study the role of aberrant B-Raf signaling in the tissue context, OTCs were treated with Vemurafenib. As result, Vemurafenib directly increased differentiation of the normal and transformed keratinocytes, resembling the hyperkeratotic skin phenotype of many melanoma patients. Remarkably, Vemurafenib stimulation of HaCaT-RAS A-5 cells caused high MMP-1 and MMP-3 expression, which correlated with Collagen remodeling and invasion into the stroma. Since both, differentiation and invasion were abolished by co-treatment with the MEK-inhibitor Cobimetinib and as patients receiving the same therapy showed highly decreased cSCC numbers, this strongly suggests that the phenotypes in in vitro as well as the rapidly developing well-differentiated cSCCs in vivo are direct results of Vemurafenib-induced ERK-activation.
Taken together, this work unraveled two novel aspects, namely Wnt/Beta-catenin deregulation in the tumor-stroma and aberrant Raf-MEK-ERK MAPK signaling in keratinocytes as oncogenic events driving cSCC progression.
Medicinal plants still represent an interesting and a challenging field of science. Plants generally contain complex mixtures of biologically active secondary metabolites with multiple target effects and often low toxicity. In this study, well-known analytical, chemical and biological methods were used in order to investigate the phytochemistry and bioactivities of secondary metabolites of 28 medicinal plant from the flora of Tajikistan and Germany. Many of these plants are endemic plants of Tajikistan and Central Asia, which are widely used in traditional medicine. For the first time, the chemical composition and bioactivity of the essential oils of Ferula clematidifolia, Galagania fragrantissima, Philadelphus x purpureomaculatus, Polychrysum tadshikorum and Salvia discolor were investigated. The main component of the essential oils of Ferula clematidifolia, Galagania fragrantissima, Philadelphus x purpureomaculatus, Polychrysum tadshikorum and Salvia dsicolor were analysed by GLC-MS and identified as pinene, 2E-dodecenal, viridiflorol, terpinen-4-ol and trans-caryophyllene, respectively. The antioxidant activity of essential oils are strongly dependent to the presence of phenolic metabolites. Essential oil of Origanum tyttanthum which contain phenols (carvacrol and thymol) exhibited a high antioxidant activity with an IC50 value of 0.1-0.3 mg/ml. Also the antioxidant activity of methanol extracts is directly proportional to their phenol contents. Galagania fragrantissima essential oil (GFEO) was very active against gram-positive bacteria (methicillin resistant Staphylococcus aureus). MIC and MBC values were 0.04 and 0.08 mg/ml respectively. GFEO shows high anti-inflammatory activity, it inhibited 5-LOX enzyme with an IC50 value of 7.3 μg/ml. Essential oils from Origanum tyttanthum, Galagania fragrantissima and Mentha longifolia exhibited a high cytotoxic effect against five human tumour cell lines (HeLa, CaCo-2, MCF-7, CCRF-CEM and CEM/ADR 5000). Their IC50 values ranged between 7.5-78 μg/ml. The combinations of doxorubicin with essential oils of Mentha longifolia, Anethum graveolens, Origanum tyttanthum, Galagania fragrantissima and Artemisia absinthium exhibit synergistic activity. The ratio of IC50 values of doxorubicin could be enhanced in dual combinations with essential oils 3-15 fold. Synergistic effects of essential oils seem promising area for future research. Methanol extracts from Polychrysum tadshikorum and Tanacetum parthenium were most cytotoxic against CCRF-CEM and CEM/ADR 5000 cell lines. Their IC50 ranged between 7.3-32.5 μg/ml. Results of cytotoxicity for the total extracts show that CCRF-CEM cells, which do not express ABC transporters were more sensitive than the derived CEM/ADR5000 (multidrug resistant) cells. This indicate that some components of the extracts are substrates of ABC transporters. In general, essential oils from Galagania fragrantissima, Origanum tyttanthum and methanol extracts from Polychrysum tadshikorum and Tanacetum parthenium are interesting candidates for a use in phytotherapy.
The study of emotional body language has been the effort of many scientists for more than 200 years, from areas such as psychology, neuroscience, biology, and others. A lot of work has focused on the analysis of the kinematics, while the study of the underlying dynamics is still largely unexplored. In this thesis we model human walking as a nonlinear multi-phase optimal control problem to investigate the dynamics of full-body emotional expressions in human locomotion. Our approach is based on rigid multibody dynamics, a highly parameterized mathematical model of the human locomotion system, and the direct multiple-shooting method to analyze the dynamics of recorded kinematic motion capture data.
Modeling the dynamics of a human rigid multibody model results in a set of highly complex differential algebraic equations that require automated methods to derive and evaluate. We created a new rigid multibody dynamics software package to model and numerically evaluate kinematic and dynamic quantities of rigid multibody systems expressed in generalized coordinates, including modeling of external contacts and discontinuities arising from contact events. Our package evaluates components of the equation of motion for multibody systems using recursive algorithms that are based on Featherstone's 6-D spatial algebra notation. Our package is specifically tailored for the use in numerical optimal control and carefully designed to exploit sparsities and reduction of redundant computations by selectively reusing computed values. By doing so we are able to achieve and partially exceed performance that is otherwise only available with source code generation modeling approaches.
We created a highly parameterized 3-D meta model for the human locomotion system. This rigid multibody model is based on biomechanical data for kinematic and inertial parameters and enables us to create subject-specific dynamic models by adjusting segment dimensions, joint locations, and inertial parameters. To describe the contact between the human model and the ground, we created a non-holonomic rigid body contact model specifically for human walking movements that approximates the foot geometry using a sphere for the heel and a line segment at the ball of the foot during forefoot contact.
Transforming motion capture marker data to rigid multibody motion is a difficult problem due to unknown joint centers, redundant marker movements, and non-rigid movement of markers as a result of skin and tissue movement. In this thesis, we developed and implemented a semi-automatic method in which we manually adjust the model to approximate the recorded subject and then compute joint angles by solving a non-linear least-squares optimization problem. Our approach is independent of the used motion capture marker set and directly maps onto the joint space of the model.
We formulate two types of multi-phase optimal control problems for human walking: an inverse reconstruction problem and a gait synthesis problem that both have the differential equations of the rigid multibody dynamics as a constraint and can be used for different purposes.
The reconstruction problem computes the unknown joint actuations from purely kinematic motion capture data. Applied to the recorded motion capture data, the reconstructed joint actuations show emotion specific features that are also found in the recorded muscle activity. This validates our model and approach to use optimal control problems as a tool to study emotional body language in a new way.
Our gait synthesis formulation allows the generation of walking motions solely based on mathematical and physical principles. It can be applied in computer animation, robotics, and predictive gait analysis. We have generated a wide range of motions by adjusting objective function and gait parameters. A long-term goal of this formulation is to investigate optimality criteria of emotional walking motions. For this, we aim to use hierarchical optimal control problems in our future works.
The interferon system functions as a first line of defense against viral infections. The cellular recognition of viruses leads to the production of interferons (IFN) by the infected cells. Secreted IFN stimulates an antiviral response in an autocrine and paracrine manner: While autocrine IFN action inhibits virus production in infected cells, paracrine IFN signaling induces an antiviral protective state in naïve cells. Although central molecular components of the IFN system have been characterized, our quantitative understanding of its dynamics remains limited. In particular, it is not precisely known which molecular processes are decisive for the outcome of virus-host interactions.
Together with our experimental cooperation partners, we have studied virus-induced IFN signaling at single-cell resolution in communicating cell populations after infection with a non-spreading and a spreading virus. On this basis, we established two complementary mathematical models. First, we developed a stochastic multi-scale model accounting for the intracellular dynamics in individual cells and the cell-to-cell communication via secreted IFN. Second, we constructed a delay differential equation model to analyze the competition between viral spread and IFN-induced antiviral defense in a cell population. Both models were parameterized on the basis of original experimental data and the numerical analyses of the models aimed at deriving testable predictions for new experiments.
By live-cell imaging, we showed that key steps of the IFN pathway including virus-induced signaling, IFN expression, and induction of IFN-stimulated genes are stochastic events in individual cells. To relate the single-cell data after primary infection to antiviral protection at the cell-population level, we established a stochastic model which combines the heterogeneous IFN signaling in single cells with the intercellular communication through released IFN. The parameters describing the virus-induced activation of the transcription factors of the IFN gene were estimated from a distribution of observed single-cell activation times using as objective function Neyman’s chi-squares statistic. The minimization of this objective function by simulated annealing revealed that virus-induced signaling is cooperative. Moreover, fitting of the measured time delays between transcription factor activation and IFN gene induction in individual cells with a gamma distribution by applying the maximum-likelihood method implies that IFN gene induction downstream of transcription factor activation is a slow multi-step process. Notably, mathematical modeling and experimental validation indicate that reliable antiviral protection in the face of multi-layered cellular stochasticity can be achieved by paracrine propagation of the IFN signal. Therefore, a few IFN-producing cells are able to protect a large number of naïve cells (Rand, Rinas et al. 2012).
To investigate the competition between viral spread and IFN-induced antiviral defense, we examined virus-host interactions after infection with spreading Dengue virus. For this purpose, our experimental partners generated data showing the antiviral response dynamics of fluorescent reporter cells after infection with a fluorescently labeled Dengue virus. Based on these kinetic data, we established a delay differential equation model with time delays for virus replication, virus production and IFN secretion. Using data-driven least-squares fitting and profile likelihood analysis, we identified the model parameters within narrow confidence bounds and found that the timing of virus production and IFN secretion after infection are almost identical. This direct competition together with the highly heterogeneous IFN response in single cells fosters the coexistence of IFN-induced protection of naïve cells and viral spread in non-protected cells. To analyze which components of the antiviral IFN system have the greatest influence on viral spread, we compared the infection dynamics of the wild-type Dengue virus with the attenuated spread of the vaccine candidate Dengue virus E217A mutant. We quantified the differences between wild-type and mutant Dengue virus infections using data-driven parameter optimization constrained by the determined wild-type virus related parameter values. In this way, we identified two mutant virus specific parameters, which explain the attenuation of the mutant through a reduced virus production rate and an accelerated IFN secretion taking place much earlier than virus production. By mathematical modeling and validation experiments, we predict that rapid IFN action curbing virus production in infected cells is critical for the attenuation of the Dengue virus E217A mutant. Thus, a fast acting autocrine IFN signal could limit viral spread in such a way that accelerated paracrine IFN response has only a minor impact on the spread of Dengue virus (Schmid, Rinas et al. submitted).
In conclusion, our work demonstrates that mathematical modeling is an essential tool to integrate data and mechanisms from the molecular to the cell-population level. The research on understanding which molecular mechanisms shape virus-host interactions might inform the development of new antiviral therapies and vaccines.
Mechanotransduction plays an important role in the regulation of muscle growth and metabolic signalling in striated muscle. Muscle disuse reduces mechanical input to the muscle, which results in a loss of muscle mass. Here I describe how titin's mechanically activated kinase domain affects muscle growth and metabolism via p62 and Akt signalling. I also demonstrate how changes in metabolic and growth signalling in hibernating grizzly bear help maintain muscle mass under conditions that induce muscle loss in humans and mice. I validated some of these changes in murine myotubes demonstrating that increasing non-essential amino acid levels, increasing Pdk4 levels or suppressing Serpinf1 has a positive effect on growth signalling that extends to non-hibernators.
In this thesis we develop a multi-component multi-phase reactive transport simulator to facilitate the investigation of a large variety of phenomena in porous media including component transport, diffusion, microbiological growth and decay, cell attachment and detachment and phase exchange. The coupled problem is solved using operator splitting approach. This approach enables us to use higher-order schemes and reduce numerical diffusion, which can result in an overestimation of phase exchange and reaction processes. Furthermore, this approach allows a flexible adaptation of the solution strategy to the concrete problem. We conduct an in-depth comparison of the fully-coupled and splitting approaches in order to derive criteria for the most efficient scheme depending on the relative importance of advection, diffusion and reaction.
In dieser Arbeit wurde die Machbarkeit der In-vivo-39K-MR-Bildgebung am menschlichen Oberschenkel sowie am Kopf mit einer nominellen Auflösung von 1 cm3 in einer Messzeit von 30 min bei einer Grundmagnetfeldstärke von 7T gezeigt. Zur Optimierung der Sequenzparameter wurden sowohl die globalen Relaxationszeitkonstanten bestimmt.
In Untersuchungsregionen mit hohem Muskelanteil, wie im Ober- und Unterschenkel, ist die Resonanz von 39K in ein Triplett aufgespalten. Die beiden Satellitenresonanzen sind etwa achsensymmetrisch zur Zentralresonanz. Der Betrag ihrer Frequenzverschiebung ist vom Winkel zwischen dem Bein und dem statischen Magnetfeld B0 abhängig. Bei zu B0 paralleler Ausrichtung des Beins sind die Satellitenresonanzen gegenüber der zentralen Resonanz etwa um 200 Hz verschoben. Der Empfang von 39K-Signal nach Doppelquantenfilterung mit magic angle Anregung stützt die These, dass die 39K-Resonanz im Muskelgewebe aufgrund der Wechselwirkung mit einem nicht fluktuierenden elektrischen Feldgradienten in einer anisotropen Umgebung aufgespalten ist.
In this dissertation we investigate several aspects of non-perturbative quantum field theory. Two main parts of the thesis are concerned with non-perturbative renormalization of quantum gravity within the asymptotic safety scenario. This framework is based on a non-Gaussian ultraviolet fixed point and provides a well-defined theory of quantized gravity. We employ functional renormalization group (FRG) techniques that allow for the study of quantum fields even in strongly coupled regimes. We construct a setup for the computation of graviton correlation functions and analyze the ultraviolet completion of quantum gravity in terms of the properties of the two- and three point function of the graviton. Moreover, the coupling of gravity to Yang–Mills theories is discussed. In particular, we study the effects of graviton induced interactions on asymptotic freedom on the one hand, and the role of gluonic fluctuations in the gravity sector on the other hand. The last subject of this thesis is the physics of the quark-gluon plasma. We set-up a general non-perturbative strategy for the computation of transport coefficients in non-Abelian gauge theories. We determine the viscosity over entropy ratio in SU(3) Yang–Mills theory as a function of temperature and estimate its behavior in full quantum chromodynamics (QCD).
In this thesis we study various aspects of the chiral phase transition of quantum chromodynamics (QCD). This transition is characterized by very different degrees of freedom at different energy scales. We therefore address the question how hadrons on low energy scales emerge from the underlying quark-gluon dynamics at high energies. To this end, we utilize the functional renormalization group which allows for the description of this dynamical transition within an unified framework. With this method at hand, we are able to identify the relevant degrees of freedom and to deepen our understanding of the underlying mechanisms that drive the transition from quarks and gluons to hadrons in QCD, including the mutual back reaction of these distinct phases. As a result, the properties of vector mesons, which play a prominent role for experimental investigations of the phase transition, can be derived from QCD. These insight are the foundation for the development of a low-energy model that aims at the effective description of the QCD phase diagram at finite temperature and density. There, our focus is on the quantitative precision of our results based on well-controlled expansion schemes and the effect of quark-meson scattering processes on the chiral phase transition.
In this work a scenario with two subsequent periods of inflationary expansion in the very early Universe is examined. The model is based on a potential motivated by symmetries being found in field theory at high energy. For various parameter sets of the potential the spectra of scalar and tensor perturbations that are expected to originate from this scenario are calculated. Also the beginning of the reheating epoch connecting the second inflation with thermal equilibrium is studied. Both is done in comparison with standard potentials leading to accelerated cosmic expansion. Perturbations with wavelengths leaving the horizon around the transition between the two inflations are special: It is demonstrated that the power spectrum at such scales deviates significantly from expectations based on measurements of the cosmic microwave background (CMB). This supports the conclusion that parameters for which this part of the spectrum leaves observable traces in the CMB must be excluded. Parameters entailing a very efficient second inflation correspond to standard small-field inflation and can meet observational constraints. Particular attention is paid to the case where the second inflation leads solely to a shift of the observable spectrum from the first inflation. A viable scenario requires this shift to be small.
Pancreatic ductal adenocarcinoma (PDAC) is by far the most common type of pancreatic cancer. It constitutes about 90% of tumors of the exocrine pancreas. The aggressive nature of PDAC along with a lack of diagnostic markers contributes to high lethality of this disease, which is nearly identical to its incidence. Studies from malignancies such as hepatocellular carcinoma and cervical cancer, along with the fact that liver and pancreas are in a close proximity, provided a plausible basis for the hypothesis of virus association in PDAC tumor development. However, there have been no established reports about virus(es) associated with pancreatic cancer.
The present study identified a new cancer-associated virus in human PDAC samples, called Meleagrid herpesvirus-1 (MeHV-1), or also known commonly as herpesvirus of turkeys, by two different and independent approaches: experimental genomic subtraction and digital microRNAome subtraction between healthy and PDAC patients. In the first approach, a genome-wide experimental comparison of DNA from PDAC tissues to DNA from tissues of healthy individuals was performed by representational difference analysis (RDA). Using this technique, differences in sequence composition were selectively isolated and amplified with very high sensitivity. Virus sequences associated with the occurrence of pancreatic cancer were detected by this process. The second approach, performed in parallel, involved a sequence analysis of the complete microRNA (miRNA) content of PDAC tissue samples. The sequencing data was digitally compared to databases of human and viral sequences so as to identify viral miRNAs. Because of the limited number of molecules, this analysis form did not need any experimental selection and amplification in order to achieve a sufficiently enough sensitivity to find viral microRNAs. The common results of the two analyses strongly suggested that MeHV-1 plays a crucial role in PDAC tumor progression. One of the viral microRNAs – hvt-miR-H14-3p – was studied in detail at the functional level by both in vitro and in vivo experiments in order to define the molecular mechanism of action with regard to its effect on pancreatic tumor carcinogenesis.
The key findings from this work include:
1. Identification of MeHV-1 DNA sequences in the PCR difference products (DPs) resulting from RDA on genomic DNA from PDAC and healthy tissues. 2. A tumor-specific MeHV-1 signature was also identified in the miRNA sequence analysis of tumor DNA, using an independent methodological approach. 3. RT-qPCR analyses showed that hvt-miR-H14-3p from MeHV-1 was expressed at significantly higher levels in PDAC and chronic pancreatitis (CP) tissues – CP being a chronic inflammation of the pancreas and a well-known risk factor of PDAC – than in healthy tissues. This observation was further verified using independent digital PCR platforms. 4. Metastatic and non-metastatic PDAC cell lines overexpressing hvt-miR-H14-3p showed a significant increase in migration and invasion compared to the respective controls, interestingly, without any significant change in proliferation. 5. Hvt-miR-H14-3p was found targeting cellular p27, down-regulating its expression. 6. The functional consequences of viral sequences identified in vitro could also be confirmed in vivo in NOD scid gamma mice.
In conclusion, this study is very significant in elucidating functional consequences of viral sequences in PDAC for the definition of relevant molecular effects responsible for carcinogenesis.
Despite more than 30 years of epidermal stem cell research, it is not yet understood how tissue homeostasis is balanced in the interfollicular epidermis (IFE). This is mainly because of the lack of distinct markers to identify human epidermal stem cells. In the hair follicle and the follicle-poor mouse ear epidermis stem cells have been described as slow-cycling cells, also called label-retaining cells (LRCs) (Bickenbach, 1981; Braun et al., 2003; Cotsarelis et al., 1990). It is suggested that these LRCs are responsible for maintaining tissue homeostasis. To address this stem cells need to be identifiable which has yet to be accomplished. Suggesting, that slowly cycling is a characteristic also of epidermal stem cells in the IFE, the aim of this study was the isolation and characterization of LRCs. Since investigation on human skin is restricted, we employed a human in vitro 3D organotypic skin model (Muffler et al., 2008). For the detection of LRCs we utilized an inducible Tet-Off-H2B-GFP lentiviral reporter vector. Thus, a method was developed to successfully infect normal human adult keratinocytes (NHEK) with the lentiviral vector and these keratinocytes were further propagated in organotypic cultures (OTC) to establish long-term skin equivalents. In pulse-chase approaches (2 weeks pulse, 6 weeks chase), viable LRCs of the IFE could be functionally identified as slow-cycling cells and isolated as GFP positive cells from OTCs. To further characterize these cells, microarray analysis was performed of the isolated basal LRCs and their respective non-labeled basal cell fraction. This analysis revealed 102 genes to be differentially regulated in the LRCs. We could show that a high fraction of upregulated genes in LRCs were ECM or ECM remodeling genes which could be verified by qRT-PCR. Further characterization on protein level is in progress. From this profile it is suggested, that LRCs actively modulate their local microenvironment, referred to as niche, by secreting unique ECM components. This distinct ECM may well serve for enhanced adhesion of LRCs to the niche as well as a reservoir for growth factors regulating cell signaling. Ingenuity pathway analysis further suggested a role for TGFβ signaling in this regulation. Accordingly, stimulation experiments provided first evidence that TGFβ-1 stimulates upregulation of those genes in NHEKs in 2D cultures that were highly upregulated in LRCs in their tissue context. Thus, we propose that LRCs exhibit a distinct molecular profile that compared to all other basal cells allows them to establish their specific regulatory niche. Their functional role as epidermal stem cells has now to await further investigations.
Accessing the relative changes in protein abundance is essential for a proper understanding of the various processes underlying disease progression and development. Nowadays, mass spectrometry-based proteomics allows for the identification of several thousand proteins in a single analysis. Unfortunately, mass spectrometry is inherently not quantitative, which is why additional techniques for protein quantification have to be developed. To measure quantitative changes in protein abundance, biological samples need either to be labeled using stable isotopes or protein abundances have to be computed using so called label-free techniques. Label-based quantification approaches are costly and the number of samples that can be quantified against each other is limited. Furthermore, depending on the sample, the introduction of the labels can be elaborate. Label-free quantification is not confronted with these limitations; principally, an unlimited number of samples can be quantified without the introduction of isotopes. Yet these advantages have their price: The development of label-free quantification algorithms is not trivial and requires profound knowledge both in bioinformatics and mass spectrometry. Namely the design of systems flexible enough to quantify data deriving from different mass spectrometric systems and proteomic workflows require additional experience and time. In order to quantify data acquired by LC-MALDI-MS, a novel software suite termed MSQBAT was developed and evaluated. MSQBAT is a platform independent software suite for MS1-based, label-free protein quantification. In contrast to other software solutions, MSQBAT is highly flexible and suited for the quantification of mass spectrometric data from various instrumental setups and proteomic workflows, such as (Ge)LC-MALDI-MS and (Ge)LC-ESI-MS. Quantification capabilities were evaluated using spike-in experiments analyzed using both different proteomic workflows and instruments. Human proteins were spiked in variable concentrations into a complex E.coli back-ground proteome and processed using both an LC-MS and a GeLC-MS approach. Samples were chromatographically separated on a nanoACQUITY UPLC system using a 120 minutes gradient and subsequently analyzed by an AB SCIEX TOF/TOF 5800 system and an AB SCIEX QTRAP 6500 system. Furthermore, a publicly available quantification benchmark data set has been used to evaluate LC-ESI-MS quantification capabilities. Obtained results show that MSQBAT can be applied to quantify data deriving from both LC-/GeLC-MALDI-MS and LC-/GeLC-ESI-MS workflows with high accuracy. Therefore, this software suite has a range of application outperforming all currently available solutions.
The resonant-Auger -- interatomic Coulombic decay (RA-ICD) cascade was recently proposed as a very efficient means of controlling the generation site and energies of slow ICD electrons. The control mechanism was verified in a series of experiments where both the energy of the photons producing the initial core excitation, and the neighbouring species were varied. The aim of this thesis is to provide a detailed theoretical investigation of the RA-ICD cascade in rare-gas dimers and give a first insight into the course of the cascade in aqueous medium.
The potential energy curves (PECs) of ionisation satellites are key ingredients in the theoretical description of electronic decay cascades. In the first chapter, we conducted a study on the PECs of the ionisation satellites of the ArHe dimer with a view to modelling such PECs in heavier dimers. Our results show that the complex valence structure in the rare-gas atom leads to the mixing of different electronic configurations of the dimer, which prevents one from assigning a single dicationic parent state to some of the ionisation satellites.
In the second part of the thesis, we present and analyse the ICD-electron and kinetic-energy-release (KER) spectra following different resonant core excitations of Ar in the rare-gas dimers Ar$_2$ and ArKr. We demonstrate that the manifold of ICD states populated in the resonant Auger process consists of fast- and slow-decaying ionisation satellites, and that the accurate description of nuclear dynamics in the latter ICD states is crucial for obtaining theoretical electron and KER spectra in good agreement with the experiment. We also show that by varying the neighbouring atom one can tune the energies of the emitted ICD electrons and even control the ICD yield.
Finally, as a first step towards the investigation of the RA-ICD cascade in aqueous medium, we present and discuss the X-Ray absorption spectra of microsolvated clusters of Na$^{+}$ and Mg$^{2+}$ at the metal 1s threshold. In this case it is important to investigate the nature of the core-excited states prior to studying the RA-ICD cascade, since in a solution the excited electron is delocalised and may ionise within the lifetime of the core hole, thus changing the course of the cascade. Our findings show that for a complete first solvation shell, the excited electron becomes spatially extended towards the water molecules.
In this thesis a fundamental physical process, radiative transfer, is modeled numerically. The implementation as a code module for the hydrodynamical simulation code Flash 4 is presented. The coupling to an efficient chemical network that explicitly tracks the three hydrogen species H, H_2, H+ and the two carbon species C+ and CO is described as well as the modeling of all relevant thermal stellar feedback mechanisms, i.e. photoelectric heating, pumping of molecular hydrogen by UV photons, photoionization and H_2 dissociation heating. These modeled processes coupled to the chemical network, make it possible to capture the non-equilibrium time-dependent thermal and chemical state of the present-day interstellar medium and dense molecular clouds affected by radiative feedback of massive stars. All included radiative feedback processes are extensively tested. The results obtained with this code module are compared to ones calculated from dedicated photo-dissociation region (PDR) codes. Good agreement is found in all modeled hydrogen species once the radiative transfer solution reaches equilibrium. In addition, it is shown that the implemented radiative feedback physics is insensitive to the spatial resolution of the simulation mesh and under which conditions a well-converged evolution in time can be obtained. The last test cases explore the robustness of the developed numerical scheme in treating the combined ionizing and non-ionizing radiation. In a follow-up study, different simplified numerical radiative transfer models are compared in the context of ionization front instabilities. The growth of unstable modes is found to be strongly dependent on the coupling of the thermal state to the ionization state. Depending on the implemented model, radically different conclusions can be drawn. For an equilibrium ionization model with a bimodal temperature structure for ionized and ambient gas, the swept up surrounding shell is found to be unstable. However, if the temperature of the ionized gas is calculated from the equilibrium ionization heating rate no instability is found. Finally, a damped ionization front instability is obtained from the newly implemented code module, which is unable to impact and perturb the shell sufficiently for it to break up.
In this thesis, a novel technique for the volumetric quantification of the longitudinal relaxation time T1 by ultra high field (UHF) magnetic resonance imaging (MRI) is introduced. It is based upon the prediction of the MR signal disturbances, due to static magnetic and RF field inhomogeneities as well as readout effects by the imaging process itself, and the corrections resulting hereof. For this reason, the mathematics of the magnetization’s equation of motion and the Bloch equations are implemented into a new simulation framework and regarded for by the evaluation algorithms. Furthermore, different MR signal simulation strategies additionally considering the k-space filters and various correction approaches are investigated. The introduced SIMBA IR and SIMBA DESPOT1-HIFI methods are capable of quantifying T1 with respective maximum deviations to the nominal values of (-0.42±1.23)% and (1.99±1.58)% within a T1 range of 1100 ms to 3300 ms. A minimization of the repetition time TR within the SIMBA IR experiments shortens the measurement time by up to 50% and further improves the accuracy. The use of a non-adiabatic preparation reduces the SAR exposure by up to 70% and allows examinations near organs of risk. Eventually, the even faster SIMBA DESPOT1-HIFI method was applied on a volume of 256×256×176mm3 with an isotropic resolution of 1mm within less than 30 min. A study of the whole human brain revealed a clearly differentiated soft tissue contrast and T1 values of (1917±95) ms for the gray and (1246±56) ms for the white matter. In a study on the human calf muscle, T1 was quantified to a value of (1877±92) ms. All T1 values are in a strong agreement with literature values.
Intercellular signaling is a defining property of multicellular organisms, yet the spatio-temporal dynamics remain poorly understood. The subject of this work is the design of an efficient numerical algorithm for simulations of intercellular signaling in multicellular 3D environments modeled by coupled systems of partial differential equations (PDE) and ordinary differential equations (ODE).
The PDE part of these systems consists of reaction-diffusion equations and describes the concentration distribution of diffusible messengers, e.g. cytokines. Intracellular dynamics are described by a small number of ODEs per cell. Thus, every single iteration of a commonly used decoupling scheme has similar computational costs than solving the coupled PDE/ODE system at once. We therefore develop an efficient multilevel preconditioner for the coupled system. The computational cost of both coupled and decoupled solution methods are investigated for model problems of different coupling strength.
To keep the computational costs of the 3D simulations moderate, we use methods for adaptive mesh refinement. We discretize the system by different time meshes for the PDE and the ODE part to reduce the number of computationally expensive PDE time steps. Reliable a posteriori error estimations for coupled PDE/ODE systems are derived by means of the ’Dual Weighted Residual’ (DWR) method. The discretization error is split into the contributions of the PDE and the ODE part. We compute local error indicators in space and time and set up an efficient adaptive mesh refinement method. The described methods are validated by numerical tests for several biologically motivated model problems.
We apply the developed numerical methods and simulate cytokine signaling between T cells in lymph nodes, which regulates the adaptive immune response in the human body. The numerical results show that, despite the high diffusivity of cytokines, highly localized cytokine concentrations with large gradients occur, which enables short-range cell-to-cell communication.
Microfluidic devices allow precise control to manipulate fluids within micrometer sized channel networks. In single phase microfluidic systems where miscible fluids are infused, laminar flow can be generated. This means liquid streams can flow parallel to each other without convective mixing. In two phase microfluidic systems where two immiscible fluids are infused, droplets are generated. Using this system, uniformly sized aqueous micro compartments can be generated in oil. This dissertation describes the development of novel microfluidic devices based on single phase and two phase systems to monitor responsiveness of cells and organisms to different chemical cues. Firstly, the possibility to apply a specifically designed single-phase microfluidic chip to study zooplankton ecology has been demonstrated. Zooplankton perceive their surrounding using chemical cues and rely on these cues for development and survival. However, with the current rapid global climatic changes affecting the ocean chemistry, it is unclear on how plankton, which form the base of the marine food chain, are coping. So far, measurements on zooplankton ecology have been hampered by technical impracticalities of exposing actively swimming plankton species to different chemical conditions simultaneously while monitoring their behavior on an individual level. Using the microfluidic device, first measurements on behavioral preferendum of zooplankton species to changes in pH and salinity could be made with a precision that additionally allowed estimating the “responsiveness”, which is the minimum change in concentration required for the plankton to elicit a response, to an environmental stimulus. Platynereis dumerilii, cosmopolitan model plankton were more sensitive to changes in pH than salinity. In addition, comparing different species lead to the observation that Euterpina acutifrons, a copepod species showed a narrower pH preferendum than P.dumerilii. These measurements allow making predications on sensitive and resilient species. Furthermore, the ability to study the interaction of zooplankton with their prey and predators and perform functional studies on identifying cell types responsible for a sensory response has been demonstrated. For cell-based screening assays however, the high-throughput offered by droplet-based systems outcompetes single-phase systems. But, generating chemical diversity in droplets that can allow screening entire chemical libraries while being able to track the sample identity remains to be demonstrated. Here a novel approach has been devised that allows generating sample barcoded combinatorial mixtures. In addition the approach has been optimized to suit for screening rare vi and sensitive cells like mouse embryonic stem (mES) cells. The ability to maintain viable mES cells in droplets for a period of 48 h has been demonstrated and in addition the possibility to differentiate them by encapsulating them together with 10-8 M retinoic acid (RA) has been shown. Lastly, a new microfluidic approach combining the advantages of single phase and two phase microfluidics has been described. This approach allows high-content cell-based screening with freely accessible cells allowing regular tissue culture handling and potentially, immunostaining experiments which are not possible when cells are encapsulated. To maximize the throughput per chip, chemicals were encapsulated in droplets and were allowed to locally diffuse through the chip material to the cells. The usability of this approach has been demonstrated by localized induction of GFP in tetracycline inducible HeLa-TRexTM cells. In conclusion, different microfluidic approaches have been described in this thesis and used for applications ranging from analyzing cells to organisms. The good spatial resolution, precise control over liquids, possibility of assays on the individual level and low cell number/reagent quantity requirement, enabled by microfluidics makes the devices an advantageous tool for biological applications.
Throughout our entire life, memory is central to our ability to carry out everyday tasks. Therefore, diseases that affect cognition have severe consequences, that influence patients' daily lives. However, not only do deficits in forming or recalling memories impact peoples' lives but also the inability to forget traumatic events or fear towards specific stimuli, such as with post-traumatic stress disorder (PTSD), anxiety disorders and phobias. Hence, it is essential to understand the cellular and molecular mechanisms in order to help people with any of these disorders. In the first study of this thesis, I investigated if nuclear calcium, which is known to control cAMP response element binding protein (CREB)/CREB binding protein (CBP)-mediated transcription is required for the formation of memory. Indeed, I found that inhibition of hippocampal nuclear calcium impairs memory formation in two hippocampus-dependent tasks. I further investigated the possible mechanisms that contribute to these deficits and found that dysregulation of nuclear calcium leads to alterations in dendritic morphology. I was able to rescue these morphological alterations via overexpression of the vascular endothelial growth factor D (VEGFD). VEGFD is regulated by nuclear calcium under basal conditions and is known to be important for the maintenance of dendritic morphology. Additionally, I could show that overexpression of VEGFD rescued memory deficits caused by nuclear calcium inhibition, probably due to the restoration of the dendritic architecture. In the second study, we aimed to investigate whether DNA methyltransferases (DNMTs) are required for memory formation. We found that DNMT3a2 is regulated by nuclear calcium and is induced upon neuronal activity. Additionally, we were able to show that the level of DNMT3a2 determines memory performance in mice. Downregulation of DNMT3a2 caused memory deficits and, in addition, a decrease in DNMT3a2 expression was associated with age-dependent memory decline. Further, restoring the level of DNMT3a2 in aged mice rescued memory impairments. In addition, we showed that DNMT3a2 expression correlates with global methylation levels, and we were able to identify two of the target genes of DNMT3a2, namely, activity-regulated cytoskeleton-associated protein (ARC) and brain-derived neurotrophic factor (BDNF). In the third part of this thesis, I studied if nuclear calcium is also required for fear memory extinction, which is routinely used as a model of PTSD. It is widely accepted that during fear extinction a new memory is built up that inhibits the previous, acquired memory. Though it is known that memory formation and fear extinction share common mechanisms, studies investigating the role of transcription in fear extinction are partly controversial. Here, I have shown that nuclear calcium is involved in fear extinction, pointing to a requirement of gene transcription. As I have shown in the first study of this thesis, nuclear calcium, which maintains the dendritic architecture primarily via alterations in the dendritic tree, probably also contributes to fear memory extinction. In summary, I have shown in this thesis that nuclear calcium mediates two forms of cognition, memory formation and fear extinction. I provide evidence that nuclear calcium-regulated VEGFD maintains the dendritic structure, which is important for the permissiveness of the neuron to process information required for long-term adaptations. Additionally, nuclear calcium regulates DNMT3a2, and we show that the level of DNMT3a2 has an impact on memory formation. Overexpression of DNMT3a2 restored the level of DNMT3a2 in aged mice and rescued age-dependent memory deficits.
Chromosomal instability (CIN) comprises an elevated rate of chromosome missegregation and correlates with the presence of extra centrosomes. Bipolar anaphases with clustered supernumerary centrosomes have been identified as a mechanism contributing to CIN via the formation of transient multipolar spindle intermediates, which promote merotelic kinetochore-microtubule attachment errors. However, the contribution of this model and that of potential additional mechanisms in human malignancies has not been addressed. Here we show that centriole rosettes, defined as multiple procentrioles engaged to a single parent, generate spindle asymmetry that favors kinetochore-microtubule attachment errors without centrosome clustering and ultimately results in CIN. Furthermore, we demonstrate that centriole rosettes, but not the progeny of clustered mitoses, are a common finding in primary human malignancies. Centriole rosettes are capable of arranging bipolar mitotic spindles but cause an increased frequency of anaphase lagging chromosomes and chromosome missegregation. The CIN phenotype is aggravated when spindle pole have asymmetric centriole numbers and it is not rescued by enhancement of kinetochore-microtubule attachment correction. Furthermore, by immunostaining for an array of centrosomal proteins, we find that, in primary human malignancies, centrosome amplification is characterized by supernumerary procentrioles forming rosettes around a single pair of parental centrioles, strongly arguing for a major contribution of this mechanism in the generation of CIN in vivo. Our results indicate that asymmetric centriole rosettes produce unbalanced microtubule numbers on mitotic half-spindles, thereby skewing the chance of binding microtubules from the more prominent spindle pole, resulting in impaired correction of merotelic kinetochore attachments and subsequent chromosome missegregation. We propose that centriole numbers at spindle poles must be carefully controlled to ensure chromosome segregation fidelity and disruption of this mechanism is an important source of CIN in human cancer.
Sixty-eight percent of human solid tumors are aneuploid, which is classically associated with poor patient prognosis. Several mouse models disturbing the spindle assembly checkpoint (SAC) have been developed to study the consequences of chromosome instability (CIN) and aneuploidy in vivo. Current knowledge suggests that aneuploidy can promote tumorigenesis or act as a tumor suppressor. Mad2 is found over-expressed in human tumors, and Mad2 over-expression in mice induces the development of aneuploid tumors and facilitates KrasG12D lung tumor relapse. A proposed mechanism of tumor acceleration by CIN is the facilitation of tumor suppressor loss of heterozygosity. In how far Mad2 over-expression influences Kras driven lung tumorigenesis in a p53 heterozygous background, remains hitherto unclear. In this thesis, I show that Mad2 over-expression increases p53(+/–);KrasG12D mice survival by delaying tumor initiation and progression. Different tumor populations (expressing low, intermediate and high levels of Mad2) have co-evolved from an original population of Mad2-expressing type 2 pneumocytes. My data suggest that high Mad2-expressing lung nodules are selected against during early tumorigenesis and are mainly composed of instable aneuploid cells. Using time-lapse microscopy on mouse embryonic fibroblasts, I analyzed the effect of Mad2 over-expression in the context of p53 heterozygosity. Upon Mad2 over- expression, the inactivation of one copy of p53 rescued mitotic cell death by inducing mitotic slippage and polyploid cells. In vivo, high Mad2 levels impaired S phase entry in tumor cells. Moreover, p53(+/– )KM high nodules strongly induced p21 in a p53-dependent manner. This data suggests that one copy of p53 can induce G1 cell cycle arrest in tumors. Although Mad2 over- expression generates aneuploidy, it does not accelerate p53 loss of heterozygosity (LOH), since Mad2 down-regulation occurs prior to LOH. Importantly, Mad2 over- expression together with p53 heterozygosity also delayed EFGRL858R-induced lung cancer.
Breast cancer is one of the most common malignancies with increasing incidence every year and a leading cause of death among women. Although early stage breast cancer can be effectively treated, there are limited numbers of treatment options available for patients with advanced and metastatic disease. The breast cancer associated antigen NY-BR-1 was identified by a serological screening strategy (SEREX). NY-BR-1 is expressed in the majority of breast tumours (>70 %) as well as in metastases, in normal breast tissue, in testis, and occasionally in prostate tissue. Due to its restricted expression pattern, its immunogenicity and its subcellular localization to the cytoplasm and plasma-membrane, NY-BR-1 represents a promising target for immunotherapeutic approaches in breast cancer patients. The aim of this thesis was the molecular and functional characterization of NY-BR-1 to reveal its biological function in normal and tumorigenic breast tissues. To examine the effect of NY-BR-1 over-expression on cellular processes, such as proliferation, apoptosis and the cell cycle, HEK293, HEK293T, MCF-10A, and MCF-7 were transiently transfected with NY-BR-1. The results showed that NY-BR-1+ cells do not proliferate, do not have an increased apoptosis rate and accumulate in the G1 phase compared to the control cells. This finding is supported by IHC stainings of normal breast tissue sections for NY-BR-1 and Ki-67 (proliferation marker) displaying NY-BR-1+/Ki-67- cells. Another approach to clarify the biological function of NY-BR-1 was the identification of specific protein interaction partners by co-immunoprecipitations with lysates of NY-BR-1 transfected cells (HEK293, MCF-10A, MCF-7) followed by mass spectrometry. The protein identified in all three analysed cell lines is the tubulin beta-4B chain, which amongst other is involved in mitosis, underlining a crucial role of NY-BR-1 during the cell cycle. To investigate the transcriptional regulation of the NY-BR-1 gene, an in silico prediction for potential transcription factor binding sites in the NY-BR-1 promoter region was performed with the Alibaba 2.0 algorithm. Tissue pieces as well as isolated epithelial cells from healthy breast tissue and tumour cells from pleural effusions were treated with different hormones, a demethylation agent and histone deacetylase inhibitors. The NY-BR-1 expression was analysed via qPCR. Several estrogen receptor binding sites were mapped in the NY-BR-1 promoter region and within the gene (up to Intron 21) by ChIP-Seq. The NY-BR-1 protein and the estrogen receptor (ER) are mainly co-expressed in normal breast tissues as assessed by IHC stainings and in silico analyses of RNA-seq data showed that NY-BR-1 is predominantly expressed in ER positive luminal A and B breast cancers. However, the stimulation of normal breast cells as well as tumour cells from pleural effusions with estrogen, tamoxifen or progesterone did not systematically show an up- or down-regulatory effect. On the other hand, short term stimulations of tumour cells from pleural effusions with progesterone, estrogen, vitamin D3 and retinoic acid lead to an up-regulation of NY-BR-1 expression (up to 8 fold) compared to the untreated controls. VPA treatment also induced NY-BR-1 expression. Of note, the combination of VPA with the above mentioned hormones only showed a weak up-regulation of NY-BR-1 expression (up to 3 fold) compared to the controls. These results suggest that NY-BR-1 expression is further regulated on histone level and/or non-histone proteins such as transcription factors modified by acetylation. Treatment of the cells with the demethylation agent 5´Aza-deoxycytidine did not show a distinct induction or up-regulation of NY-BR-1 expression. Moreover, the methylation status of selected promoter regions containing the predicted CpG islands was assessed in a selection of treated samples, but no association between methylation patterns and NY-BR-1 expression was observed. This finding could be confirmed by an in silico analysis of the TCGA database. Strikingly, a decreased NY-BR-1 expression in cultured normal breast tissue pieces and isolated epithelial cells versus snap-frozen cells was seen, which was not observed in breast tumour cells from pleural effusions cultured in conditioned medium. An in depth analysis of the conditioned medium containing the cell free effusion supernatant as well as cell culture experiments with normal breast epithelial cells in conditioned medium will reveal, which factors, e.g. cytokines, chemokines, hormones or growth factors, lead to a sustained NY-BR-1 expression. Considering the complex regulation of NY-BR-1 gene expression, the mosaic-like protein expression in normal breast tissue and the inhibitory effect of NY-BR-1 over-expression on proliferation and the G1 phase cell cycle arrest in normal cells it was hypothesized, that NY-BR-1 may be expressed by a specific breast progenitor cell population. Thus, mammospheres from isolated normal breast epithelial cells were generated and analysed by immunofluorescent staining and qPCR for NY-BR-1 expression and the presence of progenitor cell markers. In four analysed patients, NY-BR-1 was expressed in primary spheres and co-expression with integrin-α 6, HER2, GATA-3 and FOXA1 could be observed whereas no ER or progesterone receptor (PR) mRNA was detected. IHC staining with ER and NY-BR-1 in normal breast tissue showed that as well ER+/NY-BR-1+ as ER-/NY-BR-1+ cells in the mammary gland are existing. These results suggest that NY-BR-1 is expressed in the ER+/ER- luminal progenitor cells of the mammary gland. The NY-BR-1 gene was investigated regarding genetical variations to receive a better understanding of splice variants. Thereby, 69 damaging nsSNPs within the coding region of NY-BR-1 gene and 39 potential splicing SNPs could be identified by using in silico anlaysis. Taken together, NY-BR-1 is expressed mainly in well differentiated hormone sensitive estrogen receptor positive breast cancer subtypes and it is likely that NY-BR-1 expression is influenced by ERα and/or PR expression, but the association of NY- BR-1 expression and ER signaling still needs to be elucidated. Furthermore, transiently NY-BR-1 expressing cells show an inhibited proliferation rate and accumulate in the G1 phase. In vivo, endogenous NY-BR-1 is expressed in non proliferating (Ki-67 negative) cells in normal breast tissue. The protein potentially interacts with the tubulin beta-4B chain suggesting a crucial role during mitosis. Moreover, NY-BR-1 was shown to be expressed in progenitor cells of the mammary gland. The phenotypic characterization of these progenitor cells and the question whether the protein is also expressed in cancer stem cells is part of ongoing studies. In summary, the presented work could show for the first time that NY-BR-1 is a progenitor cell marker in the mammary gland and is influences the mitotic process.
Cardiovascular disease causes the highest morbidity and mortality worldwide. Moreover, the prevalence of cardiac hypertrophy and failure dramatically increases with aging, most notably in women. In this context, impairment of the ubiquitin-proteasome system (UPS) is supposed to be a pivotal element in the reinforcement of aging. Moreover, an involvement of the UPS, which accounts for the preservation of cardiomyocyte structure and function by the targeted degradation of structural, functional as well as signaling proteins, in the development of cardiac hypertrophy has been suggested. However, the number of systematic studies with regard to mechanisms underlying proteasome regulation in the heart and their role in cardiac remodeling, especially in the aging heart, is currently limited. Ongoing studies in our group reveal that cardiac hypertrophy due to continuous β-adrenergic stimulation in the mouse is exaggerated by knockout of the proteasome subunit β1i. The present study therefore addresses the question whether it is the absence of β1i incorporation into active cardiac proteasome complexes or its deficiency per se, which augments cardiac hypertrophy in β1i deficient mice upon treatment with isoprenaline. Reintroduction of β1i into cardiac proteasome complexes of β1i deficient mice by way of cardiac specific (serotype 9) adeno associated viral gene transfer resulted in reduced hypertrophy development, comparable to the level in isoprenaline-treated wild type mice, and prevention of a manifest systolic and diastolic dysfunction. Presumably, the augmented hypertrophy development in the isoprenaline treated β1i deficient mice is not related to a disturbance of the sarcomeric structure or a dysregulated degradation of contractile proteins. However, reassembly of β1i into active cardiac proteasome complexes seems to be associated with higher intracellular troponin I levels compared to the control group, which may contribute to the maintenance of cardiac function. In contrast to previous studies, marked alterations of the cardiac proteome associated with strongly increased 26S proteasome activities were detected in aging mouse hearts. Moreover, this increase in 26S proteasome activities seemed adaptive with respect to the extent of the alterations in the cardiac proteome, which may be associated with the age related deterioration of the general health status of these animals. This increase in 26S proteasome activities may thus serve to maintain protein homeostasis and cardiac function during aging. In conclusion, the present study revealed for the first time that increased incorporation of β1i into active cardiac proteasome complexes following induction of hypertrophy seems to be an important protective mechanism in maladaptive cardiac remodeling. Moreover, regulation of cardiac proteasome function during aging seems more complex than expected. Collectively, the current findings highlight how important it is to gain a better insight into mechanisms that regulate proteasome function in the context of aging and the pathogenesis of heart disease.
Saponins are widely distributed among flowering plants and some marine invertebrates and serve in defense for these organisms. Their amphiphilic molecules are composed of a lipophilic aglycone and one or more hydrophilic sugar moieties, giving them a high degree of structural diversity. Their biological and pharmacological activities range from antimicrobial, antifungal, anticancer, to immunomodulatory, etc. The most prominent feature of saponins is linked to their effects on cell membranes; they strongly affect cell membrane structure and integrity by different mechanisms depending on their chemical structure. The ability of saponins to increase membrane permeability can be used to facilitate the passage of drug molecules or other natural products through the cell membrane. The ability of saponins to affect cell membrane structure and integrity makes them interesting natural products in pharmacological and medical research and therapy, in particular as agents for enhancing drug efficacy. Saponins are known to interact with cholesterol in cell membranes by forming complexes. Until recently, there has been limited information on their potential as cytotoxicity-enhancing agents and on their molecular mechanisms of action on the membrane. This study explores the mechanisms of action of saponins on membranes and their effects in enhancing the cytotoxicity of certain anticancer drugs/toxins as applied to various cancer cell lines. Two different kinds of saponins were chosen (digitonin, a steroid saponin, and quillaja extract, a triterpenoid saponin mixture). These were investigated in combination with the anticancer drugs berberine, cisplatin, doxorubicin, dexamethasone, and mitomycin C, as well as with the polar toxin ricin (extracted from castor beans), on HeLa, COS-7, MIA PaCa-2, and PANC-1 cancer cell lines. The associated molecular mechanisms of action on membranes were investigated by employing a series of bioanalytical/ biophysical techniques: 1) MTT assay (a formazan test) which measures cell viability; 2) hemolysis (microscopic screening of erythrocytes) to measure the degree of membrane destruction; 3) dynamic light scattering (DLS) on large unilamellar vesicles (LUVs) to observe and quantify membrane leakage; 4) fluorescene/confocal microscopy of giant unilamellar vesicles (GUVs) to visualize membrane permeability; 5) quartz crystal microbalance with dissipation (QCM-D), dual polarization interferometry (DPI), and high-energy specular X-ray (XRR) showing structural changes in supported lipid bilayers (SLB), and 6) differential scanning calorimetry (DSC) which reveals thermotropic features of membranes resulting from saponin action. Digitonin and quillaja extract both enhance the cytotoxicity of the selected anticancer drugs in several cancer cell lines, the effect being either synergistic or additive. Quillaja saponin exerts a stronger cytotoxicity-enhancing effect than digitonin. The highly toxic monodesmosidic digitonin causes complete disruption of membranes at very low concentrations. The membrane activity of saponins strongly depends on the presence and amount of cholesterol in the membrane. Digitonin destroys GUVs, while quillaja saponin rather leads to pore formation. The relatively stable pores formed by the quillaja saponin-cholesterol complexes have a diameter of about 1 nm, only allowing passage of small-size molecules. Digitonin removes cholesterol from the inner membrane layer with formation of an additional layer on the outside, which eventually leads to membrane disruption. The quillaja saponins penetrate into the inner membrane layer forming complexes with cholesterol. The stabilized complexes form pores, which allow passage of water and other small molecules. Both sugar chains of the bidesmosidic quillaja saponins play an important role in stabilizing the pore formation in the membrane. Here, and for the first time, we report the occurrence of monodesmosides in the employed commercial quillaja saponin extract. The presence of both bidesmosides and monodesmosides in the quillaja saponin extract may be responsible for its bioactivity and pharmacological effects.
Compared to childless (nulliparous) women, the breast cancer risk of mothers can be 50% lower. The cause of the protective effect of parity is not yet clarified. However, it has been shown that immune cells of mothers show an enhanced cytotoxicity against tumor cells. Hence, it was assumed that T cells mediate the anti-cancer effect via the recognition of tumor-associated antigens (TAA). Moreo-ver, regulatory T cells (Treg), being essential for the prevention of pathological immune responses, can also suppress anti-tumor immunity. In breast cancer patients, the intratumoral accumulation of Treg cells is associated with a lower overall survival and an increased risk of relapse. Previous studies have revealed the presence of TAA-specific T cells in the blood of breast cancer patients. On the downside, the anti-tumor immunity of these patients is frequently suppressed by TAA-specific Treg cells. The aim of this study was to investigate the induction and characteristics of spontaneous T cell re-sponses against TAA during pregnancy, which may form a lifelong immune protection against breast cancer. Furthermore, we asked whether previous pregnancies may also have an impact on the anti-tumor immunity of breast cancer patients. We isolated peripheral blood T cells from nulliparous and primigravid women, mothers and breast cancer patients and analyzed their TAA specificity, functional properties and phenotype. IFN-γ ELISpot analyses with long synthetic peptides were performed to detect the frequency of TAA specif-ic T cells in blood samples. The impact of TAA-specific Treg cells on anti-tumor immunity was as-sessed by performing the IFN-γ ELISpot analyses before and after depletion of CD4+CD25+ T cells. By applying the Treg-specificity assay, we analyzed the antigenic specificities of these Treg cells. The results of this study indicate a spontaneous induction of anti-tumor immune responses during the first trimester of pregnancy. Highly functional TAA-specific memory T cells were detected in 85% of healthy mothers, but in none of the nulliparous controls. Furthermore, we found that a lifelong immunological memory is formed, that positively impacts on anti-tumor immunity, since TAA-specific T cell responses in mothers with breast cancer were significantly stronger and less impaired by Treg cells, as compared to nullipara. The findings obtained in this thesis support the implementation of a prospective study, which corre-lates the prevalence of TAA-specific T cells with the incidence of breast cancer. Based on this study, the potential benefit of a preventive vaccine is to be estimated, which may be applied to nulliparous women.
31P-NMR-Spektroskopie ermöglicht die nicht-invasive Beobachtung des Energiestoffwechsels im lebenden Gewebe. In dieser Arbeit wurde eine echo-planare 31P-spektroskopische Bildgebungsmethode (31P-EPSI) für In-vivo-Messungen bei einer Magnetfeldstärke von B0 = 7T entwickelt und validiert. Die Methode nutzt eine segmentierte Aufnahme der spektralen Dimension, eine Aufnahmegewichtung und den nuklearen Overhauser-Effekt (31P-{1H}-NOE). Bildartefakte der echo-planaren Auslese wurden eingehend untersucht und konnten erfolgreich vermindert werden. Zur Optimierung der Signalausbeute von 31P-EPSI wurde ein theoretisches Modell, das die Sensitivität (SNR) der Sequenz und die Dynamik des 31P-{1H}-NOE beschreibt, erstellt und in Modellmessungen validiert (Signalgewinn in vivo bis zu 35%). Die Technik wurde in Messungen an Wadenmuskulatur und Gehirn von Probanden angewendet. Die In-vivo-31P-EPSI-Spektren sind von vergleichbarer Qualität wie In-vivo-31P-Spektren mit konventionellen Aufnahmetechniken. Schließlich wurde die 31P-EPSI-Sequenz für funktionelle 31P-spektroskopische Bildgebung unter Belastung der Wadenmuskulatur eingesetzt. Die Veränderungen der Signalintensität von Metaboliten wie Phosphokreatin oder Änderungen des Gewebe-pH-Wertes konnten dabei mit hoher zeitlicher Auflösung (15-30s für einen 16x16-EPSI-Datensatz) verfolgt werden. Die 31P-EPSI-Technik bei B0 = 7T eröffnet Anwendungsmöglichkeiten für nicht-invasive Untersuchungen der Biophysik des Energiestoffwechsels.
This thesis presents a novel flavour tagging algorithm using machine learning techniques and a precision measurement of the B0-AntiB0 oscillation frequency delta m_d using semileptonic B0 decays. The LHC Run I data set is used which corresponds to 3 fb^-1 of data taken by the LHCb experiment at a center-of-mass energy of 7 TeV and 8 TeV. The performance of flavour tagging algorithms, exploiting the b Anti-b pair production and the b quark hadronization, is relatively low at the LHC due to the large amount of soft QCD background in inelastic proton-proton collisions. The standard approach is a cut-based selection of particles, whose charges are correlated to the production flavour of the B meson. The novel tagging algorithm classifies the particles using an artificial neural network (ANN). It assigns higher weights to particles, which are likely to be correlated to the b flavour. A second ANN combines the particles with the highest weights to derive the tagging decision. An increase of the opposite side kaon tagging performance of 50% and 30% is achieved on B^+ to J/Psi K^+ data. The second number corresponds to a readjustment of the algorithm to the B0_s production topology. This algorithm is employed in the precision measurement of delta m_d. A data set of 3.2x10^6 semileptonic B0 decays is analysed, where the B0 decays into a D^-(K^+ pi^- pi^-) or D^*- (pi^- AntiD0(K^+ pi^-)) and a mu^+ nu_mu pair. The nu_mu is not reconstructed, therefore, the B0 momentum needs to be statistically corrected for the missing momentum of the neutrino to compute the correct B0 decay time. A result of delta m_d = 0.503 +- 0.002 (stat.) +- 0.001 (syst.) ps^-1 is obtained. This is the world's best measurement of this quantity.
The detection of galaxy clusters needs to be reliable and transparent in order to be relevant for cosmological purposes. In this work, a detection algorithm based on the optimal matched filtering technique is introduced and tested. For each of seven combinations of optical observables a filter type is constructed. Based on these filters signal-to-noise maps are computed on a redshift-mass grid. Given some signal-to-noise thresholds the expected detection completeness rates are computed for each filter type. It is concluded that the combination of all observables performs best. The completeness prediction for this filter choice is tested against numerical simulations. Because in this simulation field and cluster galaxies are produced separately also the contamination rate below 2 per square degree could be estimated. Completeness and purity rates for the detections are measured by relating detections to simulated clusters. The measured rates give insights into complications that can be within simulations and in the choice of limits and convolution scale of the filter. Lastly, the filter is applied to the W1m0p1 field from CARS. Resulting detections are reduced to 31 by merging. 15 Detections can be related to detections from literature. It can hence be concluded that the filter may be applied to data.
Spherical void models of Gpc-scale have widely been discussed in the literature as a possible alternative to the spatially homogeneous Friedmann models with dark energy. In this framework, the local universe is modeled by an exact solution of Einstein's field equations, the so-called Lemaitre-Tolman-Bondi (LTB) metric, which constitutes a spherically symmetric spacetime that is solely filled by pressureless dust. In extension to recent multi-probe analyses of void models in a cosmological context, we study the evolution of linear, gauge-invariant perturbations on top of LTB backgrounds starting from a full spectrum of Gaussian initial conditions. The relativistic framework of perturbation theory on radially inhomogeneous spacetimes is substantially more complicated than in standard homogeneous models of FLRW type, because the spacetime is intrinsically dynamical already at first order which causes gauge-invariant perturbations to couple. As shown by Clarkson et al. in 2009 (\cite{clarkson_perturbation_2009}), their evolution is constrained by a system of linear partial differential equations which need to be integrated numerically. We present a new numerical scheme based on finite element methods to solve this equation system and generate appropriate scalar initial conditions in the homogeneous asymptotic limit of the LTB patch. In this context, we involve realisations of Gaussian random fields with an underlying power spectrum for the Bardeen potential. After spherical harmonic decomposition, the initial fluctuations are mapped to the corresponding LTB gauge-invariant variables and those evolved into the radially inhomogeneous LTB regime. Estimates of angular power spectra of each gauge-invariant quantity are computed as functions of redshift on the past null cone. This enables us to analyse the coupling strength in a statistical way. We find significant couplings up to $25\%$ for large and deep voids of Gpc scale as required to fit the distance redshift relations of SNe. As a major complication, LTB gauge-invariant perturbations are abstract mathematical objects that, although in principle observable, cannot feasibly be transformed to physically meaningful quantities. We therefore adapt a relativistic framework of light propagation to perturbed LTB models that allows to map the combined contribution of gauge-invariant metric and matter perturbations to sources of the optical tidal matrix. The corresponding Sachs equation is derived for generically perturbed LTB spacetimes and numerically investigated in case of negligible perturbation coupling.
Recent studies ascribed to the lateral entorhinal cortex (LEC) an important function in object recognition and novelty detection and confirmed the involvement of the LEC in odor processing. In this thesis, I investigated the contribution of LEC layer II neurons in sensory processing. Using immunohistochemical staining methods I could show that excitatory neurons in LEC layer II can be distinguished based on the expression of two marker proteins, namely Reelin and calbindin (CB). In combination with retrograde tracer injections, I revealed distinct projection patterns of these two excitatory cell types, with Reelin+ neurons projecting to the hippocampus and CB+ neurons providing feedback to structures of the olfactory system. Inhibitory GABAergic neurons in layer II of the LEC comprise a variety of molecularly defined subtypes. My goal was to analyze the participation of the defined cell classes in stimulus-triggered network activity. Therefore, I implemented in vivo two-photon imaging of genetically encoded calcium indicators in the LEC of anesthetized mice. This approach allowed me to investigate the activity of small neuronal networks in response to olfactory stimulation. I demonstrated that Reelin+ excitatory neurons transmitting information directly to the hippocampus respond with high selectivity to different odors. A markedly less selective response profile is exhibited by excitatory CB+ neurons that convey feedback to upstream targets in the olfactory pathway. It was possible to contrast these response patterns of excitatory neurons with that of their inhibitory counterparts. Thus, GABAergic neurons responded the least selective to various odors. Furthermore, we established in vivo whole-cell patch-clamp recordings under visual guidance. This enabled us to particularly target excitatory and GABAergic odor-responsive cells and to characterize them based on electrophysiological and morphological criteria. In summary, I defined and characterized here different neuronal subtypes in the LEC that are functionally involved in the processing and transmission of odor information.
Protein misfolding and aggregation perturbs cellular functions and is involved in aging and numerous medical disorders. In cells, the first line of defense is the association of deleterious aggregating proteins with small Heat shock proteins (sHsp). These oligomeric, ATP-independent chaperones sequester misfolded proteins into complexes and facilitate subsequent substrate solubilization and refolding by ATP-dependent chaperones. The cytosol of S. cerevisiae contains two sHsps: Hsp42 is constitutively active, while Hsp26 is activated at elevated temperatures. In my thesis, I wanted to elucidate how sHsps change the structure of aggregates, facilitating substrate reactivation. To this end, I studied the impact of Hsp26 and Hsp42 incorporation on the architecture of heat-induced aggregates by amide hydrogen exchange (HX). I established the experimental conditions for HX of heat-induced protein aggregates using thermolabile malate dehydrogenase (MDH) as model substrate. My data show that the formation of heat-induced Hsp26/MDH or Hsp42/MDH complexes has profound impact on the MDH structure. In the aggregated state formed in absence of sHsps, almost the entire MDH polypeptide becomes accessible to HX, reflecting global, large misfolding. In contrast, a more protected form of MDH is detected when complexed with Hsp26 or Hsp42. I observed that the mass spectra of many MDH peptides derived from sHsp/MDH complexes exist as a mixture of two populations after HX: a native-like and an aggregate-like population. Higher excess of sHsps promoted the native-like state. Single-molecule experiments confirmed the binding of sHsps to near native substrate folds. Furthermore, FRET experiments showed that sHsps increase the spacing between MDH molecules in sHsp/MDH complexes, preventing intermolecular contacts of misfolded MDH species. Finally, crosslinking approaches identified peripheral, surface-exposed MDH sites showing high HX as major sHsp binding sites. Summarized, these findings indicate that sHsps capture early unfolding intermediates of substrates and keep parts of the protein in a native-like state. This activity of sHsps might facilitate chaperone-dependent disaggregation. I then investigated how the two sHsps of yeast interact with their substrates. The N-terminal extensions (NTE) of both yeast sHsps were found to be the major substrate interaction sites. Compared to all known sHsps, the NTE of Hsp42 is unusually elongated and it was shown to be involved in the organized deposition of misfolded proteins at CytoQ (cytosolic quality control compartment). Hsp42 NTE harbors the two prototypes of intrinsically disordered domains (IDD): a prion-like and an unstructured subdomain. IDDs play important roles in the formation of membrane-free compartments due to their ability to self-associate and to coalesce into inclusions. In this study, the roles of both NTE subdomains in CytoQ formation and Hsp42 chaperone activity were investigated. We found that the prion-like domain of Hsp42 has a dual function: It binds misfolded substrate proteins and triggers CytoQ formation. The unstructured domain is dispensable for CytoQ formation, but it has a regulatory function, controlling Hsp42 localization and CytoQ numbers. Deletion of the unstructured domain increases Hsp42 substrate interaction and holdase activity, i.e. the prevention of tight contacts between misfolded species. Together, the presented data show that the prion-like domain of Hsp42 is essential for CytoQ formation, extending the role of prion-like domains in inclusion formation from RNA granules to protein aggregates and emphasizing their crucial contributions to protein phase transitions. In a second part of my thesis I studied how the Hsp70 chaperone system interacts with RepE, a dimeric replication initiation protein in E. coli. The disassembly of RepE seems mechanistically related to the disaggregation process. As a dimer RepE represses its own transcription, as a monomer it initiates the replication of the mini-F plasmid. Monomerization is mediated by the DnaK chaperone system. So far, it remained elusive, how components of the DnaK chaperone system interact with RepE and how they change its structure, leading to the disassembly of the RepE dimer. In this study the binding of DnaK and DnaJ to dimeric RepE wt and to RepE54, a constitutively monomeric variant, was studied by HX. HX analysis of RepE wt revealed a putative DnaK binding site and conformational changes induced by chaperones. Only dimeric RepE wt, but not monomeric RepE54, interacts with DnaJ. In contrast, both oligomeric states of RepE were able to bind DnaK – at least in absence of DNA. In presence of their respective DNA-binding elements, the binding of DnaK was prevented, most likely due to sterical hindrance as the DNA and the putative DnaK binding sites in RepE are in close proximity. The binding of DnaJ probably occurs in aa 96-116, and it destabilized parts of the DNA binding region in RepE, indicating conformational changes. Although interaction with DnaJ was shown to enhance the binding affinity of RepE to DNA, the DnaJ-induced conformational change might enable DnaK to access its binding site. Crosslinking experiments, however, showed that DnaJ binding is not sufficient to allow for interaction of DnaK with DNA-complexed RepE wt. Only concomitant presence of DnaJ and GrpE enabled DnaK to interact with DNA-bound RepE wt. HX revealed, that concerted binding of DnaJ and DnaK causes substantial conformational changes in RepE: Destabilization of the C-terminal region and stabilization in helix α4 near the dimer interface. The latter might be implicated in the monomerization of RepE wt. In summary, my results provide major contributions to elucidate the chaperone-mediated RepE monomerization process.
Motivated by the ischaemic brain stroke research, this work is devoted to the description of the osmotic swelling of a brain cell due to the absorption of the extracellular fluid during the formation of the cytotoxic (cellular) oedema. A physically motivated mathematical model describing the interaction between a single swelling cell and the extracellular fluid surrounding it is developed. In particular, the dynamics of the interaction is approximated by the coupled Biot-Stokes equations, resulting in a free boundary interaction problem. The Biot equations derived using homogenization techniques are considered and it is shown, that for the relevant data range, the temporal pressure derivative term of the Biot equations is negligible. Filtering effects of the cell membrane and the driving force of the transmembrane osmotic pressure difference are reflected in the Biot-Stokes coupling condition relating the normal fluid flux to the total pressure difference across the membrane. The analysis of the relevant experimentally obtained data for the considered biological system suggests that certain effects and processes included into the developed general coupled model can be neglected. As a result, a simpler (reduced) mathematical model is obtained and numerically implemented. The reduced Biot-Stokes coupled problem is discretized using FEMs (in space) and the implicit Euler scheme (in time), and solved following an operator-splitting approach. The numerical implementations of the (pure) Biot problem are verified by comparing the analytic and numerical solutions, and are available for two and three dimensions. The simulation results for the reduced mathematical model parametrized with the estimated experimental parameters showed good agreement with the experimental observations. The sensitivity of the Biot problem solution to the variations of the key parameters and domain geometry, as well as the overall effect of the Stokes domain solution on the solution of the coupled Biot-Stokes problem are tested and analysed.
Myb-binding protein 1A (MYBBP1A) is a nucleolar protein implicated in stress response and carcinogenesis. However, its functional contribution to cellular senescence as well as the clinical relevance in head and neck squamous cell carcinoma (HNSCC) have not been addressed so far. In the present study, a cell culture model of genotoxic senescence, which was induced by the topoisomerase II inhibitor VP-16, was established to unravel the role of MYBBP1A in cellular senescence. Interestingly, in response to DNA damage, MYBBP1A first translocate from the nucleolus to the nucleoplasm and subsequently, its protein levels decreased in senescent cells. Loss-of-function approaches in tumor cell lines provided further evidence that silencing of MYBBP1A was not sufficient to trigger senescence, but that it modulated the efficacy of genotoxic-induced senescence and augments resistance to irradiation. Although, the precise molecular mechanism by which MYBBP1A regulates DNA damage-induced senescence remains elusive and warrants further investigation, this study revealed an inverse regulation of MYBBP1A and AKT(Ser473) phosphorylation to be a characteristic feature of senescent tumor cells. Most strikingly, immunohistochemical analysis of tissue microarrays with tumor specimens from primary oropharyngeal squamous cell carcinoma (OPSCC) patients (n=61) revealed that a MYBBP1AlowpAKT(Ser473)high staining pattern serves as an independent marker for poor clinical outcome. Remarkably a significant correlation with progression-free or overall survival was not found considering pAKT(Thr308) levels, suggesting a more critical role of the mTOR/AKT pathway for the clinical behavior of OPSCCs with low MYBBP1A levels. In summary, these data demonstrate that tumor cells with a MYBBP1Alow but pAkt(Ser473)high protein pattern have a senescent-like phenotype and might critically contribute to tumor progression due to the emergence of highly malignant and/or therapy resistant tumor cells. Moreover, the abundance of MYBBP1AlowpAKT(Ser473)high senescent tumor cells in primary tumors as well as following tumor relapse could serve as a reliable biomarker for treatment decision making and to stratify HNSCC patients at high-risk for treatment failure. Thus, restoration of MYBBP1A function or pharmacological inhibition of the PI3K/mTOR/AKT pathway is an attractive new concept not only to sensitize tumor cells for available treatment options, but also to establish new strategies for targeted therapy with the potential of elimination of senescent cells.
Die vorliegende Arbeit stellt ein neuartiges Verfahren der dreidimensionalen Particle Tracking Velocimetry vor. Durch die Erweiterung der existierenden zweidimensionalen Particle Tracking Velocimetry auf die dritte Raumdimension sind erstmals dreidimensionale Strömungsmessungen bei hoher räumlicher Auflösung mit lediglich zwei Kameras möglich. Der Schwerpunkt der Arbeit lag in der Entwicklung von Algorithmen zur Korrespondenzanalyse sowie der Kamerakalibrierung.
Die Genauigkeit aller Teilalgorithmen des Verfahrens wurde anhand von synthetischen und gemessenen Daten untersucht. Die Abhängigkeit der Kalibrierung von der Positionsgenauigkeit der gefundenen Kalibrierpunkte auf der Bildebene wurde untersucht. Die Stabilität der Kamerakalibrierung konnte durch einen neuen Algorithmus zur Merkmalsextraktion verbessert werden. Dabei konnten die Meßfehler auf die zugrundeliegende zweidimensionale Particle Tracking Velocimetry zurückgeführt werden. Abschließend wurde das Verfahren an Strömungsmessungen im Heidelberger Wind-Wellenkanal eingesetzt. =============================
This thesis presents a novel approach to the area of three dimensional particle tracking velocimetry. In extending existing algorithems for two dimensional particle tracking velocimetry to the third spatial dimension, it becomes feasible for the first time to measure three dimensional flow fields with high spatial accuracy by employing two cameras only. The work of this thesis was mainly focussed on the development of algorithms for correspondence detection and camera system calibration.
The accuracy of each step constituting the algorithem was determined using synthetical as well as real world data. The dependence of the quality of the calibration on the accurate determination of the position of the calibration marks on the image plane has been analyzed. Hence it was possible to enhance the precision of the three dimensional particle tracking velocimetry based on the two dimensional counter part. Finally the technique was deployed in the wind/wave facility of the department of environmental physics.
Specific structures and cell types in the organization of the liver are the key for its variant functions, like protein production, glucose homeostasis and detoxification. In the present work, liver damage from an acute toxic injury caused by intraperitoneal injection of a mixture of CCl4 and mineral oil in Balb/c mice and its subsequent recovery was studied using different methods to investigate specific cellular functions in the liver. The analysis by in situ hybridization and RT-qPCR showed how expression of liver specific enzymes and proteins in mouse hepatocytes is changed over a period of 6 days following injection. The genes investigated included Albumin, Arginase, Glutaminase2, Glutamine synthetase, Glucose-6-phosphatase, Glycogen synthase2, Gapdh, Cyp2e1 and Glucagon receptor genes. Interestingly, a significant change in gene expression of enzymes involved in nitrogen and glucose metabolism and their local distribution in different areas of the liver were observed following CCl4 injury. Cyp2e1, an essential metabolizing enzyme in CCl4 metabolism, was strongly expressed in the pericentral zone during recovery. In comparison to hepatocytes in livers from untreated mice, liver cells from treated animals displayed distinct gene expression profiles in the damaged area around the pericentral vein during the analyzed time course and showed a complete recovery with strong albumin production at day 6 post CCl4 injection. The results obtained indicate that despite of the severe damage, liver cells in the damaged area do not simply die but instead locally adjust gene expression to deal with the damage effect and thereby ensure survival. In order to optimize the preparation of cRNA hybridization probes and enable the rapid synthetize of the large number of probes used in this study, a new rapid method for antisense cRNA preparation was established. The development of this rapid and efficient protocol for the generation of labeled cRNA probes was an important pre-requisite for the project. The new protocol is based on the preparation of DNA templates in vitro by PCR using primers that include RNA polymerase promoter sequences and size based purification of PCR fragments containing the target gene specific cDNA and promoter elements for T7 and SP6 RNA-polymerase. Purified PCR fragment based in vitro transcription enables the preparation of in situ hybridization probes, which can be used for the detection of the respective gene and visualization of the distribution of gene expression in tissue slices for any gene of interest. The optimized synthesis and purification protocols ensure high transcription efficiency and target specificity of the labeled cRNA and the obtained cRNA hybridization probes are compatible with established in situ hybridization protocols. This study proved that with a single dose of CCl4 injection in mouse, liver pericentral hepatocytes are the main cell type responsible for neutralizing the toxic agent, and the main consequence of this damage is not simply to induce cell death due to apoptosis, but instead these damaged hepatocytes seem to reduce any unnecessary activities in favor of processes needed for recovery from damage.
The maintenance of genomic stability and the repair of DNA damage are essential for the survival of all cells. Despite diverse pathways for repair of DNA lesions, different mutations can arise, ranging from Single Nucleotide Variants (SNVs) to larger Structu- ral Variants (SVs). The processes that play a role in the formation of these alterations are not fully understood. In this thesis, I present two complementary approaches for accumulating genomic variants and for identifying pathways involved in the suppression of mutation formation using Saccharomyces cerevisiae (budding yeast) gene knockout strains. First, using next-generation sequencing, I studied neutral variants through a mutation accumulation assay for up to 1800 generations. I used 47 yeast strains with known defects in DNA replication, repair and recombination pathways. In all strains, small insertions and deletions (indels) were more common than larger SVs (>50bp). Most mutations occurred in repetitive sequences, implicating replication based mechanisms and homologous recombination in the formation of genomic variants. Furthermore, the knockout of MSH2 produced a hypermutable strain that acquired the highest number of indels. Moreover, the knockout of the genes SWR1 and ISW1, involved in chromatin remodeling, resulted in strains with high number of deletions. These results suggest that defects in establishing a correct chromatin architecture may play a role in the formation of genomic variants. I further performed a genome-wide screen for genes that suppress deletion formation under different drug treatments in the presence or absence of homologous repeats by using designed constructs. As expected, deletions occurred more often between repeats, in support of the frequent involvement of homologous recombination in the formation of chromosome rearrangements. In addition, I identified genes whose knockout led to incre- ased levels of deletions. Among these, IOC4 is of particular interest given that it belongs to the same chromatin remodeling complex as ISW1, identified in the neutral mutati- on accumulation assay. This provides further evidence that chromatin remodeling may be involved in preventing the occurrence of SVs. Furthermore, several meiosis-related mutants also showed increased levels of deletions, suggesting that meiosis proteins may have additional roles in the maintenance of genomic stability during vegetative growth. By performing additional experimental validations, I verified the higher vulnerability of meiosis gene knockouts to acquire deletions, especially in their diploid stages. In the last chapter, I briefly describe the results of several side projects in which I applied computational methods learned through the above mentioned projects, to identify and characterize genomic rearrangements in different human cancers. In summary, I have found that genome-wide approaches can provide interesting insights into the understanding of genomic variants in yeast and human cancers. In particular, given the evolutionary conservation of the ISWI chromatin remodeling complex and meiosis-related genes, the results presented here point to potentially novel functions of these proteins in the maintenance of genomic stability.
Genomic variants play an important role in phenotypic variation and have significant impact on a disease development. Due to the technology limitations, inference of genomic variants and their potential consequence on phenotype was until recently restricted. Only with the advent of next-generation sequencing (NGS) approaches, could a vast majority of genomic variants be successfully identified for the first time. In my PhD Thesis I will present my work on structural variants (SVs), their formation mechanism and their functional impact. The first part of my Thesis focuses on structural variants in non-human primates, studies of which using NGS have not been pursued prior to the research studies we carried out. In order to inspect the origin and functional impact of SV formation mechanisms, we constructed a comprehensive SV map based on the fibroblast-derived DNA from three different species: chimpanzee, orangutan and rhesus macaque. We noted striking differences in the activity of homology-related SV formation mechanisms between the great apes and rhesus macaques, with a third of the chimpanzee and orangutan SVs inferred to be formed by non-allelic homologous recombination compared with only 2% of the macaque SVs. One additional key finding was the presence of a markedly higher mobile element activity in macaques compared to the other non-human primates studies. Additionally, we could show that long L1 elements surpassed Alu activity in chimpanzee and orangutan as opposed to macaque where AluMacYa3 dominates the genomic landscape causing a burst of relatively short SVs. In addition to inserting into genome, active L1 elements possess the ability to mobilize 3’ flanking DNA to different genomic loci as transductions. By combining translocation and L1 discovery pipelines we further developed a novel computational methodology, termed TIGER, for the discovery of polymorphic L1-mediated 3’ transductions. We employed TIGER to a deeply sequenced human genome and to aforementioned non-human primates species to characterize transductions. TIGER enables studying germline L1-mediated 3’ transductions, making a relevant structural variation class amenable for population and disease studies for the first time. In the second part of my Thesis, I discuss the differences in the formation mechanisms of both germline and somatic SVs in the human genome. Our de novo mechanism classification analyses performed on four previously published SV datasets revealed that almost half of germline human SVs are due to mechanisms independent of homology, followed by homology-related DNA repair, mobile elements and variable number of tandem repeats. We also investigated the formation of somatic SVs in four medulloblastoma brain tumor patients with a germline TP53 mutation (Li- Fraumeni syndrome). In contrast to the germline SVs, our analyses of rearrangement breakpoints in medulloblastoma in the context of mutated TP53, rather support a model of massive DNA double strand breaks known as chromothripsis, followed by exclusive homology-independent repair.
DNA Doppelstrangbrüche (DSB) können sowohl natürlich als auch nach Bestrahlung auftreten und stellen eine große Gefahr für die Stabilität des Genoms und Zellviabilität dar. Wird ein DSB nicht effizient repariert, können Veränderungen im Genom wie z.B. Deletionen und Vervielfältigungen auftreten, die ein Markenzeichen von Krebszellen sind. Ist der DNA Schaden allerdings zu groß, kann die Zelle Apoptose induzieren, was das Ziel der Strahlentherapie zur Krebsbehandlung ist. Derzeitige Methoden zur Bestimmung von DSB basieren vor allem auf der Markierung von DNA Reparaturproteinen wie ATM (Ataxia telangiectasia mutated), 53BP1 (53 binding protein 1) und H2AX (phosphoryliertes Histon H2AX) mittels Fluoreszenz‐markierten Antikörpern. Diese Proteine bilden innerhalb weniger Minuten nach der DSB Entstehung mikroskopisch‐sichtbare Zentren an der DSB Stelle, sogenannte Strahlen‐induzierte Zentren (RIF). Obwohl diese Methode Erkenntnisse über die DSB Entstehung und Reparaturkinetik geliefert hat, ist sie limitiert. So kann keine Aussage über die genaue Position des Strangbruchs getroffen werden, da die RIF auf mehrere Megabasen um die DSB Stelle herum vergrößern. Außerdem verschwinden die RIF innerhalb von 24 Stunden nach Bestrahlung, und ermöglichen somit keine weitere Analyse der reparierten DSB Stellen in überlebenden Zellen. Ebenso erlaubt die Immunfärbung keine Aussage über einen möglichen Zusammenhang zwischen der DSB Stelle, der Reparatur und des Schicksals der Zelle. Daher gibt es kaum Informationen darüber, wie Strahlen‐induzierte DSB im Genom verteilt sind, wie solche Schäden zur Radioresistenz führen sowie die Entwicklung von einer normalen Zelle hin zu einer Tumorzelle und die Krebsentstehung beeinflussen. Deshalb stellen wir die Hypothese auf, dass DSB im Genom nicht zufällig verteilt sind und dass die Analyse der genom‐weiten DSB Verteilung nach Bestrahlung in überlebenden Zellen auf Einzelnukleotidebene unser Verständnis über die Mechanismen, die zur Bestrahlungsresistenz führen, verbessert. Um dieses Ziel zu erreichen, wurden in dieser Arbeit neue Methodenansätze basierend auf der Beobachtung entwickelt, dass Non‐Homologous End Joining (NHEJ) Reparatur zum Einbau von DNA Molekülen ins Genom an DSB Stellen führen kann. Für die DSB Markierung wurden daher Integrase‐defiziente lentivirale Vektoren (IDLV) in Zellen eingebracht, die nach Bestrahlung an transient‐auftretenden DSB Stellen ins Genom stabil eingebaut werden. Diese Integrationsstellen wurden anschließend durch LAM‐PCR und Sequenzierung amplifiziert und identifiziert. Insgesamt wurden somit mehr als 20,000 DSB Stellen im Genom bestrahlter Zellen identifiziert und mit natürlich‐auftretenden und Doxorubicin‐induzierten DSB Stellen verglichen. Die DSB Verteilung wurde mit der Transkriptionsaktivität, verschiedenen Chromatin‐Bereichen und Genfunktionen verglichen. Die Analyse ergab, dass in überlebenden Zellpopulationen das DSB‐Verteilungsmuster nicht zufällig ist. Therapie‐induzierte DSB wurden vermehrt in kleinen genomischen Bereichen, sogenannten Hotspots, die in Grenzregionen von Eu‐ und Heterochromatin und in oder in der Nähe von Onkogenen, Tumorsuppressorgenen und Enhancer Regionen liegen, nachgewiesen. Die Ergebnisse dieser Arbeit zeigen, dass Strahlen‐induzierte Mutationen in überlebenden Zellpopulationen in bevorzugten Bereichen auftreten oder Zellen mit Mutationen in solchen Bereichen selektiert werden. Diese Hotspots können eventuell die Wahrscheinlichkeit für die Entstehung von Therapieresistenzen erhöhen.
Hematopoietic stem cells (HSCs) sustain the life-long production of blood and maintain the integrity of the hematopoietic system. Therefore, they possess self-renewal capacity and are able to differentiate into all the mature blood cell lineages. These are finely tuned processes, which constantly involve dynamic HSC fate decisions and an imbalance may result in misregulation associated with cellular transformation or other types of diseases. In the hematopoietic disorder Fanconi Anemia (FA), such an imbalance in the HSC pool results in bone marrow failure, a collapse of the entire hematopoietic system. In addition, FA patients are susceptible to developing hematologic malignancies such as acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). This is the result of a loss of function in the FA DNA repair machinery, which renders HSCs genetically unstable as they are unable to repair certain forms of DNA damage. Murine knockout models for individual FA pathway members fully recapitulate the FA HSC DNA repair defect seen in patients and demonstrate a severe engraftment defect in competitive transplantation experiments. We could recently show, that in response to proliferative stress, such as during transplantation and reconstitution, FA HSCs accumulate DNA damage and are quickly lost due to apoptotic cell death, which is a key driver of bone marrow failure. FA HSCs are also subject to cellular transformation, which may result in the outgrowth of a leukemic founder clone. The identification of novel therapeutic targets would likely provide insight into the underlying mechanism of the FA HSC defect and the associated pathology of FA. In this respect, forward genetic screens using insertional mutagenesis have proven to be a powerful screening method for the identification of genes with the potential to influence stem cell kinetics when upregulated or disrupted. We have employed retroviral insertional mutagenesis in the context of FA HSC biology using a murine transplantation model in order to try and identify novel factors with the potential to rescue the inherent FA HSC transplantation defect. In this respect, we show that retroviral vector integrations trigger the expansion of nonmalignant dominant FA clones in transplanted mice and allow the retrospective identification of nearby genes whose deregulation had caused clonal expansion in the face of genomic instability. We identified four candidate drivers of clonal dominance in the FA HSCs: Osgin1, Evi1, Taf1b and Grhl1, which we further characterized in the context of HSC biology under homeostatic and stress conditions as well as during development. In this respect, we identified the oxidative stress-induced growth inhibitor (Osgin1) as a promising candidate target gene, which is expressed in FA and WT HSCs under homeostatic conditions as well as in hemogenic endothelium/HSPCs during developmental specification from normal embryonic stem cells. Furthermore, we provide insight into the hematopoietic role of Osgin1, which displays differential expression levels during the individual steps of HSC commitment and their response to physiologic stress. We conclude that Osgin1 is an essential factor for hematopoiesis and we will further characterize Osgin1 using the tools and assays that we have developed to assess whether it impacts on FA HSC fate decisions in vitro and during hematopoietic reconstitution in vivo. Furthermore, we provide some insight into how the FA HSCs may become a dominant clone and suggest likely candidate mechanisms with the potential to compensate the inherent FA HSC defect. According to our hypothesis, the identified candidate target genes may either prevent the acquisition of DNA damage or repair it or, alternatively, block the apoptotic cell death, which is a likely cell fate outcome for FA HSCs. Our findings hold great potential for HSC research with implications for normal as well as FA HSC biology.
Thanks to revolutionary developments in microscopy techniques such as robotic high-throughput setups or light sheet microscopy, vast amounts of data can be acquired at unprecedented temporal and spatial resolution. The mass of data naturally prohibits manual analysis, though, and life scientists thus have to rely more and more on automated cell tracking methods. However, automated cell tracking involves intricacies that are not commonly found in traditional tracking applications. For instance, cells may undergo mitosis, which results in variable numbers of tracking targets across successive frames. These difficulties have been addressed by tracking-by-assignment models in the past, which dissect the task into two stages, detection and tracking. However, as with every two-stage framework, the approach hinges on the quality of the first stage, and errors propagate partially irrevocably from the detection to the tracking phase.
The research in this thesis thus focuses on methods to advance tracking-by-assignment models in order to avoid these errors by exploiting synergy effects between the two (previously) separate stages. We propose two approaches, both in terms of probabilistic graphical models, which allow for information exchange between the detection and the tracking step to different degrees. The first algorithm, termed Conservation tracking, models both possible over- and undersegmentation errors and implements global consistency constraints in order to reidentify target identities even across occlusion or erroneous detections. Wrong detections from the first step can hence be corrected in the second stage. The second method goes one step further and optimizes the two stages completely jointly in one holistic model. In this way, the detection and tracking step can maximally benefit from each other and reach the overall most likely interpretation of the data. Both algorithms yield notable results which are state-of-the-art.
In spite of the distinguished results achieved with these methods, automated cell tracking methods are still error-prone and manual proof-reading is often unavoidable for life scientists. To avoid the time-consuming manual identification of errors on very large datasets, most ambiguous predictions ought to be detected automatically so that these can be corrected by a human expert with minimal effort. In response, we propose two easy-to-use methods to sample multiple solutions from a tracking-by-assignment graphical model and derive uncertainty measures from the variations across the samples. We showcase the usefulness for guided proof-reading on the cell tracking model proposed in this work.
Finally, the successful application of structured output learning algorithms to cell tracking in previous work inspired us to advance the state-of-the-art by an algorithm called Coulomb Structured Support Vector Machine (CSSVM). The CSSVM improves the expected generalization error for unseen test data by the training of multiple concurrent graphical models. Through the novel diversity encouraging term, motivated from experimental design, the ensemble of graphical models is learned to yield diverse predictions for test data. The best prediction amongst these models may then be selected by an oracle or with respect to a more complex loss. Experimental evaluation shows significantly better results than using only one model and achieves state-of-the-art performance on challenging computer vision tasks.
This thesis examines the influence of the morphology of organic semiconductors on their electronic properties using analytical transmission electron microscopy (TEM). For the first time, the dielectric properties of organic materials are derived from electron energy loss spectroscopy. A correlation between electron diffraction and current transient measurements of the solution processed polymer poly(3-hexylthiophene) indicates that the semicrystalline structure of the polymer leads to anisotropic charge transport. The study further investigates the structural properties of co-evaporated small molecule blends of fluorinated zincphtalocyanine (F4ZnPc) and fullerene (C60) on different substrates. Energy filtered TEM reveales strong fullerene agglomeration for all substrates considered here, which is shown to be facilitated by an elevated substrate temperature (100°C). These agglomerates are still largely amorphous if the blend is grown on a pristine F4ZnPc layer as required for non-inverted solar cells. However, on an underlying C60-layer, as is typically used for inverted devices, a pronounced order of the fullerene is observed. Device characterization indicates that the highly pure and crystalline C60 domains improve free charge carrier generation and solar cell efficiency. This thesis therefore provides a novel explanation why record efficiencies in small molecule solar cells are only achieved in inverted devices.
The enzyme Purple Acid Phosphatase (PAP) is an important target for the development of new anti-osteoporotic drug leads. The major goal of this thesis is to better emulate the synergy that occurs between the primary and secondary coordination sphere within the active site of phosphatase enzymes, such as PAP. This was perceived by development of synthetic methods for new biomimetics, i.e. low-molecular weight metal complexes. Therefore, asymmetric dinucleating ligands which mimic the peptide backbone of the active site of PAP including the ability to form hydrogen bonds with a phosphoester substrate or a nucleophile were designed and synthesized. Using these ligands, more accurate model systems for the enzyme active site were achievable as they combine the two essential structural features known to influence the catalytic activity towards the hydrolysis of phosphoesters, i.e. the asymmetry of the dinuclear active site and the capacity for hydrogen bond formation. The latter was verified by the observation of hydrogen bonds in the X-ray structures of dizinc(II) and diiron(II) complexes. Moreover, two asymmetric dizinc(II) complexes were formed with two different Zn(II) sites and an unusual hydroxido co-ligand, representing two important features in the active site of PAP, the difference of the metal sites and the nucleophile needed for the phosphoester hydrolysis. In addition, hydrogen bond formation detected in these X-ray structures was accounted for the stabilization of the hydroxido co-ligand. In this work, a new synthetic approach towards more sophisticated model systems for the active form of mammalian PAP was developed. Chemical oxidation of the diiron(II) complex of an asymmetric ligand results in the generation of the Fe(III)Fe(II) complex that has been specifically designed to both satisfy the requirement of a heterovalent diiron core and to mimic the second coordination sphere of the active site of PAP. Similarly, more accurate model systems for the active site of plant PAPs have been generated in the form of heterovalent heterodinuclear complexes of asymmetric ligands bearing, adjacent to the Ga(III)Zn(II) core, functionalities capable of forming hydrogen bonds. Analysis of the complexation behavior of the respective ligands revealed the selective formation of the Ga(III)Zn(II) complexes in solution. The model complexes described above imitate successfully the extensive hydrogen bond network that is formed by the second coordination sphere within the active site of PAP as well as in a structural and functional similar phosphatase, Alkaline Phosphatase. Thus, those complexes allow to study the impact of hydrogen bonds on the reaction mechanism. The main impact of the secondary interactions in the dizinc(II) complexes was found to be the increased substrate affinity. This catalytic parameter was shown to be dependent on both the hydrogen bonding sites and the type of the hydrogen bonding groups. Although the substrate affinity of the Ga(III)Zn(II) complex was revealed to be lower compared to the dizinc(II) complex, a 50-fold faster hydrolysis rate and a 6-fold increased efficiency was detected for the heterodinuclear complex. Moreover, the mechanism previously proposed, in which the phosphoester is activated by the Zn(II) center and the Ga(III) being accountable for providing the hydroxide nucleophile at near physiological condition, was supported in this study and an accelerating effect by the interplay of the two metal ions was detected. However, the inhibition was found to be favored in the Ga(III)Zn(II) complex compared to the respective monogallium(III) complex, most likely due to bridging coordination of the hydrolysis product, additionally stabilized by coordination of the adjacent pivaloyl-amide residue. The proposed arrangement in the catalyst-hydrolysis product adduct derives from a structure of a stable phosphoester-bridged dizinc(II) complex bearing the same ligand backbone. However, the Ga(III)Zn(II) complex is the first heterodinuclear model complex that mimics the essential function of PAPs, the ability to cleave phosphomonoesters and therefore supports the crucial impact of the second coordination sphere in the active site of PAP.
In this thesis we develop a method for the estimation of the flow behaviour of an incom- pressible fluid based on observations of the brightness intensity of a transported visible substance which does not influence the flow. The observations are given in a subregion of the flow as a sequence of discrete images with in- and outflow across the image boundaries. The resulting mathematical problem is ill-posed and has to be regularised with information of the underlying fluid flow model. We consider a constrained optimisation problem, namely the minimisation of a tracking type data term for the brightness distribution and a regularisation term subject to a system of weakly coupled partial differential equations. The system consists of the time- dependent incompressible Navier-Stokes equations coupled by the velocity vector field to a convection-diffusion equation, which describes the transport of brightness patterns in the image sequence. Due to the flow across the boundaries of the computational domain we solve a boundary identification problem. The usage of (strong) Dirichlet boundary controls for this purpose leads to theoretical and numerical complications, so that we will instead use Robin-type controls, which allow for a more convenient theoretical and numerical framework. We will prove well-posedness and investigate the functionality of the proposed approach by means of numerical examples. Furthermore, we discuss the connection to Dirichlet-control problems, e. g. the approximation of Dirichlet-controls by the so-called penalised Neumann method, which is based on the Robin-type controls for a varying penalty parameter. We will show via numerical tests that Robin-type controls are suitable for the identifi- cation of the correct fluid flow. Moreover, the examples indicate that the underlying physical model used for the regularisation influences the flow reconstruction process. Thus appropriate knowledge of the model is essential, e. g. the viscosity parameter. For a time- independent example we will present a heuristic, which, beside the boundary identification, automatically evaluates the viscosity in case the parameter is unknown. The developed physics-based optical flow estimation approach is finally used for the data set of a prototypical application. The background of the application is the approximation of horizontal wind fields in sparsely populated areas like desert regions. A sequence of satellite images documenting the brightness intensity of an observable substance distributed by the wind (e. g. dust plumes) is thereby assumed to be the only available data. Wind field information is for example needed to simulate the distribution of other, not directly observ- able, substances in the lower atmosphere. For the prototypical example we compute a high quality reconstruction of the underlying fluid flow by a (discrete) sequence of consecutive spatially distributed brightness intensities. Thereby, we compare three different models (heat equation, Stokes system and the original fluid flow model) in the reconstruction process and show that using as much model knowledge as possible is essential for a good reconstruction result.
Schwerionenstrahlung entfaltet bei gleicher physikalischer Dosis eine höhere biologische Wirksamkeit als Photonenstrahlung. In dieser Arbeit wurden die Ursachen dafür anhand der DNA-Schadensantwort in U87 Glioblastomzellen untersucht. Ausschlaggebend sind hierbei DNA-Doppelstrangbrüche (DSBs), deren Induktion und Reparatur durch Messung des DSB-Markers γH2AX (phosphoryliertes Histon H2AX) im Kontext des Zellzyklus verfolgt wurde. Weiter wurde untersucht, ob es strahlenspezifische Unterschiede in der Wahl des DSB-Reparaturmechanismus gibt, und welche Folgen beim Scheitern der Reparatur eintreten. Die Untersuchungen ergaben, dass 12C-Ionen-Strahlung schwerer zu reparierende DSBs erzeugt als Photonenstrahlung, die etwas verzögert und langsamer repariert werden. Dies bewirkte stärker ausgeprägte und länger anhaltende Zellzyklusblockaden, vorwiegend am G2/M-Übergang, sowie eine höhere Apoptoserate bei 12C-Ionen-Strahlung. Autophagozytose als alternativer Weg des programmierten Zelltods spielte bei beiden Strahlenarten keine Rolle. Die Wirkung der 12C-Ionen hing weniger stark von der Zellzyklusphase ab als bei Photonenstrahlung. Dies zeigte sich besonders deutlich anhand der DSB-Reparaturgeschwindigkeit in der S- und G2-Phase. Die Zellen waren bei 12C-Ionen-Strahlung stärker auf die Reparatur durch homologe Rekombination (HRR) angewiesen als bei Photonenstrahlung, auch wenn in beiden Fällen vermutlich der Mechanismus der Nicht-homologen End-zu-End-Verknüpfung dominierte. Die Ursache dafür, dass 12C-Ionen- gegenüber Photonenstrahlung schwerer zu reparieren-de DSBs erzeugte, die langsamer und eher durch HRR repariert wurden, war höchstwahrscheinlich eine stärkere Clusterung der DSBs aufgrund der höheren Ionisationsdichte von 12C-Ionen-Strahlung. Die mikroskopische Untersuchung von immunfluoreszent markiertem γH2AX zeigte, dass die DSB-Reparaturfoci bei 12C-Ionen-Strahlung größer waren und mehr γH2AX -Moleküle enthielten (stärkere Fluoreszenz), obwohl ihre Anzahl vor Abschluss der Reparatur geringer war. Neben den Foci wurde auch ein schwächeres pan-nukleäres γH2AX -Signal beobachtet, das linear mit der Strahlendosis anstieg und bei 12C-Ionen-Strahlung stärker ausgeprägt war als bei Photonenstrahlung. Pan-nukleäres γH2AX als Folge von ionisierender Strahlung wird mit kleinen DNA-Fragmenten in Zusammenhang gebracht. Diese Indizien sprachen dafür, dass die Clusterung der DSBs, und nicht allein ihre Anzahl, aus-schlaggebend für die hohe relative biologische Wirksamkeit der 12C-Ionen-Strahlung ist. Weitere Hinweise auf geclusterte DSBs nach 12C-Ionen-Strahlung lieferten Untersuchungen der Reparaturfoci nach Immunfärbung von γH2AX und dem HRR-Enzym pBRCA1 mit Hilfe superauflösender Mikroskopie. Hierzu wurden die Zellen mit einzelnen bis einigen wenigen 12C-Ionen unter einem flachen Winkel bestrahlt. Durch Einsatz von strukturierter Beleuchtung (SIM) gelang es die Foci und ihren Kolokalisationsgrad im Verlauf der Reparatur mit einer hohen Genauigkeit jenseits der klassischen Auflösungsgrenze in 3D zu vermessen. Neben den primär entlang der Ionenflugbahn induzierten Foci traten nach 26h neue Sekundärfoci auf. Diese waren abweichend von der Ionenflugbahn verteilt und gingen vermutlich im Zuge der DNA-Replikation aus verbleibenden Nicht-DSB-Schäden hervor. In Übereinstimmung mit der Abhängigkeit solcher Sekundärfoci von der Reparatur durch Bruch-induzierte Replikation, einer Sonderform der HRR, wiesen die sekundären γH2AX Foci einen besonders hohen Kolokalisationsgrad mit pBRCA1 auf. Die innere Struktur der γH2AX Foci wurde mit SPDMPhymod, einer weiteren superauflösenden Mikroskopietechnik untersucht. Hierbei stellte sich heraus, dass die Foci, die Größen bis über 1,5 µm³ erreichen, nicht homogen aufgebaut sind, sondern über eine feine Substruktur verfügen. Demnach bestehen die Foci aus vielen Subfoci in der Größenordnung von 100nm mit teilweise länglicher Gestalt. Passend zur Organisation des Chromatins in dicht gepackten Schlaufen mit Interchromatin-Lücken ließen sich unterschiedliche Verläufe und Abstände zwischen den Subfoci erkennen.
In mammals, the balance of energy storage and breakdown is essential for the maintenance of whole body energy homeostasis. An imbalance between energy intake and expenditure as well as an impaired adaption of the central metabolic organs to changes in nutrient availability (so-called metabolic inflexibility) is tightly associated with the onset and progression of obesity and type II diabetes (T2D) and represents a hallmark of the metabolic syndrome. The metabolic syndrome is associated with a spectrum of liver abnormalities described as non-alcoholic fatty liver disease (NAFLD). Thereby, intracellular fat accumulation leads to hepatic lipotoxicity, cellular stress and dysfunction. While the liver is recognized as a key organ contributing to systemic metabolic control, the molecular mechanisms of liver metabolism in coordinating metabolic flexibility remain still puzzling.
In order to determine the molecular mechanisms involved in metabolic flexibility and to find genes which display certain ‘inflexibility’ in conditions of metabolic dysfunction, a liver transcriptome analysis was performed on obese-diabetic db/db and wild type mice under fasting and fed conditions. Array results have uncovered one ‘inflexibility’ gene in the liver: GADD45β, a member of the “growth arrest and DNA damage-inducible 45” (GADD45) gene family. In particular, we have observed a strong upregulation of liver Gadd45b expression upon food deprivation in healthy animals, which was markedly less pronounced in db/db mice. While GADD45β is reported to be involved in hyperplasia of hepatocytes and in liver regeneration, there are no studies examining the role of GADD45β in metabolic function.
In this work, we have characterised GADD45β as a nutrient starvation-induced gene, which is differentially expressed in disease and ageing. Congruently, liver Gadd45b mRNA levels were lower in fasted men with T2D than in healthy individuals. Gadd45b induction was liver specific and not compensated by an induction of the other GADD45 family members. Furthermore, we could show that the Gadd45b induction is reversible after re-feeding and intrinsic to hepatocytes.
In the present work we describe for the first time a novel role for hepatic GADD45β in adaptive metabolism under the stress of food deprivation and nutrient overload. A whole-body deletion of the gene caused an accumulation of triglycerides and cholesterol in the liver under fasting conditions and in a mouse model for steatosis. Also, GADD45β KO mice were more insulin resistant after 4 months on high fat diet. Inversely, a liver-restricted GADD45β overexpression in systemic GADD45β KO mice could partially reverse their dysregulated fasting lipid metabolism, and GADD45β overexpression in db/db mice could partially reverse their diabetic phenotype.
While the exact mechanism(s) by which GADD45β mediates its effects remain unclear we conclude that GADD45β may be a missing link between lipid and glucose homeostasis under conditions of nutrient stress, thereby protecting from metabolic dysfunction.
The endothelial to hematopoietic transition (EHT) is a key developmental event leading to the formation of blood stem and progenitor cells during embryogenesis. A small subset of cells called hemogenic endothelial cells (HE) undergoes the EHT by becoming pre-hematopoietic stem and progenitor cells (Pre-HSPC). Eventually after losing all their endothelial characteristics they become HSPC. Despite extensive studies on this process, there are several questions remaining: What are the differences between hemogenic and non hemogenic endothelial cells? How different is the EHT process in the aorta-gonad-mesonephros (AGM) generating mostly blood stem cells (self-renewing and generating all cell types) and the yolk sac (YS) producing mostly blood progenitors (non self-renewing and generating only a few cell types)? To address these questions, I used single cell transcriptomics because of the scarcity of the HE and the Pre-HSPC within the endothelial population in the AGM and YS. I examined the cells isolated at E9.0, E10.5 and E11.0 mouse embryos. I showed that the major endothelial population of AGM and YS is different from each other, which might be linked with their distinct hematopoietic program. I showed that the pre-HSPC in AGM and YS are transcriptionally alike suggesting that the different hematopoietic program between AGM and YS could be due to the microenvironment. Additionally, I identified a new population detected only in YS at E10.5 co-expressing endothelial and erythroid genes. The molecular mechanism of the EHT is still not understood. Since the TGFβ signaling triggers a similar event during heart development called endothelial to mesenchymal transition (EndMT), we hypothesized that TGFβ activity may play a similar role in EHT. When I activated the TGFβ signaling by adding TGFβ2 during in vitro EHT differentiation, I observed surprisingly a complete block of the hematopoiesis. When I inhibited it by adding the Tgfbr1 inhibitor, it enhanced blood development. Additionally, the mRNA profile of the treated cells confirmed that inhibition of Alk5 is promoting the EHT, while the TGFβ activation results in cells with a phenotype closer to cardiac and mesenchymal cells. Consequently, despite the fact that both EndMT and EHT lead to a loss of endothelial cell identity and the generation of mobile cells, our study suggests that the signaling events initiating both processes are different.
Ziel dieser Arbeit war die genetische und brutbiologische Untersuchung einer Brutpopulation des Sibirischen Trauerschnäppers (Ficedula hypoleuca sibirica). Hierfür lagen 1.969 Blutproben aus 250 Nestern einer kompletten Brutpopulation eines Beprobungsareales (Experimental Plot, Control Plot, South Plot) in den Waldgebieten von Tomsk aus dem Jahr 2005 vor. Es handelte sich zumeist um komplett beprobte Familien (Nester mit Vater, Mutter und Jungtieren). Zum Vergleich lagen noch 339 Proben aus Gärten und Parkanlagen der Innenstadt von Tomsk vor, die ebenfalls zumeist aus komplett beprobten Familien bestanden. Bei den Proben aus der Tomsker Innenstadt handelte es sich überwiegend um Verdachtsfälle polygyner Männchen. Die Alttiere wurden vor Ort morphologisch gesext. Nach der Isolierung von DNA aus den Blutproben wurden alle Individuen zudem einem genetischen Sexing unterzogen. Acht Mikrosatellitenloci (FHY336, FHY427, FHY403, FHY452, FHU1, FHU2, FHU3, FHU5) wurden für die genetische Untersuchung aus der Literatur ausgewählt. Über Multiplex-PCR mit jeweils vier analysierten Loci und anschließender Kapillargelelektrophorese (MEGABace) wurden alle Individuen untersucht. Für jeden Lauf an der MEGABace wurde mithilfe der Software Genetic Profiler Elektropherogramme erstellt und ausgewertet. Anschließend erfolgte eine Auswertung mithilfe spezieller Software (Excel Microsatellite Toolkit, CERVUS). Es überwogen monogame Familien. Daneben wiesen 23 % aller Nester Jungtiere außerhalb des Paarbundes (Extrapair Youngs, EPY) auf. Fast 10 % aller Nestlinge insgesamt waren EPYs. In 43 Fällen konnten die Väter der EPYs innerhalb der beprobten Männchen ermittelt werden. In 18 weiteren Fällen wurde das Vorkommen von floatern, d. h. Männchen ohne eigenes Revier, als Väter von EPYs diskutiert. Durch die genaue Dokumentation mit Daten zur Distanz zwischen den Nestern und der Brutdichte, konnte auf mögliche Gründe für EPYs eingegangen werden. Daneben wurden auch 8 Fälle von polyterritorialer Polygynie bei einigen Trauerschnäppermännchen festgestellt. In allen Fällen handelte es sich ausschließlich um bigynes Verhalten. Ein als trigyn vermutetes Männchen in der Tomsker Innenstadt konnte nicht bestätigt werden. Bei bigynen Fällen konnte zwischen simultaner (1 Fall) und sukzessiver Bigynie (7 Fälle) unterschieden werden. Ein Zusammenhang zwischen Nestern mit polyterritorialer Bigynie und der Schädigung solcher Nester durch EPYs konnte nicht festgestellt werden. Seltener als polyterritoriale Polygynie konnten auch 6 Fälle von monoterritorialer Bigynie nachgewiesen werden, bei denen zwei Weibchen im gleichen Nest ihre Jungen aufzogen. 13 Fälle von „Brutparasitismus“ tauchten ebenso im Datensatz auf wie 17 Fälle von „Adoptionen“ (Female Replacement bzw. Parent Replacement). Mögliche Gründe für „Adoptionen“ und „Brutparasitismus“ sind wahrscheinlich „verlegte Eier“ und „überbaute Nester“. In Kombination von monoterritorialer Bigynie gelang auch ein zweimaliger Nachweis von biandrischen Weibchen. Der Bruterfolg aller monogamen Männchen und Weibchen wurde mit denen verglichen, die durch die zuvor genannten Fälle betroffenen waren. Daneben waren Gelegegröße, Anzahl der geschlüpften Nestlinge sowie der flüggen Jungtiere gut dokumentiert worden, so dass auch hier Vergleiche zwischen den Nestern der unterschiedlichen Fälle vorgenommen werden konnten und auf signifikante Unterschiede geprüft wurden. Signifikante Unterschiede im Bruterfolg (flügge Nestlinge) von Weibchen monogamer Männchen und Sekundärweibchen polyterritorial-bigyner Männchen konnten festgestellt werden. Zudem konnte in Zweitnestern gegenüber Erstnester polyterritorial-bigyner Männchen eine signifikant erhöhte Jungtiersterblichkeit nachgewiesen werden.
Ziel der vorliegenden Arbeit war die Entwicklung der Synthese und die Charakterisierung neuer Derivate von [2.2.2]Parayclophan-1,9,17-trien mit unterschiedlichem Substitutionsgrad des bekannten Rückgrats. Durch ringöffnende Metathese-Polymerisation (ROMP) wurden aus den Monomeren Poly(para-phenylen)vinylen-Derivate (PPVs) hergestellt, deren Topologie mit der Substitution des Monomers variierte. Die neu entwickelte Syntheseroute der Paracyclophantriene verläuft über den Aufbau eines Phenylen-Ethinylen-Rückgrats, welches durch ein Grignard-Reagenz zur Phenylen-Vinylen-Einheit reduziert wird. Der Ring wird anschließend mittels McMurry-Reaktion geschlossen. Die Flexibilität der Synthesestrategie gewährt durch die Kombination der Molekülbausteine einen Zugang zu einer Vielfalt an Derivaten in guten Ausbeuten. In der ROMP wurden zweifach, vierfach und sechsfach homo-substituierte Derivate als Monomere eingesetzt und sowohl die Polymerisation, als auch die Polymere selbst untersucht. Die Polymerisation von zweifach und vierfach substituierten Monomeren wurde erfolgreich optimiert und hohe Molekulargewichte erzielt. Die Polydispersitäten waren etwas höher als nach lebender Polymerisation zu erwarten. Das Hauptaugenmerk lag auf dem Einfluss des jeweiligen Restes und dessen Fähigkeit die Löslichkeit im Polymer zu beeinflussen. Bei zweifach substituierten PPV-Derivaten wurde ein Zusammenhang zwischen der Art der Substitution und dem erreichbaren Molekulargewicht des Polymers gefunden. Ebenfalls wurde ein Zusammenhang zwischen dem Molekulargewicht und der Absorption in Lösung, sowie der Lage der Energieniveaus im Festkörper festgestellt. Die PPV-Derivate aus vierfach substituierten Paracyclophantrienen zeigten aufgrund der zusätzlichen Substitution keine Beschränkung des Molekulargewichts durch die Löslichkeit. Die Absorption und Emission verschieben sich bathochrom in Abhängigkeit des Substitutionsgrades. In dünnen Filmen vergrößert sich die optische Bandlücke (hypsochrome Verschiebung) und die Energie des emittierenden Zustands wird herab gesetzt (bathochrome Verschiebung). Dies ist Änderungen der Konformation beim Übergang von Lösung zum festen Zustand zuzuschreiben.
This thesis presents a measurement of indirect CP asymmetries in the charm system. The indirect CP violation for the charm-meson system is expected to be small in the Standard Model and has not been observed experimentally so far. Effects of beyond Standard Model physics phenomena but as well perturbatively non-calculable processes can manifest themself in a potential non-zero CP violation. The knowledge of the size of CP violation makes a significant contribution for the understanding of the Standard Model and has a direct impact on the effective parameters in the charm system. The LHCb experiment has collected the world largest data samples of charm mesons. The proton-proton collisions data used in this thesis have been recorded in the first run period in 2011-2012. They correspond to an integrated luminosity of 3.0 fb−1 . The measurement uses the singly Cabibbo-suppressed D0->K−K+ and D0->pi−pi+ decays. The charm mesons exploited in this analysis are produced in semi-muonic B-meson decays. Thereby, the charge of the created muon determines the flavour of the charm meson at production time. To perform this measurement, a robust method is developed that reaches a sensitivity at sub-permille level. The indirect CP asymmetry manifests itself in the asymmetries of effective lifetimes, AGamma , of the D0 and D0bar decays and is measured for both decays AGamma(K−K+) = (−0.134 ± 0.077 +0.026 )% , AGamma(pi−pi+) = (−0.092 ± 0.145 +0.025 )% . where the first uncertainty is statistical and the second systematic. The results are compatible with previous measurements and with the zero hypothesis of no CP violation. This analysis provides a significant contribution to the effective description of the charm meson system and to the world average. The results have been submitted for publication to the Journal of High Energy Physics (JHEP) [1].
Argumentative thought experiments are structurally conditional clauses. They can hence be formalized by means of the principle of modus ponendo ponens, as well as of modus tollendo tollens. In contrast to the practice in formal logic, exponents of argumentative thought experiments claim that the logical validity of a conclusion drawn within the framework of a particular conditional argument also holds beyond the particular conditional in question. In this paper, I articulate the criticism that this claim is wrong by arguing that the counterfactual scenario sets itself the most determinant premise. If the counterfactual scenario sets the initial conditional premise of the argument, then its true conclusion holds only as a counterfactual truth. The present paper illustrates this criticism using Frank Jackson’s thought experiment, the so-called knowledge argument, as a concrete example.
Melanoma is the deadliest form of skin cancer. Traditional therapeutic options include surgery, radiation, chemotherapy and immunotherapeutic options. Typically, around 20% of patients develop metastases as the disease progresses, which reduces therapeutic options to provide only palliative benefit in the majority of cases. The genomes of a large number of primary and secondary melanomas were recently sequenced, leading to the identification of melanoma driver mutations. Targeting pathways, activated by cancer-specific genetic alterations, enabled researchers to develop novel therapeutic drugs. Although these drugs effectively fight melanoma cells, cancer cells develop various resistance mechanisms regulated by epigenetic changes, which leads to cancer recurrence in most patients. Nuclear factor-based reprogramming was implemented in melanoma cells to test whether malignant cancer cells can reacquire developmental pluripotency, and moreover to analyze reprogramming-associated epigenetic changes on the tumor cell phenotype. The results showed that the constitutive overexpression of Oct4, Sox2, and Klf4 reprograms melanoma cells into a murine embryonic stem cell-like state. In contrast to fibroblasts, melanoma cells do not require exogenous c-Myc for the induction of a pluripotent stem cell state, characterized by the reactivation of endogenous pluripotency markers and loss of the transcriptional profile of melanoma cells. However, continuous transgene expression is required to maintain an undifferentiated state. When injected into immunocompromised mice, melanoma-derived reprogrammed cells formed teratoma-like tumors containing cell types of all three germ layers, and despite their oncogenic mutations, rarely contained melanoma-like structures. In vitro directed differentiation into neuronal-like and fibroblast-like cells demonstrated that reprogrammed tumor cells acquired the potential to execute terminal differentiation pathways. Although most melanoma cell lines are highly depended on MAPK signaling, reprogrammed tumor cells and their differentiated daughter cells became resistant against BRAF- or MEK-targeting inhibitors, suggesting that epigenetic remodeling processes facilitated therapy resistance against targeted melanoma therapy. Furthermore, global gene expression profiling demonstrated that nuclear reprogramming and subsequent differentiation induced deregulation of tumor suppressors and oncogenes. In conclusion, reprogramming cancer cells allows the investigation of a cancer genome in the context of a specific epigenetic cell state and might help study how alterations in the epigenetic signature control the biological behavior of tumor cells and their response to therapy.
The interaction of complex molecular systems with light has great relevance in nature as well as for many of the latest technological developments. The process of photosynthesis converts light into chemical energy, thereby providing the primary energy source for life on Earth. Photovoltaic devices, as the technological implementation of this principle, constitute one of the most promising sources of electrical energy for the 21st century, whose application has increased tremendously in the past decade. The reverse process, the controlled emission of light from electronically activated (excited) molecules, is central to many modern technologies, the most prominent of which are presumably the ever smaller yet higher resolving display screens of hand-held computers.
For all these applications, a fundamental understanding of the processes taking place at the atomistic scale is of key relevance to allow for a rational design and improvement of new technologies. However, due to the ultra-short time-scales on which the elementary steps of most light-induced phenomena occur and their inherent complexity, an exclusively experimental investigation is often tedious, in particular concerning the interpretation of the results. Here, the combination of experimental techniques and theoretical models can help to gain insights into the involved processes. For this purpose, the electronic structure of ground and light-activated (excited) states of the involved molecules as well as the interaction with their environment has to be approximated, which is the central topic of this work.
In the first part, namely chapters 2-4, I present applications of the quantum-mechanical methodology introduced in chapter 1 to study light-induced processes in molecular systems. The so-called caged compounds studied in chapters 2 and 3 constitute an attempt to employ the remarkable spatio-temporal light control of modern lasers to control chemical reactions. For this purpose, the investigated, prototypical molecules nitro-phenylacetate (NPA) and ortho-nitrobenzylacetate (oNBA) serve as precursors for the active compounds CO2 and acetate, respectively. Upon irradiation with UV light, the active compound is released within nano- to microseconds, and may e.g. trigger subsequent reactions. In the above-mentioned sense, my theoretical investigation accompanied and guided an experimental study, which allowed to shed light on the molecular processes and to resolve the detail of the mechanism responsible for the light-induced reactivity.
The common structural motif of NPA, oNBA and many other photo-active systems is the nitroaromatic moiety in the form of its smallest representative nitrobenzene (NB). Due to this prototypical character, the photochemistry of NB is relevant for many photochemical applications. In chapter 4, I report an extensive theoretical investigation of ground and excited states as well as the non-radiative decay of NB, which due to its small size and high symmetry allows for an application of a hierarchy of state-of-the-art quantum-chemical methods. Surprisingly, I found this small molecule to pose a serious challenge to electronic structure theory and consequently, some rather sophisticated ab initio methods fail to afford an accurate description, e.g. with respect to the photochemically very important ordering of the lowest triplet states. Nevertheless, I determined the mechanism of non-radiative decay in good agreement with experimental findings and, moreover, suggested an experiment to test my hypothesis.
Although there exist a number of accurate and reliable quantum chemical methods that allow for an investigation of the ground and excited states of isolated systems with the molecular size of NPA, oNBA and NB, the environment often plays a crucial role and may decisively influence the light-induced processes, as e.g. in NPA. Hence, the approximate modeling of molecular environments for quantum-chemical problems in condensed phase is a very active field of research, which culminated in the 2013 Nobel Price for Chemistry, which was awarded to Karplus, Levitt and Warshel for their pioneering developments in the field of multiscale models for complex chemical systems. To enable a quantum-chemical description of photo-chemical excitation processes in condensed phase, I extended and implemented a quantum-classical polarizable-continuum model (PCM) for calculation of vertical excitation energies, which is described in chapter \ref{part:pcm}. In general, PCMs allow for an efficient computation of the often dominating electrostatic portion of the solute-solvent interaction by means of the macroscopic descriptors epsilon (dielectric constant) and epsilon_opt = n^2 (optical dielectric or squared refractive index, respectively). The implementation of the method was realized in such a way that its application to any quantum-chemical model that affords electron densities for ground- and excited-states is straightforward. For the systematic evaluation of the method, I composed the first set of experimental Benchmark Data for Solvatochromism in Molecules (xBDSM), and part of the data points were measured by myself. Comparing calculated gas phase to solvent shifts to the xBDSM set, I was able to demonstrate the convincing accuracy of my approach in combination with various levels of electronic structure theory and could shed light on the relation of different flavors of excited state PCMs. Moreover, a close examination of the contributions to the calculated shifts revealed general patterns, which are essential regarding any evaluation of calculated solvent shifts by comparison to the experiment. The implemented methodology will be released with one of the next versions of the Q-Chem quantum-chemical software package.
We live in a data-rich environment where massive amounts of data such as text messages, articles, images, and search queries are continuously generated by users. In this environment, new opportunities to discover and utilize knowledge about the real-world arise, such as the extraction and description of places and events from social media records, the organization of documents by spatio-temporal topics, and the prediction of epidemics by search engine queries. Major challenges addressed in these data- and application-specific works arise from the unstructured and complex nature of the data, and the high level of uncertainty and sparsity of the attributes.
Despite the evident progress in utilizing specific data sources for different applications, there remains a lack of common concepts and techniques on how to exploit the data as high-quality sensors of geographic space in a general manner. However, such a general point of view allows to address the common challenges and to define fundamental building blocks to deal with problems in fields like information retrieval, recommender systems, market research, health surveillance, and social sciences.
In this thesis, we develop concepts and techniques to utilize various kinds of user-generated data as a steady source of information about geographic processes and entities (together called geographic phenomena). For this, we introduce a novel conceptual data mining framework, called geographic feature mining, that provides the foundation to discover and extract highly informative and discriminative dimensions of geographic space in a unifying and systematic fashion. This is achieved by representing the qualitative and geographic information in the records as geographic feature signals, each constituting a potential dimensions to describe geographic space. The mining process then determines highly informative features or feature combinations from the candidate sets that can be used as a steady source of auxiliary information for domain-specific applications.
In developing the framework, we make contributions to several fundamental problems: (1) We introduce a novel probabilistic model to extract high-quality geographic feature signals. The signals are robust to noise and background distributions, and the model allows to exploit diverse kinds of qualitative and geographic information in the records. This flexibility is achieved by utilizing a Bayesian network model and the robustness by choosing appropriate prior distributions. (2) We address the problem of categorizing and selecting geographic features based on their spatio-temporal type, such as feature signals having landmark, regional, or global semantics. For this, we introduce representations of the signals by interaction characteristics and evaluate their performance in clustering and data summarization tasks. (3) To extract a small number of highly informative feature combinations that reflect geographic phenomena, we introduce a model that extracts latent geographic features from the candidate signals using dimensionality reduction. We show that this model outperforms document-centric topic models with respect to the informativeness of the extracted phenomena, and we exhaustively evaluate how different statistical properties of the approaches affect the characteristics of the resulting feature combinations.
More and more experiments show that the CCCTC-binding factor (CTCF), a multi-Cys2His2 (mC2H2) zinc finger protein, plays a key role in the spatial organization of chromatin and gene regulation in the nucleus of eukaryotic cells. In this context an important problem is to uncover the underlying mechanism of how CTCF shapes the chromatin structure. In this thesis, models on different scales, from atomistic scale to coarse-grained scale, are studied to better understand the conformational and dynamical properties of both the unbound CTCF and CTCF-DNA complexes.
Using homology modeling, an atomistic model of CTCF is constructed to study the conformational properties of unbound mC2H2 zinc finger proteins. To enhance the computing and sampling efficiency an atomistic pivoting algorithm and a mesoscale model for mC2H2 proteins is developed. It is shown that the conformations of unbound mC2H2 proteins, like CTCF, can be explained with a worm-like chain model. For proteins of a few zinc finger, an effective bending constraint favors an extended conformation, which is consistent with experimental findings. A self-avoiding chain model applies only to proteins of more than nine zinc fingers.
As a subsequent step, a mesoscale model is designed to study how a mC2H2 zinc finger protein binds to and searches for its target DNA loci. Statistical sequence-dependent interactions between the proteins and DNA are derived. Molecular dynamics simulations of this model reproduce several kinetic properties of mC2H2 zinc finger proteins, such as the rotation coupled sliding, the asymmetrical roles of different zinc fingers and the partial binding partial dangling mode. An application to CTCF in complexes with one of its target DNA duplex shows that CTCF binds to DNA only by using its central zinc fingers. It asymmetrically bends the DNA duplex but does not form DNA loops. Other CTCF-assisted DNA looping mechanisms, like a bridged DNA loop organized by a CTCF homodimer, could be further studied with this model.
Motivated by the non-covalent binding of polypeptides to DNA, I study the adsorption of a flexible polymer to a rigid polymer with periodic binding sites, both in 2d and in 3d. Analysis of Monte Carlo simulation results show that the phase transition, from non-adsorbed to adsorbed with increasing adsorbing strength, is a second order transition in 2d, and higher order transition in 3d. Compared to the adsorbed monomers, successive non-adsorbed monomers contribute more to the winding of the flexible polymer around a rigid polymer, showing the importance of the linkers in mC2H2 zinc finger proteins to wrap around DNA.
This thesis is concerned with the analysis and modeling of effects which cause errors in passive stereo and Time-of-Flight imaging systems. The main topics are covered in four chapters: I commence with a treatment of a system combining Time-of-Flight imaging with passive stereo and show how commonly used fusion models relate to the measurements of the individual modalities. In addition, I present novel fusion techniques capable of improving the depth reconstruction over those obtained separately by either modality. Next, I present a pipeline and uncertainty analysis for the generation of large amounts of reference data for quantitative stereo evaluation. The resulting datasets not only contain reference geometry, but also per pixel measures of reference data uncertainty. The next two parts deal with individual effects observed: Time-of-Flight cameras suffer from range ambiguity if the scene extends beyond a certain distance. I show that it is possible to extend the valid range by changing design parameters of the underlying measurement system. Finally, I present methods that make it possible to amend model violation errors in stereo due to reflections. This is done by means of modeling a limited level of light transport and material properties in the scene.
In this thesis, we use a functional quantum field theoretical approach to investigate the non-equilibrium time evolution of an Anderson quantum dot with the main focus on the Kondo regime. We employ a real-time Keldysh path integral formulation to find an effective action. From the two-particle irreducible effective action, we derive, from the variational principle, the exact real-time Kadanoff–Baym equations of motion for the full propagator. We study these dynamic equations for the single impurity Anderson model, which decribes a quantum dot coupled to two finite-temperature leads. We take the tunnelling to the leads into account exactly. In order to solve the Kadanoff–Baym equations numerically we have to approximate them. For this purpose, we make a non-perturbative approximation by summing an infinite number of Feynman diagrams in the direct (s)-, particle-particle (t)-, particle-hole (u)-, and stu-channels. The aim of our investigation is to analyse the non-equilibrium realtime evolution of the quantum dot after a hybridisation and interaction quench into its stationary state. The main focus is on the narrowing of the Kondo resonance and the formation of the Hubbard side bands. Following on from this, the main achievement is the transient, as well as the stationary electrical current, through the quantum dot and the investigation of the dependence on temperature and magnetic field. We compare our results with other methods, such as functional renormalisation group and iterative sum of path integrals, and find a very good agreement in most situations.
The search for a second genesis of life outside Earth is now well and truly underway with the first rocky exoplanets detected in the central star’s liquid water habitable zone. Recent results based on population studies show that small planets are abound in our galaxy. With the next generation of space- and ground-based telescopes on the horizon, it is critical to determine the best candidate exoplanets to follow up on for potential habitability and life. This PhD thesis shows how colors of extreme Earth-like planets can be used as a first characterization when prioritizing exoplanets for spectroscopic follow up. We build a strong interdisciplinary link between geomicrobiology and observational astronomy, by exploring the color signatures of extremophiles as well as the various extreme niches that those organisms inhabit on Earth. In addition, we provide the first database of surface signatures of terrestrial life for a broad range of pigmented microorganisms, including ones isolated from Earth’s most extreme environments. Our spectral library provides a broader and more realistic guide for the search for surface features of extraterrestrial life. The work presented in this thesis provides a first step toward characterizing a second Earth, in preparation for the next generation of space- and ground-based instruments, which will increase the chances of detecting life.
The performance of fuel cells is significantly affected by “loss mechanisms”. This work is devoted to developing concepts for the efficient numerical computation of the diffusion polarization in the porous anode of a solid oxide fuel cell (SOFC). The following topics were covered:
The first part of this work is focused on the numerical verification of coupling conditions for effective viscous flows over a porous medium. It is generally accepted that the “Beavers-Joseph-Saffman slip law” holds true for a main flow direction which is tangential to the interface. However, the interface law for the effective stress has been a subject of controversy. We provide a confirmation of the “pressure jump law”, which has been recently derived by Marciniak-Czochra and Mikelic, for a range of configurations using a direct numerical simulation of the flow at the microscopic level.
The second part of this work is about the derivation of a goal-oriented, a posteriori error estimator for the finite element approximation of elliptic homogenization problems based on the “Dual Weighted Residual method” of Becker and Rannacher. In general, the solution of the macroscopic equation in the homogenized model depends on effective coefficients which in turn depend on the solutions of some additional auxiliary equations. Therefore, the accuracy of the physical goal functional is influenced by the discretization error of the macroscopic and the auxiliary solutions. By employing the error estimator developed in this work we can estimate the contribution of the discretization of each sub-problem (effective model and auxiliary problems) onto the overall error. These contributions are then balanced within a successive refinement cycle to set up an efficient discretization. Local error indicators are used to steer an adaptive mesh refinement for the macroscopic problem as well as the auxiliary problems.
We demonstrate the functionality of this algorithm on some prototypical homogenization problems and on an effective model developed in this work to simulate the gas transport in the anode of an SOFC. In the latter, the diffusion polarization is the quantity of interest. For a given accuracy, the application of the local mesh refinement based on the adaptive algorithm in this context decreases the number of degrees of freedom and computation time significantly compared to the global mesh refinement.
A molecular scale model for charge transport in organic semiconductors based on quantum chemistry is presented. It is formulated as a stochastic model on the integer lattice in three dimensions. The model is treated with probabilistic methods, which allow to prove a continuous scaling limit, especially suited for intermediate regiemes, i.e. low temperatures, large energetic disorder and small devices. The scaling limit is connected to a certain integro-differential equation. Both, the microscopic model and the scaling limit are computationally studied and it is demonstrated that the proposed model can explain dispersive effects in thin film organic semiconductor devices.
This study was originally designed to investigate the role of global cellular processes like the cell cycle in mediating signal from the circadian clock to rhythmic growth and development in Neurospora crassa. In a first attempt clock control over growth and cell cycle related genes was tested by real-time monitoring of gene expression with a luciferase based assay. It was observed that under conditions, which restrict growth and conidiation, the target genes showed no or poor rhythmicity. In contrast, under native growth conditions or in cultures which overcome growth repression by aging rhythmic gene expression could be observed in any of the target genes. Surprisingly, this included also typical “housekeeping” genes like tubulin, histoneH1 or actin for example. A further hallmark of these rhythms was that they were equal in phase even in genes which had previously been reported to be expressed antiphasic (Sancar et al., 2011). These observations suggest that the luciferase assay may interfere with clock controlled processes which are related to native growth. With a further project the role of clock control over metabolism for rhythmic growth should be analyzed. For this purpose a mutant was characterized which had been shown to be defective in the trehalose synthase clock controlled gene 9 (ccg-9RIP) (Shinohara et al., 2002). The mutant displays the loss of clock control over conidiation and a severe defect in vegetative growth and asexual development. These observations strongly suggested a link between clock control over carbohydrate metabolism and circadian growth. Changes in the transcriptome during conidiation were analyzed in ccg-9RIP. The majority of misregulated genes are related to metabolic functions. However, genotype and phenotype of ccg-9RIP did not cosegregate during backcrossing. A knockout of ccg-9 by gene replacement grew rhythmic and did not show any defect during vegetative growth and asexual development under several conditions. These results demonstrate that ccg-9 does not have any role in circadian growth. Furthermore, the phenotype of ccg-9RIP must be caused by disruption of other genes than ccg-9.
Kreatin spielt eine wichtige Rolle im Energiemetabolismus der Zellen. Die nichtinvasive Detektion von Kreatin im lebenden Gewebe kann mit Chemical Exchange Saturation Transfer (CEST)-Magnetresonanz-Bildgebung (MRI) realisiert werden. Bei CEST wird die Magnetisierung austauschender Protonen in Metaboliten selektiv gesättigt, durch anschließenden chemischen Austausch mit Wasserprotonen wird Sättigung im Pool der freien Wassermoleküle akkumuliert. Daraus resultiert eine messbare Verminderung des Wassersignals. Im Gewebe existieren diverse Metaboliten mit austauschenden Protonen – eines davon ist das Guanidinderivat Kreatin mit vier austauschenden Protonen, deren Austauschrate im intermediate exchange regime liegt. Um deren Austauschprozesse mit CEST zu detektieren, werden hohe Sättigungsamplituden B1 benötigt. Dabei werden jedoch gleichzeitig auch Protonen des freien Wassers und Protonen in Makromolekülen gesättigt, welche den CEST-Effekt überlagern und sogar verfälschen. Ziel dieser Arbeit war die Korrektur solcher Einflüsse, um eine quantitative Kreatin-Bildgebung im Muskel zu realisieren. Unter Nutzung der höheren Selektivität (chemische Verschiebung) bei B0=7T konnte eine korrigierte Intermediate-exchange-CEST-Methode entwickelt und anhand von Kreatinmodelllösungen verifiziert werden. Es zeigte sich, dass der korrigierte CEST-Effekt nicht nur unabhängig von der Wasserrelaxation ist, sondern zudem Linearitätseigenschaften besitzt, die es ermöglichen, simultan Kreatin-Konzentrations- und pH-gewichtete-Bilder zu erzeugen. Angewendet an der Wadenmuskulatur vor und während Muskelkontraktion zeigten die Methoden einen Anstieg der Kreatinkonzentration und einen leichten pH-Abfall bei Muskelarbeit. Diese Techniken ermöglichen nun ortsaufgelöste, quantitative Untersuchungen des Energiemetabolismus im lebenden Gewebe.
The concept of RESOLFT was introduced to break the resolution limit in fluorescence microscopy, which is set by the physical phenomenon of diffraction. It enables the separation of fluorophores inside a focal volume by driving reversible optical transitions between two discernible states. The family of reversibly switchable fluorescent proteins (RSFPs) with long-lived on- and off-states allows for RESOLFT imaging at low light levels. Up to now, RESOLFT involving RSFPs as fluorescent markers has been exclusively demonstrated to enhance the resolution in the lateral dimension. In this work, a novel RSFP-based RESOLFT microscope is presented, that, for the first time, breaks the diffraction barrier in the axial direction by switching fluorophores in the volume of a light-sheet. It is realized with the optical arrangement of a selective plane illumination microscope (SPIM), that illuminates only a thin section of a sample perpendicular to the detection axis and thus reduces the overall light exposure in volume recordings. The symbiotic combination of RSFP-based RESOLFT and SPIM, the so called RESOLFT-SPIM nanoscope, offers highly parallelized, fast imaging of living biological specimens with low light doses and sub-diffraction axial resolution. Compared to the diffraction-limited SPIM analogue an improvement in axial resolution by more than a factor of 12 is demonstrated.
Image segmentation constitutes one of the elementary tasks in computer vision. Various variations exists, one of them being the segmentation of layers that entail a natural ordering constraint. One instance of that problem class are the cell layers in the human retina. In this thesis we study a segmentation approach for this problem class, that applies the machinery of probabilistic graphical models. Linked to probabilistic graphical models is the task of inference, that is, given an input scan of the retina, how to obtain an individual prediction or, if possible, a distribution over potential segmentations of that scan. In general, exact inference is unfeasible which is why we study an approximative approach based on variational inference, that allows to efficiently approximate the full posterior distribution. A distinguishing feature of our approach is the incorporation of a prior shape model, which is not restricted to local information. We evaluate our approach for different data sets, including pathological scans, and demonstrate how global shape information yields state-of-the-art segmentation results. Moreover, since we approximatively infer the full posterior distribution, we are able to assess the quality of our prediction as well as rate the scan in terms of its abnormality. Motivated by our problem we also investigate non-parametric density estimation with a log-concavity constraint. This class of density functions is restricted to the convex hull of the empirical data, which naturally leads to shape distributions that comply with the ordering constraint of retina layers, by not assigning any probability mass to invalid shape configurations. We investigate a prominent approach from the literature, show its extensions from 2-D to N-D and apply it to retina boundary data.
Recent data acquisition techniques permit an improved analysis of living organisms. These techniques produce 3D+t information of cell developments in unprecedentedly high resolution. Biologists have a strong desire to analyze these cell evolutions in order to find similarities in their migration and division behaviors. The exploration of such patterns helps them in understanding how cells and hence organisms are able to ensure a regular shape development. However, the enormous size of the time-dependent data with several tens of thousands of cells and the need to analyze it in 3D hinder an interactive analysis. Visualizing the data to identify and extract relevant features provides a solution to this problem. For this, new visualization approaches are required that reduce the complexity of the data to detect important features in the visual analysis.
In this thesis, novel visual similarity analysis methods are presented to interactively process very large 3D+t data of cell developments. Three main methods are developed that allow different visual analysis strategies. The usefulness of them is demonstrated by applications to cells from zebrafish embryos and Arabidopsis thaliana plants. Both data sets feature a high regularity in the shape formation of the organs and domain experts seek to research similar cell behaviors that are responsible for this development. For example, the identification of 3D division behaviors in plants is still an unresolved issue. The first method is a novel visualization approach that can automatically classify cell division types in plant data sets with high memory and time efficiency. The visualization is based on the generation of newly introduced cell isosurfaces that allow a quantitative and spatial comparison of cell division behaviors among individual plants. The method is applied to cells of the lateral root of Arabidopsis plants and reveals similar division schemes with respect to their temporal order. The second method enables a new visual similarity analysis for arbitrary 3D trajectory data in order to extract similar movement behaviors. The algorithm performs a grouping of thousands of trajectories with an optional level of detail modification. The clustering is based on a newly weighted combination of geometry and migratory features for which the weights are used to emphasize feature combinations. As a result, similar collective cell movements in zebrafish as well as a hitherto unknown correlation between division types and subsequent nuclei migrations in the Arabidopsis plants are detected. The third method is a novel visualization technique called the structure map. It permits a compact and interactive similarity analysis of thousands of binary tree structures. Unique trees are pre-ordered in the map based on spectral similarities and substructures are highlighted according to user-selected tree descriptors. Applied to cell developments from zebrafish depicted as trees, the map achieves compression rates up to 95% according to spectral analysis and facilitates an immediate identification of biologically implausible events and outliers. Additionally, similar quantities of feature appearances are detected in the center of the lateral root of several Arabidopsis plants.
Being able to provide accurate forecasts of future quantities has always been a great human desire and is essential in numerous situations in daily life. Meanwhile, it has become routine to work with probabilistic forecasts in the form of full predictive distributions rather than with single deterministic point forecasts in many disciplines, with weather prediction acting as a key example.
Nowadays, probabilistic weather forecasts are usually constructed from ensemble prediction systems, which consist of multiple runs of numerical weather prediction models differing in the initial conditions and/or the parameterized numerical representation of the atmosphere. The raw ensemble forecasts typically reveal biases and dispersion errors and thus call for statistical postprocessing to realize their full potential. Several ensemble postprocessing methods have been developed and are partly recapitulated in this thesis, yet many of them only apply to a single weather quantity at a single location and for a single prediction horizon. In many applications, however, there is a critical need to account for spatial, temporal and inter-variable dependencies.
To address this, a tool called ensemble copula coupling (ECC) is introduced and examined. Essentially, ECC uses the empirical copula induced by the raw ensemble to aggregate samples from predictive distributions for each location, variable and look-ahead time separately, which are obtained via existing univariate postprocessing methods. The ECC ensemble inherits the multivariate rank dependence pattern from the raw ensemble, thereby capturing the flow dependence.
Several variants and modifications of ECC are studied, and it is demonstrated that the ECC concept provides an overarching frame for existing techniques scattered in the literature.
From a mathematical point of view, it is shown that ECC can be considered a copula approach by pointing out relationships to multivariate discrete copulas, which are introduced in this thesis and for which relevant mathematical properties are derived.
A generalization of standard ECC is introduced, which aggregates samples from not necessarily univariate, but general predictive distributions obtained by low-dimensional postprocessing in an ECC-like manner.
Finally, the SimSchaake approach, which combines the notion of similarity-based ensemble methods with that of the so-called Schaake shuffle, is presented as an alternative to ECC. In this technique, the dependence patterns are based on verifying observations rather than on raw ensemble forecasts as in ECC.
The methods and concepts are illustrated and evaluated based on case studies, using real ensemble forecast data of the European Centre for Medium-Range Weather Forecasts. Essentially, the new multivariate approaches developed in this thesis reveal good predictive performances, thus contributing to improved probabilistic forecasts.
Hypertension is one of the leading causes of morbidity and mortality worldwide, serving as a major risk factor for cardiovascular events such as stroke, myocardial infarction and diabetes. Although clinically well studied, relatively little is known about the cellular mechanisms at the onset of this disease. A chronic increase in arterial wall tension in hypertension translates into elevated levels of biomechanical stretch experienced by vascular smooth muscle cells (SMCs). This leads to prominent changes in gene expression initiating a complex remodelling process driven by a shift in phenotype of otherwise contractile vascular SMCs to an activated, synthetic state. Therefore, mechanotransduction in vascular SMCs in response to altered hemodynamic forces is a crucial step in the progression of hypertension-induced maladaptive remodelling. In this context, we and others have identified the focal adhesion protein zyxin as a putative mechanotransducer which regulates the cellular adaptation to exaggerated biomechanical stretch. As such, we hypothesized that zyxin might play an important role in hypertension-induced cardiovascular remodelling. This work addressed the functional consequences of loss of zyxin during pathological remodelling of arteries and the heart in hypertensive mice. Microarray analysis revealed a dramatic alteration of stretch-regulated gene expression in zyxin-null vascular SMCs. A comparison of vascular SMCs from wild type and zyxin-null mice revealed a growth-promoting, pro-migratory, anti-apoptotic and poorly contractile phenotype of zyxin-null vascular SMCs. This could be attributed to an activation of the RhoA-MRTF-A signalling axis partially driving stretch-induced gene expression in the zyxin-null SMCs. Induction of experimental hypertension led to a significantly lower increase in systolic and diastolic arterial blood pressure in zyxin-null mice particularly in older animals, an outcome that could be attributed to structural changes in the remodelling arteries. This response was paralleled by a reduced resistivity in the femoral artery of these animals likely caused by a loss of extracellular matrix (ECM) integrity observed in older zyxin-null mice. Hemodynamic overload further induced pronounced cardiac interstitial fibrosis, apoptosis and resultant cardiac dysfunction in zyxin-null mice, owing to a major shift towards a pro-fibrotic gene expression pattern within the myocardium, most likely derived from the cardiac fibroblasts. Lastly, using a ratiometric calcium imaging method, this study confirmed a TRPC3 channel-dependent stretch-induced zyxin activation in vascular cells that is mediated through activation of the beta 1 isoform of phospholipase C. Collectively, these findings highlight a novel role of zyxin in hypertension-induced cardiovascular remodelling and underscore the importance of mechanotransduction in the pathophysiology of this highly prevalent cardiovascular disease.
Pancreatic ductal adenocarcinoma (PDAC) is among the ten most frequent cancers in the western world, and also one of the most lethal. The mortality rate approximately equals the incidence rate and the five-year survival rate is only around 5%, mainly due to advanced stage at diagnosis, non-resectability, and frequent chemotherapy resistance. Epidemiological studies show that obesity and/or type 2 diabetes increase the risk of PDAC. Both conditions have been increasing world wide during the last 20–30 years, and have become a global health hazard.
The transcriptional co-regulators TBL1X (transducin-beta-like 1, X-linked) and TBL1XR1 (transducin-beta-like 1X-related protein 1) mediate the exchange of co-repressors to co-activators on target gene promoters. They have been shown by previous work in our lab to regulate lipid metabolism in liver and white adipose tissue. Furthermore, they interact with Wnt/β-catenin signaling, a pathway frequently altered in cancers. Recent studies have shown a growth-regulating role of TBL1XR1 in various cancer entities, but not in pancreatic cancer.
The aim of the present study was therefore to investigate whether TBL1X and/or TBL1XR1 play a role in pancreatic cancer and might link tumor initiation or progression with obesity or type 2 diabetes. While the latter could not be confirmed, the present study was able to show that TBL1X and TBL1XR1 were highly expressed in human and murine pancreatic cancer. The expression was specific for PanIN precursor lesions and carcinoma cells, while healthy tissue showed little to no expression. In human patients, multiple genes involved in metabolic processes showed a high correlation with TBL1X and TBL1XR1 expression. In vitro studies revealed a growth-promoting effect of TBL1X and TBL1XR1 in pancreatic cancer cells and gene expression microarrays indicated cell cycle and p53 signaling as the most prominently regulated pathways. Furthermore, both proteins, especially TBL1X, affected tumor cell glucose metabolism and cellular response to glucose withdrawal. Application of a syngeneic subcutaneous allograft mouse model confirmed the growth-regulating effect of Tbl1x in vivo. Ablation of Tbl1x additionally sensitized murine Panc02 tumor cells to gemcitabine, the most commonly used chemotherapeutic agent for pancreatic cancer. Tbl1x-deficient cells had markedly reduced levels of PI3 kinase, a major regulator of cell growth and metabolism, as well as downstream mediators. TBL1X was confirmed to bind to PI3 kinase promoter region and its expression correlated with PI3 kinase in human patients.
Taken together, this study is the first to show a role of TBL1X in cancer, and of TBL1XR1 in pancreatic cancer, both in humans and in mice. The control of PI3 kinase by TBL1X on the transcriptional level is a plausible explanation for the observed sensitization to gemcitabine, making TBL1X an attractive target for future cancer therapies to enhance treatment response and patient survival.
Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in adults of the Western world. It is a malignancy characterized by an accumulation of CD5 positive B-cells in blood and lymphoid organs. CLL is a very heterogeneous disease, where molecular subgroups display striking differences in treatment response and prognosis. A greater BCR signaling capacity and a loss of p53 signaling activity confer a poor prognosis. While the higher BCR signaling activity seen in CLL with unmutated IGHV genes supports tumor cell survival, p53 aberrations mediate resistance towards standard therapy. The aim of this work was to characterize the involvement of non-coding RNA in these two key signaling pathways of CLL cell survival and resistance. Small RNA sequencing was applied to comprehensively assess microRNA (miRNA) and other non-coding RNA expression in peripheral blood mononuclear cells of 35 CLL patients. miRNAs were identified that display IGHV mutation status dependent expression, and the transcript levels of 15 miRNAs predicted IGHV mutation status with 82% accuracy. By abrogation of BCR signaling in vitro using the small-molecule inhibitor ibrutinib, the expression of miR-320c, miR-1246, miR-484, miR-17-5p, miR-155-3p and miR-27a-5p was found to be BCR signaling dependent, suggesting a role in mediating CLL cell survival. The basal expression of 10 miRNAs was associated with ibrutinib sensitivity in vitro, implicating an involvement of these miRNAs in the regulation of BCR signaling. It was hypothesized that p53-dependent ncRNAs could be identified by comparison of CLL samples with or without TP53 mutation/deletion for their ncRNA expression changes upon DNA damage-triggered p53 induction. In addition to miR-34a, a set of further miRNAs was found to be TP53 status dependently induced (particularly miR-182-5p, miR-7-5p and miR-320d/c). Beyond miRNAs, the present data demonstrate p53-dependent expression of the long non-coding RNAs lincRNA-p21 (long intergenic non-coding RNA p21) and NEAT1 (nuclear enriched abundant transcript 1) upon DNA damage and direct p53 activation with nutlin-3. p53-dependent induction of expression was further proven in a panel of Burkitt’s lymphoma (BL) cell lines including cell lines with genetically engineered knockout or knockdown of p53. p53 ChIP demonstrated direct binding of p53 to the NEAT1 promoter. This provides first evidence of p53-dependent regulation of long non-coding RNAs in CLL and BL. The discovery of p53-dependent NEAT1 induction, which is an integral part of nuclear paraspeckles, paves the way for further research on the role of paraspeckles in tumor cell apoptosis and resistence. The current work identifies additional components of the p53-dependent DNA damage response in lymphoma. The results of these studies provide new insight into the involvement of miRNAs and lncRNAs in two key signaling pathways regulating cell survival and treatment resistance in CLL and lymphoma.
Mehr als zwei Drittel der Erdoberfläche ist mit Wasser bedeckt. Das ist eine gigantische Kontaktfläche mit dem zweiten großen Klimasystem: der Atmosphäre. Forscher wie der Heidelberger Umweltphysiker Bernd Jähne wollen herausfinden, wie der Austausch zwischen Ozean und Atmosphäre genau funktioniert.
Der Beitrag erschien in der Sendereihe "Campus-Report" - einer Beitragsreihe, in der über aktuelle Themen aus Forschung und Wissenschaft der Universitäten Heidelberg, Mannheim, Karlsruhe und Freiburg berichtet wird. Zu hören ist "Campus-Report" montags bis freitags jeweils um ca. 19.10h im Programm von Radio Regenbogen. (Empfang in Nordbaden: UKW 102,8. In Mittelbaden: 100,4 und in Südbaden: 101,1)
This thesis is devoted to mathematical modeling of acute leukemias, which form a heterogeneous group of severe blood cancers. New models of dynamic behavior of blood forming (hematopoietic) and leukemic cells are developed and studied analytically. Bone marrow aspiration data contributed from the University Hospital of Heidelberg (Prof. Dr. A. D. Ho) and clonal tracking experiments from literature serve as a test scenario for the proposed models. To reflect the compartmental architecture of the hematopoietic and leukemic cell line, the models are represented by systems of nonlinear ordinary differential equations. Different possible modes of interaction between healthy and leukemic cells are proposed such as competition for environmental signals or autonomous leukemic cell growth and competition for marrow space. Extensive analytical studies of system dynamics and the derived criteria for coexistence and out-competition of the different cell types result in biologically meaningful characterizations of the cancer stem cell state by dynamic cell properties. Numerical studies allow to investigate the impact of different cell parameters on the clinical course and patient prognosis. A model-based prognostic marker for survival of relapsing acute myeloid leukemia patients is developed and tested based on clinical data. The obtained results underline the strong impact of leukemia stem cell behavior on the clinical dynamics. Extensions of the models including multiple leukemic clones allow to link experimental observations of clonal evolution to yet not measurable but clinically meaningful cell parameters at different stages of the disease. The models derived in this thesis depend on a quasi-steady state approximation describing the dependence of cytokine concentrations on mature cell density. In the last part of this work it is rigorously shown that solutions depending on the quasi-steady state approximation are close to solutions of a singular perturbation problem including dynamics of the signal molecules as a separate ordinary differential equation that is scaled with a small parameter. L-infinity bounds for the difference of solutions based on the quasi steady state approximation and solutions of the singular perturbation problem are established for the infinite time interval.
The application of X-radiography in ceramic studies is becoming an increasingly valued method. Using the potential of industrial X-ray computed tomography (CT) for non-destructive testing as an archaeometric or archaeological method in pottery studies, especially regarding aspects such as manufacturing techniques or pottery abrics, requires controlled data-acquisition and post-processing by scientific computing adjusted to archaeological issues. The first results of this evaluation project show that, despite the difficulties inherent in CT technology, considerable information can be extracted for pottery analysis. The application of surface morphology reconstructions and volumetric measurements based on CT data will open a new field in future non-invasive archaeology.
Ribosomes are complex macromolecular machineries responsible for protein synthesis (translation) in all living cells. In yeast, they are composed of four rRNA species assembled with 79 ribosomal proteins to form the small (40S) and the large (60S) subunit. To reach their final translation-competent form, they go through a complex, highly dynamic and coordinated process termed ribosome biogenesis. In eukaryotes, more than 180 transiently associating non-ribosomal factors (assembly factors) and 70 small nucleolar RNAs (snoRNAs) are involved in rRNA processing and modifications, as well as in the assembly of r-proteins (Henras et al., 2008; Lafontaine and Tollervey, 2001; Staley and Woolford, 2009). Several of the 60S ribosome biogenesis factors belong to the superfamily of GTPases, including Nug1. Nug1 is a circularly permuted GTPase and an essential trans-acting factor in ribosome biogenesis. It co-purifies with various nucleolar and nucleoplasmic pre-ribosomal particles and exhibits RNA-binding properties (Bassler et al., 2001; Bassler et al., 2006). However, several questions remained open regarding the exact role of Nug1 in ribosome biogenesis, including the regulation of its enzymatic GTPase activity, its binding site on the pre-ribosome, as well as a possible role in the recruitment and/or release of other 60S assembly factors. During my PhD studies, I performed a series of in vitro GTPase and nucleotide binding assays using the C. thermophilum (CtNug1) orthologue to address Nug1’s enzymatic activity. With these, I showed that CtNug1 exhibits a low intrinsic GTPase activity that can be stimulated by potassium ions, rendering Nug1 a cation-dependent GTPase. I’ve also generated a series of point mutations in the G-domain that specifically inhibit GTP hydrolysis or nucleotide binding. The orthologous mutations in the yeast Nug1 GTPase domain were subsequently tested for their effects on ribosome biogenesis. Early 60S assembly factors including Dbp10, Spb1, Nop2 and Mrt4 associated less with affinity purified pre-ribosomal particles, when the Nug1 nucleotide-binding mutant (D446N) was expressed or when Nug1 was depleted. Interestingly, no growth defects or biochemical differences in pre-ribosomal particle composition were observed for the catalytic (G339A) mutant, suggesting that the GTP hydrolysis is not essential for Nug1’s function. From the early assembly factors affected, only the essential RNA helicase Dbp10 was genetically linked to Nug1 (Bassler et al., 2001). In collaboration with Dr. Emma Thomson, we identified the binding sites of Nug1 and Dbp10 onto the pre-ribosome using the CRAC technique. Both proteins were found to bind in close proximity to each other on the interface of the 60S subunit at the PTC area. Further, in vitro binding assays confirmed a physical interaction between Nug1 and Dbp10. Together the findings from my PhD thesis show that Nug1 affects the dynamic interplay of assembly factors including those localizing to the PTC area (Dbp10, Sbp1, Nop2, Nsa2), as well as factors involved in the P-stalk formation (Mrt4, Yvh1, Rpp0, Rpl12). In this interplay, the Nug1 binds at the base of helix 89 and may act as a molecular GTPase switch that mediates the crosstalk between the maturation of PTC and the P-stalk, two distinct and essential hallmarks of the 60S subunit.
Schwach koordinierende Anionen spielen heutzutage eine wichtige Rolle, sowohl in der Stabilisierung hoch reaktiver Elektrophile, als auch in Übergangsmetall-katalysierten Reaktionen. Entscheidend ist dabei die geringe Nukleophilie des Anions, wodurch eine freie Koordinationsstelle an dem Metallzentrum des kationischen Katalysatorkomplexes generiert wird. Der Großteil, der in der Literatur zu findenden schwach koordinierenden Anionen, besteht aus chlorierten oder fluorierten Verbindungen. Halogensubstituenten fuhren zu erhöhter Stabilität gegenüber reaktiven Elektrophilen, erhöhter Redoxstabilitat sowie hoher Löslichkeit in organischen Solvenzien. Aus ökologischer Sicht allerdings führt die Verwendung der Halogensubstituenten zu erheblichen Problemen. Zum einen entstehen durch Verwendung halogenierter Verbindungen hohe Herstellungs- und Entsorgungskosten, zum anderen sind die Abbauprodukte dieser persistenten Verbindungen sehr toxisch. Ein typisches Oxidationsprodukt fluorierter Kohlenwasserstoffe ist Trifluoressigsaure, dessen Salze sich langsam zu Trifluormethan zersetzen, welches ein extrem potentes Treibhausgas ist. Aus diesen Gründen wird auf eine Verwendung dieser Verbindungen in industriellem Maßstab weitestgehend verzichtet.
In dieser Arbeit wird die Entwicklung, Charakterisierung und Anwendung von wasserstabilen, halogenfreien, lipophilen, nicht persistenten Aluminat-Estern vorgestellt, die zudem aus kostengünstigen Ausgangsstoffen und in wenigen Syntheseschritten mit hohen Ausbeuten verfügbar sind. Die Strategie dieser Arbeit war die Weiterentwicklung des von M. Wrede in unserer Arbeitsgruppe im Jahr 2010 entwickelten „Altebat“-Anions, das zwar gute Löslichkeit in unpolaren Losungsmitteln aufweist; jedoch wird die Anwendung dieses Anions durch die Empfindlichkeit gegenüber wässrigen und alkoholischen Losungen stark eingeschränkt. Durch Verwendung von Bisphenolaten mit einem Dimetylmethylen- bzw. Ethylen-Linker wird die Abschirmung des Tetraoxoaluminiumkerns und die daraus folgende Hydrolysestabilitat deutlich erhöht. Durch Einführung des Dimethylmethylen-Linkers wird das „Almebat“-Anion erhalten. Dieses ist nahezu stabil in wässrigen neutralen und wässrigen alkalischen Losungen in wässrigen, sauren Losungen wird es jedoch noch sehr schnell hydrolysiert. Die Einführung des Ethylen-Linkers führt zu dem „Aletbat“- Anion. Dieses ist in methanolischer, neutraler und alkalischer wässriger Lösung über Wochen stabil. In schwach saurer Losung wird es sehr langsam innerhalb von Wochen hydrolysiert; in stark saurer Losung innerhalb von Tagen. Derivate des „Aletbat“-Anions, wie das „Aletpat“-Anion, sind ebenfalls in neutraler und alkalischer wassriger Losung uber Wochen stabil.
Die Lithium- und Natrium-Kationen der Alkalimetall-Aluminat-Ester lassen sich leicht durch organische Kationen wie Tetraphenylphosphonium-, Imidazolium- und Tetrabutylammoniumkationen durch Salzmetathesen in verschiedenen organischen Losungsmitteln in hohen Ausbeuten ersetzen. Diese Komplexe konnten durch zahlreiche Röntgenstrukturanalysen charakterisiert werden, wodurch die geringe Koordinationstendenz der Anionen belegt werden konnte. Hier weisen vor allem Komplexe mit dem „Aletbat-Anion“ eine hohe Tendenz zur Kristallisation auf, was dieses Anion ideal zur Isolierung und Reinigung von Salzen macht. Die synthetisierten Tetrabutylammonium-Aluminat- Ester sind zudem hervorragend in organischen Losungsmitteln löslich, was die Lipophilie dieser Anionen unterstreicht. Eine herausragende Löslichkeit wird vor allem fur das „Tetrabutylammonium- Aletpat“ erzielt. Selbst in unpolaren Losungsmittel wie Diethylether (cmax = 664 mg/mL), Toluol (cmax = 620 mg/mL) und auch Cyclohexen (cmax = 240 mg/mL) ist die Löslichkeit herausragend. Durch Entfernen der koordinierten THF-Moleküle der entsprechenden Na(thf)2-Aluminat-Ester können zusätzlich noch aktivere Natrium-Aluminat-Ester hergestellt werden, mit denen es möglich ist, koordinierte Chlorid-Liganden von Übergangsmetall-Komplexen, wie [Ir(PHOX)(cod)Cl] und IPrAuCl, zu abstrahieren. Die Ir(PHOX)(cod)-Aletbat-Komplexe konnten erfolgreich in der enantioselektiven katalytischen Hydrierung von Iminen und Enonen getestet werden. Durch Verwendung des „Aletbat“- Anions, als Gegenion fur den Katalysatorkomplex, konnten vergleichbare Ausbeuten und Enantiomerenüberschusse wie mit korrespondierenden Katalysatorkomplexen mit fluorierten schwach koordinierenden Anionen erhalten werden.
Der Bewuchs künstlicher Oberflächen im Kontakt mit Meerwasser, das marine Biofouling, stellt aufgrund seiner ökonomischen und ökologischen Folgen seit jeher ein Problem für die Marineindustrie dar. Aufgrund des Verbotes ehemals erfolgreicher aber hochtoxischer Biozide und einem zunehmenden Umweltbewusstsein ist die Entwicklung umweltverträglicher Beschichtungen, die die initiale Anlagerung der entsprechenden Organismen beeinflussen, in den Fokus der Forschung gerückt. Da die Adhäsionsprozesse der so genannten Fouler von einer großen Bandbreite an Oberflächen¬eigenschaften beeinflusst werden, macht man sich in diesem Zusammenhang Modellsysteme mit reduzierter Komplexität zunutze, um selektiv einzelne Variablen untersuchen zu können. Gegenstand dieser Arbeit war die Präparation und Charakterisierung stark hydratisierter Modelloberflächen und die biologische Evaluierung ihrer Fouling-Resistenz. Als biologische Modellsysteme wiederum dienten stellvertretende Spezies aus allen Stadien des komplexen Biofouling-Prozesses: Cobetia marina für marine, biofilm-bildende Bakterien; Diatomeen für die schleimbildenden Mikrofouler; Zoosporen der Grünalge Ulva linza für weiche Makrofouler und Cyprislarven der Seepocke Balanus amphitrite für die harten Makrofouler. Da im realen marinen Umfeld eine Vielzahl von Faktoren ineinander greifen, wurden außerdem Feldstudien durchgeführt, um einen Überblick über diese zu gewinnen. Zwitterionische Substanzen werden aufgrund von elektrostatischen Wechselwirkungen der vorhandenen entgegengesetzten Ladungen hydratisiert. Um den Einfluss verschiedener Ladungen und ihrer Kombination auf die biologischen Modelsysteme näher zu analysieren, wurden selbst-assemblierende Monolagen (SAMs) aus Mischungen von Alkanthiolen mit unterschiedlichen geladenen Gruppen auf Gold präpariert. Die Adsorption von Testproteinen und die Anzahl adhärenter Organismen im Feldexperiment wurden durch die gleichzeitige Präsenz der entgegengesetzten Ladungsträger an unterschiedlichen Molekülen reduziert; die Ablösbarkeit der Kieselalgen erhöht. Zoosporen der Grünalge Ulva linza wiesen dagegen komplexere Adhäsionspräferenzen auf, die von der exakten Terminierung der Oberflächen abhingen. Polysaccharide sind hydrophile Biopolymere, deren Hydratation über Wasserstoffbrückenbindungen zustande kommt. Drei strukturell leicht variierende Vertreter dieser Klasse, Alginsäure (AA), Hyaluronsäure (HA) und Chondroitinsulfat (CS), wurden kovalent auf Oberflächen immobilisiert und anschließend an ihren Säuregruppen mit einem fluorierten Amin modifiziert. Dadurch wurden einerseits freie Carboxylgruppen für die Wechselwirkung mit zweiwertigen Kationen blockiert und andererseits amphiphile Eigenschaften in den hydrophilen Polymernetzwerken etabliert. Die zugrundeliegende Hypothese, dass die verminderte Fähigkeit zur Komplexierung von Ca(II)-Ionen die inerten Eigenschaften der Polysaccharidfilme im marinen Medium verbessert, konnte für AA und HA für die Adhäsion von C. marina, das Besiedlungsverhalten von U. linza und B. amphitrite und im Feldversuch bestätigt werden. Entgegengesetzt dazu verfügte das sulfatierte Polysaccharid CS im unmodifizierten Zustand über bessere antiadhäsive Eigenschaften. Als dritte Materialklasse wurden Poly(Hydroxyethylmethacrylat)-Filme, die ebenfalls durch ihr chemisches Grundgerüst eine wasserstoffbrückenbasierte Hydratation aufweisen, charakterisiert und bezüglich ihrer Proteinresistenz getestet. Für die durch oberflächeninitiierte RAFT-Polymerisation hergestellten Substrate wurde eine Schichtdickenabhängigkeit der Resistenzeigenschaften demonstriert.
In this thesis we analyze and develop methods based on model order reduction (MOR) for the solution of optimization problems constrained by time-dependent partial differential equations (PDEs). The methods combine a direct solution approach with model reduction via proper orthogonal decomposition (POD) and the discrete empirical interpolation method (DEIM). The reduced-order models (ROMs) are used to approximate the high-dimensional dynamic systems originating from a spatial discretization of a PDE. However, when used in an optimization algorithm, conventional POD/DEIM ROMs often lack the ability to give adequate approximations of the gradient. We propose methods for a suitable enhancement of the ROMs for the optimization purpose which are based on the inclusion of derivative information in the POD and DEIM subspaces. We distinguish two types of error between quantities evaluated with the high-dimensional model and its ROM approximation in dependency on the optimization variable q: The reconstruction error which is evaluated with the same q0 which is used constructing the ROM and the prediction error which assesses approximations at q with a ROM constructed at q0 different to q. The novel reconstruction results we present include estimates for solutions of the adjoint equation and the sensitivity equations as well as for the gradient of the objective function. Based on the estimates we explain how the POD and DEIM bases should be extended with either adjoint or sensitivity information. The enhanced ROMs allow control of the reconstruction error for the objective and its gradient up to machine precision. Moreover, we propose a POD prediction estimate for the objective of the optimization problem in a neighborhood of q where the ROM is constructed. In case of sensitivity-extended POD and DEIM bases we give an analogous result for solutions of the states. The derivative-extended ROMs are then used to develop adaptive algorithms for the solution of optimal control and parameter estimation problems which results in great runtime improvements for the optimization while ensuring high approximation quality of the solution of the original problem. For the parameter estimation case a novel a posteriori error estimate is proposed which assesses the quality of suboptimal solutions obtained with the ROM. A further fundamental contribution is a discussion of discretize-then-optimize (DTO) vs. optimize-then-discretize (OTD) approaches in the context of MOR for optimization. We analyze advantages and disadvantages of both approaches and discuss to which extent our methods exhibit properties of either strategy. We also give examples of representative optimization problems in which standard POD/DEIM ROMs show an inacceptable behavior and can be successfully solved by derivative-extended ROMs. We have further implemented the developed methods emphasizing an efficient realization which is important for the investigation of the MOR potential. We showcase the practical performance of the proposed algorithms and the superiority of derivative-extended over conventional ROMs on two academic and one industry-relevant application which exhibit a variety of challenges for the model reduction approach in optimization.
The success of human gene therapy - the treatment of hereditary or acquired diseases with a genetic cause - depends on potent, safe and specific gene delivery vectors. Amongst the large variety of currently available non-viral or viral vectors, those derived from Adeno-associated viruses (AAV) are particularly attractive due to a unique combination of assets: the virus/vector is apathogenic, poses little risk of insertional mutagenesis, has a broad host and cell range, and can easily be modified and produced in large quantities. However, no natural AAV fulfills all the requirements for clinical use in humans, raising a need for new technologies and strategies to engineer synthetic “designer” vectors. Accordingly, the central aim of this work was to improve and apply two fundamental methods for molecular AAV vector evolution, DNA family shuffling and peptide display. The first technology relies on fragmentation and homology-based recombination of capsid genes from closely related AAV serotypes, resulting in libraries of AAV chimeras from which capsids with desired properties can be enriched via subsequent selection. Here, we initially assessed and optimized the key steps for AAV shuffling, culminating in a robust and standardized new protocol for AAV library generation. Next, we used this protocol to shuffle AAV2, 8 and 9, and exploited the resulting library to analyze two major parameters for AAV selection - helper virus and anti-AAV-antibodies. Interestingly, we found that AAV capsids selected in the presence of a helper virus give stronger gene expression, implying that their intracellular processing is enhanced. Moreover, comparative analyses of AAVs isolated under various conditions with human antisera showed that the degree of negative selection pressure determines the balance between infectivity and immunity of the viral particles. Finally, we also performed a helper virus-free in vivo biopanning with a library comprising AAV1, 5, 6, 8 and 9 in murine pancreas. Intriguingly, gene expression from the single clone emerging after three selection rounds was low, supporting our conclusion that the presence of a helper virus is key for potent AAV vector evolution. Unlike shuffling, AAV vector improvement through peptide display starts with a single viral serotype (traditionally AAV2) whose capsid is expanded by insertion of 7-9 aa into an exposed loop, with the aim to alter vector tropism towards desired target cells. Here, we extended this strategy to 11 alternative AAV serotypes and demonstrate their tremendous, previously unrecognized potential as scaffolds for viral peptide display. Therefore, we first implemented a simple PCR protocol for rapid cloning of peptide-encoding sequences into AAV capsid genes, which replaces the original stepwise mutagenesis. We then used our new approach to insert 6 distinct peptides into all 12 AAV serotypes, resulting in a collection of 84 YFP-encoding vectors (12 wildtypes & 72 mutants). While screening this panel in a large array of human and non-human cell lines and primary cells, we made three important observations: i) AAV vector transduction is not determined by the peptide alone but largely depends on the capsid context; ii) alternative AAV serotypes with certain peptides frequently outperform the AAV2 prototype and its peptide derivatives; and iii) some serotype-peptide combinations even allow to transduce cells previously considered refractory to AAV infection. Subsequent analysis of further peptides showed that a common motif, NxxRxxx, is enriched in the best performing candidates and particularly enhances AAV1, 7-9 and rh.10. Based on these findings, we assembled a “Master panel” of vectors including the superior serotype-peptide combinations from our various screens, and used it in collaborative studies to select potent new AAV vectors in clinically relevant cell types, such as myeloid cell lines, primary human myeloma cells, or dorsal root ganglia and proprioceptive neurons. As a whole, the work in this thesis makes a number of essential contributions to the fields of AAV biology, AAV vector development and human gene therapy. First, it resulted in optimized protocols and tools that substantially simplify, standardize and accelerate the future generation of tailored AAV capsids for vector engineering. Second, it also yielded a wide variety of new AAV variants - either in the form of shuffled libraries or as panels of specific capsid-peptide combinations - that can now be screened in further cell lines, primary cells or even directly in vivo. Third, the data obtained in this thesis with the various wildtype, shuffled or peptide-modified capsids greatly improve our knowledge of fundamental steps in cellular AAV infection. Most importantly, our results consistently exemplify that the function of AAV capsids is not determined by single residues, but rather results from very complex interactions of different regions that are dispersed throughout the viral shell. Altogether, the present thesis fuels the belief that AAV is one of the most versatile, powerful and robust viral vector systems available today and that it provides unique benefits for clinical translation in humans.
EMP3 has been proposed as a potential tumor suppressor gene in neuroblastomas, gliomas and other solid tumors, due to its differential methylation and expression pattern. In these tumors EMP3 is often transcriptionally silenced by promoter hypermethylation. The biological function of EMP3 itself is largely unknown. Based on homologies to other members of the protein family, it was presumed that EMP3 is involved in the regulation of proliferation, apoptosis and cell-cell-interactions. The aim of this work is the functional characterization of EMP3 and its role in glioma formation and progression. To this end we analyzed the methylation and expression pattern of EMP3 in gliomas, non-neoplastic tissues and cell lines. In addition we studied the effects of RNA-interference-mediated knockdown of EMP3 on proliferation, migration and apoptosis in an in vitro cancer cell line model. Furthermore we utilized different interaction assays to identify novel protein-protein interaction partners of EMP3. EMP3 is expressed in almost all analyzed normal tissues, with the highest levels in leukocytes, neurons and astrocytic cells. In gliomas, the EMP3 protein levels are highest in glioblastomas, of which over 80% show very strong EMP3 expression, while in low-grade gliomas only 20% show elevated levels of EMP3. High levels of EMP3 also correlate with shorter progression-free and overall patient survival. RNA-interference-mediated repression of EMP3 significantly reduces the proliferation and migration of cancer cells in vitro and increased their susceptibility to induced cell death. This is in part caused by decreased phosphorylation and activation of the EGFR, AKT and ERK signaling kinases. EMP3 is part of a complex interaction and signaling network, of which we could identify 10 novel interacting proteins. Through this network EMP3 is possibly involved in the regulation of several important signaling and trafficking pathways. EMP3 is therefore likely to be a mediator and regulator of intracellular trafficking and signal transduction. The data support a role for EMP3 in the progression of cancer, but, at least in glioma, not as a tumor suppressor. Nevertheless EMP3 could be a valid prognostic and possibly even predictive biomarker in the diagnosis of glioma as well as a potential novel therapeutic target in different tumors and other diseases.
Understanding molecular and cellular events influencing the neural progenitor mode of cell division and fates of daughter cells is central to Developmental Biology. Members of the cytokinetic machinery have received increasing attention as possible molecular mediators of this event: their inheritance or retention appears to influence proliferation versus differentiation of daughter cells in cancer and stem cells. I therefore took under investigation the F‐actin binding protein Anillin, shown to be essential for cytokinesis progression and completion in invertebrate model systems. I found Anillin downregulation to be mediated by the bHLH transcription factor Atoh7, which is essential for the generation of Retinal Ganglion Cells (RGCs). I therefore first investigated the expression and in vivo dynamics of Anillin in the zebrafish neural retina. This analysis revealed that Anillin expression correlates with proliferative states of retinal progenitor and, concomitantly, Anillin knock down promoted RGCs and early born retinal cell type generation at the expense of the late born ones, such as bipolar and muller glia cells. These effects on cell fate were due to change in mode of cell division promoted by Anillin knock‐down Anillin morpholino injected clones tend to undergo symmetric neurogenic division, generating two Atoh7 positive cells or two RGCs, while the control clones tend to undergo asymmetric division. These results reveal a role for Anillin in neurogenesis by affecting cell division outcomes. I then asked if anillin knockdown effect on cell division outcome correlates with putative roles in cytokinesis. By developing a zebrafish transgenic line recapitulating Anillin‐GFP expression in the in vivo developing embryo, I could analyze Anillin distribution at cellular level. Anillin protein highlights the progression of the cytokinetic furrow, as well as midbody formation and postmitotic inheritance in dividing retinal progenitor cells. Research in invertebrates and vertebrates suggests a colocalization of Anillin and its main interactor, F‐actin, during cytokinesis progression. Accordingly, Anillin and F‐actin co‐localized at the cleavage furrow and midbody of cycling retinal progenitors. Midbody and F‐actin spot remnant are both inherited by the daughter cell that differentiates as RGC, suggesting for the first time a correlation between apical F‐actin/midbody distribution and fate. I further aimed at elucidating the effect of Anillin knockdown on F‐actin dynamics during cytokinesis. Results show that symmetric outcome of cell division produced by Anillin knock down correlate with symmetric apical F‐actin inheritance at the daughter cell’s apical domain. These results indicate that Anillin links cytokinesis progression with apical F‐actin distribution between the daughter cells. In addition to slowing down cytokinesis progression, Anillin appears to influence the basal to apical interkinetic nuclear migration of progenitor cells, which I found slowed down in Anillin morpholino‐injected embryos, along with an increase in the progenitors cell cycle length. This results in an longer permanence of the nuclei close to the basal side of the neuroepithelium namely, far away from proliferative signal. Likewise, longer permanence of nuclei in this basal side of the neuroepithelium have been associated with symmetric neurogenic divisions of retinal progenitors. Controversial studies showed how correlation between mode and orientation of cell division in vertebrates is not clear. In the present study I found that increase in symmetric fate outcome of daughter cells (in term of Atoh7 expression) correlates with a shift in the orientation of cell division from radial to circumferential. The biological significance of this switch remains to be investigated. These functional studies combined with in vivo imaging provide evidences indicating that Anillin affects neurogenesis by maintaining the progenitor pool and influencing the mode of cell division. By directly assessing correlations between F‐actin, midbody inheritance, orientation of cell division and daughter cell fate, our study also provides insight into the mechanism whereby this might operate in vivo.
Release of synaptic vesicles (SV) is a process that is orchestrated by proteins present in the presynaptic terminus called the active zone (AZ). Knowledge of the placement of proteins is necessary to understand how SV release occurs. There is limited information on the location of the AZ proteins from studies of biochemical assays or immuno-electron microscope. Developments in fluorescence light microscopy are capable of reaching subnanometer resolution and therefore can be used to image multiple proteins of the AZ. For instance, a technique called direct Stochastic Optical Reconstruction Microscopy (dSTORM) can reach a resolution of 20 nm in the x-y plane, which is an order of magnitude greater than conventional light microscope. This work is devoted to developing techniques, which enables the use of dSTORM on thick brain tissue samples. In this respect, two thick tissue handling techniques have been explored, namely tomoSTORM and Tokuyasu’s ultracryotomy. Using tomoSTORM, we could construct a super-resolution 3D structure of the calyx of Held synapse. In addition, we also demonstrate multicolor capability by being able to localize the abundantly distributed mitochondria to the synaptic compartment of the calyx of Held. Due to antibody staining limitations, Tokuyasu’s ultracryotomy was explored. Using this approach we gathered dual-color super-resolution data in the calyx of Held on the distribution of Bassoon with respect to Piccolo. In agreement with the standardresolution microscopy, overview image of Bassoon and Piccolo show that both proteins exist together in the majority of the AZs. In addition we can show at the nanoscopic level in a given AZ that the two proteins not only exist as separate entities but are also found to be colocalized. We also gathered data on the distribution of Septin 5 and Piccolo and found that at P7 Septin 5 and Piccolo colocalize while at P17 they do not colocalize. This observation is consistent with the finding that Septin 5 may cluster voltage gated calcium channels at P7 at the AZ. In addition, as dSTORM is limited to photoswitching of 2 dyes, efforts were made to extend this. To this extent, we show efficient photoswitching of phalloidin conjugated to ATTO 488, TRITC and BODIPY 650. 8 In summary, this thesis is focused on adapting dSTORM to thick tissue samples and developing multicolor photoswitching probes to explore multiple protein distribution in the synaptic compartments of mammalian brain tissue.
Systemic iron homeostasis is essential for human health. Its maintenance critically depends on the interaction between the hepatic hormone hepcidin and the sole known iron exporter ferroportin (FPN) predominantly expressed in hepatocytes, duodenal enterocytes and macrophages. Hepcidin binding leads to FPN internalization and degradation resulting in cellular iron retention. Iron is an essential nutrient also for pathogens and plays a central role in host-pathogen interactions. The innate immune system fights infections by sequestration of iron in macrophages of the reticuloendothelial system. The resulting hypoferremia represents a major host defence strategy. A current model posits that hepcidin is the crucial effector of this response, as its release from macrophages and hepatocytes provokes FPN protein decrease and, consequently, tissue iron retention. The aim of my PhD project was to identify novel cellular regulators of hepcidin-mediated ferroportin (FPN) degradation, a fundamental process that controls systemic iron homeostasis. To reach this aim I generated a HeLa cell line expressing a hFPN-renilla fusion protein, which was used for a focused high-throughput RNAi screen targeting kinases and related proteins. Out of 779 genes tested, the screen identified 71 putative regulators of FPN protein stability. Validation experiments confirmed the phenotype of 24 genes. Interestingly, most validated regulators of FPN expression conferred hepcidin-independent FPN regulation. From these I selected 14 genes associated with immune processes for further characterization in murine bone marrow-derived macrophages (BMDMs). Finally, my studies focused on Toll-like receptor 6 (TLR6) as an effective regulator of FPN expression in BMDMs and I investigated how the TLR6 activation pathway modulates iron regulation in the inflammatory context. TLR2/6 ligation by the synthetic lipoprotein derived from Mycoplasma: FSL1 triggered a profound decrease in FPN mRNA and protein expression in BMDMs as well as in the liver and the spleen of mice. Unexpectedly hepcidin expression remained unchanged. Hepcidin-independent FPN down regulation was a conserved response to different microbial lipopeptides and elicited a fast, hepcidin-independent hypoferremia pathway. These findings were further confirmed in C326S FPN knock-in mice with a disrupted hepcidin/FPN regulatory circuitry. This work challenges the prevailing role of hepcidin in inflammatory hypoferremia and suggests that rapid hepcidin-independent FPN down regulation may represent the first line response to restrict iron access to pathogens.
In this work we examine different numerical methods for the simulation of Maxwell's equations in 3D with the application to ground-penetrating radar. In particular we consider an edge-based finite element and a discontinuous Galerkin method, both in the time domain. We implement these methods using the finite element framework Dune and the discretization module Dune-PDELab and test the implementations using two example problems. Finally, we apply them to a ground-penetrating radar problem derived from the ASSESS-GPR test site and compare the results to actual measurements made on the site.
A novel, high resolution Imaging Slope Gauge (ISG) at the Heidelberg Aeolotron is presented. The instrument allows measurements of the two-dimensional slope of short wind-driven water waves at unprecedented temporal and spatial resolution. Wave number spectra are measured up to k = 13000 rad/m with a temporal resolution of more than 1500 Hz. The high sampling frequency eliminates aliasing up to wave numbers of about k = 2660 rad/m. A new non-linear calibration and data processing work flow has been implemented to convert raw camera images into wave slope in the range of \pm 0.96. The average statistical measurement error is estimated to be \Delta s_rms = 0.018, a significant improvement compared to previous Color Imaging Slope Gauge setups.
Spectrally resolved measurements of the influence of various surface-active materials (surfactants) on small-scale waves are reported. The wave damping effects of different substances are analyzed. It is shown that, except for low wind speeds, gas transfer velocities across the air-sea boundary layer can be parametrized with the mean square slope of the waves, independent of the specific type of surfactant that is used.
Regulatory T (Treg) cells are generated in the thymus and are involved in the regulation of self-tolerance, immune homeostasis and immune responses. So far the mechanisms behind the Treg cell differentiation and their function are not fully understood. The work of this thesis aimed to gain insight into these processes. We used a quantitative differential proteomics approach and identified 164 proteins that were differentially expressed in Treg cells compared to conventional T cells. Among these transcription factor 7 (TCF7) was identified as a promising candidate for further investigations of its role in Treg cells. Using a Tcf7-deficient mouse model we observed elevated frequencies of Treg cells among CD4 single-positive and CD4 CD8 double-positive thymocytes in the absence of TCF7. Quite surprisingly, we also found a fraction of CD4 CD8 double-negative cells that expressed the Treg cell transcription factor forkhead box P3 (FOXP3) independent of previous T cell receptor engagement, indicating that TCF7 prevents premature FOXP3 expression. In vitro Treg precursor differentiation assays showed that the reduction of TCF7 levels was beneficial for the Treg cell generation capacity and that TCF7 activation through the β catenin/Wnt pathway had the opposite effect. Furthermore, our results indicated that FOXP3 supports down regulation of TCF7, as FOXP3 expression in thymocytes was accompanied by decreased levels of TCF7 and enforced expression of FOXP3 in T cells caused reduced expression levels of TCF7. In addition to its role in thymic Treg cell differentiation, we found that TCF7 was involved in regulating the balance between Treg and TH17 cells and the survival of NKT cells. TCF7 deficiency also changed the typical expression patterns of Treg and T cell markers. In summary, we propose that TCF7 is involved in limiting the access into the Treg cell lineage and that TCF7 influences peripheral T cell subsets.
Marine biofouling is a global problem with a negative impact on several industrial and maritime branches, causing immense costs. Alongside with the economic consequences of colonization of surfaces, the environmental aspects should not be underestimated. Because of the predominant use of heavy-metal based antifouling coatings in the last decades, the most prominent component of which is tributyltin, TBT, several unacceptable environmental `side effects' on non-targeted species have emerged. This led to a global ban of the use of TBT-containing coatings in 2008. Since then, the research efforts focus on the development of surfaces which solely rely on physicochemical properties to mitigate the undesired accumulation of biomass. For identification of surface properties which influence and guide settlement, a better understanding of the colonization mechanisms of the target organisms is required. The purpose of this work was the investigation of the exploratory behaviour of barnacle larvae (cyprids) of Balanus amphitrite. This species was selected because it is widely spread in several geographic regions and contributes significantly to biofouling pressure. To enable the extraction of three-dimensional swimming trajectories, a stereoscopic setup was developed. It was utilized to quantify the pre-settlement behaviour of cyprids on surfaces with different wettability, hydration or charge. The analysis of the data allowed the identification of basic reoccurring patterns in the motion of cyprids, i.e. spiraling, swimming, sinking, rotating and walking. A detailed evaluation of the distribution of these patterns revealed that swimming was most frequently observed, followed by sinking which seemed to be its counterpart. Furthermore, it could be shown that most surface contacts emerged after sinking, while an active swimming towards the substrate was seldom observed. In addition, the general distribution of cyprids in the water column demonstrated that there was a clear preference for interfacial regions, and that the main occurrence of cyprids concentrated in three distinct zones above surfaces - the lower, bulk and upper swimming region, denoting the distance of the larvae from the substrate. It was revealed that the settlement preferences of cyprids were reflected in the motility parameters of exploration, i.e. linearity of the locomotion, mean velocity, swimming depth and extent of explored area. Once on a surface, cyprids initiate a close surface inspection, involving bipedal walking. The quantification of this mode of movement showed that the step duration and step velocity correlated with the attractiveness of the surface for settlement. Moreover, an analysis of the complete exploration process - from swimming via close surface inspection to interface interactions, confirmed literature reports that the number of surface touchdowns and the amount of organic deposit left behind were guided by the attractiveness of the substrate for biofouling. In addition to this organic deposit, macromolecules and other compounds accumulate on surfaces, immediately after these have been placed in an aquatic environment. The influence of this `conditioning' film on the exploratory behaviour was investigated and it was proven to cover the initial chemical end groups of different substrates. This masking process provoked cyprids to explore non-attractive conditioned surfaces in the same manner as attractive ones. Except on model surfaces, the exploratory behaviour was also investigated on commercially available hydrogel-based coatings and it was found that the motility of cyprids started to decrease gradually immediately after surface contact and after 4h it completely stopped, which was contributed to mortality of the organisms caused by the paints. Finally, the stereoscopic setup was modified to allow measurements in situ, underwater. The results of the field experiments at two test sites are discussed with respect to the applicability of the system for measurements in the natural environment and its capability to reveal colonization dynamics of marine organisms.
Ein großer Teil aller therapeutisch wirksamer Arzneistoffe sowie deren Metaboliten werden durch die Nieren aus dem Körper ausgeschieden. Neben der glomerulären Filtration und passiven Transportpro-zessen sind dabei vor allem aktive, durch Arzneistoff-Transporter vermittelte, Transportprozesse in den proximalen Tubuli maßgeblich von Bedeutung. Daher kann die renale Exkretionsrate zahlreicher Arzneistoffe sowohl durch Wirkstoff-Wirkstoff-Interaktionen als auch durch Veränderung der Expres-sionsstärke oder der Aktivität einzelner Arzneistofftransporter beeinflusst werden. Derartige Änderun-gen können aufgrund verschiedener Faktoren wie Nierenerkrankungen, Alter, Schwangerschaft, Ge-schlecht, Ethnizität oder durch Polymorphismen der entsprechenden Transportergene hervorgerufenen werden. Während der Entwicklung neuer Wirkstoffe ist es daher von großer Bedeu-tung, die beteiligten Transportprozesse in den proximalen Tubuluszellen zu identifizieren. Für die Cha-rakterisierung von Arzneistoff-Kandidaten in Hinblick auf die aktive Sekretion wäre ein entsprechendes zellbasiertes In-vitro-Modell daher von großem Nutzen. Im Rahmen der vorliegenden Arbeit wurden verschiedene zellbasierte Modellsysteme im Hinblick auf ihre Eignung zur Darstellung der aktiven renalen Eliminierung untersucht. Dazu gehörten 1) frisch iso-lierte primäre proximale Tubuluszellen aus der Ratte, 2) für 5 Tage kultivierte proximale Tubuluszellen aus der Ratte, sowie 3) die immortalisierten Nierenzelllinien MDCKII, NRK-52E, IHKE-1 und Caki-1. Zunächst wurde eine Isolationsmethode für primäre proximale Tubuluszellen aus der Ratte etabliert. Die Bestätigung der Isolation wurde durch den Nachweis der proximalen Tubulusmarkerproteine alka-line Phosphatase (AP), Alanin-Aminopeptidase (ALPL), Villin 1 (VIL1) und γ-Glutamyltransferase 1 (GGT1) erbracht. Darüber hinaus konnte die mRNA von 18 in den proximalen Tubuli der Niere expri-mierten Aufnahme- und Efflux-Transportern aus der ABC-, SLC- und SLCO-Familie nachgewiesen wer-den. Auf funktioneller Ebene zeigten die Zellen außerdem eine aktive Aufnahme des organischen An-ions p-Aminohippurat (PAH), sowie des organischen Kations 1-Methyl-4-phenylpyridinium (MPP+). Im nächsten Schritt wurden die isolierten primären proximalen Tubuluszellen kultiviert und anschlie-ßend charakterisiert. Parallel wurde die Charakterisierung auch mit den immortalisierten Nierenzellli-nien durchgeführt und die Daten mit denen der Primärzellen verglichen. Da eine wichtige Vorausset-zung für bidirektionale Transportstudien die Dichtigkeit der Zell-Monolayer ist, wurden die kultivierten Zellen auch im Hinblick auf deren Barriere-Eigenschaften untersucht. Die kultivierten proximalen Tu-buluszellen zeigten eine Epithelzell-typische Morphologie und bildeten dichte Monolayer aus. Durch mRNA-Expressionsanalysen und immunohistochemische Untersuchungen konnte jedoch gezeigt wer-den, dass sich das Expressionsmuster der untersuchten Tight-Junction-Proteine deutlich von dem der frisch isolierten proximalen Tubuluszellen unterschied. Dieses von der In-vivo-Situation abweichende Expressionsmuster wurde ebenfalls in den untersuchten Nierenzelllinien beobachtet. Zudem zeigten die NRK-52E-, IHKE-1- und Caki-1-Zellen eine hohe Permeation von Mannitol, so dass diese für bidirek-tionale Transportstudien nicht geeignet sind. Ein deutlicher Unterschied der Expressionsmuster zwischen den kultivierten proximalen Tubuluszellen und den frisch isolierten Zellen wurde jedoch nicht nur im Fall der Tight-Junction-Proteine, sondern auch bei den proximalen Tubulusmarkerproteinen, sowie bei den untersuchten Transportern beobach-tet. Es wurde daher davon ausgegangen, dass sich die kultivierten Primärzellen in einem Status der Dedifferenzierung befanden. Auch die untersuchten Nierenzelllinien MDCKII, NRK-52E, IHKE-1 und Caki-1 spiegelten nicht die Charakteristika der proximalen Tubuli wider und es wurden von den frisch isolierten Primärzellen abweichende Expressionsmuster der untersuchten Markerproteine und Trans-porter gefunden. Im letzten Teil der Arbeit wurden daher verschiedene Ansätze untersucht, die Transporter-Expression in den kultivierten Primärzellen zu erhöhen. Die Behandlung der Zellen mit Modulatoren des Wnt/β-Catenin- und des Notch-Signalwegs, sowie die Behandlung mit den Steroidhormonen Testosteron und Hydrocortison hatte keinen signifikanten Einfluss auf die Aufnahme von PAH und MPP+ in die Zellen. Der Einsatz der Wachstumsfaktoren HGF, FGF-1 und EGF führte zu einer leichten Erhöhung der PAH-Aufnahme, während durch den Einsatz von 10 ng/ml EGF eine erhöhte MPP+-Aufnahme beobachtet werden konnte. Eine deutliche Erhöhung der Aufnahme von MPP+ konnte durch Behandlung der kul-tivierten Primärzellen mit 25 mM Kreatinin erreicht werden. Möglicherweise führt die erhöhte Krea-tinin-Konzentration durch einen Rückkopplungsmechanismus zu einer Induktion der Transporter-Ex-pression.
The XNAP project develops a demonstration system for a spatially resolving detector with timing capabilities in the nanosecond range. A dense array of avalanche photodiodes is combined with multiple readout ASICs to build the detector hybrid. On an area of nearly 1 cm2, single photons can be counted within each of the 1k pixels. After 20 years of continuous improvements during operation, the ESRF Synchrotron is going to be upgraded substantially by the replacement of major parts of the source and the beamlines. For experimental techniques that will benefit from the increased brilliance, research into X-ray detectors is required. The requirements for the novel detector are composed of the distinguished properties of multiple state-of-the-art detector systems, shifted towards technical limits. The specification is transferred into the design of the sensor, ASIC, interposing structure and the readout system. A smaller prototype detector is built to resolve implementation challenges ahead of its large-scale accomplishment. Emphasis is put on the ASIC, and parallel approaches for the interconnecting technology and the readout system are carried out. The usability of the smaller prototype system is demonstrated with measurements of microfocus X-ray and Synchrotron light. Parts of the final detector are characterized at the laboratory prior to its commissioning.
This thesis reports on a systematic experimental study on bubble-mediated gas transfer. Trace gas concentrations were measured using a quadrupole mass spectrometer with two silicone membrane inlets in the gas and water phase of the gas tight bubble tank. Fourteen trace gases with a wide range of solubilities and diffusivities – SF6, Neon, N2, HD, D2, O2, Krypton, Pentafluoroethane, Xenon, N2O, C2H2, CH3Cl, Benzene and DMS – were used to investigate the dependency on these two physico-chemical parameters. Bubbles were generated by a water jet with adjustable kinetic energy, which entrained a controllable gas volume flux into the water tank. Bubble size distributions in a radius range from 10 μm to 5000 μm and velocity distributions in a range from 70 μm to 3000 μm were measured at 60 positions by a telecentric optical setup. Invasion and evasion experiments with a variety of conditions were conducted including salt water (1.75% NaCl), the addition of the soluble surfactant Triton X-100, n-butanol and glycerol. All used additives tend to increase the fraction of small bubbles generated and therefore increase the transfer velocity at constant gas volume flux and energy input. The addition of salt had the strongest effect, leading to an enhancement of the transfer velocity up to a factor of approx. 4. The increase of the transfer velocity adding Triton X-100 was found to be lower than 5 %. The transition between diffusivity controlled and solubility controlled transfer occurs at solubilities ranging from 0.05 to 0.44. The transition is shifted to lower solubilities by a factor of approx. 3 by the addition of salt. Existing models for bubble mediated gas transfer are tested. Simple power law dependencies turn out to be incapable to describe the transfer for the whole range of solubilities and diffusivities. An extension of the parametrization proposed by Woolf requiring 4 parameters fits the data best. A simple model using only 2 parameters is proposed. Its performance is almost as good as the extended Woolf model.
Autor: Swen Seeland Institut / Klinik: Institut für Molekular- und Zellbiologie der Hochschule Mannheim Doktorvater: Prof. Dr. Mathias Hafner
The basic mission of the pharmaceutical industry is to understand disease and to bring safe and effective drugs to patients. Starting with the drug discovery process, for any particular disease a first step involves selecting a disease-specific target, then finding a suitable assay to determine the activity of molecules in relation to the selected target. This path is difficult and, lacking proper technology, is similar to the proverbial search for the needle in a haystack. However, once a molecule emerges as a successful candidate in the drug discovery process, it enters into drug development. The drug development process provides safety data prior to "first-in-man" trials. Drug discovery and development are extended, expensive, and inefficient processes with a success rate of only 0.01%. Placing these difficulties into perspective, it remains the desire of the pharmaceutical industry to develop novel, economical, reliable in vitro technologies to meet the above challenges. In this PhD thesis, a multiparametric cytosensor system was used for real-time identification of physiological parameters in living cells, and to meet the above-mentioned challenges in drug research. This technology allows us to simultaneously monitor extracellular acidification (pH changes), cellular respiration (oxygen consumption), and cellular morphology and adhesion (impedance measurements). All work done by a cell and all of the activities of life in general necessitate energy, commonly in the form of adenosine triphosphate (ATP). The regeneration of the consumed ATP leads to increased oxygen consumption and an excretion of acidifying side products (lactate and carbon dioxide), which can be monitored and used to deduce changes in physiological pathways. The first project of this thesis explored the influence of the P-glycoprotein (ABCB1) transporter on drug-drug-interactions, drug resistance, as well as drug absorption and distribution, all of which are important factors to be considered during the development of new drugs. Thus, the early identification and the exclusion of compounds that show a high affinity to P-glycoprotein can help to select drug candidates. The aim of this first project was to use the multiparametric cytosensor system for the label-free identification of P-glycoprotein substrates in living cells by monitoring extracellular acidification and cellular respiration upon stimulation with substrates of P-glycoprotein. Using L-MDR1 cells, a human P-glycoprotein-expressing cell line, the influence of P-glycoprotein activity was determined for seven different compounds, demonstrating the applicability of the system for P-glycoprotein substrate identification. Effects were concentration dependent, as shown for the P-glycoprotein substrate verapamil, and were associated with cellular acidification and respiration. P-glycoprotein ATPase activation by verapamil was able to be described by a Michaelis-Menten type kinetics profile showing saturation at high substrate concentrations. Control experiments using a P-glycoprotein inhibitor indicated that the observed effects were related to P-glycoprotein ATPase activity. In contrast, wild-type LLC-PK1 cells that did not express P-glycoprotein were not responsive to stimulation with different P-glycoprotein substrates. Summarising these findings, the microsensor system used is a generic system suitable for the identification of P-glycoprotein substrates. In contrast to other biochemical P-glycoprotein assays, activation of the drug efflux pump can be monitored on-line and label-free in living cells in order to identify P-glycoprotein substrates and to study the molecular mechanisms of ATP-dependent active transport. The second project of this thesis explored the pharmacology of the purinergic P2X7 receptor and its influence on changes in metabolic activity after ATP treatment. The purinergic P2X7 receptor plays a prominent role in cell metabolism and possibly determines downstream effects based on its interactions with extracellular ATP. Adenosine triphosphate, a key agent in physiology, provides energy in numerous reactions and acts as a neurotransmitter. Extracellular ATP concentrations are known to rise under pathological conditions, thereby triggering immune system responses that lead to pro-inflammatory states and immune modulation, to the extent of initiating cell death. These adverse effects have been linked to the purinergic P2X7 receptor, which triggers downstream signalling when levels of extracellular ATP are high. The purinergic P2X7 receptor is also involved in modulating cellular responses that include membrane depolarisation, secondary messenger activation, Ca2+ influx, and activation of the mitogen-activated protein kinase pathway. Moreover, it features a unique ability to form a large, non-selective pore, allowing molecules up to 900 Daltons to enter the cell, with potentially deleterious consequences. In addition, the P2X7 receptor purportedly regulates many metabolic processes inside the cell, while little is known of how extracellular ATP triggers these P2X7-mediated metabolic effects. In this study, the stimulatory effects of exogenously applied ATP on metabolic activity and the associated morphological changes in cells in relation to the P2X7 receptor were explored using the multiparametric cytosensor system for a deeper view inside the cell. Analysis of cell physiological parameters revealed that ATP-induced metabolic stimulation was detectable. Furthermore, based on signal patterns of the multiparametric cytosensor system, it was possible to detect ATP-induced oxidative stress to cells. Experiments with rodent brain cells that express P2X7 receptors demonstrated similar activation effects. Exploring and elucidating the evident relationship between the P2X7 receptor and extracellular ATP concentrations leads to the hypothesis that high levels of ATP reflect a pathological state and lead to an increase in metabolic activity in the cell. The third project of this thesis explored liver toxicology and used the multiparametric cytosensor system to detect these adverse effects. The liver plays a pivotal role in the biotransformation and detoxification of drugs and is consequently vulnerable to potential injury as a consequence of significant drug exposure. Drug-induced liver injury (DILI) is of considerable concern in drug discovery and development, placing emphasis on the need for predictive in vitro technologies that identify potential hepatotoxic side effects of drugs. A label-free, real-time, multiparametric cytosensor system has therefore been established for in vitro assessment of drug-induced toxicity. The system is based on monitoring cellular respiration, metabolic activity, cell morphology, and adhesion of human hepatocarcinoma-derived HepG2 cells. The read-out derived from the multiparametric cytosensor system has been optimised and permits sensitive, reliable, and simultaneous recording of cell physiological signals, such as metabolic activity, cellular respiration and morphological changes, and cell adhesion upon exposure to a drug. Analysis of eight prototypic reference drugs revealed distinct patterns of drug-induced physiological signals. Effects proved to be rigidly concentration-dependent. Based on signal patterns and reversibility of the observed effects, compounds were able to be classified as triggering mechanisms of oxidative or metabolic stress or as leading to cell death (necrosis-like and apoptosis-like). A test-flag-risk mitigation strategy is proposed to address potential risks for drug-induced hepatotoxicity. Concluding all three projects, the general concept of monitoring the physiological parameters of cells with an in vitro technology to overcome obstacles in drug research has clearly been shown to be viable. It was possible to develop a successful, novel assay for reliable, real-time, label-free identification of potential drug-drug interactions based on the identification of P-glycoprotein transporter substrates. Furthermore, the pharmacology of the purinergic P2X7 receptor after application of extracellular ATP was characterised and, finally and importantly, drug-induced liver effects were detected by on-line monitoring of liver HepG2 cells exposed to drugs.
In diesem Beitrag werden kurz die Grundprinzipien des Standards EMVA 1288 beschrieben: offen, nicht einschränkend, modellbasiert und Kamera als Komponente. Wenige Parameter sind notwendig, um flexibel und applikationsspezifisch eine optimale Kameraauswahl zu treffen. Dies wird an vergleichenden Beispielen illustriert. Weiterhin werden verfügbare Testgeräte vorgestellt und am Ende kurz dargestellt, wie der Standard auf 3D-Kamerasysteme erweitert werden könnte.
Eine Beschreibung einer 3D-Szene mittels Lichtfeldern führt nicht nur zu einer umfassenden Theorie ALLER möglichen optischen 3D-Aufnahmesysteme, sondern ermöglicht auch neue Ansätze, die auch die Schätzung der optischen Eigenschaften der Oberflächen integrieren. Weiterhin werden damit die Grenzen bisheriger optischer Messtechniken gesprengt. Am Beispiel von drei aktuellen Forschungsarbeiten aus dem HCI wird das Potenzial dieser Techniken aufgezeigt: a) die Bestimmung von Tiefenkarten und Bildaufnahme mit erweiterter Tiefenschärfe mittels hyperchromatischer Bildaufnahme, b) die Gewinnung von Tiefenkarten mittels plenoptischer Kameras nicht durch Korrespondenzanalyse, sondern durch Orientierungsanalyse im Lichtfeldes und c) Stereomessungen an spekular reflektierenden Oberflächen mittels aktiver Beleuchtung.
Transcription Factor IIIC (TFIIIC) is a six-subunit protein complex that recognises highly conserved promoter elements within genes transcribed by RNA polymerase III. These promoter elements, called ‘A box’ and ‘B box’, are recognised by two subcomplexes of TFIIIC, termed ‘τA’ and ‘τB’. The τA subcomplex is formed from three subunits: τ131, τ95 and τ55. The τB subcomplex is also composed of three subunits: τ138, τ91 and τ60. The binding of TFIIIC to these promoter elements leads to the recruitment of another transcription factor, TFIIIB, which subsequently coordinates the recruitment of RNA polymerase III to the transcription start site. In this thesis, I present my work on the characterisation of a novel interface between the τA and τB subcomplexes, which is located between the subunits τ131 and τ138. Chemical cross-linking coupled with mass spectrometry provides evidence for an important interaction between these two essential proteins, which I have mapped in detail using biochemical, biophysical and structural methods. The structure of a highly conserved tetra-trico peptide repeat (TPR) domain within τ131 is presented and is shown to bind with high affinity to a disordered region of τ138. In addition, I present the first high resolution structure of an extended winged helix domain of τ138 that I have solved by X-ray crystallography. Finally, I present work detailing efforts to produce the TFIIIC complex recombinantly in insect cells. The work presented here will further our understanding of the architecture of TFIIIC and provide insight into TFIIIC structure and function.
Alternative treatment strategies for pancreatic cancer are urgently needed as all available therapies fail to work efficiently. Oncolytic adenoviruses are promising agents as they can be engineered to specifically replicate in tumor cells and subsequently lyse them (oncolysis). Oncolytic adenoviruses have shown an excellent safety profile in phase 1 and 2 clinical trials. However, these studies revealed a need for improvement of the therapeutic efficiency of the viral agents. The critical issues were the limited efficiency of tumor cell lysis and the poor delivery of the virus to the tumor site after systemic administration due to clearance by neutralizing antibodies and liver sequestration. The delivery problem was approached by using mesenchymal stem cells (MSCs), isolated from human bone marrow, as carrier cells to shield the virus from nonspecific uptake and neutralization. MSCs are established carriers to mediate targeted delivery of oncolytic adenoviruses to tumor sites in animal models. In this study, genetic modifications of oncolytic adenovirus were explored for improvement of the viral agent in the context of MSC delivery. The overall aim of the study was to identify those modifications, which mediated optimized adenovirus transduction and replication in MSCs, allowed unimpeded migration of infected MSCs for the time required for tumor homing, and mediated improved killing of the pancreatic cancer cells. From a set of capsid variants, performed transduction experiments identified the fiber chimera 5/3, derived from adenovirus serotype 5 fiber with the cell binding domain of serotype 3, to consistently mediate most efficient transduction in MSCs as well as in established and low-passage pancreatic tumor cells. Ad5/3 served as capsid background in the subsequently investigated set of modified, replication-competent, oncolytic adenoviruses. The modifications included: i. the deletion of the viral gene E1B19K aiming at improved virus replication and release kinetics, ii. the transgene insertions of the shedded death ligand TRAIL to mediate bystander killing, and iii. the transgene insertion of the prodrug-converting enzyme FCU1, which mediates conversion of the systemically administered prodrug 5-Fluorocytosine to the chemotherapeutic agent 5-Fluorouracil, for additional tumor cell killing. Infection of MSCs with the E1B19K-deleted or TRAIL-modified viruses resulted in dramatically improved virus replication and release kinetics compared to a matching control virus. Further, it was shown that MSCs infected with the E1B19K-deleted or TRAIL-modified viruses maintained their migration properties over 2 days. This time span corresponds to the reported time MSCs need in vivo to home to a tumor after systemic administration. From these observations it was concluded that the investigated modifications have the potential to improve MSC-mediated virus delivery. Subsequently, the E1B19K-deleted, TRAIL-expressing, and FCU1-expressing viruses were tested in established pancreatic tumor cell lines for improved killing. All modified viruses showed enhanced tumor cell killing in a subset of cell lines. Further, virus spread and killing was investigated in low-passage pancreatic tumor cell cultures, as a clinically relevant model, and in MiaPaCa-2 spheroids, to mimic viral behavior in a 3D tumor structure. Also in these models, the results indicated that the analyzed virus modifications are suited to achieve more efficient tumor cell killing. In the course of this study, virus mutants were identified, which possessed improved replication and release kinetics in MSCs, allowed unimpeded MSC migration, and showed enhanced pancreatic tumor cell killing abilities. Therefore, strategies to improve oncolytic adenoviruses for MSC-mediated delivery to pancreatic tumor sites were derived, which can contribute to improve clinical efficiency of adenovirotherapy.
Tumor-associated antigens (TAAs) are promising targets for immunological therapeutic intervention in cancer therapy. Particularly, mutated proteins are a source for true tumor-specific antigens (TSAs) because they are exclusively expressed in the tumor and are not shared with normal tissue. Moreover, TSAs reduce the risk of autoimmunity and increase the chance to overcome tolerance compared to non-mutated protein sequences. In this study we sought to show for the first time, that a mutation-specific multi-peptide vaccine, which targets simultaneously common mutations in gastrointestinal tumors in a human HLA context, is capable to induce multifunctional and polyvalent CD4+ and CD8+ effector T cell responses with a tumor-protective capacity. Furthermore, we aimed to investigate whether this strategy results in immune-suppressive counter-reactions, like the induction of regulatory T cells (Treg). As target-antigens, we created a panel of peptides with sequences derived from the most frequently mutated variants of the tumor suppressor p53 and the oncogenes Kras and Braf described for colorectal (CRC) and pancreatic carcinomas. More precisely, the peptides represent wild-type (wt) or mutated sequences and have a length of 28-35 amino acids to facilitate a presentation of MHC I and II epitopes. In the presented study we analyzed the potency of the long peptide panel for active vaccination and its tumor-protective capacity in a murine cancer model system. For this purpose we utilized C57BL/6J mice, as well as an HLA-class I/II humanized mouse strain (HLA.A2/HLADR1 transgenic), in a multi-peptide vaccination setting. T cell responses of immunized mice were monitored by flow cytometry measuring cytokine secretion after antigen-specific in vitro re-stimulation. Thereby, we observed simultaneous, polyvalent CD8+ cytotoxic and CD4+ helper T cell responses against the majority of the peptides. Moreover, the peptide-specific T cells possessed a multifunctional cytokine-secretion profile and CD4+ T cells displayed a TH1 like phenotype. Notably, two of the mutation-comprising long peptides (Kras G12V and p53 R248W) induced a significantly higher secretion of cytokines than the corresponding wt sequences in both CD4+ as well as CD8+ T cells, which implied mutation-specificity. For these two peptides we were able to revalidate (Kras G12V) and identify (p53 R248W) HLA.A2 and HLA.DR1 restricted mutation-comprising epitopes. To investigate the tumor-protective capacity of the vaccination approach syngenic fibrosarcoma cell lines were generated in the HLA-class I/II transgenic mouse model by carcinogen-induced tumorigenesis. In tumor challenge experiments we employed cell lines carrying intrinsic Kras/Tp53 mutations and cell lines which were engineered to express the most immunogenic mutations found in our vaccination studies. Vaccination with mutated long peptides resulted in delayed tumor outgrowth compared to vaccination with wt counterparts regarding tumors with intrinsic mutations. However, animals vaccinated with highly immunogenic wt and mutated peptides showed a strong increase of immunosuppressive, peripheral Treg numbers correlating with an accelerated outgrowth of transgene-expressing tumors. In conclusion, we showed that long peptide vaccination targeting multiple mutated oncogenes and tumor suppressor genes is capable of eliciting polyvalent, multifunctional, and mutated antigen-specific effector T cells responses, which have the potential to eradicate tumor cells. Furthermore, we suggest the induction of immune-suppressive, possibly mutation-specific regulatory T cells as a critical issue for the success of this therapeutic approach.
The apicomplexan parasite Plasmodium falciparum is the causative agent of the most severe form of malaria with more than 600 000 death cases per year, mainly among young children and pregnant women. The development of the parasite within human erythrocytes is accompanied by extensive remodelling of the host cells. The establishment of nutrient acquisition pathways, the genesis of membranous structures within the red blood cell (RBC) cytosol and the formation of a cytoadherence complex on the surface of the host cell represent important examples of these modifications that contribute both to parasite survival and virulence. For this purpose P. falciparum exports hundreds of effector proteins into the RBC with the majority of them bearing a characteristic pentameric sequence termed Plasmodium export element (PEXEL), which is cleaved in the parasite after the third and N-terminally acetylated at the fourth amino acid position. To gain a better understanding of this important export signal, a mutagenesis screen on the PEXEL motif (48RLLAQ52) of a GFP-tagged model protein (STEVOR1-80) was conducted in this study. The localization of the mutant proteins to different compartments of the parasitized erythrocyte was determined by fluorescence microscopy. In addition, mass spectrometric analysis of representative mutants confirmed a correlation between processing of the export motif and protein trafficking. In summary, amino acid replacements within position 1 (R48) and 3 (L50), representing the most conserved amino acids, had detrimental effects on PEXEL cleavage and protein export. In contrast, positions 2 (L49) and 5 (Q52) proved to be more permissive towards mutations and mostly maintained the wild-type export phenotype. Special attention was paid to position 4 (A51), which is acetylated after cleavage and appeared more restricted than originally assumed. One interesting finding was the deficiency of non-acetylated mutant proteins to be exported into the host cell indicating N-terminal acetylation as a prerequisite for proper targeting of PEXEL proteins. Consequently, the second part of this study focussed on the characterization of a putative N-acetyltransferase (PfNAT) that might be responsible for this allegedly important post-translational modification. The localization of the candidate transferase to the parasite endoplasmic reticulum, the compartment of PEXEL processing, was confirmed by single crossover integration of a GFP tag into the endogenous locus. Despite the use of two different approaches, the attempted deletion of the gene for loss-of-function studies was unsuccessful. However, a conditional downregulation of protein levels by ~50% was achieved using a destabilization domain, which facilitated further investigations.
Acute myeloid leukaemia is a malicious disease. Although the initial chemotherapeutic treatment often leads to a complete remission (a disappearance of all manifestations of disease), the effective survival rate is only (30-40) % over 4 years due to a high relapse rate. This relapse is attributed to leukaemic stem cells residing in the protective environment of the bone marrow niche. There are two major approaches aiming at achieving better long-term therapeutic results. The first is to make the leukaemic stem cells more susceptible to chemotherapeutic agents and the second is to increase the efficiency of haematopoietic stem cell transplants, which are used to regenerate the haematopoietic system after failure due to chemotherapy. When searching for a receptor-ligand pair suitable as target for therapeutic agents, the prerequisite is that it must exhibit differences between the interaction it mediates in healthy and leukaemic cells. A detailed understanding of the mediated interaction and the differences would then allow exploitation of these to selectively mobilise the leukaemic stem cells increasing their susceptibility for chemotherapeutic drugs. In this work the flow-induced rolling interaction of leukaemic cells with hyaluronic acid was studied in detail using a suspension and an epithelial model cell line. It could be demonstrated, that the flow induced rolling interaction on hyaluronic acid observed for these cells was solely mediated by the cell surface receptor CD44 and that it was independent of the cell type tested. Next to a detailed validation and characterisation of this dependency and the properties of the interaction, the relevance of this interaction for the haematopoietic system and for leukaemic cells was evaluated. Therefore, the CD44 mediated interaction with hyaluronic acid of healthy haematopoietic progenitor cells from umbilical cord blood, mobilised peripheral blood and the bone marrow with that of leukaemic blasts was compared. Throughout the cell types tested two forms of interaction with hyaluronic acid were observed; a flow induced rolling and an immobile adhesion. It could be shown that while the rolling interaction was comparable for all cell types tested, the immobile adhesion to hyaluronic acid and its susceptibility to a monoclonal CD44 antibody (clone BU52) were not. The immobile adhesion was found predominantly in leukaemic cells, only playing a subordinate role in the interaction of healthy cells with hyaluronic acid. It could be demonstrated that a vicinity of the cells to the bone marrow upon isolation was directly correlated to an incomplete suppression of the immobile adhesion by BU52. Furthermore, this incomplete suppression could be linked to a non-response to induction chemotherapy and subsequently to a poor therapeutic outcome. Besides investigating the interaction with surfaces artificially coated with hyaluronic acid, the possibility of using surfaces covered with mesenchymal stromal cells isolated from the bone marrow as more realistic binding partners was explored. Furthermore, the effect of a routinely used mobilisation reagent, namely Plerixafor®, on the migration and cell-surface interaction under flow was investigated. It is not only of great interest to understand the mechanisms of retention in the niche, but also to develop more sophisticated methods of in vitro stem cell expansion. In this context the slow and continuous release of e.g. cytokines or growth factors is of great interest. The cavities in porous materials present the unique opportunity of achieving just that by being pre-loaded with such agents. These can then under the right conditions be released to the cells. Amongst the porous materials the metal-organic frameworks protrude due to their high structural and chemical flexibility. In this work a novel 2-D metal-organic framework structure, namely SURMOF 2, was tested towards its biocompatibility and smart-release properties. It could be shown that SURMOF 2 was highly stable in protein free aqueous media and that its building units did not impair the growth of rat embryonic fibroblasts. Although the stability in cell culture medium is still limited, the water stability and the biocompatibility of the components are the starting point for future SURMOF 2 cell culture applications. A first application of SURMOF 2 as a smart-release matrix was achieved with the marine bacterium Cobetia marina under salt water conditions. The results demonstrated the general applicability of SURMOF 2 as bioactive substrates with responsive properties. For the future, fine-tuning of the stability of SURMOFs will allow to tailor drug release systems for cytokine or growth factor delivery in in vitro stem cell cultures.
The aim of this work is to explore the origin of magnetic fields in the Universe. We claim that the turbulent or small-scale dynamo, which amplifies weak seed fields on short timescales in the presence of turbulence, plays an important role in the evolution of cosmic magnetic fields. The theoretical model for the turbulent dynamo is generalized for various astrophysical environments, with a focus on different turbulence spectra. We derive analytical solutions for the dynamo growth rate in the kinematic phase and discuss the subsequent non-linear evolution as well as saturation. In the history of the Universe turbulence is expected to be driven efficiently at the latest during the formation of the first stars and galaxies, where gravitational energy is converted into chaotic motions as the dark matter halos accrete gas from the environment. We model these processes semi-analytically and implement magnetic field amplification by a turbulent dynamo. Our results show that unordered magnetic fields, with strengths comparable to the ones in local galaxies, were already present in the primordial Universe. A potential observational test for magnetic fields in young galaxies is suggested to probe our proposed scenario for the evolution of cosmic magnetic fields.
In this thesis we introduce a novel coupled two scalar field model of the dark sector motivated by higher dimensional dilatation symmetric theories of gravity, which could potentially provide a solution to the cosmological constant problem. This work is split up into four parts. First, we motivate the model and discuss its evolution at the background level. We find a quintessence-type scaling solution in the early universe and a coupled cold dark matter scenario for later times. At the level of linear perturbations we introduce an effective fluid description of this model and implement it into a Boltzmann-code to recover the linear power spectrum, which exhibits a characteristic suppression of power at a Jeans scale. In a third step we analyze superhorizon perturbations in the early universe in some detail, with particular focus on the issue of the stability of the adiabatic perturbation mode. Finally we apply the extended Press-Schechter formalism to predict substructure abundances within a typical galaxy like the Milky Way and use the results to obtain parameter bounds for our model.
The behavior and dynamics of complex systems are the focus of many research fields. The complexity of such systems comes not only from the number of their elements, but also from the unavoidable emergence of new properties of the system, which are not just a simple summation of the properties of its elements. The behavior of dynamic complex systems relates to a number of well developed models, the majority of which do not incorporate the modularity and the evolutionary dynamics of a system simultaneously. In this work, we deploy a Bayesian model that addresses this issue. Our model has been developed within the Random Finite Set Theory's framework. We introduced the stochastic evolution diagram as a novel mathematical tool to describe the evolutionary dynamics of complex modular systems. It has been shown how it could be used in real world applications. We have extended the idea of Bayesian network for non-stationary dynamic systems by defining a new concept "labeled-edge Bayesian network" and providing a Bayesian Dirichlet (BD) metric as its score function.
The coupling of endoplasmic reticulum (ER) and plasma membrane (PM) is crucial for calcium (Ca2+) homeostasis. STIM1 and STIM2 are type I membrane proteins of the ER and function as Ca2+ sensors in a process known as store-operated calcium entry (SOCE). They sense a drop in luminal Ca2+ concentration and undergo conformational changes and oligomerization. The active oligomerized STIM proteins translocate to ER-PM contact sites, where they bind to phosphoinositides (PIPs) at the inner leaflet of the PM via their lysine (K)- rich domains and activate Orai1, a pore-forming Ca2+ release-activated Ca2+ (CRAC) channel subunit in the PM. I found that STIM2, but not STIM1, contains a di-lysine ER-retention signal. This signal restricts the function of STIM2 as Ca2+ sensor to the ER while STIM1 can reach the PM via the classical secretary pathway. The intracellular distribution of STIM1 is regulated in a cell-cycle-dependent manner with cell surface expression of STIM1 during mitosis. Efficient retention of STIM1 in the ER during interphase depends on its K-rich domain and a di-arginine ER retention signal. SOCE enhances ER retention, suggesting that trafficking of STIM1 is regulated and this regulation contributes to STIM1’s role as multifunctional component in Ca2+-signaling. In contrast to mitotic cells, interphase cells retain most of their STIM1 intracellularly. Under resting condition, the ER-resident STIMs are preferentially located in PI(4,5)P2 containing preexisting ER-PM contact sites, which are expanded upon ER Ca2+ depletion. The lipid-binding, K-rich domains are required to localize STIM proteins in preexisting ERPM contact sites. Moreover, STIM2 recruits ER more efficiently to the PM. This is consistent with the fact that STIM2 has higher lipid-binding affinity and lower activation threshold than STIM1 and that STIM2 functions as a regulator of basal Ca2+ homeostasis. Finally, I studied the role of microtubules in ER-PM contact site formation. I observed that STIM1 aligns along microtubules. Alignment of STIM proteins with microtubules is a conserved process. In addition to accumulation of STIM1 at microtubule plus ends, STIM1 moves along microtubules in an EB-1-independent manner. I identified two EB-1- independent microtubule-binding sites located within the C-terminus of STIM1 and found that oligomerization increases the EB-1-independent microtubule-binding affinity of STIM1. However, the physiological function of this EB1-independent microtubule binding activity remains elusive.
In past few years, genome-wide RNAi screens have identified many novel genes involved in diseases for many viruses such as Human Immunodeficiency Virus-1 (HIV-1), Hepatitis C virus (HCV), West Nile Virus (WNV) and Influenza virus (IV). However, due to difference in experimental conditions, usage of different viral strains and inherent biological noise, these screens have shown low number of common or overlapping hits for a virus. Moreover, this overlap gets poorer for similar studies on viruses of different families. Although these overlaps are significant, their lower size restricts a comprehensive insight from a comparative analysis. Thus, a direct comparison of gene hit-lists of RNAi screens may not always give meaningful results. To address this problem we propose an integrative bioinformatics pipeline that allows for network based meta-analysis of viral HT-RNAi screens. Initially, human protein interaction network (PIN) generated by collating data from various public repositories, is subjected to unsupervised clustering to determine functional modules. Those modules that are significantly enriched in host dependency factors (HDFs) and/or host restriction factors (HRFs) are then filtered based on network topology and semantic similarity measures. Modules passing all these criteria are then interpreted for their biological significance from enrichment analyses. With our approach we could predict Tankyrase-1 as a potential novel hit within the functional subnetworks, within the human PIN for Hepatitis C virus (HCV). and Human Immunodeficiency Virus-1 (HIV-1), based on HDFs and HRFs identified in the corresponding genome-wide RNAi screens of these viruses. Thus, our approach allows for a network based meta-analysis of genome-wide screens to develop plausible hypotheses for novel regulatory mechanisms in virus-host interactions based on RNAi screens.
T cells accurately discriminate between antigens that have only moderately different affinities. The decision of whether the cell is activated or not is made during the initial stages of membrane-proximal signaling triggered by the T cell receptor (TCR). Several mechanistic models have been proposed to explain the highly specific and sensitive recognition of peptide-MHC ligands. Prominent among them is the kinetic proofreading model that accounts for the high specificity of the ligand discrimination by the TCR based on ligand dwell time. Open questions of central importance are (1) whether kinetic proofreading is indeed realized in TCR signal transduction and (2) how information about ligand binding to the TCR is transduced to the cell interior. In this work, we addressed these questions quantitatively by means of two data–based models. Our models describe the dynamics of two core modules of TCR activation: the TCR signaling module and the Src–family kinase regulation module. The model of TCR signaling accounts for the reversible phosphorylation of im- munoreceptor tyrosine–based activation motifs (ITAMs) in the TCR complex by the kinase Lck and the phosphatase CD45, and the subsequent recruitment and phosphorylation of the pivotal kinase ZAP70. We parameterize the model using ki- netic measurements of phosphorylation status and protein-protein interactions. The model constrained by these data allows for kinetic proofreading of ligand dwell time; however, this capacity depends on the mechanism of signal initiation. Neither an enhancement of phosphorylation nor a reduction of phosphatase activity alone allow the TCR to discriminate ligand dwell times, whereas a combination of both effects yields kinetic proofreading behavior. TCR signaling is driven by the Src–family kinase Lck: thus we investigated the dynamic regulation of Lck activity in the second model. The parameterization of this model is based on dose–response data of wild type Lck and Lck chimeras with altered membrane targeting. These data allow us to determine the model parameters within narrow confidence bounds enabling us to make quantitative predictions with the model. We find that Lck activity is regulated jointly by its subcellular localization, trans–autophosphorylation of the activating tyrosine residue and the activity of the phosphatase CD45 in the proximity of Lck. Mechanistically, CD45 mediates both activating and inhibitory dephosphorylations of Lck. Physiologically, we find that CD45 has a solely inhibitory effect on Lck activity. Interestingly, this inhibitory action could synergize with the mechanisms modeled in the TCR signaling model to support kinetic proofreading. In summary, our results show how data-driven mathematical modeling, can be used to dissect the complexity of TCR activation on a quantitative basis.
Spermatogonial stem cells (SSCs) are located on the basal membrane in seminiferous tubules of the testis and are responsible for sperm production during the male’s life. Although the morphology and kinetics of spermatogonia has been extensively characterized, there is still a lack of specific cellular, morphological and biochemical criteria for SSCs. However, Fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), matrix selection and morphology-based selection are some of the useful methods for the isolation of SSCs. In the current study, we demonstrated that using cell sorting approaches are dispensable for the generation of mouse SSCs cultures. We identified a simple and highly reproducible protocol for the isolation of SSCs with morphology-based selection and successfully established two types of SSCs (Type I and Type II). Using co-culture systems with different mitomycin-C treated feeder layers in long-term culture, we established the optimal conditions for the cultivation and gene expansion of these two types of SSCs. We successfully expanded Type I SSCs on mouse embryonic fibroblasts (MEFs) and Type II SSCs on both SNL and primary testicular stroma cells (TSCs) feeders. Electron microscopic analysis revealed that Type I SSCs display a similar morphology with SSCs in-vivo high nucleus/cytoplasm ratio, while Type II SSCs have a different morphology small nucleus/cytoplasm ratio. Immunocytochemistry, FACS and Fluidigm real-time RT-PCR results showed that Type I SSCs clearly express germ cells markers while Type II SSCs only partially express the typical germ cell profile of SSCs. Following the transplantation of Type II SSCs to busulphan-treated NOD/ SCID mice, we observed a localization of GFP-labeled cells in the basal compartment of the seminiferous tubule. Furthermore, GFP labeled sperms were detected in epididymis. Among the most obvious molecular differences between Type I and Type II SSCs were their reprogramming capacity to mouse embryonic stem cells-(ES-) like cells that occurred only on the critical time window after initiation of Type I SSCs culture. Although testis-derived ES-like cells have been obtained in several studies, the time window for the shift to pluripotency was not clearly determined. In our experiments, we observed that the spontaneous appearance of germline-derived ES-like cells from both neonate and adult Type I cells occurred only during a special time window (41 until 125 days) after initiation of Type I cells from neonate and adult promoter-reporter Oct4-GFP transgenic mice. The generated ES-like cells expressed pluripotency markers, differentiated into all three germ lineages, formed complex teratoma after transplantation in SCID mice and produced chimeric mice. Although the exact mechanism of the development of ES-like cells from GSCs is still unclear, this new information could provide an ideal strategy for scheduling natural conversion mechanisms of ES-like cells from mouse testes. Therefore, the two different types of SSCs could provide an ideal cell system for studying both pluripotency and in-vitro differentiation of SSCs to sperm and also provide a new strategy for isolation of SSCs from neonatal and adult mice by morphology-based selection.
Brine shrimps Artemia (Crustacea, Anostraca) are a group of cosmopolitan extremophile microcrustaceans which are composed of a complex of six sexual species and numerous Eurasian Haplotype Complex (EHC) lineages. In the present study, we analyzed a partial sequence of the mitochondrial cytochrome c oxidase subunit I (COI) and nuclear Internal transcribed spacer1 (ITS1), as well as genomic fingerprints by ISSR-PCR (inter-simple sequence repeats) for a large set of Artemia specimens (N ~ 600) from various geographical localities (N = 102) across Eurasia and America. Asian lineages have revealed a mixture of paraphyletic and polyphyletic groups of Artemia. A. urmiana and A. tibetiana represent a species complex with multiple genetic lineages. EHC lineages (Eurasian and Africa) showed a star-like haplotype pattern, which had more genetic similarities to other sexual Asian species, except A. sinica. Bayesian analysis of COI was used to estimate the time of divergence of Asian Artemia species. The Bayesian analysis indicated that Asian taxa are relatively young, particularly EHC lineages. A. urmiana recorded as the oldest species originated in the Pleistocene and could be considered as a major source of its expansion to its modern habitats in Eurasia. Molecular dating analyses using a relaxed bayesian molecular clock depicted that A. sinica has been diverged from other Asian lineages in the Miocene, around 20 million years ago (Mya). A. urmiana was the dominant sexual species in Urmia Lake exhibiting a high genetic diversity with a low level of genetic structure in the entire lake. This species was essentially homogeneous indicative of panmixia. A correlation between population differentiation and geographical and ecological differences was not observed. The invasive A. franciscana have permanently colonized 31 geographical localities along the southern and eastern coastal regions of Asia. EHC lineages were observed in 39 inland geographical localities in Asia. Asian A. franciscana generated a signature of haplotype diversity as compared to the source population from Great Salt Lake (GSL, USA). The high genetic diversity of Asian A. franciscana is probably attributed the numerous, human-mediated, dispersal events and multiple introductions from GSL & San Francisco Bay (SFB, USA) and eventually indirect introductions from other native localities in the Americas. Our results indicate that biological invasion do not necessarily lead to reduced genetic diversity, particularly if multiple source populations, each with distinctive genetic composition, contribute to the founding populations. EHC lineages showed low genetic diversity which is in contrast to the restricted geographic distribution, strong genetic structure, and regional endemism of sexual Artemia lineages in Asia. A. franciscana in GSL revealed the same condition as observed in A. urmiana. It showed a high genetic diversity with an evidence of panmixia throughout the lake.
70 Nachwuchswissenschaftler aus aller Welt befassen sich zurzeit in Heidelberg mit einer Frage, die uns alle betrifft: Dem Klimawandel, und wie wir ihm begegnen können. Die Verminderung von Treibhausgasen spielt dabei natürlich eine zentrale Rolle, doch auch technische Maßnahmen können einen wichtigen Beitrag im Kampf gegen die globale Erwärmung (Climate Engineering) leisten, so der Mitorganisator der Summer School Ulrich Platt.
Der Beitrag über Climate Engineering erschien in der Sendereihe "Campus-Report" - einer Beitragsreihe, in der über aktuelle Themen aus Forschung und Wissenschaft der Universitäten Heidelberg, Mannheim, Karlsruhe und Freiburg berichtet wird. Zu hören ist "Campus-Report" montags bis freitags jeweils um ca. 19.10h im Programm von Radio Regenbogen. (Empfang in Nordbaden: UKW 102,8. In Mittelbaden: 100,4 und in Südbaden: 101,1)
In dieser Arbeit untersuchen wir die Erzeugung fermionischer Teilchen außerhalb des Gleichgewichts mittels moderner Gittermethoden. Die vorgestellten Anwendungen reichen vom Preheating nach der kosmologischen Inflation im frühen Universum über Prä- Thermalisierungsdynamik in Schwerionenkollisionen bis hin zur Paarerzeugung und Stringbrechung in einem niedrigdimensionalen Modell der Quantenchromodynamik. In Instabilitäten aufweisenden skalaren Modellen beobachten wir eine stark erhöhte Fermionproduktion in Anwesenheit bosonischer Ü berbesetzung. Als mögliche Szenarien fürs Preheating nach der Inflation untersuchen wir parametriche Resonanz und tachyonische Instabilität. Wir finden, dass sowohl die qualitativen als auch die quantitativen Eigenschaften der resultierenden Fermionverteilung weitgehend von einem effektiven Kopplungsparameter bestimmt werden. Um Fermionen in drei räumlichen Dimensionen simulieren zu können, wenden wir einen effzienten, stochastischen Gitter-Algorithmus an, welchen wir durch einen Vergleich mit exacten Gitterrechnungen und mit auf einer Kopplungsentwicklung basierten funktionalen Methode verifizieren. Im massiven Schwinger-Modell analysieren wir die Erzeugung von Fermion/Antifermion Paaren durch homogene und inhomogene elektrische Felder und beobachten den Aufbau von Strings zwischen den Ladungen. Nachfolgend studieren wir die Dynamik der Stringbrechung und beschreiben einen Zwei-Phasen-Prozess, welcher aus der anfänglichen Teilchenproduktion sowie der folgenden Ladungsseparation und Abschirmung besteht. In Quantenchromodynamik liegt unser Fokus auf den Eigenschaften des Quarksektors während der turbulenten bosonischen Energiekaskade sowie auf der Isotropisierung der Quarks und Gluonen, ausgehend von unterschiedlichen Anfangsbedngungen.
Microvascular perfusion is an important parameter of high clinical value as it provides important information on tissue viability and function. In contrast to dynamic contrastenhanced magnetic resonance imaging (DCE-MRI), arterial spin labeling (ASL) is a method to assess quantitative perfusion values non-invasively by MRI. The main goal of this work was the development of a 3D ASL technique that allows the quantitative perfusion measurement within a whole volume. Further, the diagnostic signifcance of ASL was tested by applying it in dedicated renal disease models. ASL yielded significantly (P < 0.01) different values in healthy kidneys ((500+-91) ml/100g/min) compared to kidneys with acute kidney injury ((287+-83) ml/100/min) as well as for acutely rejected transplanted kidneys compared to chronically rejected grafts. A comparison to DCE-MRI showed no signifcant differences. While literature reports on 3D ASL sequences show a prevalence for spin-echo based data acquisitions, the presented sequence employs a 3D balanced steady-state free precession (bSSFP) readout that has the ability to overcome some of the drawbacks of spin-echo based 3D sequences. The developed 3D ASL technique includes several features, each of which has been optimized separately. Special emphasis was put on design, simulation, and implementation of a slice-selective adiabatic inversion pulse. In an initial measurement, the whole-brain perfusion of a healthy volunteer was assessed with an isotropic resolution of 3mm. Mean perfusion values were f_GM = (51+-17) ml/100g/min and f_WM = (24+-8) ml/100/min for gray and white matter, respectively, which are in very good agreement with findings from the "gold-standard" method of 15O-positron emission tomography. The diagnostic value of ASL in combination with the possibility to assess absolute perfusion values in a whole volume makes it a promising technique for future clinical diagnosis.
This thesis investigates different aspects of strongly interacting ultracold Rydberg gases coupled by light, including the excitation of correlated structures called Rydberg aggregates and dipolar energy transport. For this purpose an experimental apparatus has been constructed. We study coherent population trapping (CPT) and electromagnetically induced transparency (EIT) involving Rydberg states. To study the formation of Rydberg aggregates we use full counting statistics (FCS) which makes it possible to infer the size of Rydberg aggregates. Dephasing during the excitation is found to have a dramatic effect on the formation process, leading to sequential growth around an initial grain. Rydberg state EIT is used to realize a new direct optical imaging scheme for Rydberg atoms which combines highly sensitive detection with high spatial and temporal resolution. Applying this technique we observe dipole-mediated energy transport processes between Rydberg atoms. We study classical transport in the presence of strong measurement induced decoherence. The strong interactions are seen to mediate long-range hopping significantly beyond nearest neighbors. We present first indications for coherent transport in this system. These experiments shed light on the role of correlations in the excitation and evolution of strongly interacting quantum systems and e.g. could possibly be used to model energy transport in biological systems.
Among the different biofouling species, barnacles resemble a specific threat as they are difficult to remove, able to damage fouling release coatings and increase the drag force of ships. Additionally, barnacles are a good model system for research on permanent underwater adhesion strategies. This study aims to understand and compare the spatial organization and the chemistry of the adhesive secreted by two different species (Balanus amphitrite and Balanus improvisus) of cyprid larvae and juvenile barnacles for settlement. Raman spectromicroscopy and synchrotron based X-ray microprobe fluorescence analysis have been applied for the in-situ and ex-situ investigation of juvenile barnacle cement chemistry. Confocal Raman spectromicroscopy revealed the chemical heterogeneity of the barnacle baseplate and allowed to distinguish three regions of various chemical compositions. The adhesive of cyprids was different from the one of larvae and analyzed in detail from the metamorphosis to the age of fourteen days. The results of these studies provided information on the chemical composition and morphological structure of both barnacle species at different life stages.
Haematopoietic stem cells (HSCs) are the best-characterised adult stem cells and ensure life-long maintenance of the blood system. Under homeostatic conditions most of the HSCs are found in a quiescent state, but can be activated in response to stress (Wilson et al., 2008). Infection is a major source of natural stress to the haematopoietic system, leading to exhaustion of large amounts of immune cells during the defence against invading pathogens. Reestablishment of homeostasis following infection depends on the replacement of immune cells by haematopoietic progenitors. Interestingly, recent studies have indicated that HSCs themselves can be activated in response to infection (Essers et al., 2009; Sato et al., 2009; Baldridge et al., 2010; Takizawa et al., 2011). However, whether HSCs are able to directly sense the infectious state remains unclear. In this work, we have investigated the in vivo short-term response of HSCs to lipopolysaccharide (LPS), a major cell wall component of Gram-negative bacteria. Low dose administration of LPS to mice induces a rapid TLR4 dependent proliferation of even the most dormant HSCs, without impairing their long-term repopulating potential. This activation is accompanied by upregulation of stem cell antigen 1 (Sca-1) and dependent on the expression of Sca-1 on HSCs. Both in vitro and in vivo data demonstrate that activation of HSCs via LPS is not a direct effect of LPS on HSCs. Instead, LPS treatment stimulates myeloid cells in the BM, most probably inflammatory monocytes, to produce inflammatory cytokines, which in turn induce the activation of HSCs. Furthermore, our results suggest that the activation of HSCs is mediated by initial cell-cell-interaction of these myeloid cells with HSCs. Using genetic mouse models we show that the activation of HSCs is regulated by combined IFNa, IFNg and TNFa signalling. Moreover, TNFa alone induces a Sca-1 dependent activation of HSCs in vivo, making these cells susceptible to chemotherapy. In addition to IFN and TNFa signalling also IL-1b signalling is necessary to mediate the LPS-induced activation of HSCs. Interestingly, IL-1b itself leads to increased proliferation of HSCs, most likely through direct activation of IL-1 signalling in these cells. Thus, our work provides novel cellular and genetic insights into the identity of LPS induced cytokine production, leading to HSC activation. Thus, providing a way to rapidly induce the production of mature immune cells upon infection, leading to a fast restoration of homeostasis upon bacterial infections. Furthermore, our work provides new insights into the role of the pro-inflammatory cytokines TNFa and IL-1b in the regulation of HSC function.
This thesis treats optimum experimental design for the parameter estimation problem of mobility parameters in charge transport models of organic semiconductors. The models consist of the van Roosbroeck system, a quasi-electrochemical potential defining equation, and the Extended Gaussian Disorder Model and the Extended Correlated Disorder Model both describing the mobility. The arising problems are very ill-conditioned. The essential points of this work are: • The robust numerical solution of the model equations w.r.t. varying parameters, control parameters, boundary values and initial guesses for iterative methods. • The computation of exact derivatives up to order two, which are necessary for the optimum experimental design problem. This includes derivatives of the model functions and implicitly given derivatives of the solution. The Scharfetter-Gummel scheme is applied to the spatial discretization in one dimension, whereas in two dimensions bilinear finite elements are used. The numerical simulation of the discretized equations is done by a hybrid simulation method consisting of Gummel’s method with a special, problem-adapted stabilization term, a contraction based damping strategy, and a full step Newton method in the end for quadratic convergence near the solution. These strategies are independent of the spatial discretization and are applied to the simulation of a polymer nano-chain attached to the cathode. The simulation of the one dimensional problems are used for the optimum experimental design. The derivatives are computed with automatic differentiation exactly up to machine precision. Therefor we use software tools for the computation of the derivatives of the model functions and solve tangential and adjoint equations of the problem for the parameters and control parameters. With optimum experimental design we plan experiments for newest organic materials, like NRS-PPV and a-NPD. The confidence region of the parameters are reduced by a factor of 100 for NRS-PPV.
Hermann Helmholtz lehrte von 1858 bis 1871 als Physiologieprofessor an der Heidelberger Universität. Der vielseitige Gelehrte befasste sich in Heidelberg intensiv mit den Grundlagen der Geometrie.
Die Nachrufsammlung enthält: 1.) Theodor Wilhelm Engelmann (Universität Utrecht) 2.) Emil du Bois-Reymond (Physikalische Gesellschaft Berlin) 3.) Otto Bütschli (Naturhist.-med. Verein zu Heidelberg) 4.) Wilhelm von Bezold (Wissenschaftliche Vereine in Berlin) 5.) Albert Waag (Stadt Heidelberg) 6.) Emil du Bois-Reymond (Akademie der Wissenschaften zu Berlin) 7.) Briefe von Anna von Helmholtz
Außerdem wurde die von Hermann von Helmholtz gehaltene Tischrede anl. seines 70. Geburtstages beigefügt.
Neuromorphic hardware is a promising tool for neuroscience and technological applications. This thesis addresses the question to what extent such systems can benefit from advances in CMOS scaling using the existing BrainScales Hardware System as a reference. A 65 nm process technology was selected and basic characteristics were evaluated using prototype chips. A system providing a large number of programmable voltage and current sources, based on capacitive storage cells, was developed. A novel scheme for refreshing the cells is presented. This system has been characterized in silicon. Two components required in a concept for synapse implementation, consisting of primarily digital circuits, were developed and tested in a prototype chip. One is an orthogonal dual-port SRAM with a specialized structure where every 8 bit word stored in the memory can be accessed by a single operation from either port. The second is an 8 bit current DAC which is used for generating postsynaptic events. Finally the analog neuron implementation from the existing system was transfered to the 65 nm process technology using thick-oxide transistors. Simulations suggest that comparable performance can be achieved. In conclusion, modern process technologies will contribute to successful realization of large-scale neuromorphic hardware systems.
The formation of galaxies and their subsequent evolution through cosmic time is governed by a variety of complex physical processes such as gas cooling, star formation, feedback and merger events. In this thesis we use hydrodynamical simulations to study the effect of these processes on galaxy properties. We first investigate the change in galaxy morphology as they undergo mergers. We look at a wide variety of merger events from interaction between small dark matter subhaloes and galactic discs, to major mergers. In the second part of the thesis we turn our attention to the role of stellar feedback in regulating star formation. A model for short range photoheating of gas by radiation from massive stars is tested by simulating a representative volume of the Universe and comparing the statistical properties of simulated galaxies with the observed ones. Finally, we introduce a new, computationally efficient model to calculate the gas cooling rate in the presence of local radiation fields. The model uses simple assumptions for absorption of ultra-violet photons and an optically thin approximation to propagate local radiation fields throughout the entire simulation volume. Using this new method we show that local radiation has a significant effect in regulating the star formation rate of L* galaxies. It reduces gas accretion onto the disc, thereby, producing realistic galaxies without resorting to extreme feedback mechanisms.
In studies of the brain and the nervous system, extracellular signals – as measured by local field potentials (LFPs) or electroencephalography (EEG) – are of capital importance, as they allow to simultaneously obtain data from multiple neurons. The exact biophysical basis of these signals is, however, still not fully understood. Most models for the extracellular potential today are based on volume conductor theory, which assumes that the extracellular fluid is electroneutral and that the only contributions to the electric field are given by membrane currents, which can be imposed as boundary conditions in the mathematical model. This neglects a second, possibly important contributor to the extracellular field: the time- and position-dependent concentrations of ions in the intra- and extracellular fluids.
In this thesis, a 3D model of a single axon in extracellular fluid is presented based on the Poisson-Nernst-Planck (PNP) equations of electrodiffusion. This fundamental model includes not only the potential, but also the concentrations of all participating ion concentrations in a self-consistent way. This enables us to study the propagation of an action potential (AP) along the axonal membrane based on first principles by means of numerical simulations. By exploiting the cylinder symmetry of this geometry, the problem can be reduced to two dimensions. The numerical solution is implemented in a flexible and efficient way, using the DUNE framework. A suitable mesh generation strategy and a parallelization of the algorithm allow to solve the problem in reasonable time, with a high spatial and temporal resolution. The methods and programming techniques used to deal with the numerical challenges of this multi-scale problem are presented in detail.
Special attention is paid to the Debye layer, the region with strong concentration gradients close to the membrane, which is explicitly resolved by the computational mesh. The focus lies on the evolution of the extracellular electric potential at different membrane distances. Roughly, the extracellular space can be divided into three distinct regions: first, the distant farfield, which exhibits a characteristic triphasic waveform in response to an action potential traveling along the membrane. This is consistent with previous modeling efforts and experiments. Secondly, the Debye layer close to the membrane, which shows a completely different extracellular response in the form of an “AP echo”, which is also observed in juxtacellular recordings. Finally, there is the intermediate or diffusion layer located in between, which shows a gradual transition from the Debye layer potential towards the farfield potential. Both of these potentialregions show marked deviations from volume conductor models, which can be attributed to the redistribution of concentrations and associated ion fluxes. These differences are explained by analyzing the capacitive and ionic components of the potential.
In an extension, we also include myelination into the model, which has a significant impact on the extracellular field. Again, the numerical results are compared to volume conductor models.
Finally, a model study is carried out to assess the magnitude of ephaptic effects, i.e. the influence of the electric field of one cell on a neighboring cell, in a somewhat artificial geometry. While the results probably can not be interpreted quantitatively in the majority of physiological situations, the qualitative behavior shows interesting effects. An axon can elicit an action potential in a surrounding bundle of axons, given that the distance is small enough and the resistivity of the extracellular medium is significantly increased. Further results of this study are extremely large extracellular potentials with amplitudes up to 100 mV and an unusual neuronal firing mode in which the cell is not depolarized by an increase in the intracellular potential, but by a decrease in the extracellular potential. Some literature references are given that show that these observations are consistent with previous studies.
The Interatomic/ Intermolecular Coulombic Decay (ICD) as well as the Electron Transfer Mediated Decay (ETMD) are electronic decay processes, which occur in a multidude of systems ranging from noble gas dimers to biological systems. If heavy atoms are involved in these processes, relativistic effects cannot be neglected. However, their influence has so far not been investigated thoroughly. In this thesis, the influence of the spin-orbit coupling as well as scalar-relativistic effects on openings and closings of decay channels as well as on the corresponding decay widths are studied. For this purpose, asymptotic expressions for the decay widths of both ICD and ETMD are derived. They allow for analytic studies of basic properties and estimations of the decay widths based on properties of the constituting atoms or molecules of the total system. A more precise description of the decay widths required the transfer of the non-relativistically known FanoADC-Stieltjes method to the relativistic regime and its implementation into the relativistic quantum chemical program package Dirac. Using this method, small noble gas systems are investigated. Experimentally, these decay processes are usually studied in noble gas clusters consisting of 100 – 2000 atoms. These clusters are too large to be treated with ab initio methods. In order to allow for a comparison of theoretical and experimental results, the influence of the cluster environment on the secondary electron spectra are investigated. These findings are used for the development of a method for the decay width estimation of clusters based on the asymptotic expressions or calculated decay widths for a multitude of geometries. This method was implemented as the program HARDRoC and is used for the investigation of the two competing processes ICD and ETMD in ArXe clusters. Additionally, it is the foundation of a new structure determination method of heteronuclear noble gas clusters, which is exemplarily explained for NeAr clusters.
A more complete understanding of the mechanisms involved in the exchange of gases between the atmosphere and the sea is needed if we are to address various environmental issues, and is essential to improved modeling of global climate. This volume contains selected papers from the Third International Symposium on Air-Water Gas Transfer, held at the University of Heidelberg, in Heidelberg, Germany from July 24-27, 1995. The papers are arranged into seven parts: Physical and Chemical Mechanisms, Waves and Turbulence, Breaking Waves and Bubbles, Measuring Technology, Laboratory Measurements and Facilities, Field Measurements, Remote Sensing, and Global Modeling. Emphasis is given to the transfer of carbon dioxide and other radiatively important gases, reflecting current interest in potential global warming. Breaking waves and the bubbles thereby generated play a prominent role in that regard. Also featured are non-invasive measurement technologies, many of which lend themselves to remote sensing applications. Those interested in chemical engineering, fluid mechanics, hydrology, hydraulics, environmental engineering, water quality engineering, climatology, meteorology, and oceanography will find this work a valuable resource.
Reed warblers and allies (family Acrocephalidae) present a long history of classification and successive revisions. Due to a remarkable similarity in morphology and rapid radiation, the previous studies failed to solve all phylogenetic relationships between and within the genera. In this study DNA sequences from eight mitochondrial and nuclear loci (total, 6280 nt) as well as genomic ISSR fingerprinting were implemented to reconstruct the phylogenetic relationships among 35 species of reed warblers. The results are congruent with previous molecular analyses (Fregin et al. 2009; Helbig & Seibold 1999; Leisler et al. 1997) but support some of the sister-group relationships more strongly. Based on the analyses, the major clades of Acrocephalus, Iduna (except I. aedon), Hippolais, Nesillas and Calamonastides were recovered. The current taxonomic position of Calamonastides gracilirostris as a monotypic genus in the tree was supported. Although the inclusion of Chloropeta natalensis and Chloropeta similis in genus Iduna was not robustly supported, their close relationship could not be rejected. No support was found for inclusion of aedon in genus Iduna as suggested by Fregin et al. (2009), hence the resurrection of genus Phragamaticola for this species is proposed. A molecular clock analysis confirmed the hypothesis of the rapid radiation of the family from middle Miocene (12.5 MYA). This short evolutionary time causes low nodal support at the base of the some clades. Furthermore, phylogeographic patterns and evolutionary history of the Eurasian reed warbler (Acrocephalus scirpaceus) and marsh warbler (Acrocephalus palustris) were investigated. Two mitochondrial loci (cyt b and COI) and a combination of phylogeographic tools, molecular dating, and population genetic methods were employed to address several questions regarding the genetic diversity of these two long-distance migratory reed warblers across their widespread geographic distribution range. Investigation of mtDNA diversity among more than 400 individuals of A. scirpaceus from 20 sampling sites recovered three lineages. They correspond to three subspecies and split about 0.4 million years ago: one spanning Asia (A. s. fuscus); one encompassing Europe and Northern Africa (A. s. scirpaceus); and a third, including Eastern Africa and South-western Asia (A. s. avicenniae). Results from BEAST further suggested that the third clade has the basal position and diverged from its sister species African reed warbler A. baeticatus ca. 0.6 million years ago. This primitive subspecies (avicenniae) may have survived in one of the African refugia, likely low forest refugium of the Ethiopian Highlands during the Last Glacial Maximum. Moreover, DNA analysis could detect high number of identification errors between reed warblers in the field. In total, 6.8% of Eurasian reed warblers were misidentified as the wrong species. A. palustris was the most misidentified species; and at the subspecies level, it increased to 72.7% of the specimens. Based on the molecular analysis we could also report a first evidence of the Asian subspecies A. s. fuscus in Central Europe (Treysa, Germany). In contrast to the results of A. scirpaceus, the phylogeographic structure in ca. 230 individuals from 10 breeding, migrating and wintering populations of A. palustris presented a shallow divergence. The analyses based on both mtDNA loci failed to detect any population subdivision and indicate occupation of a single glacial refugium during the Pleistocene glaciation. High levels of gene flow among breeding populations (Nm = 13.69) is another factor which leads to high degree of admixture. Low nucleotide diversity, shallow phylogenetic tree, star-like haplotype network, unimodal mismatch distribution and the time to the most recent common ancestor (TMRCA = 0.45 MYA) all point to their recent origin during the last glacial periods.
The synthesis of commercially relevant organic carbonates from CO2 can contribute to a sustainable utilization of this greenhouse gas. The catalytically controlled reaction with epoxides leads to the production of cyclic carbonates and aliphatic polycarbonates. In this work, we succeeded in finding innovative homogeneous catalysts for this task. The foundation was a very variable ligand system closely related to the salen compounds with an N2O2 framework, that was converted with the metals iron, zinc and aluminum to the corresponding metal complexes. The various combinations of ligand and metal yielded 66 different potential catalysts, of which some structures were investigated via x-ray structure analysis. High-pressure experiments were carried out to test the catalysts’ performance, allowing some insights in the relationship between structure and catalytic activity. The conversions were documented by means of the test substrates propylene oxide and cyclohexene oxide. Additionally a wider range of epoxides has been tested in some promising cases.
One result is, that the control of the product spectrum via selection of the optimal epoxide-catalyst-combination is possible. The most active iron(III) catalyst was able to produce quantitative yields of propylene carbonate from propylene oxide without the addition of a cocatalyst. With the zinc catalysts the same result was possible, also under mild reaction conditions (40 ◦C, 2 bar CO2, cocatalyst Bu4NI). However, the aluminum catalysts were suitable for the quantitative conversion of cyclohexene oxide to fully alternating polycarbonates.
Another important finding is, that the contributing nucleophiles in the reaction have a decisive influence on the formation of the product, regardless if they came from the catalyst itself or from a cocatalyst. While the more nucleophile and according to the HSAB-concept softer iodide anion ismore suitable for the synthesis of cyclic carbonates, the anions bromide and chloride are rather successful in the synthesis of polycarbonates.
By the analysis of the many catalytic test results and additional kinetic measurements via in situ FT-IR spectroscopy, it was possible to postulate mechanisms for the different new catalysts of this work.
The immune system protects our organism from harmful environmental insults. Pathogens induce innate and adaptive immune responses that resolve infection and clear damaged cells. A tight balance between efficient elimination of the initial insult and the containment of the immune response, assures protection of the organism as well as the preservation of organ integrity. Disregulation of this system can lead to devastating effects like tissue damage and organ failure caused by excessive inflammation. Tissue components play a pivotal role in the regulation of immune responses. In order to develop novel therapeutic strategies for the treatment of inflammatory diseases, their properties have to be incorporated. Hence, a detailed understanding of mechanisms by which tissue cells influence immune responses is indispensable. Recently, our lab identified Dickkopf-3 (Dkk3) as an immune modulator mainly expressed by tissue cells. Thus we aimed to uncover the contribution of Dkk3 to tissue mediated immune modulation during inflammation. With the help of a transgenic mouse model we investigated how antigen-presenting keratinocytes in the inflamed skin modulate T cell reactivity. In these mice, keratinocytes present a myelin basic protein (MBP) peptide only upon skin inflammation. In the absence of systemic MBP immunization acute skin inflammation resulted in keratinocyte-mediated activation of MBP-specific CD4+ T cells that were encephalitogenic. However, chronic skin inflammation limited the encephalitogenic potential of systemically primed MBP-specific T cells. In this setting Dickkopf-3 was indispensable for the limitation of CD4 T cell reactivity. We successfully generated a transgenic Dkk3 reporter mouse that reliably indicates sites of Dkk3 expression. Furthermore, in vitro and in vivo studies identified interferon-γ (IFNγ) as a potent regulator of Dkk3 expression in tissue cell. Finally, we found that Dkk3 promotes the development of renal fibrosis in 2 different mouse models, indicated by decreased severity of fibrosis in Dkk3 deficient mice. Furthermore, we observed that Dkk3 expression is induced in tubular epithelial cells in the course of fibrosis development. Less fibrosis in Dkk3 deficient mice was accompanied by elevated levels of pro-inflammatory cytokines and increased T cell infiltration in the respective kidneys. Additionally, we observed an altered polarization of infiltrating CD4 T cells towards a Th1/Treg phenotype in dkk3-/- kidneys, which came along with decreased expression of Wnt target genes in these cells. In conclusion, Dkk3 contributes to tissue mediated immune modulation by regulation of T cell responses.
Die Weinrebe (Vitis vinifera L. ssp. vinifera) gehört zu den bedeutendsten Kulturpflanzen weltweit. Ihre hohe Anfälligkeit für eine Vielzahl von Pathogenen, insbesondere dem Echten (Erysiphe necator) und Falschen Mehltau (Plasmopara viticola), ist für den Weinbau eine besondere wirtschaftliche Bedrohung und kann zu erheblichen Ernteausfällen führen. Nicht zuletzt darum werden in Europa zwei Drittel der Gesamtmenge an Fungiziden im Weinbau eingesetzt. Um den Fungizideinsatz zu reduzieren, sollen neue resistente Rebsorten (z.B. `Regent´) gezüchtet werden, wobei es sich bei der angewendeten klassischen Kreuzungszüchtung um ein langwieriges Verfahren handelt. Die Mechanismen, welche in den resistenten Rebsorten die gesteigerte Resistenz vermitteln, sind jedoch größtenteils unbekannt. Die Identifikation sowie die funktionelle Charakterisierung von Genen, welche an der Regulation von Resistenzmechanismen beteiligt sind, könnte somit zur Verbesserung und Beschleunigung der Züchtung neuer resistenter Rebsorten beitragen. Mittels Mikroarrays konnte eine Reihe von Genen identifiziert werden, darunter auch Rezeptoren und Transkriptionsfaktoren (TF), welche in resistenten Rebsorten im Vergleich zu anfälligen Rebsorten eine schnellere und stärkere Induktion zeigten, von denen aber bislang nur vereinzelte Gene funktionell charakterisiert werden konnten. Im Rahmen dieser Arbeit wurden daher die Funktion einer Rezeptorfamilie bei der Abwehr von P. viticola nachgewiesen sowie anschließend die Unterschiede der transkriptionellen Regulation der Resistenzantwort durch TFs zwischen einer anfälligen und einer resistenten Rebsorte untersucht. Hierzu wurden im ersten Teil des Projektes die Gene der VRP1 (Vitis Resistance to Plasmopara 1) Rezeptorfamilie charakterisiert. Der in silico Vergleich der drei chimären VRP1 Rezeptoren zwischen der anfälligen Rebsorte `Lemberger´ und der resistenten Rebsorte `Regent´ zeigte keine Sequenzunterschiede. Die Lokalisation der VRP1 Proteine nach Fusion mit dem Grün Fluoreszierenden Protein (GFP) in Protoplasten einer Suspensionskultur (V. vinifera cv. `Chardonnay´) mittels konfokaler Mikroskopie konnte belegen, dass sich alle drei VRP1 Konstrukte im Cytoplasma befanden. Zusätzlich konnte mittels qPCR Analyse nachgewiesen werden, dass die Rezeptoren im resistenten `Regent´ im Vergleich zum anfälligen `Lemberger´, spezifisch durch Infektion mit P. viticola induziert wurden, was auf eine Funktion bei der Abwehr von P. viticola schließen lies. Die transiente Transformation der VRP1 Gene in Weinrebenblätter mit anschließender P. viticola Infektion zeigte, dass die Expression von VRP1-3 eine Steigerung der Resistenz um bis zu 50 % bewirkte. Darüber hinaus wurden die VRP1 Gene stabil in Arabidopsis thaliana transformiert. In Folge der Überexpression von VRP1-3 konnte ebenfalls eine Verbesserung der Resistenz der transgenen Arabidopsis Pflanzen gegen Hyaloperonospora arabidopsidis um bis zu 50 % detektiert werden. Im zweiten Teil der Arbeit wurde die Induzierbarkeit des Resistenzgens VvPR10.1 (Pathogenesis Related 10.1) durch TFs und ihre Auswirkung auf die Resistenz untersucht. Mittels Promotor Induktionsanalyse konnte gezeigt werden, dass die TFs VvWRKY33, VvERF5 und VvCZF1 den Promotor von VvPR10.1 induzieren konnten, was auf eine Rolle in der Abwehr hindeutete. Es konnte zusätzlich in vivo gezeigt werden, dass eine durch P. viticola Infektion hervorgerufene Induktion von VvWRKY33 in Weinrebenblättern von Gewächshausreben wiederum zur Steigerung der Expression von VvPR10.1 führte. Ektopische Expression der TFs in Weinrebenblättern mit anschließender P. viticola Infektion zeigte, dass die Expression von VvWRKY33 und VvERF5 eine Steigerung der Resistenz um 50-70 % bewirkte. Außerdem konnte durch Komplementierung der A. thaliana knock out Mutante wrky33-1 mit VvWRKY33 nachgewiesen werden, dass die Expression im heterologen System Arabidopsis den Phänotyp des Wildtyps, in Bezug auf die Resistenz, wiederherstellen konnte. Darüber hinaus konnte sogar eine signifikante Steigerung der Resistenz gegen H. arabidopsidis und Botrytis cinerea im Vergleich zum Wildtyp Col-0 erreicht werden. Im Rahmen dieser Arbeit konnte somit zum ersten Mal nachgewiesen werden, dass die gezielte Expression des VRP1-3 Rezeptors sowie der TFs VvWRKY33 bzw. VvERF5 in der Weinrebe, eine gesteigerte Resistenz gegen P. viticola induzieren konnten. Dies stellt eine Basis für die Aufklärung der zugrunde liegenden Resistenzmechanismen, und somit für die Entwicklung neuer molekularer Marker zur Verbesserung und Beschleunigung der Züchtung neuer resistenter Rebsorten, dar.
This work is devoted to mathematical modeling of deregulation of the Wnt/β-catenin signaling pathway in medulloblastoma resulting in abnormal dynamics of target genes. Medulloblastoma is a brain tumor, mostly diagnosed in children. It is associated with several molecular genetic alterations. Specific aberrations of chromosome 6q, leading either to the chromosome copy-number loss (monosomy 6) or gain (trisomy 6), occur in two different subtypes of the tumor. The model is a nine-dimensional system of ordinary differential equations and describes nonlinear dynamics of the key ingredients of the signaling process. The model is based on the law of mass action and accounts for a two-compartment architecture of a cell consisting of the nucleus and cytoplasm. The model helps to understand molecular differences between the two medulloblastoma mutation subtypes that are associated with different patient prognosis. Our studies are based on a collaboration with the group of Prof. Dr. med. Stefan Pfister at the Division of Pediatric Neuro-oncology Research Group of the German Cancer Research Center (DKFZ). The model is used to evaluate data from the gene expression microarray data from the clinics in Heidelberg, Boston and Amsterdam. Numerical simulations lead to new biological hypotheses related to a significant role of the regulatory loop SGK1-GSK3β-MYC, a part of the Wnt/β-catenin signaling pathway. Simulations indicate the advantage of using the pharmacological inhibitor of SGK1 in patients with copy-number gain of chromosome 6q. Finally, the simulation results suggest a beneficial use of an adjuvant therapy in a trisomy 6 treatment. Mathematical analysis of the ordinary differential equations system confirms the wellposedness of the model and provides basic properties of the solutions. Supported by numerical analysis, we conclude about global stability of a unique positive equilibrium corresponding to the homeostasis of the system. We also tackle the parameter estimation problem using statistical assessment of the results and Gauss-Newton method. Sensitivity analysis provides insight into the role of model parameters. In particular, it confirms the sensitivity of the system to the parameter of SGK1 degradation. The model provides a powerful tool to study mechanistically the underlying process and to support the experiments.
Point mutations in the isocitrate dehydrogenase (IDH) genes are an early, if not the earliest event during the development of gliomas and other tumors, such as acute myeloid leukemia (AML). Among grade II and grade III gliomas, more than 80 % carry an IDH mutation. In these entities, mutations almost exclusively affect the catalytically critical arginine residue of the cytoplasmic IDH1, leading to the amino acid exchange to histidine (R132H). This and other IDH mutations result in a neomorphic enzyme function and the production of the oncometabolite 2-‐hydroxyglutarate (2-‐HG) and thus genome-‐wide hypermethylation and malignant transformation. The work at hand demonstrates the suitability of IDH1R132H as a target for immunotherapy in an MHC-‐humanized mouse model, A2.DR1. Peptides encompassing the mutated region bound MHC class II in vitro and induced a mutation-‐specific CD4+ T helper (Th) response in vivo, whose antigen-‐specificity persisted in a specific T cell line and clone and which was accompanied by IDH1R132H-‐specific antibody production. To detect IDH1R132H-‐specific IgG in mouse and human serum, a peptide-‐coated ELISA was established. Several tested patients with IDH1R132H+ gliomas showed spontaneous IDH1R132H-‐specific antibody and CD4+ Th1 cell responses. Preventive and therapeutic IDH1R132H peptide vaccination of A2.DR1 mice bearing syngeneic IDH1R132H+ sarcomas resulted in an effective mutation-‐specific antitumor immune response capable of controlling tumor growth in a CD4+ T and B cell-‐ dependent manner. Functionality of the vaccine was evidenced by loss of IDH1R132H expression in IDH1-‐transduced sarcomas and infiltration of IDH1R132H-‐specific CD4+ T cells into the tumor bulk. Compared to therapeutic MHC II-‐mediated peptide vaccination against the well-‐established cancer testis antigen 1 (CTAG1B, NY-‐ESO-‐1), IDH1R132H is a relevant neoantigen of comparable efficacy. Given the IDH1R132H-‐mediated accumulation of 2-‐HG, the effect of this metabolite on human T cells is of potential relevance during IDH1R132H-‐targeted immunotherapy. However, neither human peripheral CD4+ nor CD8+ T cell functions from healthy subjects were affected by 2-‐HG. In conclusion, IDH1R132H represents a potentially clinically meaningful tumor-‐specific neoantigen. Conceptually, patients with low-‐grade and anaplastic gliomas with a high II prevalence of the IDH1R132H mutation represent a patient population, which may particularly benefit from a tumor vaccine, as there is currently no therapy preventing recurrence in this relatively young and immunologically competent patient population. Moreover, patient groups with other IDH1R132H-‐mutated tumors might potentially also benefit from such a vaccine.
The large processing capacity of our brain is the result of properly formed synaptic contacts and their maintenance and plasticity. Synaptic cell adhesion molecules and neuronal activity are critically involved in these processes. While synaptic cell adhesion molecules govern molecular target recognition, structural integrity and plasticity of a synaptic contact, neuronal activity is one major factor underlying dynamic and adaptive responses of neurons and neuronal networks. Neuronal activity and synaptic cell adhesion molecules jointly contribute to the establishment of normal brain function. However, little is known about their interaction and mutual dependence. Here I show that Lrrtm1 and Lrrtm2, two recently identified synaptic cell adhesion molecules, are regulated by neuronal activity in cultured hippocampal neurons of the mouse. I found that their responsiveness to neuronal activity crucially depends on nuclear calcium signalling. In addition Lrrtm2 is bound and controlled by CREB, an important factor in activity-mediated gene transcription. Using inhibitors of several calcium-dependent pathways, I demonstrate that the expression of Lrrtm1 and Lrrtm2 is mediated by calcium/calmodulin-dependent kinases. Further I show that Lrrtm1 and Lrrtm2 mRNA levels increase during development, which correlates with the maturation of the neuronal network. I can further show that knock-down of Lrrtm2 does not influence spine density, contrary to what has been reported in the literature. It does, however, influence neuronal network activity, as I demonstrate in collaboration with H.E. Freitag using microelectrode array recordings. Similar changes appear under Lrrtm1 knock-down conditions. The network behavior in these cultures reverts to nearly normal by overexpression of Lrrtm1 protein. Similar to the changes observed in Lrrtm1 and Lrrtm2 knock-down cultures, overexpression of MeCP2 causes a desynchronization of the neuronal bursting activity. MeCP2 is a transcriptional regulator which is found mutated in Rett syndrome, a rare but severe neurodevelopmental disorder in humans. I could show that the expression of endogenous Lrrtm2 is deregulated in cultures overexpressing MeCP2. This suggests that the network changes observed in MeCP2 overexpressing cultures are caused by deregulation of Lrrtm2 by MeCP2. However, overexpression of Lrrtm2 protein failed to rescue the MeCP2-phenotype. A further aim of my studies was to analyze the function of the activity-responsiveness of Lrrtm1 and Lrrtm2. Using different methods I attempted to visualize AMPA receptor trafficking to the neuronal surface and the impact of Lrrtm knock-down thereon. However, the applied methods were too insensitive to detect changes in synaptic AMPA receptor surface expression. Together, these findings connect Lrrtm1 and Lrrtm2, respectively, two members of the group of synaptic cell adhesion molecules, to synaptic activity, nuclear calcium signalling and CBP/CREB, all of which are important mediators of sustained adaptive changes in the central nervous system. The findings also give the impetus to further explore the role of the Lrrtm1 and Lrrtm2 activity-responsiveness in neurons in vitro and in vivo.
Since the development of advanced mathematical modelling techniques in biology, thermodynamics (and therefore equilibrium statistical mechanics) has played a key role in mathematically quantifying biological activities. We use this underlying notion of thermodynamic ‘micro-states’ to attempt to uncover how the hormone hepcidin under the influence of two major signalling pathways maintains systemic iron homeostasis. Systemic iron homeostasis involves a negative feedback circuit in which the expression level of the peptide hormone hepcidin depends on and controls the iron blood levels. Hepcidin expression is regulated by the BMP6/SMAD and IL6/STAT signalling cascades. Deregulation of either pathway causes iron storage diseases such as hemochromatosis or anaemia of inflammation (AI). We quantitatively analyzed how BMP and IL6 control hepcidin expression in human hepatoma (HuH7) cells. We used data from our experimental collaborators who measured transcription factor phosphorylation and reporter gene expression under co-stimulation conditions and perturbed the promoter by mutagenesis. We applied statistical data analysis and mathematical modelling to reveal possible biological mechanisms that control hepcidin expression at the promoter level. Specifically we develop a thermodynamic modelling framework that is able to simulate and predict possible molecular mechanisms that might underlie iron homeostasis by hepcidin. Our results reveal that hepcidin cross- regulation primarily occurs by combinatorial transcription factor binding to the promoter, whereas signalling crosstalk is insignificant. We find that the presence of two BMP-responsive elements in the promoter ensures high sensitivity towards the iron-sensing BMP signalling axis, which promotes iron homeostasis in vivo. IL6 stimulation reduces the promoter sensitivity to the BMP signal that may explain the disturbance of iron homeostasis in AI. We get to understand why the iron homeostasis circuit is sensitive to certain perturbations implicated in disease. Taken together, our work reveals how mathematical quantification and modelling can aid in understanding biological phenomenon that underlies gene expression.
Um Verbrennungsmotoren emissionsreduziert zu betreiben, eird eine umfangreiche Abgassensorik benötigt, mit der neben Sauerstoff und Kohlenwasserstoffen auch Stickoxide erfasst werden können. Ein vergleichsweise neuer Ansatz ist hierbei der Einsatz von chemisch funktionalisierten Feldeffekttransitoren (kurz ChemFETs) im heißen Abgas. Bei diesen sind für die Messung von wasserstoffhaltigen Gasen die grundlegenden Mechanismen hinlänglich verstanden. Mittlerweile ist es gelungen, auch NOx-sensitive Feldeffekttransitoren herzustellen, allerdings existiert bislang noch kein belastbares Modell dafür, wodurch hierbei das elektrische ChemFET-Signal hervorgebracht wird. Im Zuge der vorliegenden Arbeit wurden mögliche signalgebende Mechanismen für die Verwendung eines ChemFETs als Stickoxidsensor untersucht. Die Ergebnisse dienen der Entwicklung eines umfassenden Modells für den Signalbildungsmechanismus. So sollte ein tieferes Verständnis für die auf der nanoporösen, katalytischen Gateelektrode ablaufenden Oberflächenprozesse erlangt und die für die Signalgebung besonders ausschlaggebenden Bereiche identifiziert werden. Zu diesem Zweck wurden neben mit einer Finite-Elemente-Methode durchgeführten Simulationen umfangreiche spektroskopische in situ-Oberflächenanalysen durchgeführt. Als Hauptmethode diente die polarisationsmodulierte Infrarot-Reflektions-Absorptions-Spektroskopie (PM-IRRAS). Hierfür wurden ein spezieller Messaufbau konzipiert und geeignete Proben hergestellt, die mit zusätzlichen Methoden wie Rasterelektronenmikroskopie(REM) und Röntgen-Photoelektronen-Spektroskopie (XPS) analysiert wurden. Mittels PM-IRRAS ließen sich einzelne Adsorbatzustände von CO und NO auf verschiedenen Platin- und Rhodium-haltigen Oberflächen identifizieren und deren zeitliche Entwicklung beobachten. Die Analyse der Adsorbatzustände und der gasförmigen Reaktionsprodukte zwischen Raumtemperatur und 350°C lieferten Einblicke in die von der Gasumgebung abhängig wechselnden Oberflächenregime. Zudem wurde der nichtresonante Schwingungsuntergrund ausgewertet. Es wurde dabei gezeigt, dass dieser Rückschlüsse auf den Oxidationsgrad einer Probenoberfläche ermöglicht. Diese hier phänomenologisch als PM-IRRAS-Offset bezeichnete Messgröße zeigte eine starke Korrelation mit der Gatespannung von zeitgleich in derselben Messapparatur gemessenen, identisch beschichteten ChemFETs. Aus den gewonnenen Erkenntnissen wurde das Schema eines Signalbildungsmodells abgeleitet, das neben atomarem Wasserstoff auch atomaren Sauerstoff als signalgebende Spezies beinhaltet. Die wesentliche Signalbildung findet durch Anlagerung dieser Spezies an bzw. in der Oberfläche des Gatedielektrikums und besonders im Bereich der Dreiphasengrenzen zwischen Dielektrikum, Katalysator und Gasphase statt. Stickoxide wirken hierbei im Wesentlichen auf dem indirekten Weg der Bereitstellung bzw. des Verbrauchs von atomarem Sauerstoff bzw. Wasserstoff. Somit konnte gezeigt werden, dass die NOx-Sensitivität der betrachteten ChemFETs ein mehrstufiger Prozess mit atomarem Sauerstoff als Signalgeber ist.
Twenty years after it was proposed, STED nanoscopy has evolved into a powerful tool in far-field fluorescence imaging which achieves resolutions below 20nm. Nevertheless, the application of STED nanoscopy in life sciences on a large scale is hindered by the high effort which the method requires. This effort is in great part due to the STED laser. So far, lasers which can be utilized for STED, e.g. titanium-sapphire lasers, show both, technical and practical shortcomings, resulting in complex nanoscopy systems. Semiconductor lasers which are compact, reliable and controllable without effort, are predestinated to improve this situation if they are adapted to meet the pulse requirements for STED. Therefore, this thesis examines the potential of different types of semiconductor lasers for STED. Fabry-Pérot laser diodes in overdriven gain switched operation are demonstrated to be suitable STED lasers. Even more suitable STED pulses, with pulse energies of up to 5nJ in 1ns, are obtained from a second semiconductor laser system. Based on a tapered amplifier, this system is constructed to be operated flexibly in various modes of ns pulse generation. With this semiconductor STED laser subdiffraction imaging of fluorescent nuclear track detectors is successfully performed for the first time. Thereby the size of tracks of carbon ions and protons in this material can be determined. Furthermore, a method for all-semiconductor picosecond-pulse generation at wavelengths of 532nm, 561nm and 593nm is presented. This was so far not possible and provides a convenient source of excitation light for red emitting organic dyes, not only for STED nanoscopy. With its findings this thesis proves that STED imaging with a fivefold resolution enhancement is possible with user-friendly and affordable lasers. This paves the way to a wide spread use of STED nanoscopy.
Es ist eher ungewöhnlich, dass ein Wissenschaftler mit über 60 Jahren die Universität wechselt. Alles andere als gewöhnlich ist auch die Karriere des Österreichers Prof. Dr. Ernst Pernicka der - bisher Dozent in Tübingen - ab dem Wintersemester Geowissenschaften mit Schwerpunkt Metallurgie in Heidelberg unterrichtet. Der Spezialist für Archäometallurgie und Archäometrie bewies die Echtheit der Himmelsscheibe von Nebra und leitete sieben Jahre lang die Grabungen in Troja. Seit 2004 ist er auch Chef des Curt-Engelhorn-Zentrums für Archäometrie in Mannheim. Jetzt übernimmt er eine eigens eingerichtet Professur an der Universität Heidelberg.
Polyakov-loop–extended constituent-quark models are useful to investigate the chiral and (de)confinement phase structure and the thermodynamics of strongly-interacting matter. It is shown that taking into account the quark backreaction on the gauge-field dynamics as well as quantum and thermal fluctuations of quarks and mesons is crucial in such models to achieve results for order parameters and thermodynamics that are in line with non-perturbative calculations at vanishing chemical potential. The dependence of the results on remaining parameters is discussed. The investigations are extended to nonzero quark density and isospin. The impact of unquenching effects in the Polyakov-loop potential on the phase structure at non-vanishing quark densities is discussed. Predictions for thermodynamics at nonzero isospin are shown. Furthermore, the reliability of those models is tested by confronting its results with lattice data on the isospin dependence of the transition temperature. The phase structure of the three-dimensional temperature - isospin - quark density phase diagram is investigated. Moreover, the process of nucleation at small temperatures and large densities is investigated and the surface tension for the phase transition calculated. Some consequences of the results for the early Universe, for heavy-ion collisions, and for proto-neutron stars are discussed.
Als Myokardhypertrophie versteht man eine Zunahme der Wanddicke des Herzmuskels sowie eine Zunahme der Herzmuskelmasse im Rahmen einer Druck- und Volumenbelastung des Herzens oder im Rahmen einer regelmäßigen Trainingsbelastung. Man unterscheidet dabei grundsätzlich in physiologische und pathologische Myokardhypertrophie. Die physiologische Hypertrophie weist eine gleichbleibende oder verbesserte kardiale Funktion auf, wohingegen die pathologische Myokardhypertrophie zu einer Beeinträchtigung der Herzfunktion führt. Im Gegensatz zur physiologischen Myokardhypertrophie wird die pathologische Hypertrophie mit einer verringerten Kapillardichte im linken Ventrikel assoziiert, die zur Beeinträchtigung der Herzfunktion führt. Die genauen Mechanismen hierfür sind jedoch noch unklar. Daher sollte der Stellenwert der Angiogenese bei der physiologischen und pathologischen Myokardhypertrophie eingehend betrachtet werden. Zum einen wurde untersucht, ob die Inhibierung der Angiogenese eine physiologische in eine pathologische Hypertrophie überführt und zum andern sollte der Einfluss von CalcineurinA, einem wichtigen Mediator der pathologischen Myokardhypertrophie, auf die Regulation der Angiogenese untersucht werden. Mit dieser Arbeit konnte gezeigt werden, dass die Inhibierung der Angiogenese in der physiologischen Hypertrophie weder die kardiale Funktion beeinträchtigt, noch die Reexpression des fetalen Genprogramms, typisches Kennzeichen der pathologischen Hypertrophie, induziert. Somit war es durch Inhibierung der Angiogenese nicht möglich, die physiologische Hypertrophie in eine patholoigsche zu überführen. Desweitern konnte gezeigt werden, dass CalcineurinA den Transkriptionsfaktor HIF-1α über einen HSP90 abhängigen Mechanismus stabilisiert und die Expression proangiogener Faktoren, wie z.B. VEGF-A, induziert. Dadurch konnte, trotz einer ausgeprägten pathologischen Hypertrophie, eine Verminderung der Kapillardichte verhindert werden. Es konnte also gezeigt werden, dass CalcineurinA einen partiell protektiven Effekt auf die pathologische Myokardhypertrophie ausübt.
Recent advances in electron microscopy techniques make it possible to acquire highresolution, isotropic volume images of neural circuitry. In connectomics, neuroscientists seek to obtain the circuit diagram involving all neurons and synapses in such a volume image. Mapping neuron connectivity requires tracing each and every neural process through terabytes of image data. Due to the size and complexity of these volume images, fully automated analysis methods are desperately needed. In this thesis, I consider automated, machine learning-based neurite segmentation approaches based on a simultaneous merge decision of adjacent supervoxels.
- Given a learned likelihood of merging adjacent supervoxels, Chapter 4 adapts a probabilistic graphical model which ensures that merge decisions are consistent and the surfaces of final segments are closed. This model can be posed as a multicut optimization problem and is solved with the cutting-plane method. In order to scale to large datasets, a fast search for (and good choice of) violated cycle constraints is crucial. Quantitative experiments show that the proposed closed-surface regularization significantly improves segmentation performance.
- In Chapter 5, I investigate whether the edge weights of the previous model can be chosen to minimize the loss with respect to non-local segmentation quality measures (e.g. Rand Index). Suitable w are obtained from a structured learning approach. In the Structured Support Vector Machine formulation, a novel fast enumeration scheme is used to find the most violated constraint. Quantitative experiments show that structured learning can improve upon unstructured methods. Furthermore, I introduce a new approximate, hierarchical and blockwise optimization approach for large-scale multicut segmentation. Using this method, high-quality approximate solutions for large problem instances are found quickly.
- Chapter 6 introduces another novel approximate scheme for multicut segmentation -- Cut, Glue&Cut -- which is based on the move-making paradigm. First, the graph is recursively partitioned into small regions (cut phase). Then, for any two adjacent regions, alternative cuts of these two regions define possible moves (glue&cut phase). The proposed algorithm finds segmentations that are { as measured by a loss function { as close to the ground-truth as the global optimum found by exact solvers, while being significantly faster than existing methods.
- In order to jointly label resulting segments as well as to label the boundaries between segments, Chapter 7 proposes the Asymmetric Multi-way Cut model, a variant of Multi-way Cut. In this new model, within-class cuts are allowed for some labels, while being forbidden for other labels. Qualitative experiments show when such a formulation can be beneficial. In particular, an application to joint neurite and cell organelle labeling in EM volume images is discussed.
- Custom software tools that can cope with the large data volumes common in the field of connectomics are a prerequisite for the implementation and evaluation of novel segmentation techniques. Chapter 3 presents version 1.0 of ilastik, a joint effort of multiple researchers. I have co-written its volume viewing component, volumina. ilastik provides an interactive pixel classification work ow on largerthan-RAM datasets as well as a semi-automated segmentation module useful for acquiring gold standard segmentations. Furthermore, I describe new software for dealing with hierarchies of cell complexes as well as for blockwise image processing operations on large datasets.
The different segmentation methods presented in this thesis provide a promising direction towards reaching the required reliability as well as the required data throughput necessary for connectomics applications.
In dieser Arbeit wird ein iteratives Rekonstruktionsverfahren für die 23Na-MRT entwickelt. Neben einer totalen Variation zweiter Ordnung (TV2) wurden A-priori-Informationen aus der 1H-MRT in Form einer Trägerregion (BM) und als anatomisch gewichtete TV2 (AnaWeTV) eingebunden. Anhand simulierter Kopfdaten und In-vivo-Messungen wurde das Leistungsvermögen des Algorithmus bei verschiedenen Auflösungen, Unterabtastungen (UAF) und Rauschpegeln analysiert. In allen Bildern wurden Gibbs-Oszillationen und Unterabtastungsartefakte wirkungsvoll unterdrückt. Bei In-vivo-Messungen an acht Probanden und zwei Patienten (Hirntumor und Multiple Sklerose) konnte ein deutlich erhöhtes Signal-Rausch-Verhältnis (SNR) im Vergleich zur herkömmlichen Gridding-Methode erreicht werden (8 Probanden: +(46+-3)% bei TV2, +(25+-2)% bei BM&TV2 in weißer Substanz (WM); Multiple-Sklerose-Patient mit AnaWeTV: +133% in WM, +55% im lateralen Ventrikel). Der SNR-Gewinn hängt dabei von der Größe der anatomischen Struktur ab. Die AnaWeTV erhöht die Auflösung bekannter Strukturen und verringert Partialvolumeneffekte. In Simulationen (2mm isotrope Auflösung, UAF = 10) konnte der Intensitätsfehler in vier kleinen Läsionen von (20,3+-3,2)% (Gridding) auf (3,2+-2,3)% (AnaWeTV) reduziert werden. Nach Anwendung eines Hammingfilters betrug er (12,6+-3,3)%. Der Algorithmus ist robust gegenüber Fehlregistrierung der 1H-Bilder um (1,5-3)mm. Auch Strukturen, für die keine anatomischen A-priori-Informationen vorliegen, werden mit hohem Kontrast dargestellt. Die BM&TV2-Rekonstruktion ist vorzuziehen, wenn nicht bekannt ist, ob Strukturen im 23Na-Bild ein anatomisches Korrelat im 1H-Bild besitzen (z.B. bei Hirntumoren).
Im ersten Artikel (Siegmund Günther) würdigt der Autor die vielfältigen Leistungen, die Siegmund Günther als Abgeordneter, Geograph und Mathematiker seit 1908 erbracht hatte. Für die vorangehende Zeit verweist er auf seinen Aufsatz zum 60. Geburtstag Günthers. Vgl. HeiDOK: http://www.ub.uni-heidelberg.de/archiv/16785)
Der zweite Artikel (Das neue Geographische Institut) schildert den neuen Bau des Geographischen Instituts der Technischen Hochschule in München.
Processes initiated by photoexcitation play an important role in many biological systems as well as in technical applications. A whole variety of quantum chemical methods for the treatment of such excited states has been developed over the past years. However, many are either restricted to small or medium-sized systems or only applicable to certain types of electronic excitations. Therefore, the development of efficient quantum chemical excited states methods is one of the central aspects of modern theoretical chemistry.
In this work, different excited state approaches within the algebraic diagrammatic construction (ADC) family of methods were derived and implemented. First, the scaled-opposite spin approximation was used to develop a variant of the extended ADC(2) methods that allows for an improved treatment of doubly excited states at reduced computational cost. Additionally, the generation of spin-orbit coupling elements based on an atomic mean-field approach was implemented for the whole hierarchy of ADC methods up to third order. Test calculations and comparison with existing methods revealed very good results. Last but not least, a scaling approach for the identification of plasmons in molecules previously introduced for TDDFT has been adopted to the ADC methods. Such plasmons are of great importance in the field of organic electronics. Here, the scaling approach was shown to work efficiently for a series of linear polyenes. All three theoretical methods were implemented in a development version of the adcman module of the Q-Chem program package. Thereby, the functionality of this module has been further extended making it applicable to a wider range of molecular systems and photochemical problems. Finally, ADC methods were used in combination with experimental results to successfully unravel the photochemical relaxation network of coumarin derivatives which turned out to incorporate two parallel radiationless relaxation pathways.
The transcriptional activity of genes as well as the conformation of transcriptionally silenced genomic regions is defined by the epigenetic state of the associated chromatin fragment. Chromatin is composed of repetitive units called nucleosomes. Each nucleosome consists of a core complex of four histones and the fragment of DNA that is wrapped around it. The distinct residues of N-terminal tails of the histones that extend out from the nucleosomal core are subject to post-translational epigenetic modifications. The composition of these modifications defines the electric charge of the histone tails and, therefore, its coupling strength with DNA. Thus, the epigenetic mark composition of each individual nucleosome governs the overall conformation of the chromatin filament. In mammals, the tight conformation of chromatin in pericentric genomic regions, called pericentric heterochromatin (PCH), is important for the stability and the proper segregation of chromosomes. The epigenetic hallmark signature of PCH is di- or trimethylation of histone H3 at lysine 9 (H3K9me2/3) enriched over the entire centromere region. This epigenetic state is constitutively maintened through cell cycle progression and throughout multiple cell generations thereby preventing chromosomal breakages, missegregation and perturbed chromosomal interactions. This work investigates the specificity, propagation and the long-term autonomous memory effect for H3K9me2/3 silencing marker in PCH in mouse fibroblasts on the single cell level. We apply fluorescent microscopy techniques, high-throughput image-processing method and mathematical modeling to test the stability of the system upon changes in the expression of the chromatin-modifying enzymes. The network operating H3K9me2/3 in PCH was constructed and translated into a deterministic system of ordinary differential equations as well as formulated stochastically using the Gillespie simulation algorithm. The model incorporates the contribution of H3K9me2/3 binding protein HP1 together with H3K9 specific methyltransferase Suv39h, the H3K9 specific demethylase JMJD2 and cell cycle dependent kinase Aurora B as well as nucleosome collision processes via DNA looping. The realization that most of these chromatin-modifying processes depend on each other and also appear to be regulated by multiple positive and negative feedback loops has lead to the proposal of nonlinear stationary and dynamical features of PCH network function. The modeling simulations have revealed an increased variability in methylation degree upon increases in JMJD2 expression in the cell. This prediction was qualitatively supported by the heterogeneity observed experimentally on the single PCH foci level. However, in the experiment the response of the whole cell population remains monostable, in spite of the presence of a bistable memory element in the network. The model explains this property confirming the significant impact of the persistent silencing origins organized by the high residence time binding of HP1-Suv39h complexes that initiate the spread of the H3K9me2/3 mark. Therefore, on the population level the bistable mechanism of silencing propagation in PCH is hidden, appearing only as severe fluctuations in the H3K9me2/3 level. In summary, a consistent model of the silencing propagation in PCH was developed. Based on that model a scenario of PCH epigenetic state maintenance and robustness towards transient perturbation and intrinsic noise is established.
Out of all malignancies, breast cancer is the second most common cancer worldwide and the leading cause of cancer related death in females. In recent years it has been shown that the anti-tumor vaccination might be a feasible approach for the treatment of certain cancer types, including breast cancer. It is of great interest to develop immunotherapies which not only prevent further dissemination by the tumor cells, but also eliminate tumor tissue and impair the function of immune-suppressive cells, such as regulatory T cells (Tregs), in the tumor microenvironment. Not only antigen-specific cytotoxic CD8+ T cells (CTLs) but also CD4+ T cells show a great capacity to facilitate a specific anti-tumor immune response. Furthermore, successful immunological eradication of tumors depends on the presence of activated tumor antigen-specific CD4+ effector T cells as documented by numerous reports. The differentiation antigen NY-BR-1 has been described to be expressed in 60% of all invasive mammary carcinomas. Since NY-BR-1 protein levels are highly elevated in malignant breast tissues compared to healthy breast tissues, NY-BR-1 might represent a suitable target antigen for T cell based immunotherapy approaches against breast cancer. The aim of this project was to identify novel MHC-I- and MHC-II-restricted T cell epitopes derived from the breast cancer associated antigen NY-BR-1. A NY-BR-1-specific peptide library was utilized to screen for the presence of MHC-I and MHC-II- restricted T cells in HLA-DRB1*0301- transgenic mice (DR3tg mice) and HLA-DRB1*0401-transgenic mice (DR4tg mice), after global NY-BR-1-DNA vaccination. Splenocytes of immunized mice were screened ex vivo for a NY-BR- 1-specific T cell response against a synthetic peptide library covering the entire NY-BR-1 protein. So far, novel NY-BR-1-specific, HLA-A2-restricted CD8+ T cell epitopes could not be identified. However, the first NY-BR-1-derived, HLA-DRB1*0301-restricted peptides (BR1-1347, BR1-88, BR1-1238) and the first NY-BR-1 derived, HLA-DRB1*0401-restricted peptides (BR1-537, BR1- 1242, BR1-656/-775) were identified in DR3tg mice and DR4tg mice, respectively. Stable murine CD4+ T cell lines specific for five new epitopes could be established from peptide-immunized DR3tg mice / DR4tg mice, and HLA-DR-restriction of the cell lines was confirmed on peptide loaded T2/DR3 and T2/DR4 target cells in vitro. Furhtermore, endogenous processing of HLA-DRB1*0301-restricted NY-BR-1-derived epitopes BR1-88, BR1-1347 and of the HLA-DRB1*0401-restricted NY-BR-1-derived epitopes BR1-537, BR1-1242 could be confirmed by specific recognition of human dendritic cells loaded with cell lysates of melanoma cell line Ma-Mel73a infected with Ad5-NY-BR-1. CD4+ T cells specific for the NY-BR-1 derived, HLADRB1* 0301-restricted peptides BR1-88, BR1-1347, BR1-1238 and for the HLA-DRB1*0401- restricted peptides BR1-537, BR1-1242, BR1-656/-775 were detected among PBMCs of breast cancer patients stimulated with the respective peptide in vitro for 24 days. Furthermore, CD4+ T cells with the same specificities were also detected among PBMCs of HLA-matched healthy donors, however, frequencies of antigen-specific CD4+ T cells were higher in the peripheral blood of breast cancer patients compared to healthy donors. The findings of this thesis, such as the identified NY-BR-1-specific, HLA-DRB1*0301-/*0401- restricted CD4+ T cell epitopes, might be used to generate NY-BR-1-specific tetramers which could be applied to monitor immune-responses in breast cancer patients with a tumor expressing the NY-BR-1 antigen. Moreover, the new NY-BR-1-specific, CD4+ T cell epitopes could be applied to expand NY-BR-1-specific autologous CD4+ T cells for an adoptive T cell transfer. Cloning of high affinity TCRs, specific for the newly identified epitopes, to generate TCR-transduced CD4+ T cells for adoptive T cell transfer might be another application for the findings obtained in this work. NY-BR-1-specific therapeutic vaccines could be designed by combination of CTL and CD4+ T cell epitopes to induce NY-BR-1-specific CD8+ T cells as well as NY-BR-1-specific CD4+ T cells. Infect, CD4+ T cells are not only important to sustain a functional CD8+ T cell response, but might also target MHC-II expressing tumor associated macrophages (TAMs) presenting NYBR- 1-specific epitopes on MHC-II, thereby contributing to anti-tumor immunity.
Wnt/β-catenin signaling plays a critical role in animal development and adult tissue homeostasis, including regulation of cell fate decisions, axial pattering, organogenesis and stem cell maintenance. Deregulation of Wnt signaling causes many human diseases, including cancer and osteoporosis. Wnt proteins constitute a large family of growth factors characterized by conserved cysteine and a lipid modification at their N-terminal half. Upon transport through a specialized secretary route they act as morphogens in variety of tissues by forming concentration gradient in the extracellular space. The post-translational addition of palmitate or palmitoic acid to Wnts renders them highly hydrophobic and is believed to control their membrane localization, it is unknown, however, how Wnts are recruited from the membrane to signaling complexes and how their signaling range is regulated. Wnts bind to Frizzled (Fz) seven transmembrane receptors and LRP5/6 co-receptors, which leads to stabilization of the transcription coactivation of β-catenin and activation of target gene. The structural basis of Wnt signaling by receptor binding has long been elusive. Only recently, the crystal structure of the Xenopus Wnt8- Frizzled8-CRD complex was solved, but the significance of the interaction sites for signaling has not been assessed. In the present thesis, by using a structure-based mutagenesis approach, I present an extensive structure-function analysis of mouse Wnt3a by in vitro and in vivo evaluation. Evidence is provided for an essential role of Serine 209, Glycine 210 (site 1) and Tryptophan 333 (site 2) in Fz binding. Importantly, I discovered that Valine 337 in the site 2 binding loop is critical for signaling without contributing to binding. Mutations in the presumptive second CRDbinding site (site 3) partly abolished Wnt binding, suggesting Fz dimerization as a necessary step in signaling. Intriguingly, most site 3 mutations increased Wnt signaling, probably by inhibiting Wnt-CRD oligomerization. In accordance, it was observed that increasing amounts of soluble Fz8-CRD protein modulated Wnt3a signaling in a biphasic manner. Based on these finding, a model was developed, in which a concentration-dependent switch in Wnt-CRD complex formation from an inactive aggregation state to an activated, high mobility state, represents a modulatory mechanism in Wnt signaling gradients.
Imprinted genes in placental mammals play a critical role in regulating prenatal growth and setting the metabolic rate of development. Highly expressed in embryos and early postnatal life, imprinted genes are down regulated when the organism reaches adulthood, remaining expressed in individual cells where they control activation and plasticity. Maternally imprinted gene Pw1, also known as Peg3, recently was described as a potential adult stem cell marker in various tissues such as bone marrow, central nervous system and intestines. In skeletal muscle it marks myogenic stem cells, which are located in interstitia and can efficiently contribute to muscle regeneration after focal freeze-crush injury. Using a transgenic Pw1:nLacZ reporter mouse, I described a subpopulation of proepicardial/epicardial derived mesenchymal cells involved in maintaining homeostasis of adult mouse heart through secretion of various growth factors. Immunohistochemistry and cytometric analysis revealed that myocardial Pw1 cells express the cardiac stem cell membrane receptors Sca1, Pdgfra, Cd34 and Cd29, making them a potentially interesting component of regenerative medicine for further investigation. Based on mRNA expression profiles and patterns during embryonic development I surmise an epicardial origin of Pw1 cells and their involvement in cardiac growth, as adult Pw1 cells are highly pro-angiogenic in vitro and can induce tube formation when they are co-cultured with an endothelial cell line. To investigate the role of Pw1 cells during postnatal cardiac growth I have linked their action to heart hypertrophy during pregnancy. Using pharmacological tools I locally depleted Pw1 cells which resulted in local cardiomyocyte atrophy and fibrosis. To explore the remodelling potential of Pw1 cells in pathological conditions we are planning to investigate their involvement in disease models of myocardial infarction.
Despite remarkable progress in understanding biology and disease at the level of nucleic acids, insights into the relevant biochemical processes frequently remain preliminary, since much regulation and activity occurs at the protein level through control of gene expression and variations of protein conformation. In particular, the effect of such variations on protein interactions is critical for a better description of biology and disease. Protein microarray technology provides a means to such ends and is a growing field of proteomics, with a high potential for analytical and functional applications in biology and medicine. On the basis of sequence information from individuals, it is possible to characterize disease-specific protein isoforms that result from mutations, polymorphisms, and splice variants with personalized protein microarrays. During my thesis, I developed such a technique. As a first step, solid-phase PCR is applied to copy a particular tissue’s RNA/cDNA onto the microarray surface, using for each gene a specific primer pair that is attached to the chip surface. The generated DNA templates are firmly attached to and specifically oriented on the array surface. The solid-phase PCR successfully amplified DNA of up to 3 kb, also allowing multiplex amplification of DNA. The arrayed DNA copies then act as templates for an in situ cell-free expression, yielding a protein microarray that presents the protein content of a particular tissue of an individual person. Expression control was conducted by a multiple spotting technique (MIST). C-terminus detection showed that translation was complete, yielding full-length proteins. During the process of setting up the technique of producing individualized protein microarrays, the MIST technology was optimized concomitantly. The various steps involved were analyzed to determine optimal conditions for template preparation, protein expression and interaction detection. Protein microarrays of 3500 human proteins were produced with these procedures and their performance was tested in model studies of protein–protein interactions.
The climate of the last 150 kyr is mainly driven by orbital forcing leading to long-lasting glacial conditions framed by the short warm periods Eemian and Holocene. To understand this climate variability it is crucial to investigate the response of the Atlantic Meridional Overturning Circulation (AMOC) on the insolation and the consequent effect it has on the climate by redistributing heat and CO2. However, the mechanisms that determine changes of the AMOC are poorly understood within the investigated time period. In this study the strength and direction of the AMOC is examined by temporal high-resolution measurements of the two proxies 231Pa/230Th and eNd from the marine sediment core ODP 1063 which is situated in the North West Atlantic. Despite the short half life of 231Pa, significant 231Pa/230Th ratios were obtained until 134 kyr which extends the so far released scope by 9 kyr. During Dansgaard-Oeschger warm events variations in the northern deep water formation and circulation dynamics are found. Further, rarely detected Heinrich cold events from the early glacial (H7, H9 and H10) are identified. It is demonstrated that the Termination II lacks a Younger Dryas-like cold event due to differences in the summer insolation. Instead to the widely perceped view of a generally shallow and weak AMOC during full glacial conditions, a reduced circulation strength associated with northern sourced water in the deep Atlantic during MIS 4 and the early MIS 3 are found. Due to the significant difference of this pattern to the so far described circulation modes, the new `intermediate mode' is defined.
This work is about the analysis of 4D light fields. In the context of this work a light field is a series of 2D digital images of a scene captured on a planar regular grid of camera positions. It is essential that the scene is captured over several camera positions having constant distances to each other. This results in a sampling of light rays emitted by a single scene point as a function of the camera position. In contrast to traditional images – measuring the light intensity in the spatial domain – this approach additionally captures directional information leading to the four dimensionality mentioned above. For image processing, light fields are a relatively new research area. In computer graphics, they were used to avoid the work-intensive modeling of 3D geometry by instead using view interpolation to achieve interactive 3D experiences without explicit geometry. The intention of this work is vice versa, namely using light fields to reconstruct geometry of a captured scene. The reason is that light fields provide much richer information content compared to existing approaches of 3D reconstruction. Due to the regular and dense sampling of the scene, aside from geometry, material properties are also imaged. Surfaces whose visual appearance change when changing the line of sight causes problems for known approaches of passive 3D reconstruction. Light fields instead sample this change in appearance and thus make analysis possible. This thesis covers different contributions. We propose a new approach to convert raw data from a light field camera (plenoptic camera 2.0) to a 4D representation without a pre-computation of pixel-wise depth. This special representation – also called the Lumigraph – enables an access to epipolar planes which are sub-spaces of the 4D data structure. An approach is proposed analyzing these epipolar plane images to achieve a robust depth estimation on Lambertian surfaces. Based on this, an extension is presented also handling reflective and transparent surfaces. As examples for the usefulness of this inherently available depth information we show improvements to well known techniques like super-resolution and object segmentation when extending them to light fields. Additionally a benchmark database was established over time during the research for this thesis. We will test the proposed approaches using this database and hope that it helps to drive future research in this field.
Zusammenfassung Ziel der vorliegenden Arbeit war die Untersuchung der differenziellen Wirkung von Kohlen-stoffionen (12C) gegenüber einer Photonenbestrahlung in vivo anhand von drei verschie-denen Sublinien des syngenen Prostata- Adenokarzinoms (PCa) R3327 (AT1, HI und H) der Ratte sowie die Identifizierung Tumor- assoziierter Einflussfaktoren auf die Strahlenreaktion. Subkutane PCa- Tumore in Ratten wurden mit Photonen bzw. 12C- Ionen (75 keV/ µm) unter Verwendung aufsteigender Einzel- (1 Fx) bzw. Zweifachdosen (2 Fx) bestrahlt. Primärer Endpunkt war die lokale Tumorkontrolle nach 300 Tagen. Basierend auf Dosis- Wirkungs-kurven (DWKs) wurde mittels TCD50 (Dosis für 50% Tumorkontrollwahrschein¬lichkeit) die relative biologische Wirksamkeit (RBW) von 12C- Ionen berechnet. Zusätzlich wurden Untersuchungen durchgeführt, um mögliche Kohlenstoffionen- spezifische Mechanismen der Strahlenreaktion zu identifizieren. Hierzu wurden in vivo tumor-physiologische Verände-rungen, strukturelle und funktionelle Bestrahlungseffekte auf histologischer Ebene, strahlen-bedingte Einflüsse auf die Genexpression sowie Tumorsubpopulationen mit Tumor- indu-zierenden Eigenschaften untersucht. Für 12C- Bestrahlungen mit 1 Fx bzw. 2 Fx ergab sich eine RBW von 1,62 (H), 2,08 (HI) und 2,3 (AT1) bzw. 1,9 (H), 2,1 (HI) und 2,67 (AT1). Bestrahlungen mit isoeffektiven Dosen zeigten für Photonen und 12C- Ionen unterschiedliche Kinetiken der strahleninduzierten Effekte. Mittels Magnetresonanztomographie (MRT) und Histologie wurde im Vergleich zu Photonen eine schnellere und stärkere Gefäßpermeabilisierung durch 12C- RT festgestellt. Nach Bestrahlung wurden DNA- Reparaturmechanismen, Immunsystemkomponenten und Migration induziert, dagegen die Zellzyklusprogression, Adhäsion, Angiogenese und der aerobe Stoffwechsel reprimiert. Insbesondere die zeitliche Induktion von Signalkaskaden, Apoptose und Stressantworten zeigte sich abhängig von der Strahlmodalität. In den HI- und H-Sublinien wurden CD24+/CD45- Zellen als Tumor- induzierende Zellen (TICs) in vivo identifiziert und alle drei Sublinien in vitro durch Zellkulturetablierung weiteren molekular-biologischen Analysen zugänglich gemacht. Schlussfolgernd ergibt sich, dass 12C- RT besonders für undifferenzierte Tumore geeignet ist, da die RBW für den radioresistenten, anaplastischen AT1-Tumor am höchsten war und sich mit zunehmender Differenzierung verringerte. Ursache hierfür ist, dass die TCD50- Werte für 12C- Ionen weniger abhängig vom Differenzierungsstatus sind, als bei Photonen- RT. 12C- Ionen inaktivieren Tumorzellen und Normalgewebsanteile sowie Gefäßstrukturen schneller und effektiver als Photonen. Die strahleninduzierte Genregulation nach Photonen- und 12C- RT betrifft ähnliche Funktionen, aber der zeitliche Verlauf variiert besonders in DNA- Reparaturmechanismen und Signalwegen des Zellüberlebens. Da CD24+/CD45- - Zellen in H- und HI-Tumoren als TICs identifiziert wurden, impliziert der unterschiedliche Einfluss von 12C- und Photonen- RT auf die CD24- Expression einen ersten Hinweis auf differenzielle Effekte der beiden Strahlmodalitäten auf putative Prostatakrebsstammzellen.
Tunnel-ionization is investigated in the framework of relativistic quantum mechanics. For an arbitrary constant electromagnetic field a gauge invariant energy operator is introduced in order to identify the classically forbidden region for tunnel-ionization. Furthermore, relativistic features of tunnel-ionization are explored. A one-dimensional intuitive picture predicts that the ionized electron wave packet in the relativistic regime experiences a momentum shift along the laser’s propagation direction. This is shown to be consistent with the well-known strong field approximation. Furthermore, spin dynamics in tunnel-ionization process is discussed in the standard as well as in the dressed strong field approximation. Next, the tunneling time delay is investigated for tunnel-ionization by extending the definition of the Wigner time delay. Later, this concept is redefined in terms of the phase of the fixed energy propagator. The developed formalism is applied to the deep-tunneling and the near-threshold-tunneling regimes. It is shown that in the latter case signatures of the tunneling time delay can be measurable at remote distance. Finally, the path-dependent formulation of gauge theory is discussed. It is demonstrated that this equivalent formulation of gauge theory leads to a canonical gauge fixing, in which the Feynman path integral becomes more intuitive and the calculation of the quasiclassical propagator is considerably simplified
Schwann cells are the glial cells of the peripheral nervous system (PNS) with multifunctional roles. Myelinating Schwann cells wrap axons with multilayered myelin sheaths providing electrical insulation and rapid impulse propagation. They also play a key role in the immune response after nerve injury and play an active role during nerve repair by contributing to a surrounding growth environment that allows peripheral nerve axons to regenerate (Rodrigues, Rodrigues et al. 2012). GDF-15, a neurotrophic factor and member of the TGF-β superfamily has been shown to support nerve regeneration (Mensching, Borger et al. 2012; Charalambous, Wang et al. 2013). Schwann cells express GDF-15 and seem to be an important source of GDF 15 in peripheral nerves (Strelau, Strzelczyk et al. 2009). Interestingly, analysis of GDF-15 deficient mice showed a severe and progressive motoneuron loss accompanied by a loss of axons. Moreover, studies in our laboratory showed a hypermyelination in adult mutant mice (Dr. J. Strelau, unpublished data). Together these data suggest that GDF-15 affects Schwann cell. In this study, I first investigated the putative effect of GDF-15 deficiency on Schwann cells and showed for the first time that GDF-15 loss reduces Schwann cell numbers in adult mice. To address the question whether this observation is correlated with an up or down regulation of other important signaling molecules I analyzed several gene expression patterns in adult peripheral nerves and spinal marrow associated with Schwann cells and/or GDF-15. Interestingly, IL-6 expression, a key regulator of the immune response is upregulated in peripheral nerves. However, in vitro studies failed to proof the direct effect of GDF-15 knockdown on IL-6 expression in Schwann cells. For functional studies including GDF-15 dependent proliferation, cell death and migration of Schwann cells I next established a method to produce highly enriched adult Schwann cell cultures, showing that GDF-15 is a survival factor for Schwann cells and that GDF-15 deficient Schwann cells fail to migrate. Both defects were rescued by treatment with recombinant GDF-15. Since GDF-15 specific receptors are not unequivocally identified, I began to investigate the underlying signaling cascades well known to control myelin sheath growth, most notably the putative interaction of GDF-15 with ErbB2 receptor tyrosine kinase, expressed on Schwann cells. Here I showed the activation of the ErbB2 receptor by recombinant GDF-15 in Schwann cells for the first time. Taken together, this suggests that GDF-15 constitutes an important endogenous regulator of Schwann cell functions in the adult.
This thesis investigates methods for the creation of reference datasets for image processing, especially for the dense correspondence problem. Three types of reference data can be identified: Real datasets with dense ground truth, real datasets with sparse or missing ground truth and synthetic datasets. For the creation of real datasets with ground truth a existing method based on depth map fusion was evaluated. The described method is especially suited for creating large amounts of reference data with known accuracy. The creation of reference datasets with missing ground truth was examined on the example of multiple datasets for the automotive industry. The data was used succesfully for verification and evaluation by multiple image processing projects. Finally, it was investigated how methods from computer graphics can be used for creating synthetic reference datasets. Especially the creation of photorealistic image sequences using global illumination has been examined for the task of evaluating algorithms. The results show that while such sequences can be used for evaluation, their creation is hindered by practicallity problems. As an application example, a new simulation method for Time-of-Flight depth cameras which can simulate all relevant error sources of these systems was developed.
Cellular variability is fundamental to physiological reality but usually unattended in signaling models. This thesis introduces the new approach of cell ensemble models, which describe biochemical signal transduction networks in heterogeneously behaving cells. Cell ensemble models comprise sets of coupled ordinary differential equations describing protein concentration trajectories in different cells, which are linked by boundary conditions restricting models to physiological limitations. Simultaneous description of single-cell and population data facilitated model discrimination and improved the accuracy of parameter estimations. The approach was applied in two biochemical systems, programmed cell death and the intracellular traffic of erythropoietin receptors. An experimental method was developed to quantify the enzymatic activity of caspase-8, which initializes programmed cell death, in single cells. The analytic solution of a death receptor oligomerization model was combined with cell ensemble models of caspase-8 activation. An activation mechanism, which implies positive feedback, was predicted and experimentally validated. Simulations based on estimated multivariate log-normal distributions of initial cellular protein concentrations clarified the functional roles of involved signaling proteins. In a similar manner, a cell ensemble model was applied to characterize cell-to-cell variability in intracellular erythropoietin receptor transport. The new approach might support optimization of therapeutic applications targeting heterogeneous populations of cancer cells.
The L-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are glutamate-gated ion channels that are important for fast synaptic transmission and synaptic plasticity in the central nervous system. The AMPARs’ gating properties are tightly regulated by receptor number, subunit composition, co-transcriptional as well as post-translational subunit modifications and by their interactions with auxiliary proteins. Recently, a novel endogenous auxiliary protein of AMPARs, CKAMP44, was identified. CKAMP44 modulates the gating kinetics of AMPARs, such as deactivation, desensitization and recovery from desensitization. However, the detailed molecular mechanisms for the CKAMP44/AMPAR interaction and CKAMP44 regulation of AMPAR gating remain to be resolved. This dissertation unravels different CKAMP44 domains involved in AMPAR interaction and modulation, and investigates new roles of CKAMP44 in neuronal morphogenesis of primary neurons. In order to identify CKAMP44 protein domains that are essential for CKAMP44/AMPAR interaction, AMPAR-mediated current modulation, and CKAMP44 spine delivery, a series of CKAMP44 deletion mutants were generated, expressed in HEK293 cells, primary hippocampal neurons and in neonatal mouse brains, and analyzed by co-immunoprecipitation, immunostaining and electrophysiological recordings. Our results show that the extracellular CKAMP44 domain triggers the modulation of AMPARs’ gating properties. The intracellular Cterminal domain of CKAMP44 is required for postsynaptic localization of CKAMP44, and the R/K domain is needed for the physical CKAMP44/AMPAR interaction as well as for the efficient spine targeting of CKAMP44. The requirement of the R/K domain in CKAMP44/AMPAR binding might explain why the recombinantly expressed CKAMP44 extracellular domain failed to modulate AMPAR activity. In addition to the regulatory function of CKAMP44 on AMPAR-mediated currents, our bidirectional manipulation of CKAMP44 expression in primary hippocampal neurons of Ckamp44–/– and wild-type mice provided evidence that neuronal morphogenesis is modulated by CKAMP44. Primary neurons derived from Ckamp44–/– mice exhibited increased dendritic arborization and spine volume, but decreased spine density when compared to CKAMP44 expressing neurons. IV CKAMP44 overexpression in neurons leads to the opposite results. In addition, CKAMP44 overexpression induced irregular spine morphology and multiple synapses generated at single spines. Therefore, we suggest that, in addition to the modulation of the AMPAR gating, CKAMP44 can tune dendritic arborization and spine formation during neuron maturation.
It is well known that our genetic material influences our tendency to develop certain conditions. Finding the causes behind these predispositions assumes the understanding of mechanisms handling and maintaining the genome. While the problem is important from the biological point of view, being one of the basic riddles of life, it also poses interesting questions which may only be answered by physics. Topics include transport, reaction-diffusion, polymer physics, equilibrium and non-equilibrium dynamics and chaos, amongst others. Experimental techniques, like microscopy or molecular biology approaches provide an ever improving insight in the structure of the nucleus, however, computational and modelling approaches are still needed to explain unknown aspects of genetics.
% Evidence is accumulating that our genetic material not only influences our resemblance to relatives and the chances that we may have a tendency to develop certain diseases, but also our predisposition to contract viral infections or to develop conditions like depression, obesity or substance dependence. It has become clear that understanding how the genetic material is organized and how it is being handled might be the key to revolutionize medicine. Experimental techniques, like microscopy or molecular biology approaches provide an ever improving insight in the structure of the nucleus, however, computational and modelling approaches are still needed to explain unknown aspects of genetics.
In this thesis we tackle the problem of understanding the structure of the nucleus from the two opposite sides of the experimental ``blind-spot''. We develop alternative image modelling and analysis tools which are able to capture and recreate the ``large scale'' density patterns observed in confocal microscopy images of the nucleus. For this, we introduce a generalized Potts model which is extensively analysed also from the statistical mechanics point of view. Furthermore, we apply statistical mechanics and graph theory calculations to study patterns registered with super resolution microscopy techniques. We investigate the effect of irradiation and light stress on the structure of the chromatin, and are able to quantitatively support prior experimental observations regarding structural changes.
Understanding the interaction and classification of proteins, structures which perform vastly different functions on molecular scales, is also important to achieve the final picture. We contribute to this by elaborating a framework to assess topological similarity among these chemicals. Our approach is based on recently developed computational topology algorithms used to calculate fingerprints of the molecules. We discuss three different modifications of the framework and investigate them on real-world datasets. In addition, we recognize that the mentioned fingerprints can be used to calculate the fractal dimension of certain objects, and offer an intuitive explanation for the observed relation.
Effective prediction of diagnosis and prognosis in cancer is an important step for selection of a suitable treatment regimen. In this dissertation, the importance of using antibody microarray for effective diagnosis and prognosis of cancer was studied using bladder and gastric cancers. In the first part of study, establishment of a protein signature to predict recurrence of non-muscle invasive bladder cancer was aimed at. Antibodies against cancer-related proteins were spotted and proteins from recurrent and non-recurrent non-muscle invasive bladder cancer tissues incubated. The protein profiles of the samples were analyzed for statistical significance and differential expression of proteins among the cancer groups. After a series of analysis using bioinformatic tools, a 20 protein-signature predicting recurrence of non-muscle invasive bladder cancer was identified along with important molecular mechanisms underlying recurrence. High grade gastric adenocarcinomas are often lethal with metastasis and frequent recurrence. The second study concentrated on more personalized cancer medicine by direct comparison of healthy controls and gastric adenocarcinoma tissues from the same patient. Antibody microarray was used to study the protein profiles of gastric cancer by incubating protein samples from healthy controls in tandem with the cancer protein from the same patient. Statistically and clinically significant proteins were identified including a 16 protein signature for betterment of individual-based cancer treatment regimen. Identified biomarkers included known therapeutic targets such as VEGFA, S100A9 and newly identified markers like OCLN and TIA1. The analyses on two cancer types revealed two different protein signatures with high specificity and sensitivity. Moreover, our findings were clinically relevant and superior to many other approved available methods for diagnosis and prognosis.
Patterned ground structures characterised by strong heterogeneities in surface and subsur- face properties are a common feature of permafrost landscapes in the Arctic. In this thesis, an existing permafrost model including conductive heat transfer and the phase change of water is extended to simulate the thermal dynamics of polygonal soil structures in an Arctic tundra wetland in Northern Siberia. A two-dimensional model formulation in cylindrical coordinates is implemented and coupled to a surface energy balance model. The model performance is assessed by a comparison with analytical solutions and field measurements of soil temperature and surface heat fluxes. The heat transfer in the soil is represented well with mean absolute deviations of up to 1 ◦ C. The coupled simulations with surface energy balance calculation represent the soil temperatures with larger deviations that can be attributed to the description of the snow cover in the model. Lateral heat fluxes in the polygon only occur during limited periods and do not have a substantial impact on the thermal dynamics of the system. Simplified one-dimensional model descriptions based on averaged soil and surface parameters show a cold bias of up to 0.7 ◦ C in the mean annual temperature of the permafrost compared with the detailed two-dimensional model. The thaw depth is simulated consistently by the models of different complexity .
Tumor radiotherapy with charged particles offers superior properties in covering the tumor with radiation dose, while sparing the surrounding, healthy tissue better than standard X-rays. Thus, the success of the therapy is potentially increased. However, the application of this technique necessitates not only profound knowledge about its physical aspects, such as the uncertainties in the range of the particles which need to be taken into account in the therapy planning stage. In addition to the physical aspects, the biological effectiveness of particle radiation needs to be thoroughly understood. The effectiveness depends on many physical as well as biological quantities and is determined involving complicated models. Though, there is a close relation with the linear energy transfer (LET), i.e. the measure of the local concentration of energy deposition along a particle’s track. The higher the LET (up to a certain limit), the higher the biological effect. Furthermore, radiation resistant cells, e.g. due to a lack of oxygenation, can be effectively killed with radiation that has a high LET. The LET itself depends on the kinetic energy of the particle, just like the dose, which makes a simultaneous optimization of dose and LET possible only under some circumstances. However, this work presents a method that makes use of the dose ramp concept to beneficially re-distribute areas with high LET using protons, carbon ions and antiprotons, respectively, without notably influencing the dose distribution. In the experimental part of this work, a tissue-equivalent proportional counter (TEPC) is used to measure microscopic dose distributions in lineal energy, i.e. fluctuations of the energy concentration on sub-cellular length scales. The extent of these fluctuations as well as the magnitude of the energy concentration have, like the LET, an influence on the biological effect. In the measurements presented here it is assessed if there is a significant change in the dose distributions in lineal energy when comparing two methods of particle beam application to determine a potential impact on the biological effect.
Speleothems are recognised as valuable palaeo climate archives. This has lead to an increasing number of analysed speleothem proxy time series from caves that are distributed on a global scale. In particular speleothem δ18O and δ13C time series are investigated. A hotspot of analysed speleothem proxy time series is Europe, which makes it possible to perform spatio-temporal coherency analysis of speleothem proxy time series. For this aim a method is developed that is based on Principal Component Analysis (PCA). The method is based on a Monte Carlo approach and accounts for the speleothem age uncertainty and the different temporal resolution of speleothem proxy time series. This method is applied to compilations of European speleothem δ18O and δ13C time series. It is demonstrated that the results of the PCA for the compiled δ18O time series can be interpreted as a temperature proxy and as a precipitation/hydrology proxy for the compilation of δ13C time series. Furthermore, it is showed that the spatio-temporal coherence between the analysed speleothem δ18O and δ13C time series varied with time. Moreover, a change of the predominant pattern is observed at 4.0 ka. The second aim of this study is to analyse the change the observed speleothem δ18O gradient for European speleothems. A multi-box Rayleigh approach model is developed (Stable Isotope in Precipitation (SIP) model) that computes the stable isotope composition of precipitation, infiltrated water and calcite. The model is validated with measured precipitation δ18O and δD values from the GNIP dataset. It is demonstrated that the SIP model agrees with the observed δ18O and δD values for the analysed Central European and Northern European transect. Moreover, it is showed that the precipitation δ18O (δD) gradient depends on the North Atlantic Oscillation. This is interpreted as a change of the amount of moisture in the atmosphere for Central Europe. The application of the SIP model of palaeo climate speloethem δ18O gradients suggests that the climate was drier in the early- and mid-Holocene compared to present-day. In addition, the past δ18O gradients suggest a transition of the atmospheric circulation from a very negative NAO like pattern in the early-Holocene (11 ka) to a very positive NAO like pattern in mid-Holocene (4 ka) and that a reorganisation of the atmospheric circulation occurred at approximately 4 ka when present-day atmospheric circulation established.
Ziel der vorliegenden Arbeit war die Synthese und Charakterisierung großer, linear anellierter N-Heteroacene. Durch gezielte Derivatisierung sollten ihre Material-eigenschaften auf Basis literaturbekannter Kriterien für einen effektiven Elektronentransport optimiert werden. So konnten N-Heteroacene bis hin zu Azahexacenen über unterschiedliche Kupplungsmethoden dargestellt werden. Dabei wurde der Pd-katalysierten Aminierung eine erhöhte Beachtung geschenkt, da durch diese gleichsam junge und effektive Methode elegante Synthesewege möglich sind. Aber auch klassische Methoden fanden Einsatz, sodass eine Vielzahl neuer Verbindungen dargestellt werden konnte. Ansätze, die Klasse der Azaacene durch Azaheptacene zu erweitern, blieben dagegen bis zuletzt ohne Erfolg. Darüber hinaus wurde der Einfluss des Heteroatoms in einer Serie von Acenothiadiazolen und Acenoselenadiazolen untersucht. Dabei zeigte der Austausch des Schwefelatoms gegen das elektropositivere und voluminösere Selenatom bemerkenswerte Veränderungen in optischen und elektronischen Eigenschaften, sowie in der Festkörpermorphologie der Heteroacene. Mit dem Ziel, potente Elektronenhalbleiter darzustellen, wurden die einzelnen Fragmente der N-Heteroacene systematisch variiert und optimiert, woraus ein besseres Verständnis dieser Verbindungsklasse resultierte. Durch gezielte Modifikationen der Struktur konnten dabei Morphologie, Löslichkeit, elektronische Struktur und Stabilität angepasst werden, woraus etliche potentielle Ladungstransport¬materialien resultieren.
This thesis treats different aspects of the class of Mixed-Integer Optimal Control Problems (MIOCPs). These are optimization problems that combine the difficulties of underlying dynamic processes with combinatorial decisions. Typically, these combinatorial decisions are realized as switching decisions between the system’s different operations modes. During the last decades, direct methods emerged as the state-of-the-art solvers for MIOCPs. The formulation of a valid, tight and dependable integral relaxation, i.e., the formulation of a model for fractional values, plays an important role for these direct solution methods. We give detailed insight into several relaxation approaches for MIOCPs and compare them with regard to their respective structures. In particular, these are the typical solution’s structures and properties as convexity, problem size and numerical behavior. From these structural properties, we deduce some required specifications of a solver. Additionally, the modeling and subsequent limitation of the switching process directly tackle the class-specific typical issue of chattering solutions. One of the relaxation methods for MIOCPs is the outer convexification, where the binary variables only enter affinely. For the approximation of this relaxation’s solution, we took up on the control approximation problem in integral sense derived by Sager as part of a decomposition approach for MIOCPs with affine binary controls. This problem describes the optimal approximation of fractional controls with binary controls such that the corresponding dynamic process is changed as little as possible. For the multi-dimensional problem, we developed a new heuristic, which for the first time gives a bound that only depends on the control grid and not anymore on the number of the system’s controls. For the generalization of the control approximation problem with additional constraints, we derived a tailored branch-and-bound algorithm, which is based on the properties of the Lagrangian relaxation of the one-dimensional problem. This algorithm beats state-of-the-art commercial solvers for Mixed-Integer Linear Programs (MILPs) for this special approximation problem by several orders of magnitude. Overall, we present several, partially new modeling approaches for MIOCPs together with the accompanying structural properties. On this basis, we develop new theories for the approximation of certain relaxed solutions. We discuss the efficient implementation of the resulting structure exploiting algorithms. This leads to a deeper and better understanding of MIOCPs. We show the practicability of the theoretical observations with the help of four prototypical problems. The presented methods and algorithms allow on their basis the direct development of decision support and analysis tools in practice.
Environmental exposure such as tobacco smoke is the principal cause of most lung cancer cases worldwide. However, only a small proportion of heavy smokers develop lung cancer which suggests that other factors such as genetic and/or epigenetic interindividual variations may be responsible for individual disease susceptibility. The overall aim of this study was to determine germline copy number variations (CNVs) associated with early-onset lung cancer risk and to further investigate the genetic and epigenetic interplay of microRNAs (miRNAs) and genes located in two candidate CNVs on 8q24.3 and 11p15.5 in lung cancer. A genome wide association study (GWA) had been performed using the Illumina Infinium platform Human Hap550 BeadChip on 492 early-onset lung cancer cases and 487 population based controls. Two computational CNV detection algorithms, QuantiSNP and PennCNV, were applied to this existing data set to identify CNVs and the overlapping CNVs between the two algorithms were further analyzed for association with the disease. Ten CNVs were significantly associated with early-onset lung cancer. Two CNVs were selected for strength of association and for containing miRNAs and genes likely to be relevant for lung cancer. To assess their functional relevance in non-small cell lung carcinoma (NSCLC), qPCR based expression analysis and quantitative methylation analysis using the MassCLEAVETM assay of genes and miRNAs in these regions were performed on NCSLC and matched normal lung tissue. The expression analysis showed that miR-661 on 8q24.3 was significantly upregulated in lung tumor compared to normal. The putative miR-661 promoter was hypomethylated in tumor tissue and revealed a significant negative correlation with expression in tumor. Additionally, the loss of methylation at these sites was significantly associated with worse outcome independent from stage, histology and gender. The most significant changes in the gain CNV region on 11p15.5 were seen for miR-210 and Plakophilin 3 (PKP3) which both were significantly upregulated in NSCLC. Promoter hypomethylation at the transcription start site of PKP3 was inversely correlated with expression in NSCLC, suggesting that methylation regulates the PKP3 expression. For further functional analysis of the two miRNAs, predicted targets were identified in silico and 3´UTR luciferase reporter assays for the predicted targets and expression analysis after ectopic overexpression in A549, H1299 and H1703 lung cancer cell lines were carried out to determine whether a direct link between the miRNAs and the targets could be shown. The results showed that mitogen associated protein 3 kinase 3 (MAP3K3) and Cadherin1 (CDH1) are direct targets of miR-661, suggesting that miR-661 has oncogenic properties in lung cancer. Furthermore, miR-210 was shown to target the tumor suppressor gene Runt related transcription factor 3 (RUNX3), a transcription factor known to be involved in lung development and to be a crucial regulator of cell proliferation. The results from this study suggest that CNV analysis of GWAs data for lung cancer risk can point to functionally important regions in the genome that are deregulated in NSCLC and may contribute to lung tumorigenesis. Further investigation of the relevance of these CNVs to early-onset lung cancer risk is needed to confirm our suggested finding of two risk markers. Furthermore, additional analyses on the functional role of miR-661 in lung cancer are desirable to elucidate to what extent this miRNA contributes to tumorigenesis. Taken together, this study provides evidence that interplay between genetic variations and epigenetic deregulation plays a pivotal role in NSCLC pathogenesis.
Epigenetic factors such as DNA methylation, histone modification and noncoding RNAs are highly associated with early developmental processes, later environmental adaption and diseases development such as cancer. With the availability of current high throughput assays (microarray and next generation sequencing), one can already produce comprehensive picture of the epigenetic profile, especially the DNA methylome, in normal and tumor/diseased cells. However, managing and analyzing such vast datasets is challenging. In addition, interpretation of the observations from (epi)genetic information is also a limiting factor due to the lack of understanding epigenetic mechanisms and the interactions between genetic and epigenetic factors under environmental selection. Thus, during my PhD studies, two pipelines were developed to process genome-wide methylation data generated by Methyl-CpG-immunoprecipitation sequencing (MCIP-seq) for the ICGC early onset prostate project and whole genome bisulfite sequencing (WGBS) for the environment induced transgenerational epigenetic remodeling project. The WGBS pipeline was adjusted later for a modified WGBS protocol, tagementaion-based WGBS, which allows to investigate the whole methylome (around 27 million CpGs) at single base resolution by using only 10-20 ng of input DNA compared to 3-5 ug required for traditional WGBS. Developing these computational tools, provided an opportunity to look closely at methylation changes in prostate cancers. With an integrative meta-analysis of public prostate (epi)genomic data and a large cohort of 7682 prostate cancer specimens, BAZ2A was found to be overexpressed in a large subset of prostate tumors that are characterized by early post-operative PSA recurrence and high tumor grades. In multivariate analyses, BAZ2A was found to be an independent factor predicting recurrence. Furthermore, high levels of BAZ2A were tightly associated with a distinct molecular subtype demarked by aberrant genome-wide DNA methylation and elevated numbers of genetic alterations suggesting a CpG island-methylator phenotype (CIMP) to selectively occur in BAZ2A-upregulated tumors. In summary, this study showed the clinical impact of BAZ2A as a key epigenetic regulator linking aberrant DNA methylation and outcome in prostate cancer. In addition, epigenetic changes is not only important for the diseased individuals including cancer, but also for the healthy individuals to adapt the external environmental stimulus such as smoking. In order to investigate the interaction between the methylome and environmental factor in a human prospective mother-child study at single base resolution, tobacco smoke-induced changes to epigenetic programming during the prenatal period was studied by WGBS and targeted methylation analysis. In mothers and children a distinct, genome-wide epigenetic response is induced. While mothers showed a genome-wide hypomethylation profile, children revealed tobacco-smoke induced hyper- and hypomethylation. By focusing on chromatin regulators, differential DNA methylation with functionally deregulated histone modifiers was linked, which together induce epigenetic reprogramming upon exposure to tobacco smoking. Together with the observed deregulation of a number of disease related pathways, the identified aberrant DNA methylation was suggested as a possible molecular mechanism linking between prenatal exposure and disease outcomes later in life. In summary, comprehensive epigenomic analyses were performed on both diseased and healthy individuals in order to shed a light on how epigenetic factors influence the tumor development and interact with external environmental stimulus.
Recent advances in RESOLFT (reversible saturable optical fluorescence transitions) microscopy have enabled the non-invasive three-dimensional visualization of numerous structures in living cells at high spatial resolution. This technique, which utilizes low light intensities, has manifold potential applications in the life sciences. The work presented here envisions further broadening the applications of RESOLFT microscopy by implementing a scheme for fast image acquisition of large fields of view, applicable to thick specimen. With a pattern consisting of line-shaped intensity minima, this novel technique, called lineRESOLFT, permits fast imaging of living cells at ~40nm lateral resolution while offering strong optical sectioning. The full potential of this method is further illustrated by the achievement of continuous three-dimensional imaging of neurons in living brain slices with high spatio-temporal resolution, enabling the observation of rapid spine motility for large fields of view on the second time scale.
Die Arbeit untersucht die Synthese und die physikalischen und elektrochemischen Eigenschaften von nano- und mikroskalierten Materialien für Lithium-Ionen-Batterien, wobei neben den elektrochemischen Eigenschaften auch grundlegende physikalische Fragestellungen im Vordergrund standen. Zur Herstellung der Materialien wurden dabei sowohl die konventionelle als auch die mikrowellenunterstützte Hydrothermalsynthese benutzt. Die physikalische Charakterisierung der Materialien erfolgte vor allem mittels Röntgendiffraktion, Magnetisierungsmessungen und Elektronenmikroskopie, darüber hinaus wurden als elektrochemische Methoden zyklische Voltammetrie und galvanostatische Zyklierung eingesetzt. Als relevante Batteriematerialien wurden sowohl Oxide (TiO2, LiCoO2) als auch Phosphate (LiMPO4 mit M=Mn, Fe, Co, Ni) untersucht. LiCoO2 dient dabei als Modellsystem für die mikrowellenunterstützte Synthese, welches durch die Niedertemperatursynthese in nanoskalierter Modifikation hergestellt und mit konventionell synthetisiertem Material verglichen wurde. Des Weiteren wurden TiO2-Nanoröhren und -Nanopartikel untersucht, die beide deutlich verbesserte elektrochemische Eigenschaften als die entsprechenden Bulk-Materialien zeigen. Für die Herstellung von LiMPO4 (M=Mn, Fe, Co, Ni) wurde eine Vielzahl verschiedener Syntheseparameter untersucht, mit deren Hilfe die Morphologie und das Agglomerationsverhalten phasenreiner nano- bzw. mikroskalierter Materialien gezielt manipuliert und studiert werden konnten. Ein wichtiges weiteres Ergebnis ist die erstmalige Darstellung und Untersuchung eines neuen LiCoPO4-Polymorphs (Raumgruppe Pn21a). Für LiFePO4 und LiMnPO4 konnte ein deutlicher Einfluss der Größenreduktion bzw. Form auf die elektrochemischen Eigenschaften gezeigt werden. Zusammen mit der Anwendung eines Oberflächenbeschichtungsverfahrens können so die elektrochemischen Eigenschaften deutlich verbessert werden.
Ice nucleation in clouds has a significant impact on the global hydrological cycle as well as on the radiative budget of the Earth. The AIDA cloud chamber was used to investigate the ice nucleation efficiency of various atmospherically relevant mineral dusts. From experiments with Arizona Test Dust (ATD) a humidity and temperature dependent ice nucleation active surface site density parameterization was developed to describe deposition nucleation at temperatures above 220 K. Based on these results, a parameterization for deposition nucleation initiated by desert dusts and clay minerals is proposed. The time dependence observed during the deposition nucleation experiments with ATD seems to be relevant only at very small cooling rates. Experiments with atmospherically relevant dust samples revealed large differences in the ice nucleation efficiencies among the different dusts. Volcanic ash emitted during the Eyjafjallajökull eruption in 2010 proved to be slightly less ice-active than mineral dust particles from desert areas. In contrast, soil dust particles rich in organic matter and fossile diatomite were very ice-active particles. For the soil dust particles, the soil organic matter is probably the reason for the enhanced ice nucleation efficiency compared to desert dusts which is, however, most likely not directly determined by viable organisms such as fungi or bacteria. Coatings with secondary organic compounds led to a significant deterioration of the ice nucleation efficiency observed for fossil diatomite in the immersion freezing and the deposition nucleation mode. Deposition mode nucleation initiated by mineral dust particles was suppressed by coatings with sulfuric acid whereas there was no effect on the immersion freezing properties. The immersion freezing properties of several substrates used as ice nuclei substitutes were investigated with a cold stage setup. Muscovite initiated heterogeneous ice nucleation at approximately 250 K but was much less ice-active than mineral dusts. Droplets placed on silicon wafers froze only close to the homogeneous freezing threshold. The freezing properties of these silicon wafers could be changed signicantly by modifying the wafer surface through adding regular structures such as trenches which suggests that the surface morphology may have an influence on the ice nucleation efficiencies of atmospheric particles.
Embedded systems comprise diverse technologies complicating their design. By creating virtual prototypes of the target system, Electronic System Level Design, the early analysis of a system composed by electronics and software is possible. However, the concrete interaction between hardware modules and between hardware and software is left for late development stages and real prototype making. Generally, interaction between components is assumed to be correct. However, it has to be assumed on development implicitly because interaction between components is not considered in the functionality design.
While single components are mostly thoroughly tested and guarantee certain reliability levels, their interaction is based on often underspecified interfaces. Although component usage is mostly specified, operational constraints are often left out. Finally, not only the interaction between components but also with the environment and the user are not ensured. Generally, only functional integration tests are executed and corner-cases are left out, leaving uncovered faults that only manifest as failures later when their cost is higher. Therefore, this work aims at component interaction through specification of interfaces, test generation and real-time test execution. The specification is based on the design-by-contract approach of software that specifies semantics of component interaction in addition to the syntactical definition through functions.
In the first part of this work, a specification for the interaction between hardware modules is given. With the automatic real-time test execution, fulfillment of specified preconditions for correct component operation can be checked. In component-based design, the component is trusted and thus, its functionality is assumed to be correct when certain postconditions are specified. In a correct component assembly, component postconditions fulfill preconditions of other components resulting in an operational system.
The specification of preconditions follows the definition of environmental properties, acceptable input sequences for interfacing pins, as well as acceptable signal parameters, such as voltage levels, slope times, delays and glitches. Postconditions are defined by the description of a functionality accompanying constraints, such as timing. These parameters are automatically determined on operation by a testing circuit. Parameters that violate the specification are signaled by the testing circuit and failure is detected. The chosen parameters can give hint of the reason for the failure being an evidence of a circuit fault. In the example of an Inter-Integrated Circuit (I2C) communication system, we define contracts and show comparisons between contract violation, fault categorization and failure occurrence under signal fault injection.
To complete this work, support for fault analysis on the electronic system level design is given. For this, the data transfers between the high-level models used in the design are augmented with the defined contract parameters. With a specific interface, digital faults are generated for transactions with violating signal parameters that can be tracked by the system. This way, recovery mechanisms for synchronous communication are proposed and tested.
In the second part, the interaction between hardware and software is tackled providing special methods for developing device drivers. For this, we do not only specify the interface between hardware and software but also map the hardware control elements to software, partially generating the software interface for a device. This is necessary because drivers handle devices with internal control elements like registers, data streams and interrupts that cannot be represented on software.
This systematic composition of drivers facilitates the development of a device interface called the device mechanism. It is the lowest layer of a two-layer architecture for driver development. The device mechanism carries out the access to the device exporting a pure software interface. This interface is based on the device implementation being, thus, fully specified. Further data processing required for compliance with the operating system or application is carried out in the driver policy, the layer on top of it.
With the definition of a software layer for device control, contracts specifying constraints of this interface are proposed. These contracts are based on implementation constraints of the device and on its dynamic behavior. Therefore, an extended finite state machine models the dynamic behavior of the device. Based on it, functions of the device mechanism can be augmented with preconditions on the state or on state machine variables. These conditions are then checked on runtime. After execution of a function, its postconditions are ensured, such as timing. This guarantees that different driver policies, operating systems or firmwares, use this same device mechanism fulfilling its constraints. On the example of a Philips webcam, we develop the complete driver for Linux based on our architecture, creating contracts for its device mechanism. Following the systematic composition and the contract approach, driver bugs are avoided that otherwise violate allowed values for device data and execution orders of device protocols.
Spray drying is one of the most widely used drying techniques to convert liquid feed into a dry powder. The modeling of spray flows and spray drying has been studied for many years now, to determine the characteristics of the end products, e.g. particle size, shape, density or porosity. So far, the simulation of polymer or sugar solution spray drying has not been studied because drying behavior as well as properties are unknown. Previous studies concentrated on the systems of milk, salt solution, colloids or other materials for which the thermal and physical properties are well tabulated.
The present study deals with the modeling and simulation of polyvinylpyrrolidone (PVP)/water and mannitol/water spray flows. PVP is a polymer, widely used as a pharmaceutical excipient, and mainly manufactured by BASF under several patented names, whereas mannitol is a sugar, which is used in dry powder inhalers and tablets. Experimental studies have shown that the powder properties of PVP and mannitol are significantly influenced by the drying conditions. The growing importance of PVP or mannitol powders and the inability of existing studies to predict the effect of drying conditions on the properties of the end product have prompted the development of a new reliable model and numerical techniques.
Evaporating sprays have a continuous phase (gas) and a dispersed phase, which consists of droplets of various sizes that may evaporate, coalesce, or breakup, as well as have their own inertia and size-conditioned dynamics. A modeling approach which is more commonly used is the Lagrangian description of the dispersed liquid phase. This approach gives detailed information on the micro-level, but inclusion of droplet coalescence and breakup increase computational complexity. Moreover, the Lagrangian description coupled with the Eulerian equations for the gas phase, assuming a point-source approximation of the spray, is computationally expensive. As an alternative to Lagrangian simulations, several Eulerian methods have been developed based on the Williams’ spray equation. The Euler – Euler methods are computationally efficient and independent of liquid mass loading in describing dense turbulent spray flows.
The objective of this thesis is the modeling and simulation of spray flows and spray drying up to the onset of solid layer formation in an Euler – Euler framework. The behavior of droplet distribution under various drying conditions in bi-component evaporating spray flows is examined using, for the first time, direct quadrature method of moments (DQMOM) in two dimensions. In DQMOM, the droplet size and velocity distribution of the spray is modeled by approximating the number density function in terms of joint radius and velocity. Transport equations of DQMOM account for droplet evaporation, heating, drag, and droplet–droplet interactions.
At first, an evaporating water spray in nitrogen is modeled in one dimension (axial direction). Earlier studies in spray flows neglected evaporation or considered it through a simplified model, which is addressed by implementing an advanced droplet evaporation model of Abramzon and Sirignano, whereas droplet motion and droplet coalescence are estimated through appropriate sub-models. The assumption of evaporative flux to be zero or computing it with weight ratio constraints was found to be unphysical, which is improved by estimating it using the maximum entropy formulation. The gas phase is not yet fully coupled to the DQMOM but its inlet properties are taken to compute forces acting on droplets and evaporation. The simulation results are compared with quadrature method of moments (QMOM) and with experiment at various cross sections. DQMOM shows better results than QMOM, and remarkable agreement with experiment.
Next, water spray in air in two-dimensional, axisymmetric configuration is modeled by extending the one-dimensional DQMOM. The DQMOM results are compared with those of the discrete droplet model (DDM), which is an Euler – Lagrangian approach. Droplet coalescence is considered in DQMOM but neglected in DDM. The simulation results are validated with new experimental data. Overall, DQMOM shows a much better performance with respect to computational effort, even with the inclusion of droplet coalescence.
Before extending DQMOM to model PVP/water spray flows, a single droplet evaporation and drying model is developed, because most of the evaporation models available in the literature are valid for salts, colloids or milk powder. The negligence of solid layer formation effects on the droplet heating and evaporation is addressed, and treatment of the liquid mixture as the ideal solution is improved by including the non-ideality effect. The PVP or mannitol in water droplet evaporation and solid layer formation are simulated, and the results are compared with new experimental data, which shows that the present model effectively captures the first three stages of evaporation and drying of a bi-component droplet.
Finally, PVP/water spray flows in air are simulated using DQMOM including the developed bi-component evaporation model. Simulation results are compared with new experimental data at various cross sections and very good agreement is observed.
In conclusion, water and PVP/water evaporating spray flows, and preliminary stages of PVP/water and mannitol/water spray drying, i.e., until solid layer formation, are successfully modeled and simulated, and show good agreement with experiment.
Stinging cells or nematocytes are specialized cells that are unique to Cnidarians. They contain a highly sophisticated organelle, the nematocyst, used for locomotion, defence and capture of prey. The proteome of the nematocyst has provided unique insights into its molecular organisation. The goal of my thesis was to investigate novel molecular factors involved in aspects of nematocyst morphogenesis, structure and function. A critical part of nematocyst morphogenesis is the initiation of tubule formation by a constriction of the Golgi vesicle membrane. Nematomyosin, a newly identified non-muscle myosin type II, in the present thesis is shown to localize to a collar around the outgrowing tubule indicating an essential role in this process. Blocking of myosin II activity by Blebbistatin leads to malformed nematocyst vesicles. Tubule size control is probably facilitated by a PKD2 channel, shown to be active at the point of maximal tubule outgrowth. The nematocyst structure has been characterized by stiff and tear-resistant minicollagens, although the discharge process is accompanied by extreme volume changes of the capsule. Here, I have characterized the novel elastic protein Cnidoin that shares structural homology with the spider silk protein Spidroin-2. Cnidoin is expressed in developing nematocytes and locates to wall and tubule structures. Recombinant Cnidoin showed a high tendency to aggregate and to form linear fibres. Cnidoin thus behaves as a typical elastic protein. Being an integral part of the mature nematocyst it could provide the molecular basis for the energy stored that is released in the ultrafast discharge process. The discharge of nematocysts is triggered by chemical and mechanical stimuli that are detected by the cnidocil at the apical end of the nematocyte. The cnidocil is surrounded by a set of stereocilia, providing a similar arrangement as vertebrate hair cells. A newly identified calcium channel of the TrpA family is shown to locate to stereocilia of the Hydra cnidocil apparatus. The protein can be visualized by immunostainings during developmental stages as well as in mature capsules and thus represents a candidate for mechanosensation during discharge. Nematocalcin, a penta-EF-hand protein, was also located to the stereocilia, but at a more basal position than TrpA, where it might act as a modulatory factor associated with the mechanosensory apparatus.
Zusammenstellung der von Ludwig Boltzmann verfassten Publikationen.
Spinning, turbulent structures swirling around its centers within various flow media are known as vortices. The capability of locating and extracting vortical structures in flow data is crucial for understanding the flow. Vortices also have a strong impact on flow control and transport processes.
Real-time vortex extraction methods are presented, offering immediate notion of the shape and location of the vortex structures. Using a real-time fluid simulation based on Navier-Stokes equations presented in \cite{Stam:1999:SF}, several vortex extraction methods are interactively performed in real-time. Following vortex extraction methods are implemented using the GPU: vorticity threshold, Q criterion, $\lambda_2$ criterion, the eigenvector method via parallel vectors operator (PVO) and the eigenvector method via coplanar vectors operator (CVO).
Diffusional methods outputting flow fields with preserved/enhanced vortical structures are also presented. Such methods are useful for obtaining an alternative insight into vortices within a flow field and can also be used within the real-time simulation.
Using a number of human performed gestures for human-computer interaction, special ensemble flow fields are produced. Detecting vortices from these gesture ensemble range flows is introduced as aid for gesture classification. Gesture range data is recorded using the Microsoft Kinect device. Range or scene flow is a 3D vector field describing movement within a scene. Range data consists of images (color channels) and corresponding depth images (depth channels) in which the distance of objects is recorded as a grayscale image. Ensemble range flow is estimated from gesture videos. Ensemble flow describes the overall flow within the scene and is obtained by averaging the structure tensor throughout the scene. Vortices are extracted from an ensemble range flow of the gestures. Their number and location is offering an additional parameter for gesture classification.
Collection of methods for detecting vortices and obtaining vector fields with emphasized vortices are introduced in this thesis. Real-time execution of vortex extraction methods offers an instant notion of the nature of the flow. Diffusional methods can serve as a processing step within the real-time vortex extraction. As an additional application, gesture ensemble flow is presented. By detecting its vortices, a parameter for gesture classification is introduced.
This thesis deals with advanced models to characterize microfluidic flows from image sequences. The governing equations and boundary conditions for viscous flows are introduced as a global model in order to impose physically sound motion results. The connection between the computational fluid simulations and experimental measurement data is established by using constrained optimization. This framework also allows to introduce control variables, which are determined in agreement with the underlying data. In this context, the thesis focuses on the study of the influence of i) the image data, ii) the underlying motion and iii) the boundary conditions on the estimation of the control variables and the corresponding physical quantities. These questions are assessed by the application to synthetic images that allow to measure the induced errors. It is shown that the application of physically motivated differential equations as global motion models increase the robustness and accuracy of the motion estimation. Control variables are used to change the equations in a modeled manner, so that the solution describes the processes that are inherent in the images. The strength of global models lies in the combination with sparsely distributed information in the images, where common state-of- the-art methods have extreme difficulties to obtain reasonable results. It is demonstrated that the optimal control framework allows to relax the governing equations in order to model uncertainty of the measurement setting parameters, such as wall-slip. And finally, such a parameter model is extended to three dimensions and allows to estimate the pressure drop of the flow and the diffusion coefficient of the trace substance caged Q-rhodamine dextran in water.
The primary aim of this thesis is to investigate the lateral diffusion, correlation and interactions of proteins and peptides with cell membrane models by the combination of experimental techniques in real and reciprocal space. In Chapter 4, the characteristic distance and range of lateral correlation between non-crystalline proteins anchored to fluid lipid monolayers at high surface densities were determined by grazing incidence small angle X-ray scattering (GISAXS) for the first time. Moreover, the lateral density of membrane-anchored proteins could be quantified from Sulfur Kα emission detected by grazing incidence X-ray fluorescence (GIXF). In Chapter 5, the influence of molecular crowding on the lateral diffusion of membrane-anchored proteins was investigated by fluorescence recovery after photobleaching and single particle tracking microscopy, yielding a clear transition from free diffusion to confined diffusion. In Chapter 6, the interactions between antimicrobial peptides and bacterial membrane models are probed by GIXF that allows for the identification of the spatial localization of ions with Ångstrom accuracy. This enabled one to discriminate different "modes" of membrane-protein interactions, such as adsorption and incorporation on the molecular level. The obtained results demonstrated that the use of real and reciprocal space techniques can provide information about fine-structures, electrostatics, and dynamic correlation at biological interfaces.
In highly precise carbon ion radiotherapy, fragmentation of the primary nuclei in the patient results in a spectrum of lighter ions. Due to their radio-biological effectiveness being different from the primary ions, they need to be considered separately in therapy planning. To determine secondary ion spectra, mainly large apparatus based on scintillation detectors have been used until now, limiting the flexibility of the methods and the amount of available data. In this thesis, a novel method for ion spectroscopy based on a small pixelated semiconductor detector is presented. The used Timepix detector, originally designed for photon beam imaging, offers a high spatial resolution enabling the detection of single particles. At first, an extensive characterization of the detector response to therapeutic ion beams was performed. The detector was found suitable for energy-loss measurements on a single ion basis in proton beams between 0.55 and 221MeV, providing mean energyloss values, which deviate less than only 10% from calculations. For the investigated heavier ions, deviations of up to about 30% were observed. The presented novel experimental approach to fragment distinction with the Timepix detector is based on pattern recognition analysis of the signal created by individual ions. For designated configurations, it enables identification of all ion species in mixed particle fields resulting from 12C-fragmentation. The performance of the method was evaluated using reference data of an established technique. The relative fractions of H-, He-, Be- and B-ions agree within 1.1Δref (uncertainty of the reference). For lithium, the agreement is within 2.3Δref. In addition, applications of the method relevant for benchmarking physical models used in Monte Carlo simulations and treatment planning are presented. Providing the advantages of a small and flexible set-up, together with the further improvements suggested, the method is promising to widely expand the available fragmentation data and to complement large experimental set-ups.
Recent observations have shown that star formation is correlated with the molecular phase of the interstellar medium. Molecular gas tends to organize itself into large and roughly self-gravitating entities called Giant Molecular Clouds (GMCs). These objects should, therefore, play a key role in controlling star formation and defining its modes. However, their physical properties, formation and evolution mechanisms are still poorly understood - especially in spiral galaxies. The new PdBI Arcsecond Whirlpool Survey (PAWS) offers, for the first time, the possibility to study the molecular gas distribution in a grand-design spiral galaxy dominated by dynamical phenomena. The aim of this thesis is to evaluate the importance of galactic environments for the gas organization. Via a thorough analysis of the gas kinematics I study the structure of M51’s gravitational potential and spiral arm streaming motions. This analysis provides several insights on the differing nature of the molecular and atomic phase of the interstellar medium. I also find evidence for a kinematic m = 3 mode that explains the asymmetry of the spiral arms. To investigate the effect of the dynamical environment on the molecular gas I have generated the largest extragalactic GMC catalog to date using an automatic algorithm that accounts for the observational biases. Differences in the cloud properties suggest that environments, and in particular dynamical effects, strongly influence the organization of the gas in spiral galaxies and provide a way to discriminate between the various mechanisms of cloud formation and evolution that have been proposed in the literature.
Im Rahmen dieser Arbeit wurden erstmalig die thermischen Eigenschaften von supraleitenden massiven metallischen Gläsern im Temperaturbereich zwischen 6mK und 300K untersucht. Die Messung der Wärmeleitfähigkeit bietet die Möglichkeit fundamentale Wechselwirkungsmechanismen zu beobachten, die den Wärmetransport in Festkörpern bestimmen. Bei ultratiefen Temperaturen wurde hierfür eine neuartige berührungsfreie Messmethode verwendet. Diese basiert auf einer optischen Heiztechnik und paramagnetischen Temperatursensoren, die über SQUID-Magnetometer ausgelesen werden. Die Messergebnisse lassen sich weit unterhalb von TC durch die resonante Streuung der Phononen an atomaren Tunnelsystemen beschreiben. Oberhalb von Tc kann der phononische Beitrag zur Wärmeleitfähigkeit mithilfe eines Modells erfolgreich beschrieben werden, das neben Elektronen und Defekten lokalisierte Moden als zusätzliche Streuzentren für Phononen berücksichtigt. Zur experimentellen Erschließung des Mikrokelvinbereichs wurde ein adiabatischer Kernentmagnetisierungskryostat aufgebaut. Die Badtemperatur wurde mit einem neuartigen Rauschthermometer bestimmt, das zum ersten Mal eine kontinuierliche Temperaturmessung in diesem Bereich ermöglicht. Dafür wird das magnetische Johnson-Rauschen eines massiven Kupferzylinders gleichzeitig durch zwei SQUID-Magnetometer induktiv ausgelesen. Die anschließende Kreuzkorrelation unterdrückt das Verstärkerrauschen um mehr als eine Größenordnung. Das Thermometer wurde zwischen 42µK und 0,8K charakterisiert, wobei keine Abweichungen vom erwarteten linearen Verhalten zwischen der Rauschleistung und der Temperatur festgestellt wurde.
After almost 30 years of inertia in the field of sequencing, the emergence of a whole range of so-called "next-generation" sequencing technologies has revolutionized the way we approach genomic and genetic research. Sequencing all 3 gigabases of a human genome, once a costly task of 13 years of international efforts, can now be done within a matter of days with a coverage of 30x and more, and comes with a price tag that is affordable for a middle-sized lab. Among the different next-generation sequencing machines developed over the course of the last 6 to 8 years, four instruments from three different companies have established themselves on the market for human whole-genome sequencing: Illumina's HiSeq2000, Life Technologies' SOLiD 4 and 5500xl SOLiD, and Complete Genomics' technology.
However, these next-generation sequencing platforms are still relatively new, and a comprehensive comparative assessment of their performance is lacking. For this purpose, the DNA of two tumor-normal pairs from medulloblastoma patients was sequenced individually to 30x coverage on each of the four instruments. The resulting data was analyzed with respect to its coverage distribution and biases over the genome, in particular GC bias, and regions without coverage as well as specific genomic regions were assessed. SNP calls on the different sequencing machines were compared, and the benefits of combining read information from different instruments were evaluated. Additionally, somatic mutations were analyzed.
The most striking result is the poor coverage of GC-rich regions by SOLiD 4 and 5500xl SOLiD, discouraging their use in particular for methylation experiments and exome sequencing. In contrast, Complete Genomics seems the least affected by GC content and shows the most comprehensive coverage of many genomic regions, except for short repeats. HiSeq2000 exhibits the most even genome-wide coverage distribution and the least sample-to-sample variation, while consistently achieving the highest sensitivity in SNP calling. A combination of read data from different technologies is shown to entail limited improvement in most cases, and is advisable only for very specific applications. Finally, the comparison of somatic variation confirms that calling somatic alterations is still a big challenge, which is due in particular to low allele frequency. In summary, this comparative study illustrates the assets and drawbacks of each individual machine and can be used as a guide to find the most suitable platform for a specific experimental goal.
In the first step of planet formation micrometer-sized dust grains grow in a protoplanetary disk through collisional sticking. This growth becomes inefficient at several centimeters up to meters in size, depending on the distance to the star. The resulting agglomerates are concentrated by turbulence in the disk up to densities at which they fragment through self-gravitaty to $100\,\textrm{km}$ sized planetesimals.
In my PhD thesis I simulate the concentration of dust particles in the turbulent gas flow of protoplanetary disks. Here I treat the gas as a fluid and solve the magnetohydrodynamic equations with the {\sc Pencil code}. Dust particles are simulated as non-collisional point particles, decoupled from the grid. At first I test the particle representation of the {\sc Pencil code} by comparing a Rayleigh-Taylor instability (RTI) simulation of a dust-laden fluid with a classical two-layer fluid RTI simulation. Additionally I simulate the sedimentation of a dust clump in a fluid which can be compared with experiments.
Further I study zonal flows and the resulting long-lived axisymmetric pressure bumps that are created in magnetorotational instability simulations. Zonal flows are described by annuli of gas rotating faster or slower than the pressure-supported Keplerian rotation. They are created by temporal and spacial variances in the magnetic pressure. In a convergence study I measured a typical radial size of $5$ to $7$ vertical gas pressure scale heights with a life time of up to $50$ local orbits ($T_{\textrm{orb}} = 2 \pi \Omega^{-1}$). Particles get captured by these pressure bumps. For dust particles with a friction time $\tau_{\textrm{f}} \ge 0.1 \Omega^{-1}$ I found concentrations that are some hundred times higher than initially. Larger particles ($\tau_{\textrm{f}} \ge 0.5 \Omega^{-1}$) reach densities $10$,$000$ times higher than their initial densities, sufficient to trigger secondary instabilities such as the streaming instability and gravitational collapse.
I study the streaming instability in a zonal flow environment in simulations of higher resolution including the back-reaction drag from particles to the gas. These simulations show that the axisymmetric pressure bumps can accumulate enough particles to trigger the streaming instability, even with small particles ($\tau_{\textrm{f}} = 0.1 \Omega^{-1}$). Allowing for self-gravity dust clumps form, yet they are not stable against tidal forces. This is due to the insufficient resolution here.
For my last project I studied the final collapse of a spherical dust cloud with a much higher resolution than in the above simulations. In this study I investigate a dust cloud with an initial density ranging from Roche density $\rho\Roche$ down to $10^{-3} \rho_{\textrm{Roche}}$. Dust spheres with $0.1 \rho_{\textrm{Roche}}$, like I typically get from large scale simulations, fragment to a swarm of bound objects with a size distribution that is comparable to the observed size distribution of asteroids.
The problems of chaos and relaxation have a fundamental importance in the study of many-body classical and quantum systems. We investigate some of the issues related to these problems numerically in classical and quantum spin systems. New results reported in this thesis include: (i) A remarkably simple algorithm for discriminating chaotic from nonchaotic behavior in classical systems using a time series of one macroscopic observable. The effectiveness of this algorithm stems from the qualitative differences in the power spectra of chaotic and nonchaotic systems. (ii) A modified version of the Onsager regression relation applicable to pure quantum states. (iii) An efficient algorithm for computing the infinitetemperature time correlation functions in systems with large Hilbert spaces. (iv) Absence of exponential sensitivity to small perturbations in macroscopic nonintegrable systems of spins 1/2 . Such a behavior is contrasted with the exponential sensitivity to small perturbations in chaotic classical spin systems. (v) Accurate numerical investigations of free induction decay and spin diffusion in certain spin lattices. The consequences of these results have implications for the foundations of statistical mechanics or practical problems such as computing the long-time behavior of the free induction decay in solids.
1860 fanden Gustav R. Kirchhoff und Robert W. Bunsen in ihrem Heidelberger Labor heraus, dass das durch ein Prisma geleitete Licht erhitzter chemischer Elemente charakterische Linien erzeugt. Diese Linien erlauben die eindeutige Fernidentifizierung der beteiligten Stoffe. Die beiden Forscher publizierten ihre Entdeckung in den Annalen der Physik und Chemie.
Der Krautturm des Heidelberger Schlosses, heute als Gesprengter Turm“ bekannt, wurde 1693 im Zuge des Pfälzer Erbfolgekriegs gesprengt. Der massive Rundturm aus rotem Sandstein, der eine Mauerstärke bis zu 6,50 m besitzt, wurde dabei teilweise zerstört, wobei sich ein riesiges abgesprengtes Mauerstück heute noch eindrucksvoll gegen den Turm legt. Es schien lohnenswert zu sein, mit einem Laserscanner ein maßstabsgetreues Modell des Turms aufzunehmen, um zu testen, inwiefern es möglich ist, diesen Turm virtuell wieder zusammenzusetzen. Zu diesem Zweck wurde der Turm in drei Tagen rundum von verschiedenen Standpunkten aus gescannt. Das Projekt war eine Kooperation von Doktoranden der Fachbereiche Wissenschaftliches Rechnen, Physische Geografie und Europäische Kunstgeschichte der Universität Heidelberg und resultierte aus dem Interesse der Beteiligten, Anwendungen und Möglichkeiten der jeweils anderen Fachbereiche kennenzulernen.
Rotationally symmetric objects commonly occur at archæological finds. Instead of creating 2D images for documentation purposes by manual drawing or photographic methods, we propose a method based on digitally colored surface models that are acquired by 3D scanners, thereby including color information. We then transform these highly-detailed meshes using simple geometrical objects such as cones and spheres and unwrap the objects onto a plane. Our method can handle curved vessel profiles by dividing the surface into multiple segments and approximating each segment with a cone frustum that serves as an auxiliary surface. In order to minimize distortions, we introduce a simple quality measure based on distances of points to a fitted cone. We then extend our method to approximately spherical objects by fitting a sphere on the surface of the object and applying a map projection, namely the equirectangular projection known from cartography. Our implementation generates true-to-scale images from triangular meshes. Exemplary results demonstrate our methods on real objects, ranging from small and medium-sized objects such as clay cones from the Ancient Orient and figural friezes of Greek vessels to extremely large objects such as the remains of a cylindrical tower of Heidelberg Castle.
Die Entwicklung Nanomedizin-basierter Therapeutika (Nanomedizin = medizinsche Anwendung der Nanotechnologie) hat zum Ziel, pharmakologische Substanzen zielgerichtet zu ihrem Wirkort zu transportieren und so die mangelnde Gewebsspezifität konventioneller Wirkstoffe zu überwinden. Humanes Serumalbumin bietet sich als Basis für die Entwicklung verschiedenster Nanotherapeutika an, unter anderem da für fluoreszenz-markiertes HSA eine selektive Anreicherung im Gewebe solider Tumoren nachgewiesen werden konnte. Aufgrund der hohen metabolischen Aktivität eines Tumors und der Tatsache, dass Patienten mit progressiven malignen Erkrankungen häufig eine Kachexie und Hypoalbuminämie entwickeln, postulierten Stehle et al., dass HSA den Tumorzellen als Energie-und Stickstoffquelle dient und der Tumor selbst somit den Hauptort des Albumin-Katabolismus im Körper darstellt. In aufgereinigten Membranpräparationen von humanen Tumorzellen wurden Calreticulin und hnRNP A2/B1 als Albumin-bindende Proteine identifiziert. Zusammengenommen legt dies eine Beteilung dieser beiden Proteine an der HSA-Aufnahme in Tumorzellen nahe. In einem ersten Teil der Arbeit wurde die Anwesenheit von Calreticulin und hnRNP A2/B1 auf der Plasmamembran von lebenden Zellen durch Antikörperfärbung und FACS-Analyse überprüft. Keines der beiden Proteine konnte auf der Oberfläche von CCRF-CEM Zellen, einer humanen lymphoblastischen T-Zell Leukämie, nachgewiesen werden. Obwohl Calreticulin mittlerweile auf der Zellmembran von pre-apoptotischen, anthracyclin-vorbehandelten Zellen nachgewiesen wurde, konnte auch nach Induktion einer Apoptose durch Mitoxantron kein membranständiges Calreticulin auf CCRF-CEM Zellen detektiert werden. Zur funktionellen Analyse der zellulären Albumin-Aufnahme wurde die Beteiligung der Clathrin-vermittelten Endozytose (CME), einem gut charakterisierten Mechanismus der Rezeptor-vermittelten Endozytose, an der Internalisierung von Alexa488-BSA in A240286S Zellen, einem humanen Lungenadenocarcinom, durch FACS-Analyse untersucht. Die Hemmung der CME wurde durch hypertone Bedingungen, intrazellulären Kaliummangel, Chlorpromazin und transiente Transfektion mit siRNA gegen Clathrin Heavy Chain, einem essentiellen Protein der CME, erreicht. Die reduzierte Aufnahme von Alexa488-Transferrin, einem Protein das selektiv durch den Transferrin-Rezeptor über CME internalisiert wird, diente zur Überprüfung der erzielten Hemmung. Auch etablierte Methoden zur Hemmung einzelner Endozytose-Mechanismen sind nicht unbedingt selektiv und können einen Einfluss auf andere Mechanismen haben, zudem können sie in unterschiedlichen Zelllinien auch keine oder nur eine geringe Wirksamkeit zeigen sowie gegebenenfalls die Aufnahme des untersuchten Markers sogar verstärken. Die Ergebnisse der CME-Inhibition in A240286S Zellen bestätigen dies, da hypertone Bedingungen während der Inkubation zwar die Aufnahme von Alexa488-Transferrin und Alexa488-BSA hemmten, gleichzeitig aber auch die Endozytose von Dextran, einem Marker für die unspezifische Fluid-Phase Endozytose, reduzierten. Weder intrazellulärer Kaliummangel noch Chlorpromazin zeigten eine Einfluss auf die Alexa488-Transferrin-Aufnahme. Der erfolgreiche Knock-Down von Clathrin Heavy Chain (CHC) konnte durch Western Blotting nachgewiesen werden und führte zu einer deutlichen Verminderung der CME von Alexa488-Transferrin. Die Aufnahme von Alexa488-BSA hingegen zeigt nur eine geringe Hemmung durch siRNA CHC. Clathrin-vermittelte Endozytose leistet demzufolge entweder keinen oder nur einen geringen Beitrag zur Aufnahme von Albumin in A240286S Zellen. Zusätzlich zu der funktionalen Analyse der Albumin-Aufnahme wurde ein Protokol zur Isolation von HSA-Bindungspartnern aus der Plasmamembran von humanen Tumorzellen etabliert. Ein Teil des Protokols war die Synthese eines HSA-Konjugates, das sowohl einen photoaktivierbaren Crosslinker (SDAD) zur Konjugation an Albumin-bindende Proteine der Plasmamembran als auch einen Biotin-Tag zur selektiven Aufreinigung der Crosslinking-Produkte nach der Zelllyse enthielt. Zur Validierung des Protokols wurde ein vergleichbares Transferrin-Konjugat hergestellt, das zur Isolation des Transferrin-Rezeptors CD71 dienen sollte. Die Synthese beider Konjugate war erfolgreich, insbesondere enthielten beide Endprodukte keine hochmolekularen Verunreinigungen, reagierten nicht mit sich selbst und das Biotin-Tag war auch unter UV-Belichtung stabil. Durch FACS-Analyse konnten für beide Konjugate eine extrazelluläre Bindung und ein UV-induziertes Crosslinking nachgewiesen werden. Ein Western Blotting der Zelllysate bestätigte das erfolgreiche Crosslinking an Bindungspartner der Zellmembran. Das Protokoll zur Immunopräzipitation über die Streptavidin-Biotin-Interaktion wurde für beide Konjugate überprüft. Eine Isolation des CD71 aus A240286S Zellen durch das Transferrin-Konjugates war jedoch bisher nicht erfolgreich. Eine rezeptorvermittelte Endozytose von HSA bzw. ein diskretes Albumin-bindendes Protein auf der Zellmembran von humanen Tumorzellen konnte in dieser Arbeit nicht nachgewiesen werden. Der Schluss liegt nahe, dass die Anreicherung von HSA-Konjugaten in soliden Tumoren vor allem auf dem „Enhanced Permeability and Retention“ Effekt, wie er auch für andere Nanotherapeutika nachgewiesen wurde, und auf der langen Plasmahalbwertszeit des Proteins basiert.
Secretory membrane trafficking is essential for the homeostasis of most cellular organelles and mediating the secretion of a variety of cargo proteins, including digestive enzymes, hormones, growth factors, antibodies, extracellular matrix proteins and many other proteins. Small GTPases, particularly Rab and Arf GTPases, have been shown to play key roles in regulating the transport of secretory cargo proteins. Nevertheless, the biological role of each individual small GTPase is not fully elucidated yet. To address that, I performed microscopy-based RNAi screening to identify small GTPases that are involved in the biosynthetic transport of a secretory cargo protein ts-O45-G. From RNAi screen in HeLa cells, I identified 18 potential regulators, with 14 of them not associated with this cellular function before. One of the hits was chosen for further characterization. Rab42 is an interesting hit in that it has not been characterized and the retroposed transcript of Rab42 is expressed in HeLa cells. After having designed specific siRNAs only targeting Rab42, it is shown that specific down-regulation of Rab42 inhibited secretion of ts-O45-G from the ER to the PM. Moreover, I showed that RNAi-mediated inhibition of ts-O45-G transport can be rescued by over-expressing a siRNA-resistant form of Rab42. By invesitigating more closely which particular stage of ts-O45-G secretion is affected, I have identified that trafficking from the TGN to the PM is affected in a more pronounced manner. Immunofluorescence analysis showed that over-expressed Rab42 localized preferentially to the cytoplasm and PM, and, to a lesser extent, to the intracellular punctual structures, which are in close proximity to perinuclear region. During my thesis work, yet another interesting and poorly characterized Rab, Rab40c, was identified as a novel regulator for PC I secretion from our RNAi screens in NIH3T3 fibroblasts. Down-regulation of Rab40c inhibited secretion of PC I and ts-O45-G in NIH3T3 fibroblasts, albeit it had no consistent effect on ts-O45-G secretion in HeLa cells. I showed that PC I secretion is blocked in the early secretory pathway in NIH3T3 fibroblasts depleted for Rab40c. In addition, I observed cell type-specific localization of Rab40c by comparing the localization of Rab40c in NIH3T3 fibroblasts and HeLa cells. In NIH3T3 fibroblasts FLAG-tagged Rab40c localized to the Golgi complex, whereas in HeLa cells FLAG-tagged Rab40c localized to perinuclear tubulovesicular structures and cytoplasmic structures. Furthermore, Rab40c is an interesting Rab GTPase in that it contains a GTPase domain and a SOCS-box domain. By immunofluorescence analysis, it has been shown that SOCS-box domain is crucial for the localization of Rab40c at the Golgi complex. Taken together, my thesis work has identified novel regulators of secretory membrane trafficking, and serves as a basis for further functional characterization.
In human colon cancer only a small subfraction of all tumor cells is able to rebuild the tumor in immunodeficient mice. It has been hypothesized that the proliferative activity of these tumor initiating cells (TIC) may differ from the bulk of the tumor cells and that mitotic quiescence of TIC may contribute to chemotherapy resistance or relapse after treatment. By genetic marking, it has previously been shown that a variable proportion of all human TIC contributed to tumor xenograft formation only late after serial transplantation suggesting that these delayed contributing TIC indeed might have been quiescent in primary recipient mice. In order to investigate the cell cycle and proliferative activity of human colon TIC in vitro and in vivo, human colon cancer patient samples were dissociated and cultured under serum free conditions favoring the outgrowth of tumor spheres enriched for TIC. The CFSE label-retaining assay was used to analyze the proliferative activity of human colon TIC in vitro. It allowed discrimination of fast (F), slow (S) and rarely dividing (R) cell fractions suggesting that a rarely dividing population of human colon TIC might exist in vivo as well. Cell surface markers previously associated with tumor initiating potential (CD133, CD44, EpCAM and CD166) were equally expressed in all proliferative subfractions. A limiting dilution assay confirmed the self-renewal potential of spheroid cells. Furthermore, it revealed that the frequency of sphere forming cells (SFC) was similar in the fast, slow and rarely dividing fraction within individual sphere lines, demonstrating that the vast majority of all SFC were rapidly cycling in vitro. To assess the in vivo tumor initiating potential and self-renewal ability, equal cell numbers of sorted R, S and F cells were transplanted into immunodeficient mice. All sorted cell fractions of three patients formed tumors, irrespective of their proliferative kinetics in vitro. Moreover, the majority of cells within serially transplanted tumors originating from CFSE+ fractions lost fluorescence intensity indicating that they actively cycled after transplantation. Hoechst/Pyronin-staining of dissociated sphere cells allowed investigation of their cell cycle status. Equal numbers of G1-, S/G2/M- and G0-cells were transplanted under the kidney capsule of immunodeficient mice. Each cell fraction comprised self-renewing, human colon TIC as shown by a serial transplantation assay. In order to investigate the proliferative activity of human colon TIC within an established tumor in vivo, intra-tumoral cell divisions were tracked using a genetic high resolution label-retaining assay. A tetracycline-regulated H2B-GFP expression system was implemented into spheroid cells by lentiviral transduction prior to transplantation. H2B-GFP-expression was suppressed after establishment of the tumor microenvironment. Further cell divisions dilute the GFP-label and thereby enable Summary II analysis of the cell’s proliferative activity. FACS analysis of formed tumors revealed fast, slow and rarely dividing cell fractions in vivo. All cell fractions harbored selfrenewing, human colon TIC as shown by serial transplantation. Interestingly, only quiescent TIC showed a polyclonal contribution to tumor formation in mice. A proportion of quiescent TIC might have been activated to proliferate upon chemotherapeutic treatment. This study demonstrates that human colon cancer harbours tumor initiating cells with differing cell cycle status and proliferative activity. Self-renewing colon TIC were present in all cell cycle phases demonstrating that the tumor initiating potential is not restricted to a dormant cell cycle status. A rarely dividing population of human colon TIC derived from different patient samples exists in vitro and in vivo. However, the majority of colon TIC rapidly divided in sphere cultures as well as in vivo. Colon TIC were found to be enriched in the quiescent population and were recruited to tumor formation upon chemotherapeutic treatment. Our results provide basis for a better understanding of quiescence and proliferation of human colon TIC. This will hopefully lead to the development of innovative treatment strategies directed against colon cancer initiating cells.
Ketogenic diet is protective in models of ischemic stroke and neurodegenerative diseases. Currently, clinical trials are testing the efficacy of this diet in neurodegenerative diseases but its mode of action is still unclear. The ketone body β-hydroxybutyrate (BHB) is the endogenous agonist of the hydroxy-carboxylic acid receptor 2 (HCA2, GPR109A) which is expressed in various immune cells; therefore, we tested the potential involvement of this receptor in a mouse model of ischemic stroke. The protective effect of ketogenic diet and BHB was lost in Hca2-/- mice, demonstrating that HCA2 receptors are responsible for neuroprotection. Similarly, nicotinic acid, a HCA2 agonist, reduced the infarct size via a HCA2-mediated mechanism. Immunohistochemical analysis of immune cells in Hca2mRFP transgenic mice revealed HCA2 expression in monocytes/macrophages. Bone marrow transplantation demonstrated that HCA2 on monocytes/macrophages is required for the protective effect. Activation of HCA2 receptors induced a neuroprotective phenotype of monocytes/macrophages that depended on PGD2 production by COX-1 and the hematopoietic PGD2 synthase. Our data reveal that HCA2 activation by dietary or pharmacological means instructs monocytes/macrophages to carry a neuroprotective signal to the brain which can be used therapeutically.
Metastatic melanoma is a severe disease with a high rate of lethality. It is known to be resistant to current therapies. Since melanoma is immunogenic the development of an immunotherapy can be a promising possibility to enhance an antitumor effect in vivo. Memory T cells (MTC) have abilities to respond quicker to antigens and to release a broader spectrum of cytokines than naïve T cells. The ret transgenic mouse melanoma model was used in this study since it resembles the pathological situation of human melanoma in contrast to transplantation models. It has been previously shown that the bone marrow (BM) is a major site for the persistence of tumor-specific MTC in cancer patients. In addition, melanoma-specific MTC were also found in the BM of ret transgenic mice without macroscopic tumors. Therefore, we isolated CD3+ cells from the BM of ret transgenic mice with and without tumors. Therefore, we isolated CD3+ T cells from the BM of ret transgenic mice with and without tumors. After a 40 h ex vivo stimulation of bone marrow-derived T cells with melanoma antigen-loaded DC, which were treated with anti-PD-L1 antibody overnight, T cells revealed a higher IFN-γ production and an increased T cell activation in vitro. Moreover, activated CD8+ T cells displayed mainly a central memory phenotype and an increased level of CD69 expression after 40 h of co-culture with DC. Labeled melanoma-specific, stimulated memory T cells from ret transgenic mice migrated to skin tumor lesions, metastatic lymph nodes (LN), BM and spleen after adoptive transfer into ret transgenic tumor-bearing mice. A similar migration pattern was detected using stimulated TRP-2 TCR transgenic effector T cells. Furthermore, migrated CD8+ T cells showed an increase in effector memory (TEM) and effector phenotype at day 7 post injection and a decrease of central memory and naive CD8+ T cells within tumor lesions, whereas at day 3, central memory, effector memory, naive and effector CD8+ T cells were equally distributed. To investigate the anti-tumor activity of melanoma-specific memory T cells in vivo we adoptively transferred MTC, which were prior activated with DC, into tumor-bearing mice by i.c. injections. Mice receiving memory T cells showed a significantly longer survival compared to the control group. Mice receiving the phosphodiesterase-5 inhibitor sildenafil and adoptive transfer of MTCs displayed a significantly higher survival rate than mice treated with sildenafil or PBS only. We suggest that an adoptive transfer of melanoma-specific memory T cells activated with antigen-loaded DC, which were pre-treated with anti-PD-L1 antibodies, can enhance an anti-tumor response and therapeutic efficacy in vivo.
Lymphocytes express highly specific receptors to recognize pathogens and malignant cells. At the same time, lymphocytes recognizing self-antigens also develop. These need tight regulation in order to prevent autoimmunity. The elimination or control of self-reactive cells is ensured by central and peripheral tolerance mechanisms. A detailed understanding of tolerance induction and maintenance is indispensable for the establishment of novel therapeutic strategies to treat autoimmune diseases and to prevent rejection of transplanted tissue. Recently, we identified Dickkopf 3 (Dkk3) as a novel immune-mediator in a transgenic model of peripheral T cell tolerance. To further clarify the immune-modulatory capacity of Dkk3 we investigated the influence of this secreted protein on T and B cell function. Experimental autoimmune encephalomyelitis (EAE) is a T cell mediated mouse model of the human autoimmune disease multiple sclerosis. Dkk3 deficient mice developed more persistent symptoms of EAE with increased numbers of brain infiltrating CD4 and CD8 T cells as well as myeloid cells in comparison to wild type mice. Lack of environmental Dkk3 was responsible for the observed phenotype as shown by adoptive T cell transfers using Dkk3 deficient T cells. Disease persistence in Dkk3 deficient mice was associated with an altered local cytokine profile. Absence of Dkk3 did not change the differentiation of Th1 and Th17 T cells but led to increased levels of Th2 associated cytokines. The role of Dkk3 on B cell function was analyzed by investigating antibody and cytokine secretion. Both assay systems showed that B1 related functions were increased in Dkk3 deficient mice, such as antibody secretion to the antigens LPS and phosphorylcholine and IL-10 production. Indeed, Dkk3 deficient mice yielded increased numbers of B1 cells. B2 cell development was supported by Dkk3 at the step from the pre- to immature B cell stage, whereas Dkk3 negatively regulated B1 cell generation. The murine MRL/LPR strain, a mouse model of human systemic lupus erythematosus (SLE), was used to investigate the role of Dkk3 in a B cell mediated autoimmune disease. Treatment of these mice with a Dkk3 neutralizing antibody led to increased numbers of B1 cells and higher pathology of SLE, including enlarged lymph nodes, higher levels of autoantibodies, increased glomerulonephritis and inflammation in pancreas and lung. Among the Dkk protein family, only Dkk3 and Soggy share the unique sgy-domain. To address the question whether this sgy-domain may be responsible for the immune-modulatory capacity we investigated immune functions of Soggy deficient mice. Alterations in the T cell compartment were observed, such as decreased numbers of regulatory T cells in comparison to wild type mice. Furthermore, Soggy deficiency led to an earlier onset and stronger symptoms of EAE in comparison to the disease course in C57BL/6 and Dkk3-/- mice. In summary, Dkk3 regulates T and B cell mediated autoimmunity. In addition, we were able to identify Soggy as a novel immune-mediator suggesting that the shared sgy-domain is contributing to the immune-suppressive capacity of Dkk3 and Soggy.
This thesis deals with the remote sensing and the flux calculation of atmospheric trace gases, using Differential Optical Absorption Spectroscopy (DOAS). Since 2010, within the CARIBIC project (Civil Aircraft for the Regular Investigation of the atmosphere Based on an Instrument Container), a new DOAS instrument is installed in the cargo compartment of a passenger aircraft once per month as part of a fully automated measurement container. With this instrument, nitrogen dioxide (NO2), sulfphur dioxide (SO2), bromine oxide (BrO), nitrous acid (HONO), formaldehyde (HCHO) and ozone (O3) are measured. The results of these measurements are presented with focus on SO2 and NO2, which were observed in the downwind plumes of large industrial plants and cities. Using flux calculations, the emission of SO2 from a nickel smelter in Norilsk (Siberia) and the NO2 emission of the city of Paris are estimated. Thereby, the uncertainty factors are discussed and comparison with satellite data are performed. The question is dealt with, whether such calculations can be used to quantify further emission sources using similar instruments onboard additional passenger aircraft.
Although the process by which galaxies obtain the gas needed for star-formation is amongst the most fundamental processes related to the formation of baryonic structure in the universe, there is very little in the way of empirical evidence with which to constrain theoretical models. In particular, the postulated environmental dependencies of this process, although widely modeled, remain largely unconstrained. In this work, I present the first detailed, quantitative analysis of the environmental effects on the process of gas-fueling in which the relevant effects of the galaxy - intergalactic medium interaction have been isolated from other potential environmental in uences. In the context of this analysis, a new robust method for selecting morphologically defined samples of galaxies by photometric proxies is developed, as well a powerful new method for correcting the UV/optical emission of samples of spiral galaxies for the effects of attenuation by dust located in their disks. Combining these tools with the data from the GAMA survey, in particular the galaxy group catalog, I present a detailed analysis of the environmental dependencies of gas-fueling. The results obtained require a fundamental re-evaluation of the assumptions concerning the fueling of satellite galaxies and the effects of active galactic nuclei.
We study tensor product decompositions of representations of the General Linear Supergroup Gl(m|n). We show that the quotient of Rep(Gl(m|n),\epsilon)$ by the tensor ideal of negligible representations is the representation category of a pro-reductive supergroup G red. In the Gl(m|1)-case we show G red = Gl(m-1) \times Gl(1) \times Gl(1). In the general case we study the image of the canonical tensor functor Fmn from Deligne's interpolating category Rep (Gl m-n) to Rep(Gl(m|n),\epsilon). We determine the image of indecomposable elements under Fmn. This implies tensor product decompositions between projective modules and between certain irreducible modules, including all irreducible representations in the Gl(m|1)-case. Using techniques from Deligne's category we derive a closed formula for the tensor product of two maximally atypical irreducible Gl(2|2)-representations. We study cohomological tensor functors DS : Rep(Gl(m|m), epsilon) -> Rep(Gl(m-1|m-1)) and describe the image of an irreducible element under DS. At the end we explain how these results can be used to determine the pro-reductive group G L \hookrightarrow Gl(m|m) red corresponding to the subcategory Rep(G L, epsilon) generated by the image of an irreducible element L in Rep(Gl(m|m) red, epsilon).
Gefäßerkrankungen sind nach wie vor eine der vorwiegenden Todesursachen in den Industrienationen, wobei statistisch gesehen chronischer Bluthochdruck (Hypertension) als bedeutender Risikofaktor für der Bildung arteriosklerotischer Plaques, einen großen Anteil daran hat. Eine Charakteristik der Hypertension ist insbesondere ein chronisch erhöhter diastolischer Blutdruck, der den Anstieg des peripheren (Gefäß-)Widerstandes widerspiegelt und lokal die transmurale Druckdifferenz für arterielle Gefäße und damit deren Wandspannung erhöht. In der Folge kommt es zu einem Umbauvorgang der Gefäßwand, der dem Gesetz von Laplace folgend die Wandspannung senkt, langfristig aber zu einer Abnahme des intraluminalen Durchmessers, zur Verdickung der Gefäßwand und zu einer Zunahme der Steifigkeit der Gefäße führt. Hierbei ändert sich der Phänotyp der glatten Gefäßmuskelzellen von ruhend und kontraktil zu aktiviert und synthetisch, was eine Voraussetzung für die nachfolgende vaskuläre Remodellierung der Gefäßwand ist. Nach wie vor ist jedoch unklar durch welche Stimuli diese Anpassung des glattmuskulären Phänotyps und damit die Reorganisation der Gefäßwandarchitektur initiiert werden. Ausgehend von der Hypothese, dass die Erhöhung der Wandspannung selbst Mechanismen antreibt, die entscheidende Stabilisationsfaktoren für den kontraktilen Phänotyp außer Kraft setzen, sollte in dieser Arbeit untersucht werden, welchen Einfluss eine erhöhte Wandspannung oder Bluthochdruck auf die Funktion von Myokardin hat – einem transkriptionellen Koaktivator des Serum response Faktors (SRF), der die Expression von Genprodukten des kontraktilen Apparates wie smooth muscle actin (SMA) und Calponin reguliert. Die Bildung eines ternären Komplexes durch SRF und Myokardin stabilisiert dabei die Kontraktilität der glatten Muskelzellen. Im Zuge der Arbeiten wies die Analyse siRNA-vermittelter Inhibition der Myokardin-mRNA eine Zunahme der Proliferation und Reduktion der Kontraktilität der glatten Muskelzellen nach und zeigte damit die Bedeutung dieses Koaktivators für die Aufrechterhaltung des kontraktilen und ruhenden Phänotyps dieser Zellen. Zudem wurde erstmalig gezeigt, dass die Myokardinmenge in medialen glatten Muskelzellen remodellierender Gefäße von hypertensiven Mäusen abnimmt und gleichzeitig die Proliferation dieser Zellen zunimmt. Darüber hinaus belegt die Untersuchung isolierter, druckperfundierter Gefäße und die zyklische Dehnung (eine wichtige Komponente der Wandspannung) glatter Gefäßmuskelzellen eine Abnahme der mRNA- sowie Proteinsynthese von Myokardin. Die weitere mechanistische Analyse dieser Beobachtungen impliziert einen bis dato unbekannten Signalweg, der durch erhöhte Wandspannung zur Reduktion von Myokardin und damit der Kontraktilität der vaskulären glatten Muskelzellen führt: Die Dehnung glatter Gefäßmuskelzellen hat die Serinphosphorylierung von Myokardin durch ERK1/2 zur Folge und initiiert so die Dissoziation - 16 - von Myokardin vom SRF. Phosphoryliertes Myokardin bindet intranukleär an HDAC4/HDAC5 und wird durch das Chaperon 14-3-3 exportiert. Darauffolgend wird Myokardin im Zytoplasma durch die E3-Ligase CHIP ubiquitinyliert und durch das 26S-Proteasom abgebaut. Zusammenfassend wird durch diese Arbeit erstmalig gezeigt, dass (I) die Steigerung der Wandspannung ausreichend für die Inaktivierung von Myokardin ist, (II) die dehnungsinduzierte Myokardin-Translokation vom Kern ins Zytoplasma von ERK1/2 und Klasse II HDACs/14-3-3 abhängt und (III) in dehnungsstimulierten Gefäßmuskelzellen zytoplasmatisch lokalisiertes Myokardin proteasomal abgebaut wird (siehe zusammfassendes Schaubild). Mit diesem Mechanismus wird die Funktion eines stabilisierenden Faktors des glattmuskulären kontraktilen Phänotyps blockiert und somit die Ausbildung eines aktivierten und synthetischen Phänotyps dieser Zellen begünstigt. Diese Ergebnisse weisen Myokardin eine kritische, regulative Funktion im Rahmen der Aufrechterhaltung der vaskulären Homöostase und Unterdrückung maladaptiver, vaskulärer Remodellierungsprozesse zu und zeigen gleichzeitig neue Angriffspunkte für die Behandlung kardiovaskulärer Erkrankungen auf.
This work introduces two investigations on possible new weak lensing applications. In the first part, I present a study on the possibility of detecting baryon acoustic oscillations by means of 3d weak lensing (3dWL). Basing our analysis on a Fisher matrix approach, we quantify the uncertainty on inferring the amplitude of the power spectrum wiggles with 3dWL. Ultimately, we find that surveys like Euclid and DES should be able to detect, respectively, the first four and three oscillations, with errors reaching the 1% or 10% of the amplitude for the first two wiggles in the case of Euclid. The second part of this work focuses on the study of primordial non-Gaussianities with a classical weak lensing approach. We study inflationary bi- and trispectra, the strentgh of their signals, and the consequences of fitting data with a wrong type of bispectrum on the inferred on fNL. We conclude that contraints on fNL are not competitive with the ones from CMB, but nonetheless valuable in case of a scale-dependent fNL. Lastly, we quantify lensing ability to test the Suyama-Yamaguchi inequality, and ascertain that Euclid could give evidence in favour or against the inequality for large non-Gaussianity values (tauNL > 10^5 or fNL > 10^2).
Cerebral malaria is caused by a complicated series of immune reactions in the host, marked by inflammatory immune responses, margination of leukocytes and parasitized erythrocytes in cerebral vessels leading to breakdown of the blood-brain-barrier. Studies of the immune responses that lead to human cerebral malaria are limited since patients typically present once symptoms have commenced. Along with ethical considerations, this has led to the immunopathogenesis of cerebral malaria being studied in the rodent model. Such studies have generally overlooked the very early stage of infection, during which the malaria parasite invades the liver, despite some evidence that early immune responses and intrahepatic attenuated infections, such as caused by the RTS, S vaccine, play a role in preventing cerebral pathology.
This thesis describes the development of a model that attenuates infection at a very early stage prior to the onset of blood infection by the subtherapeutic administration of isopentaquine, an 8-aminoquinoline. Such chemical attenuation of the parasite at the very early, clinically silent liver stage suppresses parasite development, delays the time until parasites establish blood-stage infection and provokes an altered host immune response, altering immunopathogenesis and protecting from cerebral disease. This early response is a pro-inflammatory, cell-mediated one with increased T-cell activation in liver and spleen, elevated numbers of effector T cells, cytokine-secreting T cells and proliferating, multifunctional T cells producing pro-inflammatory cytokines. The response destabilizes the usual series of events that leads to cerebral pathology, by downregulating inflammatory responses and T-cell activation at late infection. Dendritic cell numbers, T-cell activation and infiltration of CD8+ T cells to the brain are decreased in later infection, mediated by the anti-inflammatory cytokine IL-10.
These data indicate that liver-stage-directed early immune responses can moderate the overall downstream host immune response and modulate severe malaria outcome. Strikingly, CD8+ T cells isolated from the spleen as early as day 2 post infection are responsible for protection. Protection can be transferred to naïve animals by adoptive transfer of lymphocytes from the spleen at very early infection, but not when CD8+ T cells are depleted.
The reliance of this phenotype on CD8+ T cells and the transferability of protection are of particular interest, especially since these cells are isolated so early on in infection. Neither attenuated infections or early T cell responses have been studied in relation to cerebral pathology before and this is the first evidence that they can influence the course of the downstream systemic immune response and alter cerebral pathology. This draws parallels with the RTS, S vaccine, which is designed to elicit strong CD8+ T cell responses against the parasite in the liver, and produces similar protection exclusively against severe malaria, including cerebral malaria.
This work has larger implications in dissecting the complex sequence of inter-related events that form the immunological basis of human cerebral (malaria) pathology and uncovers a relationship in both localization and timing of anti-parasitic T-cell responses involved in the immunopathogenesis of cerebral malaria, presenting an insight into the potential role of the preerythrocytic response in tempering downstream cerebral immunopathogenesis. These data support the notion that Th1 cellular responses represent a kill or cure response to Plasmodium that must be tightly controlled in both a spatially and temporally specific manner.
The research field of functional magnetic resonance imaging (fMRI) has made possible a remarkable progress in the understanding of the human brain enabling neuroscientists to study spatio-temporal alterations in the healthy and the diseased brain. While current theories of schizophrenia stress the critical role that plays aberrant connectivity among brain regions, other theories point towards the crucial role that plays functional excitation-inhibition (E-I) balance. Indeed, recent neuroscientific research has revealed increasing evidence that taking functional brain connectivity into account is essential to understand how the human brain works, and many studies have reviewed that serious behavioural impairments in mental disorders such as schizophrenia result from increases in the functional (E-I) balance within the neural microcircuitry.
Particularly, the connection between the dorsolateral prefrontal cortex (DLPFC) and the hippocampal formation (HF) during working memory (WM) was found to be increased in carriers of schizophrenia risk genes and patients. However, less is known about causality, i.e. which region drives the altered connection. Stochastic Dynamic Causal Modelling (sDCM) is a novel mathematical algorithm for studying the causal connectivity among higher cognitive brain regions from fMRI data. The main purpose of this study is to identify alterations on genetic risk carriers and patients from the DLPFC-HF network estimated with sDCM and describe how these alterations have an impact on the behavior. Over the study, we strive to give to the sDCM parameter estimates a neurobiological explanation by linking the concepts of causal connectivity with functional (E-I) balance.
In this work, we applied this methodology in two samples by constructing a systematic set of sDCMs describing interactions between right DLPFC and left HF. In a first sample, 180 healthy subjects were measured by fMRI during a standard working memory N-Back task at three different sites (Mannheim, Bonn, Berlin; each with 60 participants). Bayes Model Selection (BMS) revealed the same causal pattern or winning model across the three sites, with the 2-Back working memory condition as driving input to both DLPFC and HF and with a connection from DLPFC to HF. Furthermore, a genome-wide risk variant for schizophrenia: ZNF804A (rs1344706), showed a strong impact on the DLPFC-HF network. On the one hand, risk homozygotes showed higher effective connectivity or higher functional (E-I) balance between DLPFC and HF. On the other hand, risk allele carriers showed higher functional (E-I) balance on the self-connection in the DLPFC. In a second sample, 33 schizophrenia patients were measured by fMRI during the same working memory N-Back task. We pair-wise matched healthy volunteers of the first sample and patients and applied the same methodology. BMS revealed the same winning model in patients but sDCM parameter estimates differed significantly between groups. Patients showed higher functional (E-I) balance on both self-connections in comparison to healthy volunteers. In summary, we observed that risk allele carriers and patients have a higher functional (E-I) balance within the DLPFC-HF network. In view of these research findings, we hypothesized a possible biological functioning of ZNF804A (rs1344706) on the DLPFC-HF network and suggested a mechanistic model for explaining the underlying neurobiology of schizophrenia within this network. Then, we reported causal relations between sDCM parameter estimates and behavior in terms of functional (E-I) balance in both samples. On the one hand, we observed that risk allele carriers and patients require lower functional (E-I) balance on the DLPFC-HF network in order to achieve the best performance during the task. On the other hand, we found that healthy volunteers require higher functional (E-I) balance on the network in order to achieve the optimal behavior.
This study investigated the applicability of computational models like sDCM to establish the functional significance of specific genetic polymorphisms for schizophrenia and identify causal mechanisms associated with the disease in relation to the underlying neurobiology and behavior. In forthcoming studies, we plan to investigate whether subject-specific directed connections strengths between DLPFC and HF, and genotype, contain sufficiently rich information to enable accurate predictions of behavior. In order to study how temporal patterns in the neuronal ensembles and genotype convey robust information about behavior, multivariate regressors or statistical decoding algorithms will be used in both samples.
The author presents an unfitted discontinuous Galerkin method for incompressible two-phase flow applicable to dynamic regimes with significant surface tension. The method is suitable for simulations in complex geometries and a recursive algorithm is proposed, which allows the generation of piecewise linear sub-triangulations re- solving both the domain boundaries and the interface between the two immiscible phases. Hence, discontinuous finite element spaces can be employed to capture the irregularities in the solution along the interface, i.e. the jump in the pressure field and in the velocity derivatives. While the sub-triangulation is based on a linear Cartesian cut-cell approach, its resolution is decoupled from the resolution of the finite element mesh thus enabling the application of higher-order finite element spaces. The time development of the two subdomains is realized by level set methods and an unfitted discretization for the solution of the corresponding equations is described. Multiple approaches for the numerical treatment of surface tension in the context of unfitted discretizations are discussed and compared. Furthermore, these methods are extended to allow simulations with contact lines taking into account the occurrence of microscopic deformations of the contact angle. All proposed methods are verified by numerical test simulations in two and three dimensions.
Marine radiocarbon reconstructions from different intermediate depths reveal large depletions exceeding several thousand 14C years compared to the contemporaneous atmosphere during the last termination. It is suggested that very old waters from a radiocarbon-deficient abyssal reservoir previously well isolated from the atmosphere during the last glacial maximum was mixed back to the upper ocean and atmosphere during the deglaciation thereby raising atmospheric CO2. In pursuing the idea of a hypothesized isolated reservoir, measurements on sub-fossilized cold-water corals from intermediate depths off Brazil are performed. Coupled 230Th/U and 14C dates allow reconstruction of the ∆14C history of the ambient seawater for the past ∼40 ka BP. It becomes apparent that large depletions in the radiocarbon content of these depths are not a phenomenon restricted to the last termination. Injection of very old waters appear during the mid-Holocene and glacial period before the onset of the last termination with depletions comparable to other studies. Local hydrocarbon seepage activity as a possible source of 14C-dead carbon can be precluded as indicated by stable isotope measurements. Interestingly, 14C activity decrease apparently following in part the decay curve one would expect for a closed system pointing to an isolated and continuously ageing water mass, which bathed the corals. εNd isotopic composition indicate no significant changes of the water mass composition for the deeper coral sites for the last 37 ka BP supporting the assumption of a southern origin of the water also at times of large depletions in 14C. For the shallower corals, however, εNd exhibits large variations. It is suggested that local boundary exchange processes have altered the original Nd isotopic signature in these depths.
This thesis presents a novel approach for tracking a varying number of divisible objects with similar appearance in the presence of a non-negligible number of false positive detections (more than 10%). It is applied to the reconstruction of cell lineages in developing zebrafish and fruit fly embryos from 3d time-lapse record- ings. The model takes the form of a chain graph—a mixed directed-undirected probabilistic graphical model—and a tracking is obtained simultaneously over all time slices from the maximum a-posteriori configuration. The tracking model is used as the second step in a two-step pipeline to produce digital embryos—maps of cell nuclei in an embryo and their ancestral fate; the first step being the segmentation of the fluorescently-stained cell nuclei in light sheet microscopy images. The pipeline is implemented as a software with an intuitive graphical user interface. It is the first freely available program of its kind and makes the presented methods accessible to a broad audience of users from the life sciences.
Understanding molecular cloud formation is a major challenge in modern astrophysics. Although the improvements on computational power and novel astronomical instrumentation have allowed us to reach unprecedented accuracy, there are still many open questions. One key issue which helps us to understand the physics behind this problem is the correct comparison between numerical models and observations. Usually, the $^{12}$CO($J$=1-0) emission is considered to be a good tracer of the temperatures and structure of molecular clouds. However, it has been found that it may provide a biased picture of clouds, at best. In this thesis, we analyze a large set of numerical simulations with the aim of making direct comparison with observations. Using a 3D magneto-hydrodynamical simulation including time-dependent chemistry, we find that most of the CO is located at number densities greater than 100 cm$^{-3}$ and kinetic temperatures ($T_{\rm K}$) below 40 K, regardless of the mean number density ($n_0$), metallicity ($Z$) and UV radiation field strength (UV). Radiative transfer calculations are performed to analyze the $^{12}$CO($J$=1-0) rotational transition line intensity that comes out of the cloud. We then calculate the excitation temperature ($T_{\rm ex}$) considering theoretical and observational approaches and find that the gas is mostly sub-thermally excited, indicating that $T_{\rm ex}$ represents a lower limit of $T_{\rm K}$. $T_{\rm ex}$ is used for estimating the CO column density ($N_{\rm CO}$). Considering the full position-position-velocity spectrum for inferring $T_{\rm ex}$, instead of the usual way of using the maximum of the intensity along the line of sight, improves the estimates of $N_{\rm CO}$ by $\sim$30\%. Besides, when a single Milky-Way like CO-to-H$_2$ conversion factor is assumed, the total inferred mass of H$_2$ is underestimated by a factor which typically increases from $\sim$0.1 to 1, as the product $n_0\times Z$ decreases, and/or UV becomes stronger. Moreover, we propose density variance--Mach number relations for supersonic, magnetized, turbulent gas, including an isothermal and non-isothermal equation of state. These analytical relations reproduce satisfactorily the measurements made on numerical simulations. We find that the magnetic field strength scales with density in a relationship of the kind $B\propto \rho^{\alpha}$ with $0\leq \alpha< 1/2$.
The first steps of planet formation imply that dust grows from interstellar-like particles to planetesimals. Diferent physical processes play an important role in the dust evolution and its ability to coagulate and fragment, such as: radial drift, settling to the midplane and turbulent mixing. Observational evidences have shown that millimeter sized particles survive in the outer regions of protoplanetary disks in spite of the fragmentation destructive collisions and rapid inward migration of dust bodies, which are theoretically expected. The main goal of my thesis is to compare the state-of-the-art of gas and dust evolution models with current millimeter observations and look for the most favorable scenarios to have a good agreement between the two. Diferent cases have been explored: typical disks around Sun-like stars, lowmass disks as the ones around Brown Dwarfs and disk with inner gaps or holes known as transition disks. This work therefore brings new theoretical constraints to the planetesimal formation in young circumstellar disks, in direct link with the available observations.
Mammalian signal transduction pathways are highly integrated within extended networks, with crosstalk emerging in space and time. This dynamic circuitry is dependent on changing activity states for proteins and organelles. Network structures govern specificity of cellular responses to external stimuli, including proliferation and cell death. Loss of regulation virtually underlies all disease. However, while the contributions of individual components to phenotype are mostly well understood, systematic elucidation for the emergence or loss of crosstalk and impact on phenotype remains a fundamental challenge in classical biology that can be investigated by systems biology. To that end, we established a mathematical modeling platform, at the interface between experimental and theoretical approaches, to integrate prior literature knowledge with high-content, heterogeneous datasets for the non-intuitive prediction of adaptive signaling events.
In the first part of this work, we investigated high-content microscopy datasets of morphological, bio-energetic and functional features of mitochondria in response to pro- apoptotic treatment in MCF-7 breast cancer cells. Data pretreatment techniques were used to unify the heterogeneous datasets. Using fuzzy logic, we established a generalized data-driven modeling formalism to model signaling events solely based on measurements, capable of high simulation accuracy via non-discrete rule sets. Employing neural networks, a generalized fuzzy logic system, i.e. its rules and membership functions, could be parameterized for each potential signaling interaction. An exhaustive search approach identified models with least error, i.e. the most related signaling events, and predicted a hierarchy of apoptotic events, in which upon activation of pro-apoptotic Bax, mitochondrial fragmentation propagates apoptosis, which is consistent with reported literature. Hence, we established a predictive approach for investigation of protein and organelle interactions utilizing cell-to-cell heterogeneity, a critical source of biologically relevant information.
In the second part of this work, we sought to identify network evolution in the topology of MAPK signaling in the A-375 melanoma cell line. To that end, the modeling method was extended to incorporate temporal and topological structure from phosphorylation profiles of key MAPK intermediates treated with different pharmacological inhibitors and acquired over 96 hours. To increase prediction power, a parameter reduction strategy was developed to identify and fix parameters with lowest contribution to model performance. Therefore, training datasets were bootstrapped and signatures of deviation in flexibility and accuracy were calculated. This novel strategy achieved an optimal set of free parameters. Finally, a reduced multi-treatment model encoding the behavior of the full MAPK dataset was systematically trained to a sequentially increasing subset of time points, enabling time-defined identification of discrepancies in reported vs. acquired network topology. To that end, an objective function for fuzzy logic model optimization was implemented, which accounted for time-defined model training. Analysis led to the identification of emerging discrepancies between model and data at specific time points, thus characterizing a potential network rearrangement upstream of MAPK kinase MEK1, consistent with studies reporting increased resistance to apoptosis exhibited by A-375 melanoma cell line. The approach presented here was successfully benchmarked against a recently published fuzzy-logic-based analysis of signal transduction.
A Modelling and Simulation Study to Understand the Enzymatic Conversion of Waste Cellulose into Biofeuls
Flow compensated and monopolar diffusion weighting gradients are employed to determine the characteristic time scale of the incoherent blood motion causing the biexponential signal decay. A pulse sequence for diffusion weighted magnetic resonance imaging is developed, which allows one to suppress velocity encoding of imaging gradients and which is designed such that the influence of concomitant fields is reduced. It is tested with phantoms and healthy volunteers, revealing different signal attenuation curves for flow compensated and monopolar diffusion gradients in liver and pancreas. Furthermore, a dependence on the total duration of the applied diffusion gradient profile is measured. To describe the experimentally observed signal attenuation curves, a model is developed, which allows one to calculate the signal attenuation due to incoherent blood motion for arbitrary diffusion gradient profiles. Precalculated normalized phase distributions allow one to fit the model to the experimental data. For the signal attenuation curves averaged over test subjects, the characteristic timescale of the blood motion is found to be τ = 184±64 ms in pancreas and τ = 156 ± 22 ms in liver. To facilitate a pixel-wise evaluation and the creation of parameter maps, a denoising algorithm based on principal component analysis is implemented. The denoising reduces the effect of pseudo-random signal contributions allowing one to obtain parameter maps from only 33% of the originally acquired data, which are less affected by noise than the original ones.
The successful application of alpha-emitters in targeted alpha therapy (TAT) goes together with developments in radionuclide production and labelling chemistry. Especially profound understanding of the coordination chemistry of the respective metal ion-ligand system is of major importance to develop protocols for the synthesis of radioimmunoconjugates and to predict the fate of radionuclides in vivo.
Radioconjugates of the therapeutic alpha-emitter Ac-225 with polyamino-polycarboxyl ligands as chelating agents are being actively studied [1, 2, 3, 4]. In particular the macrocyclic ligand 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) has shown promise due to the high kinetic- and thermodynamic stability (log K > 20) of its complexes with trivalent metal cations [5]. The scope of the presented work was the experimental characterisation and evaluation of DOTA as a suitable chelator for trivalent actinides (An(III)), particularly in terms of stable binding of the long-lived alpha-emitting radionuclide Ac-225 to biomolecules for safe application in radioimmunotherapy (RIT). The project included not only basic studies to contribute to a better understanding of the complexation mechanisms and kinetics, but, based on these findings, was aimed at the development of a robust labelling protocol for facile and effective synthesis of an Ac-225-DOTA-MabThera® conjugate. In the course of the investigations, focus was set on in vitro testing of the obtained radioimmunoconjugate, in particular the evaluation of the kinetic stability in presence of competing agents as well as in human blood serum. Assessment of the antigen binding affinity of the antibody conjugate completed the work.
The protocol for the design and synthesis of Ac-225 radiopharmaceuticals of McDevitt et al. provided the starting point for this study [6]. This research group developed a synthetic scheme to radiolabel DOTA-proteins with Ac-225. Later, the protocol was applied to antibodies in form of a two-step synthesis, with the first step being the Ac-225 DOTA-Bn-NCS complexation, followed by the coupling of the Ac-225-DOTA-Bn-NCS to the mAb. The idea behind this two-step approach was that, when the complexation of Ac(III) with DOTA is conducted at elevated temperatures and basic pH (first step), presumably a more stable complex is formed. However, since higher temperature / pH are known to have a negative effect on the antibody efficiency, the conjugation to the biomolecule can hence only be conducted at lower temperatures in a second step. This two-step synthesis though suffers from yields below 10 %, which is assumed to be due to the competing hydrolysis reaction of the isothiocyanate moiety occurring at the pH used during the complexation step. This makes the labelling protocol an interesting subject for further studies on how this synthesis can be improved.
From the findings of McDevitt et al it was apparent that a number of variables needed to be investigated in order to improve on the low efficiency of the protocol. These studies were conducted previously and are summarised in the Diploma Thesis, S. Kannengieÿer, 2009 [7]. A one-step synthesis protocol was tested and the labelling yields for Ac-225 were found to be dependent on temperature and especially on the pH of the reaction mixture. Eventually, an optimised protocol for radiolabelling of DOTA-peptides and MabThera® with Ac-225 activities up to 2 µCi (= 74 kBq) per 100 µg mAb was established, offering labelling yields > 95 %. The present work now aimed on translation of the developed synthesis protocol to higher specific activities of clinical relevance (>10 µCi (>370 kBq) per 100 µg mAb).
To gain profound knowledge about the complexation reaction mechanism and the thermodynamic and kinetic properties of the An(III)-DOTA system, the coordination chemistry was initially studied by means of time resolved laser fluorescence spectroscopy (TRLFS). Since Ac(III) has no suitable spectroscopic properties, the metal ion complexation by DOTA was investigated with Cm(III) as substitute for the trivalent actinides. A comparable study on [Eu(III)DOTA]- has been reported before [8, 9, 10]. Besides determination of the kinetic rate constants and thermodynamic parameters (log K, deltaG, deltaH, deltaS) at labelling-relevant temperatures up to 90 °C, attention was also paid to the detection of possible intermediate species which are frequently discussed in the literature [11].
TRLFS is a powerful speciation method which makes use of the excellent fluorescence properties of Eu(III) and Cm(III), both regarded as good representatives for trivalent lanthanides and actinides. With TRLFS it is possible to detect and characterise complex species in sub-micromolar concentrations without influencing the chemical equilibrium of the system. An experimental setup was chosen which allows for adjusting the concentration of the reactive DOTA species by variation of the pH of the reaction. Due to the slow kinetics of the complexation reaction at room temperature, experiments at 45 to 90 °C were conducted to identify the complex species and quantify their relative ratios by means of peak deconvolution. Based on the potentiometrically determined pKa,n values of HxDOTA(4-x)- for the respective conditions, from these ratios the conditional complex stability constants log K of [Cm(III)DOTA]- were calculated (I = 0.1, 45 to 90 °C). Application of the van't Hoff law allowed for extrapolation of the log K at 25 °C to be 22.0±0.4. The parameters deltaG, deltaH and deltaS obtained from the Gibbs Helmholtz relation indicate that the reaction is exergonic, endothermic and driven by the change of entropy.
Identification and further characterisation of the involved complex species was done by comparison of the fluorescence lifetimes, which give information about the first coordination sphere of the metal cation. Furthermore, additional investigations with NMR were executed to identify and understand the mechanism related to the complex formation with DOTA-Bn-NCS. Based on the results of the NMR study, the complexation kinetics of DOTA and DOTA-Bn-NCS were further investigated and compared to gain insight into the involved reaction mechanisms.
Proper understanding and interpretation of the thermodynamic behaviour of the Cm(III)-DOTA complex formation allowed for facile translation of the experimental settings to the Ac(III)-DOTA system. Since no spectroscopic methods are available for this system, Chelex cation exchange resin as well as Instant Thin Layer Chromatography (ITLC) were chosen as radiochemical speciation methods and were evaluated for their feasability. Determination and refinement of the stability constant log K of Ac(III)DOTA]- for the temperature range of 25 to 90 °C was done in analogy to the Cm(III)-DOTA system, resulting in a log K25°C = 19.5±0.4. To obtain reliable results, the protocol for the radiochemical separation of Ac-225 from Ra-225 required optimisation to ensure highest purity and quality of the radionuclide.
Based on these findings, the studies on the Ac-225-labelling of DOTA-Bn-NCSMabThera® for targeted alpha therapy of Non-Hodgkins-Lymphoma were subsequently continued. The previously established protocol was modified and further optimised in order to be applicable for facile clinical synthesis of radioimmunoconjugates with higher specific activities (SA) within 20 min. In this regard, the antibody labelling kinetics were reviewed respective reaction temperature and ideal pH of the high-yield radiolabelling, which permitted further improvement of the labelling effectiveness (pH 9, 37 - 42 °C, 5 - 15 min; ave. yields 94 - 96 %, > 98 % RCP after purification). The protocol was successfully evaluated for reliability with SA up to 50 µCi (= 1.85 MBq) per 100 µg mAb. The obtained radioconjugates were assessed for their kinetic stability in different buffers as well as under physiolocigal conditions in human blood serum and proved to be satisfyingly stable over up to 30 days ( > 85 % Ac-225 still bound).
Finally, a preliminary radiobiological study with cancer cells (K422 cell line, B-cell lymphoma) was conducted to determine the antigen binding afinity of the radiolabelled CD20-antibody (SA = 1 µCi (= 37 kBq)/100 µg mAb, Bmax= 8.88 nM, Kd = 52.55 nM). The results give rise to further preclinical in vitro studies. In summary, it was demonstrated that rapid, high-yield radiolabelling of DOTAchelated mAbs is possible under alkaline conditions at rather low temperatures. Under these conditions a thermodynamically and kinetically stable radioimmunoconjugates with specific activities suitable for application in clinical TAT studies is formed while the integrity of the antibody is preserved.
Das Mischen von Pulvern ist ein wichtiger Schritt in der Herstellung von festen Arzneiformen wie z.B. Tabletten oder Kapseln. Es ist das Ziel, eine homogene Mischung zu erhalten, von der jede Probe die gleiche Komposition und gleichen Eigenschaften hat wie die andere. Jedoch kann es nach dem Mischen bei weiteren Prozessen oder Herstellungsschritten, wie z.B. Ausfließen aus einem Trichter, dem pneumatischen Transport durch Röhren (Fluidisierung) oder der Vibration der Herstellungsmaschine, zu einer erneuten Auftrennung in die jeweiligen Komponenten kommen. Dieses Phänomen bezeichnet man als Entmischung bzw. Segregation. Eine solche Auftrennung kann die Qualität, z.B. Massen- und Gehaltseinheitlichkeit, der Endprodukte maßgeblich beeinflussen und im schlimmsten Fall zur Ablehnung einer hergestellten Produktionscharge führen. Das Mischen und die Entmischung hängen unter anderem von den Eigenschaften der verwendeten Komponenten ab, genauer gesagt, wie stark sich die Komponenten voneinander unterscheiden. Deshalb wurde in dieser Arbeit untersucht, ob es möglich ist, anhand einfach zu ermittelnder Pulvereigenschaften das Entmischungsverhalten durch Fluidisierung oder Vibration von Einzelsubstanzen bzw. binären Mischungssystemen vorherzusagen. Hierfür wurden pharmazeutisch relevante Hilfsstoffe mit gängigen Methoden charakterisiert: Partikelgröße und deren Verteilung, Feststoffdichte, Schütt- und Stampfdichte, Wassergehalt, Sorptionsverhalten und Fließverhalten. Es wurde festgestellt, dass sich manche Methoden nur bedingt für eine solche Charakterisierung eignen, da die Ergebnisse entweder sehr stark vom Bediener abhängig sind (z.B. Pfrengle-Trichter) oder sich die Substanzen nicht ausreichend charakterisieren lassen (z.B. Siebanalyse und Erweka Granulat-Tester). Nach Identifizierung geeigneter Methoden (Dynamische Bildanalyse, Schütt- und Stampfvolumetrie, Helium-Pyknometrie, Ringscherzelle und Trocknungswaage) wurde die Segregation der Einzelsubstanzen durch Fluidisierung und Vibration untersucht. Anhand der statistischen Auswertungen wurden Modelle erhalten, die in der Lage sind, die Entmischung auf Basis einzelner Eigenschaften bzw. Eigenschaftskombinationen bereits sehr gut vorherzusagen. Bei der Untersuchung binärer Mischungen stellte sich die mediane Partikelgröße vor der Feststoffdichte als der maßgebende Faktor für die Entmischung heraus. Auf Basis der physikalischen Eigenschaften der untersuchten Mischungen und dem ermittelten Entmischungsverhalten wurden über lineare Regressionen mathematische Modelle zur Vorhersage der Segregation berechnet. Diese Arbeit zeigt, dass es möglich ist, mit den verwendeten Segregationstest-Methoden Entmischung bei pharmazeutisch relevanten Pulvern hervorzurufen und diese über geeignete Messmethoden zu quantifizieren. Mit einigen der berechneten Modelle ist es möglich, anhand weniger und einfach zu ermittelnder Stoff- bzw. Mischungseigenschaften, das Segregationsverhalten mit hoher Sicherheit vorherzusagen.
In this work thermodynamic properties of molten fluoride salts and salt mixtures are investigated. Fluoride salts have advantageous properties to be used in energy producing systems based on the conversion from heat to energy like i.e in Molten Salt Reactors. For this purpose it is very important to have detailed information about the heat capacity of the pure salts and salt mixtures. To get a better understanding about the heat capacity in mixtures drop calorimetry measurements of mixtures of LiF with other alkali fluorides were conducted and compared. The investigation of fluoride salts at elevated temperatures is complicated by the fact that fluoride vapour is aggressive to many materials. In order to protect our sensitive measurement equipment the salt samples were encapsulated in nickel crucibles using a laser welding technique and afterwards the whole nickel capsule was measured. This method was veried by the measurement of unmixed CsF and KF where in both examples an excellent agreement with literature data was obtained. Afterwards various intermediate compositions of the systems LiF-KF, LiF-CsF and LiF-RbF were investigated and a general trend according to the difference in cation radii could be established. In combination with literature data for the LiF-NaF system the heat capacity of the liquid state follows the order LiF-NaF <LiF-KF <LiF-RbF <LiF-CsF. An other very effective source of information about materials is their phase diagram. From such diagrams multiple data about phase transition temperatures, vapour pressures, enthalpy of fusion, enthalpy of mixing or solubilities can be deduced. Based on own measured phase diagram data using differential-scanning calorimetry (DSC) with a special encapsulation technique and on available literature data a complete description of the LiF-NaF-CaF2-LaF3 phase diagram was obtained. With the help of mathematical models the phase diagrams can be calculated and also higher order systems can be predicted. The LiF-NaF-CaF2-LaF3 system was calculated with the classical polynomial model and the quasi-chemical model in parallel in order to evaluate which of the two models provide a better extrapolation to higher order systems (ternary or quaternary) based on the related binary systems. The two models behaved very similar at the investigated system and the quasi-chemical model was chosen for further assessments of phase diagrams. This model was selected, since it considers the chemical nature of the investigated system and to simplify the integration of the obtained data in an already existing database of fluoride salt phase diagrams at the Institute for Transuranium Elements in Karlsruhe, Germany. In the Molten salt reactor technology UF3 has a big influence on the corrosion properties of the used salt mixture. But only limited phase diagram data exist regarding this compound. Therefore, the LiF-UF3 and NaF-UF3 system was measured with the DSC method and the LiF-NaF-UF3-UF4 quaternary system was mathematically assessed. The assessment was complicated by a disproportionation of UF3 during the measurements which is faster in the NaF-UF3 system due to the fluoroacidity difference of LiF and NaF. A key system for various designs of the Molten Salt Reactor is LiF-ThF4 binary system. The phase diagram of that system was reassessed based on various newly measured data in this study. In this context also a modication to the used DSC measurement technique was done in order to be able to measure enthalpies of mixing of the two components LiF and ThF4. This method was developed during this work and was veried by the measurement of the LiF-KF system where experimental data exist. A very good agreement between the measured data and the literature was obtained. Also an intermediate solid compound of the LiF-ThF4 system, namely Li3ThF7, was synthesized and the enthalpy of fusion was determined. All new experimental results were considered in the reassessment of the phase diagram. As consequence the assumption of the liquid heat capacity of ThF4 was corrected from 133.9 J ·K^-1 · mol^-1 to 170 J ·K^-1 · mol^-1. With the obtained results and several new phase diagram descriptions it was investigated, if CaF2 is a beneficial component to be used in the salt mixture of two different molten salt reactor designs. It is concluded that CaF2 has no profitable influence on the LiF-NaF-BeF2-PuF3 salt mixture in a specific transuranium burner design (MOSART concept). But it has advantageous influence on the LiF-ThF4 mixture of a thorium breeder design (MSFR) and should be subject to further investigations. During the experimental work of this thesis several scientific articles were published. Major parts of this work can be found in:
O. Benes, M. Beilmann, R. J. M. Konings, "Thermodynamic assessment of the LiF-NaF-ThF4-UF4 system", J. Nucl. Mat 405 (2010) 186-198.
M. Beilmann, O. Benes, R. J. M. Konings, Th. Fanghänel, "Thermodynamic investigation of the (LiF + NaF + CaF2 + LaF3) system", J. Chem. Thermodyn., 43 (2011) 1515-1524.
M. Beilmann, O. Benes, R. J. M. Konings, Th. Fanghänel, "Thermodynamic assessment of the (LiF + UF3) and (NaF + UF3) systems", J. Chem. Thermodyn., 57 (2013) 22-31.
O. Benes, R. J. M. Konings, D. Sedmidubský, M. Beilmann, O. S. Valu, E. Capelli, M. Salanne, S. Nichenko, "A comprehensive study of the heat capacity of CsF from T = 5 K to T = 1400 K", J. Chem. Thermodyn., 57 (2013) 92-100.
E. Capelli, O. Benes, M. Beilmann, R. J. M. Konings, "Thermodynamic investigation of the LiF-ThF4 system", J. Chem. Thermodyn., 58 (2013) 110-116.
M. Beilmann, O. Benes, E. Capelli, R. J. M. Konings, Th. Fanghänel, "Excess heat capacity in liquid binary alkali fluoride mixtures", Inorg. Chem., http://dx.doi.org/10.1021/ic302168g.
In sexual plants meiosis gives rise to recombined and reduced gametes. In diplosporous accessions of the North American genus Boechera meiotic processes are circumvented (i.e. apomeiosis) which lead to a supressed recombination and to production of clonal unreduced male and female gametes. Unreduced male gametes in diplosporous Boechera are required to produce balanced endosperm. The objective of this study was to identify and characterize candidate genetic factors for unreduced pollen formation, and to analyze their genus-wide dynamics in order to contrast the hypotheses whether apomeiosis expression was induced through interspecific hybridization or if it could be an older characteristic of the genus (i.e. pre-Pleistocene). Apomictic Boechera demonstrated high variability for reduced and unreduced meiocyte production. Early flower developmental staging and flow-cytometric analyses together led to the selection of a single pollen mother cell stage at the onset of meiosis for microarray-based comparative gene expression analyses between diploid sexual and diploid apomictic genotypes, and led to the identification of a single highly-upregulated factor (BspUPG-2) which is highly conserved among apomictic Boechera but has no homologue in sexual Boechera or in other taxa. BspUPG-2 exhibits four intron-exon structure variants which suggest alternative splicing, and lack of a prominent open reading frame and no overall sequence homology to known genes suggests that BspUPG-2 belongs to the novel class of long regulatory non-coding mRNA-like RNAs. BspUPG-2 has apparently arisen through a three-step process initiated by ancestral gene duplication of the original non-genic BspUPG-1 locus, followed by sequential insertions of segmentally duplicated gene fragments which led to its chimeric structure and neofunctionalization in apomictic Boechera. Its genesis reflects the hybridization history which characterizes the genus Boechera. Computational analysis demonstrated that portions of BspUPG-2 form secondary structures which were classified as potential primary microRNAs (pri-miRNAs) according to their minimal folding free energy index (MFEI≥0.70) and their A+U content (≥56.70%). One such structure is highly similar to the third exon of a GTP-binding elongation factor Tu/1-A family homolog from Arabidopsis (AT4G02930, E-value=7.00E-24) which could be a potential regulatory target. Two other sequence fragments at the 5`-end of BspUPG-2 are also homologous to known protein-coding genes (AT5G19960 and AT1G18260), hence supporting the hypothesized regulatory function of BspUPG-2. The apomixis-specificity of BspUPG-2 enabled a genus-wide analysis which demonstrated its ubiquity in all Boechera lineages, including the ancient AB haplotype and two single individuals each of one closely-related genus. These results attest to BspUPG-2’s hypothesized importance for unreduced pollen formation and hence for balanced endosperm formation, and point to a single origin of this factor which coincides with the root of the genus Boechera dating to the middle of the Pleistocene.
The complexity of biological systems is one of their most fascinating and, at the same time, most cryptic aspects. Despite the progress of technology that has enabled measuring biological parameters at deeper levels of detail in time and space, the ability to decipher meaning from these large amounts of heterogeneous data is limited. In order to address this challenge, both analysis and visualization strategies need to be adapted to handle this complexity. At system-wide level, we are still limited in our ability to infer genetic and environmental causes of disease, or consistently compare and link phenotypes. Moreover, despite the increasing availability of time-resolved experiments, the temporal context is often lost. In my thesis, I explored a series of analysis and visualization strategies to compare and connect dynamic phenotypic outcomes of cellular perturbations in a genetic and network context. More specifically, in the first part of my thesis, I focused on the cell cycle as one of the best examples of a complex, highly dynamic process. I applied analysis and data integration methods to investigate phenotypes derived from cell division failure. I examined how such phenotypes may arise as a result of perturbations in the underlying network. To this purpose, I investigated the role of short structural elements at binding interfaces of proteins, called linear motifs, in shaping the cell division network. I assessed their association to different phenotypes, in the context of local perturbations and of disease. This analysis enabled a more detailed understanding of the regulatory mechanisms beyond the malfunctioning of cell division processes, but the ability to compare phenotypes and track their evolution was limited. Exploring large-scale, time-resolved phenotypic screens is still a bottleneck, especially in the visualization area. To help address this question, in the subsequent parts of the thesis I proposed novel visualization approaches that would leverage pattern discovery in such heterogeneous, dynamic datasets and enable the generation of new hypotheses. First, I extended an existing visualization tool, Arena3D, to enable the comparison of phenotypes in a genetic and network context. I used this tool to continue the exploration of phenotype-wide differences between outcomes of gene function suppression within mitosis. I also applied it to an investigation of systemic changes in the network of embryonic stem cell fate determinants upon downregulation of the pluripotency factor Nanog. Second, time-resolved tracking of phenotypes opens up new possibilities in exploring how genetic and phenotypic connections evolve through time, an aspect that is largely missing in the visualization area. I developed a novel visualization approach that uses 2D/3D projections to enable the discovery of genetic determinants linking phenotypes through time. I used the resulting tool, PhenoTimer, to investigate the patterns of transitions between phenotypes in cell populations upon perturbation of cell division and the timing of cancer-relevant transcriptional events. I showed the potential of discovering drug synergistic effects by visual mapping of similarities in their mechanisms of action. Overall, these approaches help clarify aspects of the consequences of cell division failure and provide general visualization frameworks that should be of interest to the wider scientific community, for use in the analysis of multidimensional phenotypic screens.
This work is devoted to analysis and development of efficient adaptive algorithms for problems related to the transport of chemical species in the Earth’s atmosphere from data of remote-sensing instruments. Focal point of the thesis is the assessment of different types of errors by a posteriori error analysis. On the basis of these a posteriori error estimates the algebraic iteration can be adjusted to discretization within a succesive mesh adaptation process. The presented adaptive algorithms are applicable for a wide range of problems.
This thesis is brought forward to improve the understanding of peculiar aspects concerning signal and background in the XENON100 experiment, which aims at the direct detection of weakly interacting massive particles (WIMPs). These yet undiscovered particles provide a well motivated solution to the quest for dark matter in our Universe. Within three years of operation, the XENON100 detector has evolved to become the most sensitive instrument to probe spin-independent WIMP-nucleon cross-sections down to 2x10^(-45) cm^2 for WIMP masses in the range of 55 GeV/c^2. We first present an introduction to the detection principle underlying the application of liquid xenon as a target medium for rare event searches. In the following, we summarize our contributions to the suppression of anomalous radiation backgrounds, appearing in the context of reported data analysis for the dark matter searches. We devote the subsequent chapter to the investigation of naturally decaying radon as one of the most dominant sources of internal background. Conclusions drawn are relevant not only for the interpretation of the current background level in XENON100 but also for future detector generations. Finally, we aim at a coherent understanding of nuclear recoil interactions, as mediated by neutrons or potential WIMPs, in the XENON100 detector. Through comparison of neutron calibration data to a dedicated simulation of the entire detector signal response, we derive a measurement of the charge yield and light quenching in liquid xenon, both functions of recoil energy. By achieving absolute and spectral agreement in both accessible signal channels between data and simulation we further provide proof of the correctness and robustness of the interpretation of dark matter results put forward by the XENON100 experiment.
The main goal of this thesis is to evaluate constraints on the composition of interstellar dust (ISD) grain candidates, obtained via impact ionization time-of-flight-mass spectrometry with the Cosmic Dust Analyzer (CDA) onboard the Cassini spacecraft at Saturn. For this work, spectra of 13 extremely rare ISD candidates were extracted from the vast Cassini CDA data set, based on the evaluation of their dynamical and compositional properties, namely mass, speed and trajectory. The candidates show a siliceous composition. Space-based mass spectrometers need terrestrial calibration. Therefore, we accelerated a specifically manufactured orthopyroxene dust analogue from a natural rock onto the laboratory unit of the CDA, and onto the Large Area Mass Analyzer (LAMA). The dust analogue material underwent extensive geochemical analysis with scanning electron microscope (SEM) and electron microprobe analyses (EMPA) beforehand. In the course of this study we learned that the orthopyroxene separate used as dust analogue material contains five additional minor mineral species. Using classical four-isotope geochemical plots, data show clustering at orthopyroxene composition and asymmetric directional scatter towards the minor mineral endmembers. While a significant part of the stochastic scatter can well be due to experimental artifacts, the results imply that different compositions can be distinguished. It remained unclear why many particles - though being very small in the sub-micron range - seem to occur as mixtures. Mass spectra both from CDA and LAMA, can be divided into different types according to the dominating mass line within the spectra, which are in agreement with the results from the chemical analysis performed on the LAMA spectra. Further, the types of both CDA and LAMA spectra are comparable, since they show similar features. The calibration of LAMA spectra with orthopyroxene composition determined by EMPA allowed evaluation of sensitivity coefficients, and hence, to compare the chemical signatures of the in-situ spectra of the ISD candidates with typical compositions of terrestrial silicate minerals, and cosmochemically relevant reservoirs, e.g. primitive chondritic compositions resembling unaltered solar, volatile depleted or differentiated material, which experienced Fe-loss or gain due to core formation processes. A suite of Mg-Ca-rich ISD candidates tends to primitive, only slightly volatile depleted and undifferentiated matter, while a suite of Fe rich particles is similar to reduced metal or more oxidised Fe-rich silicate material.
To date, it is unclear whether adverse effects by genotoxic anthropogenic pollutants in the aquatic environment are linked to the decline of fish populations observed in European and North American freshwaters. Therefore, there is a need for investigations into the relationship between genotoxic stress and detrimental effects on development and reproduction in fish. In order to contribute to this field, the present thesis investigates effects of the alkylating genotoxin methyl methanesulfonate (MMS) on genetic integrity, histological status and reproduction in zebrafish (Danio rerio). In addition, in their unexposed offspring (F1), larval development, histological status and reproduction as well as development in the F2 generation were examined at the population level in order to identify potential inheritable effects of genotoxicity. First, methods for the use of primary gonad cells from zebrafish in the alkaline comet assay and histological sections of testis and ovary in the micronucleus test were developed. After in vivo exposure of adult zebrafish to MMS for up to two weeks, in the comet assay, concentrations-dependent genotoxic effects were detected in gonads, liver and gills. Likewise, the micronucleus frequency was elevated by MMS in all of these organs. Thus, the concentration range adequate for MMS exposure in the designated multi-generation experiment was identified. In the next step, zebrafish (F0) were exposed to MMS in vivo from fertilization until the age of one year. Mortality of F0 fish clearly depended on MMS concentrations. In exposed fish, times of first spawning were delayed and fertility was reduced. However, no unequivocal effects on growth were found. In F1 fish derived from MMS-exposed fish, teratogenic effects were increased, larval survival was reduced and sex ratio was shifted towards females. However, compared to the exposed F0 generation, fertility of the non-exposed F1 generation recovered. Development and survival rates recovered in the F2 generation. Significant genotoxic effects were found in the livers, gills and gonads of either sex of the F0 generation. Regarding histopathological aberrations and external lesions, mainly malformations of eyes, gills and liver and a number of neoplasia were observed in both the F0 and F1 generation. According to indirect measurement of MMS concentrations using ion chromatography, real concentrations in the replicate tanks were similar. Taken together, chronic exposure of zebrafish to MMS led to DNA damage in somatic and generative tissues, induced the formation of a multitude of histopathological aberrations and affected survival, reproduction and development in exposed fish and their offspring. Regarding several endpoints, cessation of exposure allowed for recover over the generations. Combining these results with data of previous studies and transferring them to the environmental situation, there is considerable evidence that anthropogenic genotoxicants play a role in the decline of wild fish populations.
This thesis explores the quantum many-body tunneling dynamics of open ultracold bosonic systems with the recently developed multiconfigurational time-dependent Hartree for bosons (MCTDHB) method. The capabilities of MCTDHB to provide solutions to the full time-dependent many-body problem are assessed in a benchmark using the analytically solvable harmonic interaction Hamiltonian and a generalization of it with time-dependent both one- and two-body potentials. In a comparison with numerically exact MCTDHB results, it is shown that e.g. lattice methods fail qualitatively to describe the tunneling dynamics. A model assembling the many-body physics of the process from basic simultaneously happening single-particle processes is derived and verified with a numerically exact MCTDHB description. The generality of the model is demonstrated even for strong interactions and large particle numbers. The ejection of the bosons from the source occurs with characteristic velocities. These velocities are defined by the chemical potentials of systems with different particle numbers which are converted to kinetic energy. The tunneling process is accompanied by fragmentation: the ejected bosons lose their coherence with the source and among each other. It is shown that the various aspects of the tunneling dynamics’ can be controlled well with the interaction and the potential threshold.
Marine biofouling, the colonization of submerged surfaces by unwanted organisms, has an important economic and environmental impact. This PhD thesis focuses on the smaller organisms involved in the biofouling process such as bacteria, diatoms and protozoa also called microfoulers. As bacteria are usually among the first organisms to settle on submerged surfaces, the characterization of their adhesion to these surfaces is essential for the development of strategies for antifouling, and in particular fouling release coatings. To this end, the adhesion of the bacterium Cobetia marina on various model systems for anti fouling coatings was investigated using a microfluidic shear stress assay which applies shear stresses covering a range of nearly six orders of magnitude from 0.01 to 5,500 dyn/cm2. For this assay, the experimental parameters such as medium, incubation time and increase of the applied volumetric flow were optimized.
In this work various surface properties relevant for bioadhesion were investigated, namely wettability, chemistry, hydration, transition from monolayers to polymeric coatings, and the controlled release properties of metal organic frameworks as a smart release coating. The surfaces used for this study were self-assembled monolayers (SAMs) with different chemical end groups and hydration levels, polysaccharide coatings with and without capping of their carboxylic groups, poly[oligo(ethylene glycol)methacrylate] (POEGMA) brushes and copper based metal organic frameworks (Cu SURMOF 2). The results showed that in general the hydration of the surface is more important for the resistance against bioadhesion than the wettability. It was demonstrated that the critical shear stress needed for removal of bacteria from a SAM system based on ethylene glycols (EGs) decreased with an increasing number of EG units which is directly related to an increment of hydration. Furthermore, good fouling release properties of polysaccharide coatings were demonstrated, especially if the free carboxyl groups of alginic acid (AA) and hyaluronic acid (HA) were capped with a hydrophobic amine. Cu SURMOFs 2 were investigated as an example of smart release coatings. When bacteria interacted with these surfaces they induced a loss of crystallinity and a harmful effect on themselves. These findings, together with the observed stability of the coatings in artificial seawater (ASW) and the integrity of the coating in areas without bacteria demonstrated a stimulus response of these surfaces upon presence of bacteria. In order to compare the performance in the field of the surfaces investigated in the laboratory assays, a set of well characterized samples were immersed into the ocean at the Sebastian test site of the Florida Institute of Technology. The aim of these field tests was to compare the results of the laboratory experiments, which solely investigated a single species under controlled conditions, with field experiments which employed a mixed species marine environment under natural conditions. The results showed that air and water temperature seemed to be an important factor for the abundance of species and composition of the fouling community. Furthermore, the level of hydration of the surfaces was found to be more important for their colonization than their wettability. Some trends that have also been observed in previous laboratory assays such as the good performance of the polysaccharide coatings and the EG SAMs, compared to other SAMs, could be confirmed in the field. Hence, the inert properties of hydrophilic hydrogels could be demonstrated in both laboratory assays and in the natural marine environment.
Viele der heute industriell eingesetzten, heterogenen Katalysatoren bestehen aus nanoskopischen Partikeln eines katalytisch aktiven Materials, die auf ein Trägermaterial aufgebracht sind. Ein Hauptproblem solcher Katalysatorsysteme stellt die Alterung beziehungsweise die Agglomeration der katalytisch aktiven Spezies bei hohen Reaktionstemperaturen dar. Dies führt zu einer Verschlechterung der katalytischen Eigenschaften, da das Oberfläche-zu-Volumen-Verhältnis verringert wird, welches in einem direkten Zusammenhang mit der Aktivität des katalytisch aktiven Materials steht. Ein Lösungsansatz dieses Problems könnte die Beimischung eines weiteren Metalls zur katalytisch aktiven Spezies sein. Bimetallische Nanopartikel vereinen die Größeneffekte der Nanopartikel mit den Kompositionseffekten der bimetallischen Spezies. Dies führt zu neuen physikalischen und/oder chemischen Materialeigenschaften, die nicht durch Anpassung der Größe oder Zusammensetzung allein erreicht werden können. Die Beimischung eines weiteren Metalls zu katalytisch aktiven Nanopartikeln stellt in der Tat eine Möglichkeit dar, die katalytische Aktivität und Stabilität zu verbessern. Um den Einfluss von Partikelgröße, Partikelzusammensetzung und Partikelstruktur auf die katalytischen Eigenschaften zu untersuchen, wurden verschiedene Herstellungsverfahren monodisperser Nanopartikel entwickelt. Die Agglomeration der Partikel bleibt jedoch ein zentrales Problem bei der Abscheidung auf festen Trägermaterialien. Die mizellare Blockcopolymer¬nanolithographie (BCMN) ist eine Möglichkeit der kontrollierten Strukturierung von verschiedenen Substraten. Im Rahmen dieser Arbeit konnte die BCMN dahin weiterentwickelt werden, dass sie die Synthese von monodispersen, thermisch stabilen, bimetallischen Nanopartikeln sowohl auf planaren als auch auf mesoporösen Katalysatorträgern und Mikrosphären ermöglicht. Die bimetallischen Nanopartikel konnten dabei kontrolliert aus verschiedenen Übergangsmetallen wie Au, Pt, Pd, Rh, Ni und Ag mit verschiedenen prozentualen Zusammensetzungen aufgebaut werden. Hierfür wurden in Toluol gebildete Mizellen aus amphiphilen Polystyrol‐block‐Poly(2‐vinylpyridin)-Diblock-copolymeren (PS b P2VP) mit zwei verschiedenen Übergangsmetallkomplexen beladen. Die Strukturierung der planaren Substrate erfolgte entweder durch Tauchbeschichtung oder Rotationsbeschichtung, während pulverförmige Substrate mit der mizellaren Lösung durchspült wurden. Zur Entfernung des Polymers und der gleichzeitigen Reduktion der Metallionen wurden die Proben anschließend mit Wasserstoffplasma behandelt. Mit dem gleichen Polymer hergestellte, bimetallische Partikel wiesen dabei gleiche Partikelgrößen und -formen auf. Mit HRSTEM-EDX-Untersuchungen konnte ein Alloy-Strukturtyp mit einer ungeordneten Verteilung der beiden Metalle nachgewiesen werden, obwohl einige der Metalle (z.B. Au und Pt) als dreidimensionaler Festkörper mit einer makroskopischen Ausdehnung weitgehend als nicht mischbar gelten und auf der Nanometerskala zur Ausbildung von Kern@Schale-(engl. „core@shell“)Strukturen neigen. Die prozentuale Zusammensetzung der Alloy-Nanopartikel konnte relativ einfach über die Beladung der Mizellen mit dem entsprechenden Metallkomplex reguliert werden. Die beiden Metalle zeigten selbst dann keine Segregation in eine Core@Shell-Struktur, wenn sie bei 750 °C für sieben Stunden bei Umgebungsdruck und Luftfeuchtigkeit getempert wurden. Darüber hinaus wurden die AuPt-, NiPt- und RhPt-Alloy-Nanopartikel teilweise in die siliziumoxidhaltigen Substrate eingebettet. Die mittels BCMN erhaltene Anordnung der Nanopartikel auf dem Substrat blieb dabei unverändert, und eine Agglomeration der Nanopartikel trat nicht auf. Die Legierung von Pt-Nanopartikeln mit einem zweiten Metall führte somit zu einer thermischen Stabilisierung, ohne dass weitere Stabilisatoren zugegeben werden mussten oder zusätzliche Stabilisierungsschritte erforderlich waren. In dieser Arbeit wird eine einfache aber effektive Syntheseroute vorgestellt, mit der thermisch stabile Nanolegierungen verschiedener Übergangsmetalle mit unterschiedlichen prozentualen Zusammensetzungen hergestellt werden können. Die so synthetisierten Nanopartikel können gezielt auf planaren und 3D-Substraten abgeschieden werden. Durch das teilweise Einsinken der Alloy-Nanopartikel in siliziumoxidhaltige Substrate während eines Temperprozesses wird sowohl eine Agglomeration der Partikel, als auch eine Segregation der beiden Metalle verhindert.
Urumqi is located in the remote center of the Eurasian continent. It is a future mega-city with rapid economic development and high population density in China's western interior. Urumqi's water resource problems are the main research objects in this thesis. Several models have been put forward to predict water demand in Urumqi and useful suggestions have been gathered to reduce water scarcity. In 2010, the average annual water resources of Urumqi were at 939.22 million m³ and the average per capita water resources were 387 m³, meaning that water resources are inadequate in Urumqi. The water consumption in Urumqi already exceeded the total amount of water resources. Furthermore, almost half of the wastewater is discharged directly into rivers and wasteland in Urumqi and as such, both surface water and groundwater are seriously polluted. Since there is also no reasonable water price system, the price of water is relative low which leads to weak awareness of water conservation. In addition, the high leakage rate of the pipe network and the backward technology of agricultural irrigation have resulted in serious water losses. In order to alleviate the scarcity of water resources and instead increase the number of resources, while at the same time improving water quality, wastewater in Urumqi and how it is reused of Urumqi was analyzed. Some suggestions about Urumqi's sewage and water reuse system were put forward. Moreover, various water scarcity assessment indexes were used to evaluate the water scarcity risk in Urumqi. Based on the results of a water scarcity risk assessment, the water scarcity decision model was built up by adopting the advanced Analytic Hierarchy Process (AHP) methodology. The measures to reduce water scarcity include a.o. adjusting industrial structures, water conservation, using unconventional water resources, implementing economic regulation measures, controlling environmental safety, improving urban functions, and the interbasin transfer of water. According to the results of the analysis of water scarcity decisions, major solutions to resolve the problem of water scarcity were identified, with water conservation as the most important step in reducing water scarcity in Urumqi. In addition, a water conservation index system was set up based on the water-saving evaluation standard in China to change the present situation of serious wastage of water resources in Urumqi. This index system can be used to reflect the problems (e.g. high leakage rate of the water supply pipe network, low water price, low conveyance efficiency of irrigation canal system, low rate of recycled industrial water and water conservation awareness) and the potentials of water conversation in each sector (agriculture, industry and domestic). The results of the index system show that there is a large potential of agricultural water conservation, and it can be achieved by several measures, such as improving the water efficiency of the canal system, promoting the usage of advanced water conservation irrigation techniques and increasing the water price for agricultural irrigation. In addition, the "quota method" and the "grey model" (used to analyze the system, which related to time includes both certain and uncertain information) were used to predict water demand. The "quota method" predicts the water demand based on indicators of socioeconomic development and the water use quota in each sector. The "grey model" was constructed according to the time series of agricultural, industrial, domestic and total water consumption in Urumqi from 2003 to 2010 by creating a sequence of first-order accumulated generating operation and differential equations. The predictions that were calculated by using the grey model show that agriculture will still be the biggest user of water in 2015. Therefore, changing the agricultural system and improving the efficiency of agricultural water use are the best ways to realize the rational allocation and sustainable use of water resources in Urumqi. In order to effectively manage Urumqi's water resources and to integrate the water demand prediction model and the water scarcity decision model, the water resources management and information system for Urumqi was built up by using various technologies (database, Web and GIS server). This system not only reflects the current situation of Urumqi's water resources but also helps users to make decisions for reducing water scarcity.
Apple proliferation (AP) disease caused by ‘Candidatus Phytoplasma mali’ is one of the most economical important diseases in the apple industry, causing tasteless undersized fruits. A project to produce AP-resistant rootstocks by crossing the natural resistant wild type M. sieboldii with commercial M. domestica apple rootstock varieties, observed severe decline and death in the progeny associated with latent apple viruses, namely Apple stem grooving virus (ASGV) and Apple stem pitting virus (ASPV). This study aimed to explain this phenomenon by gaining new information on the causative agent(s) as well as to see if there is any interaction between the different apple viruses or between virus and phytoplasma, and to study the effect of these pathogens on M. sieboldii, M. domestica and the hybrid progeny. The genome of the ASGV-AC strain, which was associated with the decline, was determined and variability and evolutionary studies were performed, revealing significant differences in the selection pressure of two variable areas in the genome. An in vitro culture system was established and ASGV, ASPV and ‘Ca. P. mali’ were successfully graft-transmitted to the different Malus genotypes as single infection or as virus-virus and virus-phytoplasma co-infection. The transmission rate was severely affected by the Malus genotype, with each genotype acting differently to the different virus, virus-virus and virus-phytoplasma combinations. A hypersensitive reaction was observed on M. sieboldii and some of the M. sieboldii-derived hybrid progeny depending on the inocula. Some hybrids showed severe resistance reactions while others recovered from the initial reaction and allowed the systemic spread of the virus(es). Quantitative Real-Time PCR was established for ASGV and ASPV and used to determine the mean virus titer per plant cell. ASGV titer was significantly lower compared to ASPV in M. domestica. The ASGV titer was lower in M. domestica compared to M. sieboldii and the hybrid progeny confirming the tolerant nature of M. domestica towards ASGV. An antagonistic interaction was observed between ASGV and ASPV, with the ASGV titer decreasing by half when co-infected with ASPV, while the ASPV titer is unchanged. An antagonistic interaction was also observed between ASGV and ‘Ca. P. mali’. While the ‘Ca. P. mali’ titer was significantly increased, the ASGV titer was decreased compared to the titer values obtained from single infections. This is the first study performed on the possible interaction between phytoplasma and virus.
The droplet size distribution and interaction of the liquid phase and the gas flow are key features in the modeling of evaporating spray flows, which are important because of their vast range of industrial and engineering applications. Two-phase effects and poly-dispersity of droplet size distributions dominate the structure of any spray and related applications such as spray flames, end products of spray drying processes, or medical applications. The spray dynamics depends on various physical processes such as droplet inertia, evaporation, and gas phase characteristics. Thus, it is important to have reliable models and numerical techniques in order to be able to describe the physics of two-phase flows, where the dispersed phase consists of droplets of various sizes that may evaporate, coalesce, breakup as well as have their own inertia and size-conditioned dynamics.
In the present thesis, an evaporating water/air spray is modeled using direct quadrature method of moments (DQMOM) and discrete droplet model (DDM) in an axisymmetric geometrical configuration. In DDM, the two-phase effects are captured by resolving the gas phase conservation equations considering the droplets as point sources. The system of conservation equations is closed using an extended k-epsilon model. The system of equations is solved using a hybrid finite volume - Lagrangian particle tracking method. DQMOM is not yet coupled to gas phase fully, rather the inlet gas flow properties are used to compute the drag force exerted on droplet velocity. For both DDM and DQMOM, appropriate initial and boundary conditions as well as the starting values for simulations are generated from experimental data, which have been carried out by the group of Prof. G. Brenn at TU Graz, Austria. The simulation results are compared with experiment and found in good agreement.
Furthermore, a turbulent methanol air jet spray flame is investigated. A detailed methanol/air combustion mechanism consisting of 23 species and 168 elementary reactions is implemented through a spray flamelet model. The process of molecular mixing is treated following probability density function (PDF) modeling, where two approaches are used i.e., presumed PDF and transported PDF. The standard beta distribution is used as the base case to describe the process of molecular mixing. Its shape parameters and distribution characteristics are known and well established.
A bivariate joint PDF of the mixture fraction and enthalpy is applied for turbulent spray flames. The PDF transport equation is deduced. The mixture fraction and enthalpy are described using an extended Interaction-by-Exchange-with-the-Mean (IEM) model and modified Curl's model. The PDF transport equation is closed through coupling with an extended k-epsilon model, and it is solved using a hybrid finite volume/Lagrangian Monte-Carlo particle method. The numerical results of the gas velocity, the gas temperature, and the Sauter mean radius are compared with experimental data from the literature and good agreement with the experiment is observed. Furthermore, the shapes of the PDF of the mixture fraction and enthalpy at different positions, which are computed by the transported PDF method, are presented and analyzed. For the sake of comparison, the presumed PDF method is also applied, where statistical behavior of mixture fraction is described using standard beta function. A comparison of the results of the transported PDF method using modified Curl's and IEM models with the standard beta function shows that the standard beta function fails to describe the statistical behavior of mixture fraction accurately. Effect of a four parameter modified beta distribution instead of a standard beta distribution are also discussed. A trivariate joint PDF of enthalpy, gas velocity and mixture fraction is proposed for future simulations, and its transport equation is derived, where the gas velocity is modeled using an extended simplified Langevin model.
The intention of this dissertation was the development of a multi-channel mixedsignal detector readout ASIC. This paper describes the whole design process that starts with the vague requirement for a readout chip for some CBM/FAIR sub-detector and that ends with the latest and actually realized system on a chip solution called SPADIC, which is mainly intended to read out the future CBM-TRD. This work comprises the design from scratch of 6 ASIC prototypes and 10 PCB readout setups as well as the development of various software and firmware components, the characterization of the designed ASICs, the development of the SPADIC concept, the design of the SPADIC website, and not at least the collaboration with the TRD physicists with the achieved goal to read out signals of chamber prototypes using different SPADICs during CERN beam-times or in the laboratory. Besides the descriptions of the most important chip details and the corresponding theoretical analyses, an overall introduction into detectors and detector physics – written on a level for engineers – is given in this paper in order to embed this technical work into its physical context. The effective output of this dissertation is the self-triggered 32-channel charge pulse amplification and digitization chip SPADIC 1.0.
Transportstudien für Efflux-Transporter sind in der pharmazeutischen Industrie weit verbreitet und dienen dazu potentielle Arzneimittel als Substrate von Efflux-Transportern zu identifizieren. Diese verhindern, dass u.a. therapeutisch wirksame Mengen von Arzneistoffen ihren Wirkort erreichen. Von besonderer Bedeutung sind dabei, auf Grund ihres breiten Substratspektrums, die beiden ABC-Transporter P-Glykoprotein (Pgp, MDR1) und Breast Cancer Resistance Protein (BCRP, ABCG2). Die Madin-Darby canine kidney Zelllinie (MDCK) und ihre, mit einem humanen Transportprotein stabil transfizierten Varianten (MDCK-MDR1, MDCK-BCRP), werden vielfach als in vitro Zellkulturmodelle für diese Transportstudien verwendet. Die Wildtyp MDCK-Zelllinie dient bei Experimenten als Kontrollpopulation, da es zwischen dem endogen exprimierten Transporter und der humanen, transfizierten Proteinvariante zu Substratinteraktionen kommt. Aus diesem Grund ist es wichtig, sich auf konstante und vergleichbare Expressionslevel der Zelllinien hinsichtlich der Transportproteine verlassen zu können, um falsch negative oder falsch positive Ergebnisse bei der Identifikation von Substraten zu vermeiden. Aus der MDCK-MDR1 und der MDCK-BCRP Zellpopulation wurden Einzelzellen präparativ sortiert, um eine neue homogene Population mit hoher und stabiler Expression des transfizierten, humanen Gens zu generieren. Gleichzeitig wurden Einzelzellen der Wildtyp-Populationen sortiert, um eine Kontrollzellinie mit verminderter Expression des endogenen Genproduktes zu erhalten, welche mit der Expression in den zuvor gewonnen Einzellzellpopulationen vergleichbar ist. Die generierten Zellpopulationen wurden molekularbiologisch mittels qRT-PCR und Western Blot analysiert und bestätigten den Erfolg des Versuchsziels. In Ergänzung dazu wurden durch funktionelle Transportversuche mit radioaktiv markierten Substraten von Pgp die gewonnenen Erkenntnisse verifiziert. Darüber hinaus wurden die Zelllinien hinsichtlich der Expression weiterer ABC-Transporter sowie Transporter aus der Gruppe der Organic Anion und Organic Cation Transportproteine näher charakterisiert, um ein tieferes Verständnis für die Transportvorgänge und mögliche Interaktionen in Transportstudien zu erhalten. Um vergleichbare Zelllinien mit möglichst geringer Expression des endogenen Pgp zu generieren, wurden Populationen der MDCK, MDCK-MDR1 und MDCK-BCRP Zellen mit Zinkfinger Nukleasen (ZFN) behandelt, um cPgp auf genomischer Ebene zu deletieren. Auch hier wurden Einzelzellen zur Etablierung einer homogenen knockout (KO) Population sortiert. Die generierten KO-Zelllinien wurden molekularbiologisch charakterisiert und auf ihre Transporteigenschaften bezüglich Pgp, insbesondere in der betriebsinternen Transportversuchsanordnung von AbbVie Deutschland GmbH & Co. KG, untersucht. Die KO-Zellen konnten verbesserte Werte für Pgp-Refernzsubstrate im Transportversuch aufweisen und auch für betriebsinterne Substanzen, im Vergleich zu den Ausgangkulturen, verlässliche Ergebnisse erziehlen.
Neuronal activity is required for proper neural development, as well as for the maintenance of neural circuits, adaptation to changing environments, learning and memory formation. Electrical activation of neurons induces transient increases in the intracellular calcium (Ca2+) concentration, which can propagate to the nucleus thereby linking synaptic stimulation to gene regulatory events. However, it is unclear if and how different spatial-temporal patterns of activity give rise to distinct genomic responses. In this thesis I used optogenetic method to investigate activity dependent regulation of signaling pathways and gene expression in cultured hippocampal neurons. The goal was to investigate whether intrinsic activity could be mimicked in a controllable way, and if yes, how this was achieved. Specifically, I used Channel- rhodopsin (ChR2), a light-gated, cation-selective, small membrane channel isolated from Chlamydomonas reidhardtii. ChR2 can be activated with blue light and functions without any addition of cofactor when expressed in mammalian systems. I used transfection and adenoassociated virus-mediated gene transfer to express ChR2 in cultured hippocampal neurons, which afterwards could be stimulated and activated with blue LED pulses to reliably induce action potential firing and Ca2+transients. A double mutant variant of ChR2, ChR2-ab, was made, characterized and its cellular functions were studied. The results indicated that ChR2-ab inherited the properties from its previous single mutant, being more sensitive, longer opening and bi-stable. Neurons expressing ChR2-ab responded to light exposure with membrane depolarization, which ultimately led to the expression of a previously characterized pool of activity-dependent genes. ChR2-ab-mediated induction of these genes was L-type voltage-dependent Ca2+ channel dependent, as it was completely blocked with nifedifine. Similarly, treatment of ChR2-ab-expressing neurons with NMDA receptor blockers MK-801 and APV inhibited the light-evoked induction of atf3, ifi202b, inhba and serpinb2. When synaptic activity was inhibited with Na+channel blocker TTX to prevent action potential fir- ing, ChR2-ab-mediated gene induction of atf3, inhba, npas4, nr4a1 and serpinb2 was also restrained. Expression of CaMBP4, an inhibitor of nuclear Ca2+/CaM complex, revealed that ChR2-ab-mediated gene induction dependent on nuclear Ca2+signaling. Interfering the function of CREB/CBP transcription complex with E1A protein dimin- ished gene expression induced with ChR2-ab. Whole-cell patch-clamp and signaling pathway analysis revealed that neurons expressing ChR2-ab had higher basal activity, with more frequent mEPSC and higher level of pCREB and pERK. However, some ChR2-ab-expressing cells died after light application on day in vitro 14. Pharmacological analysis suggested that this was due to activation of extra-synaptic NMDA receptors during ChR2-ab activation. In an alternative strategy to induce activity, ChR2 was targeted to the membrane of endoplasmic reticulum using various targeting sequences with the goal to directly trigger Ca2+release from this internal Ca2+store. Additionally, transcription analysis with catFISH method revealed that a single nuclear Ca2+ transient induced with Bicuculline treatment was capable and sufficient to initiate transcription of arc gene.
Neuroblastoma is the most common extra-cranial solid tumor of early childhood. Standard therapies are not effective in case of poor prognosis and chemotherapy resistance. To improve drug therapy, it is imperative to discover new targets that play a substantial role in tumorigenesis of neuroblastoma. The mitotic machinery is an attractive target for therapeutic interventions and inhibitors can be developed to target mitotic entry, spindle apparatus, spindle activation checkpoint, and mitotic exit. Thus, we performed a study to find genes that cause mitosis linked cell death upon inhibition in neuroblastoma cells.
We investigated gene expression studies of neuroblastoma tumors and selected 240 genes relevant for tumorigenesis and cell cycle. With these genes we performed image-based time-lapse screening of gene knockdowns in neuroblastoma cells. We developed a classifier to classify images into cellular phenotypes, using SVM, performing manual evaluation and automatic corrections. This classifier yielded better predictions of cellular phenotypes than the standard classification protocol. We further developed an elaborated analysis pipeline based on the phenotype kinetics from the gene knockdown screening to identify genes with vital role in mitosis to identify therapeutic targets for neuroblastoma. We developed two methods (1) to generate clusters of genes with similar phenotype profiles and (2) to track the sequence of phenotype events, particularly mitosis-linked-celldeath.
We identified six genes (DLGAP5, DSCC1, SMO, SNRPD1, SSBP1, and UBE2C) that cause mitosis-linked-cell-death upon knockdown in both of the neuroblastoma cell lines tested (SH-EP and SK-N-BE(2)-C). Gene expression analysis of neuroblastoma patients show that these genes are up-regulated in aggressive tumors and they show good prediction performance for overall survival. Four of these hits (DLGAP5, DSCC1, SSBP1, UBE2C) are directly involved in cell cycle and one (SMO) indirectly which is involved in cell cycle regulation. Functional association and gene-expression analysis of these hits indicated that monitoring cell cycle dynamics enabled finding promising drug targets for neuroblastoma cells.
In summary, we present a bioinformatics pipeline to determine cancer specific therapeutic targets by first performing a focused gene expression analysis to select genes followed by a gene knockdown screening assay of live cells.
At present, 170 million people are infected with Hepatitis C Virus, which is about 3 % of the world population. Currently available treatment is successful in only 50 % of treated patients. Further development of the treatment strategies requires a profound quantitative understanding of the viral lifecycle. This remains a major challenge, however, by combining the experimental and modeling approaches it has become possible to understand it quantitatively. In this thesis, we develop a mathematical model to investigate the intracellular dynamics of HCV replication. In this model, two processes, the translation of viral proteins and the replication of viral genomes are considered. This model is established by using ordinary differential equations. The established model is then calibrated by using the time course data representing the viral plus-, minus-strand RNA and the polyprotein dynamics and the steady state data reflecting the ratios among the plus-, minus-strand RNA and the non-structural proteins. Subsequently, the model is validated using the independent measurements highlighting the replication deficient and synthesis inhibited HCV RNA dynamics. Furthermore, the model is used to analyze the observed difference in HCV RNA replication in the clonal Huh-7 cell lines. We demonstrate that this difference can be explained by the differential expression of the cellular host factor which involves in the replication of viral genomes. Using the model, a role of replication vesicles with respect to viral dynamics is analyzed. A sensitivity analysis is performed on the model parameters to reveal the crucial steps in the viral replication. This analysis shows that the processes in the replication vesicles are the most crucial for HCV replication. Finally, an identifiability analysis is performed to check whether the model parameters are sufficiently estimated from the measured data.
Model reduction methods in chemical kinetics are used for simplification of models which involve a number of different time scales. Slow invariant manifolds in chemical composition space are supposed to be identified. A selection of state variables serve for parametrization of these manifolds. Species reconstruction methods are used to compute the values of the remaining variables in dependence of the parameters. We discuss theoretical results and numerical methods for an application of a model reduction method that is developed by D. Lebiedz based on optimization of trajectories. The main focus of this work is an application of the model reduction method to models of chemical combustion. The existence of a solution of the semi-infinite optimization problem, which has to be solved to obtain a local approximation of the slow manifold, is proven. A finite optimization problem for the same purpose is presented which can be solved with a generalized Gauss-Newton method. This method is used with an active set strategy. A filter framework and iterations with second order correction are employed for globalization of convergence. Families of neighboring optimization problems can be solved efficiently in a predictor corrector continuation scheme. The tangent space of the slow manifold can be computed by evaluation of sensitivity equations for the parametric optimization problem. A step size strategy is applied in the continuation scheme for efficient progress along the homotopy path. Results of an application of the presented method are shown and discussed. The test models range from simple test examples to realistic models of syngas combustion in air.
Metastasis formation is the life-threatening end stage of cancer. It is therefore vital to understand how this process is regulated so that therapeutic intervention becomes possible. ASAP1 (Arf-GAP with SH3-domains, Ankyrin-repeats and PH-domains) was discovered in an unbiased genetic screen for genes that are involved in metastasis formation. Subsequently it was shown that this protein promotes tumor cell motility and invasiveness. Loss and gain of function experiments in a pancreatic carcinoma model demonstrated a functional role for ASAP1 in regulating metastasis. In human colorectal cancer patients ASAP1 expression strongly correlates with short metastasisfree survival and poor prognosis. At the molecular level, co-immunoprecipitation experiments have shown that ASAP1 binds to both h-prune and Nm23-H1 (Non- Metastatic protein 23-H1), proteins that are also involved in the metastatic process. Using the highly metastatic breast cell line MDA-MB-231 that endogenously expresses ASAP1, Nm-23H1 and h-prune, in my PhD thesis I aimed to characterize how ASAP1 contributes to metastasis by addressing the following issues: a) the influence of ASAP1 and its interaction partners on the internalization of EGF; b) effects of ASAP1- containing protein complexes on cellular motility; c) further possible interaction partners that may modulate ASAP1 activity. My results showed that, despite their role in regulating motility and metastasis formation, neither Nm23-H1, nor h-prune influence the effects of ASAP1 on cell migration. Moreover, these two proteins also do not affect the ability of ASAP1 to regulate the internalization of EGF. I then focused my attention on other possible proteins that might contribute to ASAP1-mediated metastasis by screening for potential SH3 domain-bearing interaction partners, because the SH3 binding domain of ASAP1 is crucial for its motility-promoting activity. I thereby identified SLK (Ste-20 Like Kinase) as a new interaction partner of ASAP1, and analyzed its effects on ASAP1 function. Although SLK co-immunoprecipitated with ASAP1, and is itself involved in the control of cell motility, this protein did not affect the motility-promoting activity of ASAP1. Together, my results demonstrate the role of ASAP1 in promoting both cell motility and receptor internalization in the context of tumor cells, important biological processes that regulate metastasis formation.
Deregulation of apoptosis is a frequent alteration in benign, pre-‐cancerous lesions of the colon mucosa that has been extensively discussed as contributor to the development of colorectal cancer. Individual differences in the regulation of apoptosis are (epi-‐)genetically determined and identification of SNPs within coding/ flanking regions of genes with apoptosis relevant function could provide a basis to assess the individual risk to develop these lesions. To identify a possible association between genetic polymorphisms and tumorigenesis we selected 865 genes with reported function within the apoptosis pathways or in related (e.g. stress related) pathways. Our screening was performed on a customized goldengate Illumina chip covering 1536 single nucleotide polymorphisms in a two stage approach. Stage I was performed on 272 patients harboring hyperplastic polyps and 512 controls. Stage II aimed to validate preliminary data by screening the candidate SNPs (p<0.01) on an independent cohort of patients and controls (n=76). After the meta-‐analysis between stage1 and stage2 the false discovery rate (FDR) approach has been used to calculate the “q” of significativity in order to have a better compromise between Type1 and Type2 errors. Among the candidate polymorphisms 9 SNPs were significantly correlated with the occurrence of hyperplastic polyps. Out of these the variation rs4709583 (PARK2) presented the highest significativity level (q=0.003) and was therefore further analysed in-‐vitro to identify its potential influence on splicing in the PARK2 gene.
Head and neck cancer is the sixth most common cancer worldwide and associated with a poor clinical prognosis, due to development of recurrent tumors and metastasis. Tumor recurrence and low patient survival are strongly linked with the ability of tumor cells to invade and infiltrate the surrounding tissue. Stress-activated protein kinases (SAPK), particularly p38, are known to regulate a wide range of cellular phenotypes, including cell invasion via the activity of secreted proteases. The proliferation-associated Forkhead box protein M1 (FOXM1) transcription factor, a p38 downstream target, plays a role in the development and growth of many cancer types. However, only very little is known about the role of p38 and FOXM1 in invasive processes of head and neck cancer and the exact mechanism underlying this process. In this work we examined the downstream events of p38 signaling primarily focusing on the role of FOXM1 transcription factor in regulation of the urokinase-type plasminogen activator (uPA) gene and invasion of head and neck squamous cell carcinoma (HNSCC) cells. Using different HNSCC cell lines, we confirm that p38 regulates FOXM1 expression and provide evidence that p38 signaling driven in vitro invasion of HNSCC cells requires FOXM1 expression. Furthermore, siRNA-mediated FOXM1 knockdown is sufficient to inhibit the invasive behavior of HNSCC cells in vitro. By using reporter gene assays, bioinformatical analysis of the publically available ChIP-Seq data, chromatin immunoprecipitation assays, and transplantation-based mouse model of oral cancer, we identified the molecular mechanism of FOXM1-mediated invasion of HNSCC cells. FOXM1 controls the uPA-dependent invasion via activation of c-Fos and thus drives AP-1 activity on the uPA promoter, which enhances its expression and proteolytic activity. Further, an activated Ras signaling is necessary for a potent FOXM1-mediated uPA activity and tumor formation. The data are supported by a bioinformatical study, demonstrating concomitant up-regulation of FOXM1 and uPA in oral dysplasia and SCCs of head and neck, oesophagus, lung and cervix. In the mouse model of oral cancer we show that uPA expression is upregulated in recurrent tumors compared to primary tumors, giving further evidence for a crucial role of the p38-FOXM1-uPA axis in the development of recurrent tumors. Taken together, we conclude that the stress signalling cascade requires a FOXM1-dependent intermediate step preceding the activation of AP-1 transcription factor to enhance invasive behaviour of tumor cells. This novel mechanism promotes invasion of HNSCC and may provide a potential target for the adjuvant therapy of these highly invasive cancers.
In the last one and a half decades, the generalization of high throughput methods in molecular biology has led to the generation of vast amounts of datasets that unraveled the unfathomed complexity of the cell regulatory mechanisms. The recently published results of the ENCODE project (ENCODE Project Consortium et al., 2012) demonstrated the extend of these in the human genome and certainly more regulation mechanisms will be discovered in the future. Already, this complexity within a single cell - without taking into account cell-cell interaction or micro-environment influences - cannot be abstracted by the human mind. However, understanding it is the key to devise adapted treatments to genetic diseases or disorders, among which is cancer. In mathematics, such complex problems are addressed using methods that reduce their complexity, so that they can be modeled in a solvable manner. In biology, it led researchers to develop the concept of systems biology as a mean to abstract the complexity of the cell regulatory network. To date, most of the published studies using high throughput technologies only focus on one kind of regulatory mechanism and hence cannot be used as such to investigate the interactions between these. Moreover, distinguishing causative from confounding factors within such studies is difficult.
These were my original motivations to develop analytical and statistical methods that control for confounding factors effects and allow the integrative and comparative analysis of different kinds of datasets. In fine, three different tools were developed to achieve this goal. First, "customCDF": a tool to redefine the Custom Definition File (CDF) of Affymetrix GeneChips. It results in the increased sensitivity of downstream analyses as these bene fit from the constantly evolving human genome reference and annotations. Second, "aSim": a tool to simulate microarray data, which was required to benchmark the developed algorithms. Third, for the integrative analysis, a set of combined statistical methods and finally for the comparative analysis, a modification of the integrative analysis approach. These were bundled in the "crossChip" R package. The "customCDF" and "aSim" tools were first validated on independant datasets. The developed analytical methods ("crossChip") were first validated on "aSim" simulated data and publicly available datasets and then used to answer two biological questions. First, using two retinoblastoma datasets, the effect of genomic copy number variations on gene-expression was investigated. Then, motivated by the fact that retinoblastoma patients have a higher chance to develop osteosarcoma later in life than the average population, datasets of both these tumors were comparatively analyzed to assess these tumors similarities and differences.
Despite a rather limited number of samples within the selected datasets, the developed approaches with their higher sensitivity and sensibility were successful and set the ground for larger scale analyses. Indeed, the integrative analysis applied to retinoblastoma revealed the high importance of the chromosome 6 gain at a later stage of the disease, indicating that many genes on that chromosome are beneficial to cancerogenesis. Moreover, in comparison to standard microarray analyses, it demonstrated its efficacy at detecting the interplay of regulatory mechanisms: examples of positive and negative compensation of gene expression in lost and gained regions, respectively, as well as examples of antisense transcription, pseudogene and snRNAs regulation were identified in this dataset. The comparative analysis on the other hand revealed the high similarity of the retinoblastoma and osteosarcoma tumors, while at the same time showing that either of them take advantage of their distinct micro-environment and consequently appear to make use of different signaling pathways, PKC/calmodulin in retinoblastoma and GPCR/RAS in osteosarcoma. The developed tools and statistical methods have demonstrated their validity and utility by giving sensible answers to the two biological questions addressed. Moreover, they generated a large number of interesting hypotheses that need further investigations. And as they are not limited to microarray analysis but can be applied to analyze any high-throughput generated data, they demonstrated the usefulness of "systems biology" approaches to study cancerogenesis.
Because of its ability to catalyse oxidation/reduction reactions, iron (Fe) is an essential microelement in living beings. However, high levels of Fe in the cell can lead to detrimental effects, as this metal can also catalyse the production of harmful reactive oxygen species. Therefore, tight regulation of cellular Fe concentration is required. In this thesis, two independent genes with a putative role in Fe homeostasis were characterized in the model organism Arabidopsis thaliana. AtRAI1 is homolog to the yeast Rai1p (Rat1 Interacting protein 1), an activator of 5´-to-3´ exoribonucleases, enzymes that degrade decapped RNAs. In Arabidopsis protoplasts, AtRAI1 co-localized with the decapping enzyme DCP2 (Decapping 2) in processing bodies, which are structures where mRNAs are processed for degradation. DK9, a loss-of function mutant for AtRAI1, had phenotypic traits attributable to impaired Fe homeostasis (chlorosis, reduced size) and to homeostasis of the phytohormone auxin (curly leaves and low fertility). Assessment of the activity of the Fe-dependent anti-oxidant enzymes catalase and SOD (SuperOxide Dismutase), multi-element analysis and determination of transcript levels of genes involved in Fe homeostasis showed that AtRAI1 is an upstream regulator of Fe uptake and transport in A. thaliana. The studied protein probably activates the cytosolic exoribonuclease XRN4, which is a component of the ethylene signalling pathway that is known to control Fe uptake in roots. Since homozygous knockout alleles of the analysed gene were not viable, it is concluded that AtRAI plays an essential role for Fe acquisition and proper growth of A. thaliana. The second gene studied in this thesis is BTS2 (BRUTUS2). It encodes a protein that shares similarity with BTS, an E3 ubiquitin ligase with putative roles in Fe regulation. A loss-of-function allele was more tolerant to excess Zn, a condition that leads to physiological Fe deficiency in A. thaliana. Under this condition, the mutant accumulated less Fe in the root than wild-type plants, indicating that the protein probably induces Fe uptake under Fe deficiency conditions. Furthermore, low Zn concentrations and reduced transcript levels of marker genes for excess Zn in shoots suggest an additional function in Zn translocation to the shoot. Therefore, BTS2 may play a critical role in positively regulating Fe deficiency response in roots and Zn translocation to the shoot by targeting negative regulators for degradation.
The research presented in this thesis aims at improving the characterization of the scaling relations of the masses of central Supermassive Black Holes with bulge and total luminosities of their host galaxies. These scaling relations are significant for our understanding of the evolution of galaxies and the origins of Supermassive Black Holes. As part of this investigation, 35 galaxies with known central Black Hole mass were observed at near-infrared wavelengths. The obtained images were subjected to a thorough photometric analysis, which led to reliable bulge and total luminosities. As a result, it could be shown that the slope of the correlation between central Black Hole masses and bulge luminosities was previously overestimated. Further, it was found that the correlation with total luminosity is equally tight as the correlation with bulge luminosity. A linear regression method was devolped that enables extraction of more detailed information about the cosmic scatter in the scaling relations. In the last part of this thesis, a Black Hole mass was determined by means of dynamical modeling. The measured mass far exceeds the prediction from current scaling relations, thereby putting the universality of the scaling relations in question and principally opening up opportunities to better understand the physics of galaxy and Black Hole formation.
In their approach to higher-dimensional global class field theory, Kato and Saito define the class group of a proper arithmetic scheme \bar{X} as an inverse limit C_{KS}(\bar{X}) = \varprojlim_{\mathcal{I}} C_{\mathcal{I}}(\bar{X}) of certain Nisnevich cohomology groups C_{\mathcal{I}}(\bar{X}) taken over all non-zero coherent ideal sheaves \mathcal{I} of \mathcal{O}_{\bar{X}}. The ideal sheaves \mathcal{I} should be regarded as higher-dimensional analogues of the classical moduli \mathfrak{m} on a global field K, which induce a filtration of the idele class group C_K by the ray class groups C_K/C_K^{\mathfrak{m}}. In higher dimensions however, it is not clear how the induced filtration of the abelian fundamental group can be interpreted in terms of ramification. In view of Wiesend's class field theory, we define an easier notion of moduli in higher dimensions only involving curves on the scheme. We then show that both notions agree for moduli that correspond to tame ramification.
Das Endometriumkarzinom (EK) ist in den Industrieländern die häufi gste diagnostizierte invasive Tumorerkrankung des weiblichen Urogenitaltrakts. Bei der Klassifi zierung wird zwischen dem meist mit einem positiven Verlauf assoziierten Typ-I EK und dem aggressiven Typ-II EK unterschieden. Ergebnisse der letzten Jahre zeigen, dass dem Zelladhäsionsmolekül L1CAM, welches von gesundem Endometrium nicht exprimiert wird, eine entscheidende Rolle in der Tumorprogression zukommt. L1CAM wird dabei hauptsächlich bei hochgradigen Typ-II EKs, die als seröse und klarzellige Adenokarzinome eingestuft werden, detektiert. Es konnte bereits gezeigt werden, dass die Überexpression des L1CAM-Gens die Motilität der Tumorzellen sowie das Tumorwachstums fördert. Dabei ist ein charakteristisches Merkmal, dass die L1CAM-Genexpression oft mit der invasiven Front eines Tumors assoziiert ist. Es wird beschrieben, dass Zellen in der invasiven Front eine Epitheliale-Mesenchymale-Transition (EMT) durchlaufen und anschließend in das umgebende Gewebe invadieren. Dieser Prozess wird durch die L1CAM-Überexpression unterstützt. Derzeit ist immer noch unklar, wie es zu dieser atypischen L1CAM-Expression an der invasiven Tumorfront kommt. Die zentrale Frage der vorliegenden Arbeit war daher, welchen Regulationsmechanismen das L1CAM-Gen im endometrialen Tumorgewebe unterliegt. Um dies zu klären, wurde die transkriptionelle Regulation von L1CAM auf unterschiedlichen Ebenen untersucht. Dabei konnte gezeigt werden, dass die TGF-β1 induzierte EMT in EK-Zellen zu einer Aktivierung von L1CAM führt die durch den EMT assoziierten Transkriptionsfaktor Slug vermittelt wird. Die durchgeführten DNA-Bindungsanalysen bestätigen eine direkte Interaktion des Transkriptionsfaktors Slug mit den zwei alternativen Promotoren des L1CAMGens. Des Weiteren sind Slug und L1CAM auf mRNA-Ebene in EK-Zelllinien positiv miteinander korreliert. Neben dem EMT-Aktivator Slug konnte mit dem Repressor REST ein weiterer Regulator von L1CAM identifi ziert werden. Der Einfl uss von REST auf die L1CAM-Expression war zuvor bereits in neuronalen Zellen beschrieben worden. Der direkte Mechanismus, über den REST in die L1CAM-Expression eingreift, bleibt zu klären. Über der Ebene der transkriptionellen Regulation hinaus, konnte gezeigt werden, dass auch der epigenetischen Regulation eine bedeutende Rolle in der Kontrolle der L1CAM-Expression zukommt. So konnte nachgewiesen werden, dass sowohl ein Einfl uss der Histon-Deacetylierung als auch der DNA-Methylierung bei der Suppression des L1CAM-Gens vorliegt. Dementsprechend führte die Behandlung von EK-Zelllinien mit Inhibitoren der Histon-Deacetylasen und/oder DNA-Methyltransferasen (DNMTs) zu einer Aktivierung des L1CAM-Gens. Darüber hinaus konnte mittels einer Methylierunganalyse nachgewiesen werden, dass in EK-Zelllinien der Grad der Promoter-Hypomethylierung mit der L1CAM-Expression korreliert. Entgegen der Erwartungen führte die Behandlung von L1CAM exprimierenden Zelllinien mit DNMTInhibitoren, zu einer drastischen Abnahme der L1CAM-Expression. Die nachfolgende Analysen dieses Effekts offenbarten eine Aktivierung von microRNAs, insbesondere der miR-34a, welche sehr wahrscheinlich zu der post-transkriptionellen Regulation von L1CAM beiträgt. Zusammenfassend belegen die Daten dieser Arbeit eine komplexe Kontrolle des L1CAM-Gens auf mehreren Ebenen. So greifen sowohl epigenetische als auch transkriptionelle und post-transkriptionelle Mechanismen in die Expression von L1CAM ein. Eine Deregulation dieser komplexen Repression kann so zu einer fokalen Aktivierung von L1CAM in hochgradigen EKs führen.
Salt lakes can be found on all continents and saline soils cover 2.5% of the land surface of the earth (FAO, 2012). This thesis investigates the presence of reactive halogen species (RHS) above salt lakes and saline soils to evaluate their relevance for tropospheric chemistry of the planetary boundary layer. Ground-based MAX-DOAS and LP-DOAS measurements were conducted at salt lakes and two other sites with high halogen content. Prior to this work, RHS were found at three salt lakes with mixing ratios ranging from several pptv up to more than 100 pptv. On the basis of this work the data base on reactive halogen release from salt lakes was significantly expanded. Passive DOAS and active DOAS systems were used to examine the vertical as well as the horizontal distribution of BrO in the Dead Sea Valley. Vertical profiles of aerosols, BrO and NO2 were measured at two adjacent sites at the Dead Sea using MAX-DOAS. The first time vertical profiles of BrO and NO2 at the Dead Sea were determined and interpreted in terms of the special atmospheric dynamics in the Dead Sea Valley. The resulting ground values of BrO and NO2 at one measurement site were validated by LP-DOAS and show very good agreement. The field studies presented in this work showed, that the release of RHS in those regions is less widespread than previously assumed.
The human brain is the most complex organ of the human body and many aspects of its functioning have not yet been understood. One of the most fascinating abilities of the human brain is the skill to store and retrieve information, which is what we refer to as memory. One attempt to get a deeper insight into the functioning of memory is to analyze the complex activity pattern of the human brain that emerges while a memory task is being processed. The understanding of memory is epistemologically very intriguing since it is this ability that enables us to collect, to store and to recall ideas, emotions and thoughts - hence, it builds our own identity. This thesis analyses age-related changes in functional connectivity networks related to episodic and working memory processing. The data for this study were measured using fMRI technique and the sample set consisted of healthy individuals aging from 20 up to over 80 years. Based on the fMRI data we construct correlation networks by correlating pairwisely the measured voxel activity, the nodes of the network being brain voxels, the edges being correlations. These networks are thresholded, anatomically clustered and analyzed by computing statistical network measures, using spectral methods, computing network entropy and calculating persistent homology. The main findings are: elderly individuals exhibit expanded neural networks with less differentiation between episodic and working memory tasks. However, we observe compensatory mechanisms that accompany this dedifferentiation process. Network synchronizability is higher for elderly individuals. Network entropy increases as well with age, yielding a lower network vulnerability for elderly individuals. Aging processes leave traces in the homology pattern of the networks, whereas all brain networks exhibit different persistent homology features.
Cell shape changes are of fundamental importance during morphogenesis. These changes are often initiated by the contraction or expansion of plasma membrane domains. During differentiation the plasma membrane also undergoes more complex functional re-organization that brings about specialized function such as absorption, secretion and photo-transduction. While the role of cytoskeleton elements in controlling the structure and dynamics of the plasma membrane is well-established, little is known about the contribution of membrane trafficking in this process. The aim of this thesis was to address the contribution of membrane trafficking in controlling cell shape changes during tissue morphogenesis. More specifically I have investigated the role of endocytosis in controlling the remodeling of the plasma membrane during cellularization, the transformation of the syncytial Drosophila embryo is 6000 mononucleated cells. By following the early endocytic regulator Rab5, I identified two pools of endosomes. Early during cellularization endosomes accumulate at the invaginating furrows. Towards the end of cellularization a second pool of endosomes appeared at the apical surface. This increase in apical endosomes coincides with changes in apical morphology. Blocking endocytosis by inhibiting dynamin function prevented the re-absorption of apical protrusions and subsequent membrane flattening. Using a novel genetically-encoded cargo uptake assay I discovered that during apical surface flattening endocytosis is up-regulated of approximately five-fold. Strikingly this assay also revealed that the primary entry route for soluble extracellular cargo is through long tubular intermediates that serve as platform for the generation of Rab5 vacuolar endosomes. Blocking dynamin activity resulted in the complete inhibition of both tubular endocytosis as well as in the disappearance of Rab5 endosomes. These data collectively support a role for membrane trafficking in morphological remodeling. Surface flattening is thus an endocytosis-dependent morphogenetic process driven by the rapid internalization of large quantities of plasma membrane through tubular invagination and up-regulation of Rab5 endosome production. To further characterize the molecular machinery controlling apical endocytosis during cellularization a biochemical approach was undertaken. I performed large-scale affinity purification from 0-4 h embryos in order to identify Rab5 effectors operating during these early stages of embryonic development. This experiment led to the identification of Rabankyrin-5. Using a combination of live imaging and correlative light-electron microscopy I could show that Rabankyrin-5 controls the budding and processing of apical vacuoles from tubular plasma membrane invagination. In conclusion, in this thesis I have identified a novel endocytic pathway and linked its function to the remodeling of the apical surface during epithelial morphogenesis.
Gustav R. Kirchhoff war vom 18.4.1865 bis zum 2.4.1866 Prorektor der Heidelberger Universität. In dieser Eigenschaft hielt er am 22. November 1865 zur Jahresfeier die Rede ,,Ueber das Ziel der Naturwissenschaften''. Seine Grundvorstellung ist, dass alle Naturerscheinungen durch Bewegungen entstehen und daher durch die Mechanik beschrieben werden können. Zeitbedingt nimmt er noch an, dass das Weltall durch den Lichtaether gefüllt ist, aber die Existenz eines Wärmestoffes verneint er bereits; er hatte erkannt, dass Wärme durch Bewegung der Materie entsteht.
Erinnerungen des Arztes Adolf Kußmaul (1822-1902, Erfinder der Magenpumpe), der seinen Lebensabend in Heidelberg verbrachte, an Robert W. Bunsen, Gustav R. Kirchhoff und Hermann von Helmholtz (1. Teil des Aufsatzes) sowie an deren Jünger und andere Heidelberger Dozenten (2. Teil des Aufsatzes).
Increased awareness of the human and environmental health risks associated with perfluorinated chemicals (PFCs) has raised intensive discussions among authorities and policymakers. Yet, despite declined use of PFCs in industrial and consumer products, these emerging contaminants are still being detected in aquatic environments worldwide. In light of the persistent properties of PFCs, fairly little attention has been given to long-term effects. Another challenging topic in the context of PFCs concerns their potential to interact synergistically; a relevant matter given the complex exposure scenarios in aquatic systems. This thesis aimed at increasing the knowledge and understanding of PFCs and their toxicity towards an aquatic vertebrate model, the zebrafish (Danio rerio). Focus was on multixenobiotic resistance (MXR) and toxicity following chronic exposure. Selected PFCs were evaluated in transporter efflux assays serving to determine the synergistic potential via P-glycoprotein (P-gp) transporter inhibition. Long-term effects following single and binary exposures of perfluorooctane sulfonate (PFOS) and bisphenol A (BPA) were evaluated over two full generations with emphasis on survival, histological alterations, vitellogenin (Vtg) and reproductive success. Among the tested PFCs, PFOS induced the strongest accumulation of the standard P-gp transporter substrate rhodamine B (RhB) in zebrafish embryos. An up to fourteen-fold RhB-retention was found in PFOS-exposed embryos if compared with control animals. In comparison, the effect of PFOS on the uptake of the P-gp substrate calcein-AM by MDCKII cells overexpressing human P-gp was substantially smaller than that of the reference compound verapamil indicating that PFOS only weakly interacts with human P-gp. In the long-term study, the most prevalent effects following waterborne PFOS-exposure were decreased survival in off-spring generations and hepatocellular alterations. The hypothesis that the presence of PFOS increases the endocrine potential of BPA could not be confirmed in zebrafish. This thesis provides further evidence of the chemosensitizing potential of some PFCs in zebrafish. Although the exact mechanisms of action behind the increased uptake of P-gp substrates remain unclear, the results obtained further highlight the importance of mixture toxicity when investigating the hazardous potential of PFCs. Adverse long-term effects on liver structure and survival in zebrafish were only seen at concentrations well above ecologically relevant concentrations. Yet, the decline in survival rates following PFOS-exposure seen over generations again documents the necessity of long-term approaches within the assessment of persistent environmental pollutants.
Cells and tissues are constantly exposed to mechanical forces. Understanding how these forces act on cells to regulate essential processes like development, differentiation, tissue homeostasis and how their alteration is related to disease requires the characterization of their mechanical properties. Several methods have been developed to study mechanical properties of cells and nuclei. However, most of the established methods are not com- patible with culturing of cells in 3D substrates, a factor which plays an essential role in defining the structural and mechanical behavior of cells naturally existing in 3D environ- ments. In this work, image and model based methods have been developed to approach this problem and enable the characterization of the cells mechanical phenotype in 3D.
On a first step, a previously developed method to measure the compressibility of the nuclear interior was enhanced to enable statistical significant measurements of nuclei to perform comparative analyses between phenotypes. Optimization of both the experi- mental, as well as the image processing methods led to a robust framework that served to measure an increase in nuclear compressibility in nuclei of LMNA−/− mouse embryonic fibroblasts. This study served as a proof of principle for this contact free method, which in a subsequent step was adapted to work for cells embedded in 3D substrates.
Aiming to develop a method, in which specific forces could be applied and relate to cellular deformations, the second part of this work was centered in the development of the 3D substrate stretcher. This involved identifying and implementing the needs of the experimental and image analysis framework to ensure the required environment for the cells, while at the same time enabling the acquisition of suitable data for the mechanical analysis. The resulting experimental and analysis framework enables for the first time application and quantification of strains on cells embedded in 3D substrates.
Motivation of the 3D-culture based methods was the analysis of epithelial-mesenchymal transition (EMT) in hepatocytes. These epithelial cells undergoing dedifferentiation upon treatment with TGF-β serve not only as a preeminent example of the need of 3D cell cultures in the characterization of mechanical properties, but also as a model of malignant transformation in fibrotic diseases and cancer. Quantification of previously unobserved morphological and structural properties led to the mechanical phenotyping of these cells, where a decrease in the compressibility of the nuclear interior, an enhanced resistance to deformation and a better anchorage of the nuclei inside the cells was observed after EMT.
Der Autor, der keine Literaturliste angibt, benutzt offensichtlich die dreibändige Helmholtz-Biographie Leo Koenigsbergers (1902-1903) für den biographischen Teil seines Werkes.
Transmission electron microscopy (TEM) plays an important role in structure determination of biologi- cal or material science samples. Resolutions up to the sub- ̊Angstrøm range are attainable. Weak phase objects do not reach this resolution due to their low signal-to-noise ratio. As the structure information of weak phase objects is mainly contained in their phase contrast, the conventional method for contrast enhancement is image acquisition at high defocus. This leads to a lower attainable resolution and con- trast inversion for different spatial frequencies, due to the sinusoidal phase contrast transfer function. As a consequence of these drawbacks, the development of physical phase plates for in-focus phase con- trast enhancement in TEM is of widest interest. In this thesis the development and application of the electrostatic Zach phase plate (ZPP) is presented. The impact of ZPP positioning on single sideband image artefacts is investigated. The applicability of the ZPP for inducing invertible and tunable phase contrast is demonstrated for the first time experimentally on frozen-hydrated biological samples. ZPP contamination, resulting charging artefacts and solution for these problems are presented. Furthermore the application of the ZPP with conventional electron optics and with a microscope containing a diffrac- tion magnification unit and an aberration corrector is compared. From this new insights into the design and use of an obstruction free anamorphic phase plate (APP) can be gained.
In the first part of this thesis we develop an investment consumption model with convex transaction costs and optional stochastic returns for a finite time horizon. The model is a simplified approach for the investment in a portfolio of commodity related assets like real options or production facilities. In contrast to common models like [Awerbuch, Burger 2003] our model is a multi time step approach that optimizes the investment strategy rather then calculating a static imaginary optimal portfolio. On one hand, our numerical results are consistent with the well-known investment-consumption theory in the literature. On the other hand, this is the first in-depth numerical study of a case with convex transaction costs and optional returns. Our focus in the analyses is the form of the investment strategy and its behavior with respect to model parameters.
In the second part, an algorithm for solving continuous-time stochastic optimalcontrol problems is presented. The numerical scheme is based on the Stochastic Maximum Principle (SMP) as an alternative to the widely studied dynamic programming principle (DPP). By using the SMP, [Peng 1990] obtained a system of coupled forward-backward stochastic differential equations (FBSDE) with an external optimality condition. We extend the numerical scheme of [Delarue, Menozzi 2005] by a Newton-Raphson method to solve the FBSDE system and the optimality condition simultaneously. This is the first fully implemented algorithm for the solution of stochastic optimal control problems through the solution of the corresponding extended FBSDE system. We show that the key to its success and numerical advantage is the fact that it tracks the gradient of the value function and an adjusted Hessian backwards in time. The additional information is then exploited for the optimization.
In the following study the mouse mutant cobblestone (cbs) concerning the development of the forebrain was analyzed. The cbs mutation was uncovered by an ethyl-nitroso-urea (ENU) genetic screen, using a mouseline called tauGFP. At the beginning of the analysis of the cbs mutation, the phenotype of the cbs/cbs mouse mutant was already known, but because of the mutagenic ability of ENU, which causes random mutations, the affected gene was unclear. By applying the method of positional cloning the gene intraflagellar transport 88 (Ift88) was identified as the candidate gene. A detailed analysis of mRNA levels of Ift88 in the cbs/cbs mutant was undertaken by Northen Blot analysis as well as quantitative real-time RT-PCR. At the same time the Ift88 protein levels were also investigated by Western blot analysis. A complementation analysis by crossing cbs heterozygotes to mice heterozygous for a targeted deletion of the Ift88 gene (Ift88tm1.1Bky) (Haycraft et al., 2007) was done to ascertain, if the genetic defect in the cbs/cbs mutant is located in the Ift88 gene. In the represented study it could be shown that cbs is a hypomorphic allele of the gene Ift88, in which both Ift88 mRNA and protein levels are reduced by 70% to 80%, respectively. A detailed analysis by in situ hybridization (ISH) was followed, using different markers, which are specific for various areas of the developing telencephalon such as Ttr1 for the choroid plexus, Wnt2b for the cortical hem, as well as EphB1 and Lhx2 for the hippocampal anlage. Furthermore both the dorsal-ventral and rostral-caudal compartmental boundaries of the forebrain were investigated by ISH. cbs/cbs mutants display defects in the formation of dorsomedial telencephalic structures, such as the choroid plexus, cortical hem and hippocampus. Furthermore mutants exhibit a relaxation of both dorsal-ventral and rostral-caudal compartmental boundaries of the forebrain, resulting in the intermixture of otherwise separated cell populations. It is further demonstrated that the proteolytic processing of Gli3 is reduced in the cbs/cbs mutant, leading to an accumulation of the full-length activator isoform. In addition the cbs/cbs mutant exhibits an upregulation of canonical Wnt signalling in the neocortex and in the caudal forebrain. Primary cilia, microtubule-based organelles that protrude from the surface of most cells of the vertebrate body, are dependent on Ift88 for their formation and maintenance. The ultrastructure and morphology of cilia of the ventricle of the cbs/cbs mutants was therefore simultaneously investigated by transmission and scanning electron microscopy. Surprisingly, examined cilia are still existing and intact in the cbs/cbs mutant. Taken together, these results indicate a fundamental role for primary cilia in the development of the forebrain.
We study the étale homotopy theory of Brauer-Severi varieties over fields of characteristic 0. We prove that the induced Galois representations on geometric homotopy invariants (e.g., l-adic cohomology or higher homotopy groups) are all isomorphic for Brauer-Severi varieties of the same dimension. If the base field has cohomological dimension smaller or equal 2 then we can show more in the case of Brauer-Severi curves: There is even an isomorphism between the Hochschild-Serre spectral sequences computing cohomology with local coefficients. Further, we study homotopy rational and homotopy fixed points on Brauer-Severi varieties and their connections to genuine rational points. In particular, we show that under a suitable assumption on the first profinite Chern class map an analogue of the weak section conjecture for Brauer-Severi varieties turns out to be true. We can give a counter example to this analogue without the extra assumption over p-adic local fields.
Mechanical force alters a protein's stability not only due to its ability to unfold the biomolecule. As soon as a disulfide bond cross-linking the protein is exposed to force, its reduction rate is altered. Our first aim was quantifying the direct effect of force onto the chemical reactivity of sulphur-sulphur bonds in contrast to indirect, e.g. steric or mechanistic, influences. To this end, we evaluated the dependency of a disulfide bond's redox potential on a pulling force applied along the system. Our hybrid quantum and molecular mechanics simulations of cystine as a model system take conformational dynamics and explicit solvation into account and show that redox potentials increase over the whole range of forces probed here (30 - 3320 pN), and thus even in the absence of a significant disulfide bond elongation(<500 pN). Instead, at low forces, dihedrals and angles as the softer degrees of freedom are stretched and contribute to the destabilization of the oxidized state. We find physiological forces to be likely to tune the disulfide's redox potentials to an extent similar to the tuning within proteins by point mutations. Next, we asked how internal strain resulting from the protein structure tunes redox potentials using free energy calculations, more precisely nonequilibrium Molecular Mechanics transformations and the Crooks Gaussian Intersection method. We added a residue to the Charmm force field that models a disulfide bond in the reference state and that can be transformed into a thiol in the product state. To our knowledge, this is the first approach to open a covalent bond by means of free energy transformation. We tested our method on E. coli and S. aureus thioredoxin, and could partly reproduce relative redox potentials of the wild-type and some mutants. We discuss promising routes to improve the accuracy of these challenging calculations. Finally, we investigated the impact on force-induced unfolding by a special type of disulfide bond, a vicinal disulfide that links two adjacent cysteines. Our model system here is the von Willebrand factor (vWF) A2 domain. We observe similar stabilities in equilibrium for both the native system and its analogue with the disulfide bond broken and also similar collective motions. Application of an external force, however, induces a difference: Unfolding of the vWF A2 domain with the vicinal disulfide bond present leads to higher rupture forces than when it is missing. This indicates that the vicinal disulfide bond prevents the domain from unintentional unfolding.
Active learning is one form of supervised machine learning. In supervised learning, a set of labeled samples is passed to a learning algorithm for training a classifier. However, labeling large amounts of training samples can be costly and error-prone. Active learning deals with the development of algorithms that interactively select a subset of the available unlabeled samples for labeling, and aims at minimizing the labeling effort while maintaining classification performance. The key challenge for the development of so-called active learning strategies is the balance between exploitation and exploration: On the one hand, the estimated decision boundary needs to be refined in feature space regions where it has already been established, while, on the other hand, the feature space needs to be scanned carefully for unexpected class distributions. In this thesis, two approaches to active learning are presented that consider these two aspects in a novel way. In order to lay the foundations for the first one, it is proposed to express the uncertainty in class prediction of a classifier at a test point in terms of a second-order distribution. The mean of this distribution corresponds to the common estimate of the posterior class probabilities and thus is related to the distance of the test point to the decision boundary, whereas the spread of the distribution indicates the degree of exploration in the corresponding region of feature space. This allows for the evaluation of the utility of labeling a yet unlabeled point with respect to classifier improvement in a principled way and leads to a completely novel approach to active learning. The proposed strategy is then implemented and evaluated based on kernel density classification. The generic active learning strategy can be combined with any other classifier, but it performs best if the derived second-order distributions are sufficiently good approximations to the sampling distribution. Although second-order distributions for random forests are derived in this thesis, they do not approximate sufficiently well the sampling distribution and mainly allow only for the relative comparison of prediction uncertainty between test points. In order to combine the state of the art classification performance of random forests with the principal ideas of the first active learning approach, a related second approach for random forests is derived. It is, in addition, tailored to the demands in industrial optical inspection: bag-wise labeling with weak labels and strongly imbalanced classes. Moreover, an outlier detection scheme based on random forests is derived that is used by the proposed active learning algorithm. Finally, a new computational scheme for Gaussian process classification is presented. It is compared to two standard methods in geostatistics, both with respect to theoretical consistency and practical performance. The method evolved as a by-product when considering using Gaussian process models for active learning.
A superresolution scanning fluorescence microscope – or nanoscope – allows imaging structures smaller than the diffraction limit of light. The principal idea in targeted readout microscopy methods is the on/off-switching of a subset of fluorophores inside the focal volume with the help of different reversible switchable optical fluorescence transitions. If, in addition, the effective numerical aperture is doubled as in a 4Pi optical setup, the resolving power in axial direction of a far field microscope can be significantly increased. Due to the local field enhancement, a 4Pi augmented STimulated Emission Depletion (STED) setup exploits STED light in an effective manner. To this date, it features the smallest effective scannning volume. However, the acquisition of the obtained image information with this slender volume requires smaller scanning steps and in consequence a longer overall acquisition process. This disadvantage can be compensated for by parallel readout and is relevant for a 4Pi-STED combination, in particular. The proposed image parallelization scheme allows to trade-off acquisition speed against required resolution. In this thesis, a 4Pi augmented line parallelized STED setup is presented that is, for the first time, able to resolve 20 nm fluorescent microspheres with a resolution of about 50 nm in two dimensions with a focal volume that is 10 times parallelized as compared to a single spot confocal volume.
Motivation dieser Arbeit ist die Präparation ultradünner Nanomembranen (< 10 nm), die außerdem biokompatibel bzw. proteinresistent sind, um als Trägerfilm biologischer Proben für die hochauflösende Transmissionselektronenmikroskopie (HRTEM) Verwendung finden zu können. Als Basis werden selbstaggregierende Monolagen (SAMs) aus 4‘-Nitrobiphenyl-4-thiol (NBPT) auf einem Au(111)-Substrat eingesetzt, die durch Bestrahlung mit niederenergetischen Elektronen lateral quervernetzt und deren terminale Nitrogruppen zu oberflächengebundenen Aminogruppen reduziert werden (iNBPT). An diese lassen sich Epoxid-terminierte Polyethylenglykole (PEG) derart koppeln, dass die resultierende PEG-iNBPT Doppelschicht bei einer Gesamtdicke von nur 5 nm biokompatibel wird und der Adsorption von Fibrinogen als Testprotein widersteht. Ferner wird ellipsometrisch und röntgenspektroskopisch (X-ray photoelectron spectroscopy, XPS) untersucht, welchen Einfluss Schichtdicke und Packungsdichte des PEG-Films auf die proteinabweisenden Eigenschaften des PEG-iNBPT-Films ausüben und die Resultate mit denen eines etablierten, biokompatiblen SAMs aus Oligoethylenglykol-terminiertem Undecanthiol verglichen. Zuletzt muss der PEG-iNBPT Film vom Substrat abgelöst und als Membran über ein TEM-Grid mit freistehenden Bereichen von mindestens 10 x 10 µm² gespannt werden können, ohne dass die Folie dabei ihre Biokompatibilität verliert. Ihre Eignung als HRTEM-Trägerfilm wird anhand von Gold-Nanopartikeln und dem Eisenspeicherprotein Ferritin demonstriert. Wegen der vielseitigen Anwendungsmöglichkeiten von NBPT-SAMs wäre es interessant, ein alternatives SAM-bildendes System mit ähnlichen Merkmalen zu besitzen. In einer führen Arbeit wurde bereits aufgeführt, dass 4‘-Cyanobiphenyl-4-thiol (CBPT) Monolagen ebenfalls durch Elektronenbestrahlung quervernetzt und die terminale Nitrilgruppe zu einer Aminogruppe reduziert wird. Darauf aufbauend wird in dieser Arbeit gezeigt, dass sich CBPT-SAMs als negatives Resist-Material in Elektronenstrahllithographie, sowie als Templat in der chemischen Elektronenstrahllithographie (EBCL) eignen. In diesem Zusammenhang wird der Einfluss der Elektronenenergie im Bereich von 0.5 10 keV auf den Grad der Quervernetzung aromatischer SAMs sowie auf die Effektivität der Reduktion terminaler Gruppen beleuchtet, um so Aufschlüsse über zugrundeliegende Prozesse bei der Elektronenbestrahlung selbstaggregierender Monolagen zu gewinnen. Außerdem wir der Frage nachgegangen, ob alle durch elektronenstrahlinduzierte Reduktion entstandenen, oberflächengebundenen Aminogruppen gleichermaßen in der Lage sind, als Anker zur Kopplung weiterer Moleküle zu dienen. Die SAM-basierenden, PEGylierten Nanomembranen sind mit 5 nm zwar außerordentlich dünn, aber nur einseitig biokompatibel. Es wird in dieser Arbeit eine zweite Präparationsmethode vorgestellt, die es ermöglicht, direkt und ohne den Umweg über quervernetzte SAMs, doppelseitig biokompatible Nanomembranen variabler Dicke herzustellen, die ab 20 nm ausreichend mechanische Stabilität aufweisen, um freistehend existieren können. Als Vorstufe dient ein aus Epoxid- und Amino-terminierten, multifunktionellen Polyethylenglykolen bestehender Zweikomponentenfilm, der per Rotationsbeschichtung auf ein flaches Substrat aufgebracht und thermisch quervernetzt wird. Die 6 - 300 nm dicken PEG-Filme werden ellipsometrisch sowie XPS- und infrarotspektroskopisch charakterisiert und auf ihre filmdickenanhängige Proteinresistenz untersucht. Durch die Quervernetzung zeigt das polymere Netzwerk ein Hydrogel-typisches, reversibles Quellverhalten beim Kontakt mit Wasser, welches eingehend ellipsometrisch analysiert wird. Zudem können Gold-Nanopartikel (AuNPs) irreversibel und in hohen Dichten in der Matrix des PEG-Films immobilisiert werden und so das Quellverhalten in eine optische Antwort des Systems umwandeln. Ein zweiter Aspekt bezüglich der PEG-Filme betrifft ihre Reaktion auf die Bestrahlung mit Elektronen. Das Verhalten von Polyethylenglykolen wird diesbezüglich erstmals systematisch mit Hilfe der PEG-Filme XPS- und infrarotspektroskopisch sowie mit Röntgen-Nahkanten-Absorptions-Spektroskopie (NEXAFS) untersucht. Ferner wird rasterkraftmikroskopisch (AFM) und fluoreszenzmikroskopisch ermittelt, wie sich die dosisabhängige Elektronenbestrahlung auf die PEG-Filme in Bezug auf Quellverhalten, Topographie, Benetzbarkeit und Biokompatibilität auswirken. Zuletzt werden die PEG-Filme von ihrem Substrat abgehoben, freistehend über eine Gitterstruktur gespannt und die mechanischen Eigenschaften wie Elastizitätsmodul und Eigenspannung der Membranen bestimmt. Auch in diese können Gold-Nanopartikel eingebettet werden und somit eine Verbundmembran mit erweiterten optischen Eigenschaften hergestellt werden. Sie zeigt zudem ebenfalls ein ausgeprägtes Quellverhalten, welches zu veränderten Eigenschaften in Abhängigkeit von äußern Parametern wie Temperatur oder Luftfeuchtigkeit führt.
T cell responses in cancer patients correlate with improved prognosis. This effect can partially be attributed to cytotoxic T cells that recognize tumor antigens on cancer cells. Hence, the induction or improvement of such therapeutic tumor antigen-specific T cell responses is a major goal of tumor immunotherapy. So far the target-antigens of relevant prognostic T cell responses are unknown. In this thesis a systematic analysis was performed to identify those antigens that are able to elicit spontaneous T cell responses in patients with colorectal carcinoma. Those responses against the newly identified antigens were then compared, in a larger patient cohort, to T cell responses against established “canonical” tumor antigens that are commonly used for immunotherapy. The establishment of the proteome-based technology PF2D (Protein fractionation in two dimensions) in our group allowed the identification of protein fractions out of lysates from primary and metastatic tumor tissue that were capable to elicit strong T cell responses in ELISpot assays. With mass spectrometry and further ELISpot tests we could identify 21 so far unknown tumor-associated antigens. We could show that the T cell response was directed against different antigens in primary tumors or liver metastasis. Some of the identified antigens were selectively overexpressed on tumor stem cells. Additionally in the blood of these patients there was a higher frequency of T cells specific for the newly identified TAA in comparison with canonical TAA, that have been described in the past. In part two of this thesis we could show that patients suffering from colorectal cancer are able to induce spontaneous T cell responses against mutated forms of tumor-associated antigens. These patients harbor higher frequencies of T cells in the blood that are specific for the mutated forms compared to the corresponding wildtyp antigens and they show an increased immune response. This work shows that classical tumor-associated antigens play a minor role in the immune response against tumors. Patient-individual antigens, a result of the unique tumor development in every patient, are highly immunogenic and serve as better targets for immune monitoring and directed T cell therapies.
Ground-Penetrating Radar (GPR) is a non-invasive electromagnetic geophysical method, which is sensitive to variations of subsurface dielectric properties. With this, GPR has become a versatile tool in various fields of geophysics and the soil science. In particular the determination of field-scale soil water content has drawn considerable research interest over the past decade. However, the quantifiability of achieved results remains often contested. In this thesis, three approaches for a quantitative use of GPR in soil hydrology are presented. First, a new calibration approach is developed for quantifying near-surface soil water contents with GPR and its applicability is demonstrated in field applications. Second, the ability of GPR methods for monitoring soil water dynamics is tested in well-controlled field experiments featuring imbibition into and drainage from a known subsurface structure. Finally, GPR applications are demonstrated in the broader context of developing monitoring schemes at a set of representative sites in a highly structured watershed.
A bottle neck in malaria research is the investigation of Plasmodium falciparum liver stage parasites because of technical issues in the infection of Anopheles mosquitoes with these parasites and subsequent generation of infectious sporozoites. Therefore I, in close collaboration with a colleague, established a combined in vitro/in vivo P. falciparum life-cycle in our lab. For that a protocol was established that included the generation of sexual P. falciparum stages in cell culture that were subsequently transmitted to Anopheles mosquitoes utilizing a special membrane feeding system. Later in the life-cycle sporozoites were extracted from the mosquito salivary glands to nally infect liver cells for further studies. We were able to establish a constant mosquito-infection rate for several months to perform experiments on P. falciparum sporozoites and exo-erythrocytic forms. To help decipher the in apicomplexans so far mostly uncharacterized cellular process endocytosis I investigated the function and localization of an EH-domain containing dynamin-like protein in Toxoplasma and Plasmodium. It belongs to a family of eukaryotic Eps15-homology domain containing proteins (EHDs) that have been characterized in higher eukaryotes and especially vertebrates to be part of endocytic events such as vesicular tracking and endocytic recycling. I was able to show by an in silico analysis that in contrast to vertebrates (four dierent EHDs) there is only one protein member of this familiy existing in each apicomplexan. Nevertheless, the apicomplexan EHD-protein has similar to all other EHD-proteins a predicted characteristic ATPase-domain (dynamin-like G-domain) and the Eps15-homology domain (EH). Through a uorescent tagging approach I was able to show a dynamic localization of the Toxoplasma EHD-protein member TgRME-1 (named after its ortholog in C. elegans receptor-mediated endocytosis protein 1) within the parasites. It localized to a vesicular compartment within the parasites that did not colocalize with known organelles so far. The compartment fragmented upon cellular division and is most likely involved in vesicular tracking of supply vesicles that transport lipids or other nutrients to the newly forming daughter-cells. From the data obtained in this thesis it can be hypothesized that the TgRME-1 labelled compartment represents a storage compartment that is filled up during the non-replicative phase and during endodyogeny helps to form daughter-cells. Structural analysis of the protein by deletion of either the G-domain or the EH-domain revealed a similar architecture of the protein compared to published data on mammalian EHDs. Investigation of the Plasmodium berghei EHD (PbEHD) with an antibody generated against the protein revealed a dfferent localization in different parasite stages. Whereas the protein localized to several vesicular compartments in the sporozoite stage it concentrated to a single organelle-like compartment in liver-stages 24 hours after invasion. This compartment later (48 hours after invasion) also fragmented and was distributed to the newly forming merozoites during schizogony, similar to TgRME-1. This subcellular localization indicated that both proteins might share a similar function in tachyzoites of Toxoplasma and Plasmodium liver stage parasites. A phenotypical analysis of PbEHD via generation of a pbehd (-) parasite revealed a putative function for the protein during intrahepatic development. The pbehd (-) liver stage parasite showed a reduced growth rate in vivo and in vitro but was still able to complete the life-cycle. In vivo, C57BL/6 mice infected with pbehd (-) parasites showed a prolonged prepatency period and did not develop experimental cerebral malaria in contrast to wildtype-infected mice. I was able to narrow down this protective effect solely to both the prolonged liver-stage phase and the involvement of the immunemodulator cytokine IL-10. Even though a defined role for the EHD-protein in the apicomplexan parasites could not be determined in this thesis I was able to characterize its architecure and localization in Toxoplasma gondii and Plasmodium berghei. I was able to identify a so far uncharacterized compartment in these parasites that is most likely involved in endocytic-recycling and storage of nutrients such as lipids for the parasites. In addition, my studies showed that the apicomplexan EHD-protein is involved in processes of the cellular division. A better understanding of these and other mechanisms of endocytosis will lead to anti-parasitic strategies that may reduce the burden caused by apicomplexan parasites.
We utilized the framework of mathematical modeling to gain insights into two distinct biological systems, the JAK/STAT1 signal transduction pathway and the regulation of cell cycle decisions in neuroblastoma.!! The family of JAK/STAT signaling pathways plays a key role in immunity. In several tumors dysregulation of the JAK/STAT pathways is observed. To investigate the functionality of this signal transduction pathway and eventually understand basic building principles, we establish a databased mathematical model of the JAK/STAT1 pathway by means of kinetic rate equations. We showed that pathway activation is coupled tightly to the receptor stimulus at the cost of signal strength. The nuclear signal is sustained by a combination of fast translocation rates and short nuclear residence times of activated STAT1 protein molecules. Model simulations reveal that STAT1 dimerization kinetics have a strong impact on both efficiency of signaling and response kinetics, implying that protein-protein interactions are evolutionary constrained to ensure network functionality. Measurements of STAT1 transport mutants validated the mathematical model and showed that STAT1 activation is robust against enhanced nuclear export. By the kinetic design of the pathway input noise is suppressed, the pathway can be efficiently activated and rapid relaxation after stimulus withdrawal is ensured.!! Neuroblastoma is the most common extracranial solid tumor of infants and children. Its course of illness varies between spontaneous regression and malignant, aggressive progression. Amplification of the MYCN oncogene is predictive for poor clinical outcome in neuroblastoma. MYCN-amplified cells proliferate strongly and exhibit impaired cell cycle arrest. To rationalize the impact of MYCN on the regulatory networks, governing cell cycle progression and DNA damage response, we established mathematical models of the regulatory modules, p53-MDM2 and E2F1-pRB, by means of mass action kinetics. The inherent regulation in the p53-MDM2 module leads to an universal form of the p53- MDM2 steady state and can account for several qualitatively different behaviors upon p53 activation. We show that it is plausible that the weak G1 arrest in the MYCN- overexpressing cells is due to a MYCN-induced protein level imbalance in the p53- MDM2 module. Furthermore we argue that the bifurcation diagram of the G1-S transition model can both theoretically as well as experimentally be used as an output to analyze the restriction point behavior in neuroblastoma. It shows that for cells with relatively high MYCN level and an enhanced CDK4 signal the bistable region is shifted to low stimuli and the model stays in an activated state even under DNA damage. A mathematical framework is provided, which potentially can serve as a future standard method to extract underlying cell cycle parameters from combined FACS-measured cell cycle phase distributions and cell growth rate measurements. Analysis of measurements in the SH-EP neuroblastoma cell line showed that conditionally upregulated MYCN mainly changes the length of the G1 phase.
Within this work, a simplified model system was developed for the experimental investigation of physical mechanisms underlying cell adhesion. Cellular complexity was reduced to three essential components: the lipid bilayer, the cell adhesion molecule integrin and the cytoskeletal protein actin. Integrin was integrated in spherical lipid bilayers, called giant unilamellar vesicles (GUVs), which were additionally filled with actin. Integrin alphaIIb beta3 was purified from human platelet membranes with high yield. Initially, this integrin was reconstituted in small unilamellar vesicles (SUVs). By variation of lipid composition, vesicle size and detergent concentration, crucial parameters for efficient integration could be determined and incorporation efficiency was enhanced. Two different approaches were compared to grow GUVs: electroformation and spontaneous swelling on agarose-lipid films. Besides biochemical characterization, formation of GUVs was investigated by means of confocal laser scanning microscopy confirming successful integration of integrin into the bilayer of the giant vesicles. A novel method for the preparation of GUVs from purified platelet membranes was established. Platelet membranes were directly purified and grown to giant vesicles by electroformation. Interaction of these GUVs with nano-structured and bio-functionalized surfaces was investigated. Adhesion of these integrin-coated vesicles was characterized for the first time by reflection interference contrast microscopy (RICM), which allowed estimation of an average adhesion energy.
Molecular membrane dynamics in living cells are often obscured from the observer because of the limited spatial resolution of conventional far-field optical microscopy. The superior spatial resolution of stimulated emission depletion (STED) nanoscopy provides new insights into the dynamic processes within the plasma membrane. In combination with the high temporal resolution of fluorescence correlation spectroscopy (FCS), we record and characterize the diffusion of membrane constituents within nanoscale observation areas.
In the first part of the thesis, we compare STED-FCS data of various fluorescent lipid analogs and proteins in the plasma membrane of living cells. Our results reveal distinct modes of diffusion which can be differentiated according to the chemical structure of the molecules. Phosphoglycerolipids diffuse freely and only weakly interact with other membrane constituents. Sphingolipids exhibit a strong molecular confinement due to the formation of hydrogen bounds within the ceramide backbone or between large polar head groups. Transmembrane proteins corral in compartments built by the cellular cytoskeleton. We implement Monte-Carlo simulations to support and explain our experimental results.
In the second part of the thesis, we enhance the experimental STED-FCS setup by the integration of a fast scanning unit. This newly developed concept not only enables us to perform calibration-free measurements of slowly diffusing particles but also visualizes spatial diffusion heterogeneities along the scan trajectory.
Entwickelt und evaluiert wurde eine Messmethode zur markerbasierten photogrammetrischen Bewegungserfassung (Kapitel III). Etabliert als „Heidelberger Upper Extremity Modell (HUX)“ konnte diese Methode für zahlreiche orthopädische Fragestellungen eingesetzt werden. Die Methode zeichnet sich durch Einsatz von Schätzmethoden zur Bestimmung von Gelenkzentren und -achsen aus und erfolgte unter der Randbedingung, nur eine kleine Zahl von Markern einzusetzen. Damit wurde die Integration in die klinische Routine möglich. Erprobt wurde die neue Methode durch ihre Anwendung zur Analyse der Armbewegungen beim Gehen. Es wurden Daten von Patienten mit infantiler Zerebralparese analysiert. Verstärkte Humerus-Abduktions- und Innenrotations-Stellung sowie eine vergrößerte Ellenbogenbeugung zeigte sich als Grundmuster der ansonsten individuell sehr unterschiedlichen Armbewegungen beim Gehen (Kapitel V). In diesem Zusammenhang wurde auch die Eignung einiger unkonventioneller Analysemethoden wie z.B. Winkeldarstellung von Armsegmentschwerpunkten untersucht. Die Ausweitung der Anwendung der Methode auf Alltagsbewegungen mit großem Bewegungsumfang in der Schulter führte an die Grenzen der Anschaulichkeit konventioneller Kardanwinkel-Darstellungen. Die Winkeldarstellungen selbst wurden daher zum Gegenstand dieser Arbeit und auf Eignung besonders im Hinblick auf Anschaulichkeit untersucht (Kapitel IV). In Folge wurde eine neue Konvention entwickelt, um die Stellung der Humeruslängsachse in Öffnungswinkeln und die Außen/-Innenrotation des Humerus als adjunkte Rotation im Sinne von Codman (1934) zu beschreiben.
To predict the of Earth system dynamics, observations of the vadose zone structure and water content are of vital interest. A suited measurement technique is ground penetrating radar (GPR). In this dissertation, the constructive inversion of surface GPR data is introduced. It relies on a parameterized model of the subsurface structure and distribution of dielectric permittivity. With it, GPR measurements are simulated by numerically solving Maxwell’s equations. After detecting signals in the measured and simulated data, the residuals of the signals’ traveltime and amplitude is iteratively minimized to estimate the subsurface parameters. Then, water content is computed from dielectric permittivity. The method was applied to measurements obtained on a testbed, providing ground-truth data. A comparison with the estimation results showed an agreement for the structure within ±5 cm and for the water content, a difference less than 2 % vol. A further evaluation of field data demonstrated the method’s applicability, when representing structure and permittivity by spline functions. Additionally a time-series was evaluated with assuming a constant structure, which enabled to interpret water dynamics. Besides providing accurate information on water content distribution and subsurface structure, the method allows the future attempt to estimate hydraulic properties.
We present methods for the systematic modelling and clustering of time series. Our data is associated with behavioral studies of alcoholism in animals. We analyze multivariate time series obtained from an automated drinkometer system. Here, rats have free access to water and three alcoholic solutions (this being the baseline treatment level), which is then interrupted by repeated deprivation phases. We develop a methodology to simultaneously classify into- and characterize dynamic patterns of the observed drinking behavior. This is achieved by extending known results on generalized linear models (GLM) for univariate time series to the multivariate case. We simplify the computational fitting procedure, by assuming a shared seasonal pattern throughout individuals and implementing an estimation maximization (EM) algorithm to fit mixtures of the mentioned multivariate GLM. A partition of the data, as well as a characterization of each group is obtained. The observed patterns of drinking behavior differ in their preference profile for the three alcoholic solutions, and also in the net alcohol intake. We observe an evolution of the drinking behavior over the repeated cycles of alcohol admission and deprivation, with a clear initial preference profile and a development to one of the advanced profiles. Furthermore, to measure the alcohol deprivation effect in this 4-bottle setting, a new criterion is developed, which enables us to classify each rat into presenting ADE or not. This classification shows that the rats develop a tolerance to taste adulteration after few deprivation phases. The proposed framework can be employed to find differences in behavior between different conditions and/or groups of animals and in the prediction of alcoholism from early phases of alcohol intake. The developed methods can also be used in different fields, where the analysis of time series plays an important role (e.g. microarray analysis and neuroscience).
This thesis deals with projection methods of soil temperature and soil moisture into depth by a forward model based on near-surface time-series. In addition, thermal as well as hydraulic soil parameters are estimated by using the Levenberg-Marquardt algorithm. For soil temperature, two analytical projection methods are compared which use the transfer function and the Fourier transform approach, respectively. In each case, additional mathematical strategies are required to improve the projection results, e. g. by adding an integral over the initial profile or applying the Tukey window on the time-series. The resulting projected temperature matches field measurements very well, especially for the transfer function method a residuum down to ±0.05 �C is achieved. Further, the uniqueness of the parameter space is evaluated and the temporal evolution of the thermal diffusivity is estimated through both projection methods. The projection of near-surface soil moisture is realized numerically by a finite volume scheme due to the strong non-linearity of the system. On the basis of synthetic data the conditions are explored under which accurate estimations of the hydraulic parameters are feasible. One of these conditions is found to be that the water content range should be larger than 0.5 times of the porosity. The corresponding relative parameter error is found to be some 10−5. Furthermore, the study shows that an accurate estimation is more feasible for soils with a lower saturated conductivity, and steep functions of the hydraulic properties. This is typically the case for soils with a higher sand content. Data from a field site is then used to verify the findings of the synthetic study and to discuss limitations, e.g. ponding water at a soil layer interface.
On the present thesis the focus was set on the synthesis and structure investigation of novel subvalent oligogallanes. Since gallium has an electron deficiency and acts as a strong lewis acid it is not trivial to get low oxidation state compounds. Disproportion reactions often lead to Ga(+III) and elemental Ga(0), so it is essential to avoid oxygen, water and in many cases strong donor solvents like THF. Many reactions were observed where the mere warming to room temperature led to undesired byproducts. A convenient method to get access to gallium in low oxidation states is the sonicated “GaI”. It was used for most of the compounds presented in this work. In the first part neutral and anionic phosphorous compounds are used to stabilize and shield the sensitive gallium cores from decomposition. Suitable ligands were di-tert-Butyl-, di-Phenylphosphanes or mixtures of them combined at one phosphor atom. In this manner several molecules could be synthesized with two to four gallium atoms. Two of the maintained structures contain three connected metal atoms with the oxidation states (II)-(I)-(II). In the second part novel nitrogen ligand compounds are described with special focus on amidinates in different coordination forms. In the given literature they are usually formed by deprotonation of dialkylamidines or by nucleophilic attack of BuLi derivates on carbodiimides. In this work the nucleophilic attack of alkyl- and silylamides on nitriles is compared and thereby the formation of two types of coordination to the gallium metal.
Electrical resistivity tomography (ERT) is gaining in importance in the field of archeological site prospecting. This contrasting study examines the results of mapping archeological structures and the resulting archeological excavation results. The study aims to show the boundaries of interpreting ERT scans and possible sources of error in interpreting ERT scan results. To accomplish this, ERT scans were performed at two selected sites in South West Germany. The results were then compared with later archeological excavation findings at these sites. Studies at the quadrangular Celtic enclosure near Sinsheim-Dühren in the Kraichgau have shown that the perimeter of V-shaped ditches can be distinguished well from the surrounding bed rock and back-fill. The resolution of the perimeter is not without uncertainty but the uncertainty is on such a minor scale that it does not affect the planning of an archeological dig at the site in question. Artifacts in ERT measurement interpretations could be linked to computational difficulties in the inversion routines. The impact of such artifacts depend on the chosen calculation configuration though. As a result of the measurements taken at "Gewann Steinbock" near Sinsheim-Dühren a soil map was created. More importantly though, the unique Celtic tomb was accurately localized within the quadrangular Celtic enclosure with the help of ERT. Research at a celtic ore smelting site in the Grösseltal near Neuenbürg (Enzkreis) in the northern Black Forrest showed that the interpretation of data taken near lightly slag-rich heaps in partially rocky terrain is not possible without error. Also, localizing and determining the extent of the bloomeries could not be determined reliably via the ERT method. The ability to successfully localize the extracting traces of the celtic ore smelting site via the ERT method though were proven. After inspecting the sight for visible terraces with advancing upstream heaps, in depth ERT scans could be performed to localize access shafts, binges or other cavities in the perimeter of the terrace. Artifacts in ERT measurement could in turn be linked to computational difficulties in the inversion routines. A current limit to the ERT method lies in interlocked archeological finds in combination with the inversion routine interpretation. The ability to be able to distinguish between misinterpretations and actual archeological finds therefore is important.
The steadily increasing demand for energy worldwide has resulted in the depletion of the existing fossil energy resources and the pollution of the atmosphere by greenhouse gases such as CO2, which are responsible for global warming. To curb these problems, research activities aiming at CO2 conversion into value-added products, e.g. fuels like methanol, using sunlight have intensified over the last few years. In this work, TiO2 nanoparticles functionalized with perylene-based dyes containing either a carboxylic acid or anhydride group as molecular anchor to the TiO2 surface were studied as potential photocatalyst for solar light-driven CO2 reduction. The dye binding geometry is of particular importance since it influences the electron transfer from the dye to TiO2 and hence the photocurrent output of any given dye/TiO2 system. Two dyes, ID1157 and ID1152, structurally identical apart from their anchor group, were selected to allow direct comparison. In addition, a simple model substance bearing an anhydride group was investigated to facilitate the interpretation of the lesser known anhydride binding mode. Surprisingly, despite their structural similarity, the observed photocurrent amplitude of the ID1157/TiO2 system containing a carboxylic acid anchor was much higher than for the ID1152/TiO2 system containing an anhydride anchor. With the help of ultraviolet/visible (UV/Vis) absorption, infrared (IR), Raman and vibrational sum-frequency generation (VSFG) spectroscopy it was sought to determine whether the photocurrent signals were correlated to the dissimilar anchor groups and their binding modes. From the UV/Vis spectra it was found that the anhydride group opened upon binding. As for the IR and Raman studies it could be concluded that the carboxylic acid anchor of ID1157 and both carboxylate groups of the opened anhydride of ID1152 bound via a bidentate bridging pattern. Furthermore, it was shown from the background-suppressed VSFG spectra of the bound dyes in air and water that these adsorbed in an orderly fashion, ID1157 more so than ID1152, but with both only slightly disturbed in the presence of water. In addition, it was found that all molecules in the ID1157 dye layer were adsorbed on the TiO2 surface via chemisorption. By contrast, the ID1152 dye layer was composed of chemisorbed as well as physisorbed dyes, the latter being coordinated via a closed anhydride group. Also, ID1157 displayed a higher surface density of adsorbed molecules compared to ID1152. From a preliminary polarization analysis, it was suggested that the dyes with carboxylic acid anchor adopted a tilted binding geometry. In view of the results obtained on the binding geometry of the dyes ID1157 and ID1152 it was possible to identify some criteria important for the generation of a high photocurrent: 1) binding of the dye via chemisorption with 2) a high surface density and possibly through 3) a tilted geometry. These findings have important implications for the understanding of the mechanism of dye functionalization. Finally, methanol and CO2 adsorption on TiO2 films was investigated by VSFG spectroscopy. Only molecularly adsorbed methanol was observed which was easily displaced by water or methanol/water mixtures. However, CO2 adsorption could not be detected in the spectral range which was investigated.
Rotating, compact objects power some of the most spectacular phenomena in astrophysics, e.g., gamma-ray bursts, active galactic nuclei and pulsar winds. The energy is carried by Poynting flux, and the system is usually modelled using relativistic magnetohydrodynamics (MHD). However, in the relatively low density medium expected around some of these objects, the MHD approximation breaks down, allowing new, large-amplitude waves to propagate. We discuss the role of these waves in two astrophysical contexts:
In blazar jets, we show that a magnetic shear, launched together with a plasma from the black hole magnetosphere, begins to accelerate particles at a large distance from its source. The resulting non-thermal emission can, nevertheless, be modulated on very short timescales, which can explain the rapid variability of the TeV gamma-ray flux observed from some blazars.
In pulsar winds, we analyze the radial propagation of superluminal modes, including their damping by radiation reaction and by interaction with an external photon field. We discuss their effect on the structure of the pulsar wind termination shock, presenting new solutions in which the nonlinear wave is asymptotically matched to the constant pressure surroundings. The observational implications of these solutions are discussed for both isolated pulsars, and pulsars in binary systems.
High-precision atomic mass measurements are vital for the description of nuclear structure, investigations of nuclear astrophysical processes, and tests of fundamental symmetries. The neutron-rich A ≈ 100 region presents challenges for modeling the astrophysical r-process because of sudden nuclear shape transitions. This thesis reports on high-precision masses of short-lived neutron-rich 94,97,98Rb and 94,97-99Sr isotopes using the TITAN Penning-trap mass spectrometer at TRIUMF. The isotopes were charge-bred to q = 15+; uncertainties of less than 4 keV were achieved. Results deviate by up to 11σ compared to earlier measurements and extend the region of nuclear deformation observed in the A ≈ 100 region. A parameterized r-process model network calculation shows that mass uncertainties for the elemental abundances in this region are now negligible. Although beneficial for the measurement precision, the charge breeding process leads to an increased energy spread of the ions on the order of tens of eV/q. To eliminate this drawback, a Cooler Penning Trap (CPET) has been developed as part of this thesis. The novel multi-electrode trap structure of CPET forms nested potentials to cool HCI sympathetically using either electrons or protons to increase the overall efficiency and precision of the mass measurement. The status of the off-line setup and initial commissioning experiments are presented.
Although many studies proposed methods for the identification of enhancers, reliable prediction on a genome-wide scale is still an unsolved problem. One of the reasons for this is the highly flexible regulatory logic underlying a detectable enhancer activity. In each cell type or tissue and at any given time, a mostly unknown set of transcription factors activates specific regulatory elements by coordinated binding to the corresponding genomic region. Position, spacing, and orientation of the individual bound factors can thereby vary between different enhancers yet result in a highly similar spatio-temporal activity. Due to this inner flexibility, so-called “alignment-free” methods have been proposed for enhancer prediction, as they are able to cope with rearrangements by comparison of word profiles rather than linear sequence. However, the problems caused by allowing for permutation in sequence comparison have not been investigated so far. In this study I implemented several published alignment-free metrics and analysed, which parameters affect their ability to successfully predict regulatory regions. As results show, single point mutations and the increasing amount of spurious matches with decreasing word size pose the biggest challenge to alignment-free techniques, especially when applied on a genome-wide scale. Alignment algorithms usually solve these problems quite efficiently but cannot handle permutation. I therefore implemented a new technique for enhancer prediction that combines the advantages of both algorithm types and used it for the identification of regulatory regions in the teleost fish Oryzias latipes (Medaka) based on a set of known and validated human enhancers. Predicted medaka regions and human enhancers were subsequently used in an in vivo enhancer assay and analysed for their activity. In total, 12 predicted regions corresponding to 9 human enhancers showed clear enhancing activity in the fish. This shows that the principle implemented here is able to predict active enhancers at a high rate on a genome-wide scale even in species as diverged as human and fish. Furthermore, evidence for motif-level conservation between some of the human and medaka enhancers could be found that was invisible for most of the alignment-algorithms used for comparison.
This work describes the design of an analog circuit emulating a multi-compartment neuron model on a microchip. Initially, the single-compartment adaptive exponential integrate-and-fire neuron model is implemented as a hardware model. Therefor, the differential equations describing the model dynamics are directly translated into an electronic circuit based on operational transconductance amplifiers. Consequently a close correspondence between model and circuit is achieved enabling references to experiments done with computer simulators. 512 of these neurons are implemented on a single micro-chip. Individual control of each neuron’s biases is achieved by the use of analog floating-gate memory. In most cases, these biases directly correspondent to parameters of the model, hence simple translations are possible. The single neuron implementation has been verified on a prototype chip in several experiments. Inter alia, its capabilities of reproducing biological neuron’s behavior and the influence of fixed-pattern noise on the circuit are analyzed. To step over to a multi-compartment circuit, the neuron has been enhanced by a resistive element and a routing network to build complex dendrite structures. Furthermore, the parameterization allows compartments of different sizes covering large somatic and small dendritic compartments. A dedicated test chip has been designed for the verification of the new model. Several simulations show the enhanced behavior of the multi-compartment emulation including dendritic attenuation and active spike propagation. The neuron circuits are dedicated for a new kind of computer based on the cortex.
The mass is a unique fingerprint of each nucleus as it reflects the sum of all interactions within it. Comparing experimental mass values with theoretical calculations provides an important benchmark of how well the role of these interactions is already understood. By investigating differences of experimental binding energies, such as two-neutron separation energies (S2n), valuable indications for nuclear-structure studies are provided. The present thesis contributes to these studies providing new high-precision mass measurements especially in the heavy-mass region. Here, nuclear theory is heavily challenged due to the large number of nucleons. The data have been obtained at the Penning-trap mass spectrometer ISOLTRAP located at the radioactive-ion-beam facility Isolde at CERN. For the determination of the masses, the time-of-flight ion-cyclotron-resonance technique has been applied. While the new mass data for 122−124Ag continue existing trends in the S2n energies, the new mass values for 207,208Fr render them more precisely. In the case of the mass values for 184,186,190,193−195Tl a new interesting odd-even effect has been revealed. The comparison of the measured mass values with theoretical models furthermore demonstrates significant problems in reproducing the strength of the pairing correctly. This is of special interest for the discussion about shape coexistence in the region around the doubly-magic 208Pb.
Maintenance of the hematopoietic system is dependent on hematopoietic stem cells (HSCs). During homeoastasis HSCs are quiescent. However upon injury, HSCs can efficiently be activated, leading to repair of the system. Signals leading to the activation of quiescent HSCs are still largely unknown. Recently our group has shown that administration of IFNα leads to activation of mouse HSCs in vivo. This is mediated by activation of IFNAR/STAT1 signaling followed by the up-regulation of Sca-1, however the exact mechanism of cell cycle activation remains unclear. To get further insight into this process we performed microarray analysis on HSCs after treatment with IFNα. This screen identified several candidate genes, which are potentially involved in HSC activation, including cell cycle regulators like p57KIP2, Maged1 and Reprimo as well as cytokines like Ccl5 and Cxcl10, which are key regulators of inflammatory responses. Furthermore we identified interferon response genes like Ifitm1, Ifitm3, Iigp1, Iigp3 or Ddx58, which were previously linked to regulation of proliferation in different contexts. Along these studies we uncovered that Ifitm1 and Ifitm3 expression is highly enriched within hematopoietic stem and progenitor cells both on the RNA as well as on the protein level. Moreover expression is further induced by IFNα. However mice lacking the Ifitm family show normal hematopoiesis and normal HSC numbers and cycling behavior of HSCs in homeostatic conditions. Ifitm-deficient HSCs are capable to self-renew and differentiate similar to wild-type HSCs. This suggests that the Ifitm protein family is dispensable for HSCs during homeostasis. Notably Ifitm-deficient HSCs are also efficiently activated by IFNα, similar to wild type HSCs. Microarray analysis of HSCs from Ifitm deficient mice, both during homeostasis and after administration of IFNα, showed no differences in the expression profiles, indicating a role for the Ifitm family as terminal effectors rather than regulatory proteins within HSCs. During our study it was shown by others that the Ifitm family is a potent viral restriction factor in endothelial cells. We are currently investigating whether Ifitm proteins have a similar role in the immune defense of HSCs. Thus far it is still unclear whether human HSCs are similarly activated by IFNα as mouse HSCs. To elucidate this we established a xenotransplantation model with human cord blood cells, which allows testing of the effects of IFNα on human HSCs in vivo. Surprisingly, unlike mouse HSCs, human HSCs are not activated by IFNα in this model. Notably human HSCs in this model are already less quiescent during homeostasis compared to their mouse counterparts. In the mouse also the bacterial endotoxin LPS can induce cell cycle activation in HSCs. Surprisingly LPS similarly activates human HSCs in our model. Gene expression analysis showed a high overlap between the genes induced in mouse and human HSCs after LPS treatment, while IFNα only affected cell cycle regulatory genes in murine HSCs. One explanation for this phenotype could be an impaired interaction of murine stromal niche cells with human HSCs and we are currently investigating this in more detail. Finally we examined the effect of IFNα on quiescent leukemic stem cells (LSCs). While IFNα is known to activate normal mouse HSCs, it is unclear whether also LSCs are similarly affected. To address this we investigated the effects of IFNα on LSCs in a mouse model for chronic myeloid leukemia. Surprisingly LSC were less efficiently activated compared to normal HSCs. This can be explained by down-regulation of the IFNAR by BCR-ABL kinase activity, which was previously described in vitro. This also highlights the importance of exact timing of LSC activation and treatment in combination therapy approaches. Our group is currently using this model to further identify and optimize new possible combination therapies.
C. Binz berichtet vor allem über die Bedeutung Nicolaus Cusanus' in den weltlichen Wissenschaften: er setzte die Waage in der Heilkunst ein, erstellte eine Europakarte mit korrektem geometrischen Netz und schlug eine Reihe juristischer Reformen für das Deutsche Kaiserreich vor.
B-cell chronic lymphocytic leukemia (CLL) is the most common type of leukemia in the western world. CLL is not curable with currently available therapies. Another neoplastic entity that shares common genotypic and phenotypic characteristics with CLL is mantle cell lymphoma (MCL). Like CLL, MCL is an incurable B-cell neoplasm comprising approximately 6% of all Non-Hodgkin lymphomas (NHL). MicroRNAs (miRNAs) are small single stranded RNA molecules with a length of ~21 nucleotides, which regulate the stability and translation of protein coding messenger RNAs (mRNAs). It is estimated that 30% of all protein coding genes of an organism are regulated by miRNAs. Within recent years, evidence arose that the deregulated expression of miRNAs plays a pivotal role in cancer. MicroRNA-155 and the miR-17-92 cluster are aberrantly expressed in various cancers including B-cell lymphomas. The present work aimed to identify transcripts regulated by these aberrantly expressed miRNAs. To this end, current standard techniques like quantitative real-time reverse transcription PCR, luciferase sensor assays and Western blot analyses were performed. As the results of these studies revealed just minor effects, an alternative screening method was established, which is based on the immunoprecipitation (IP) of RNA induced silencing complexes (RISC) with subsequent sequencing of precipitated mRNAs (RIP-Seq). As a model system for RIP-Seq, HEK293T cells with stable, ectopic expression of miR-155 were generated. In comparison to the control cell line, these cells showed enhanced cell proliferation. RIP-Seq experiments using these cells revealed an enrichment of 67 mRNAs predicted as miR-155 targets, including related to cell proliferation. Some of these, like the transcription factor CEBPB, are well established miR-155 target genes. Others like the RNA destabilizing protein ZFP36 are frequently reduced in human cancers. Expression profiling further revealed significant changes in the transcriptome and miRNome of these cells, presumably as a secondary consequence of ectopic miR-155 expression. For instance, the cell cycle gene CCND1 was severely up-regulated after miR-155 overexpression. Furthermore, the cancer associated ETS transcription factor family PEA3 was highly up regulated as well. Using RIP-Seq, the targetomes of two B-cell lines representing CLL and MCL were identified. These targetomes comprise more than 1,000 target genes each, with more than 600 genes shared by both cell lines. The putative tumor suppressor gene BTG2 was one of the most prominent miRNA target genes identified by RIP-Seq in both cell lines. BTG2 was predicted to be simultaneously regulated by more than 15 of the 30 most highly expressed miRNAs within these cell lines. MicroRNA profiling of IP and corresponding total lysate fractions generated by RIP-Seq, uncovered a recurrent disproportional presence of several miRNAs, including miR-155 and members of the miR-17-92 cluster, within the AGO2 IPs. However, the underlying mechanisms, explaining the bias in miRNA binding to AGO2 remain elusive so far.
Hermann von Helmholtz reiste 1893 als Vertreter Deutschlands zum Internationalen Elektrischen Kongress in Chicago. An den Kongress schloss sich eine Amerika-Rundreise an. Auf der Rückfahrt stürzte Helmholtz, da er kurzzeitig das Bewusstsein verloren hatte, die Kabinentreppe des Dampfers hinab. Nach scheinbarer Genesung erlitt er im Juni 1894 einen Schlaganfall, an dessen Folgen er am 8. September 1894 verstarb.
Dissertation von Hermann Helmholtz: Helmholtz entdeckte mit Hilfe des Mikroskops, dass die Nervenfasern aus den Ganglienzellen entspringen. Enthält auch: Lebenslauf [PDF S. 16] und Thesen [PDF S.17]
"Campus-Report" heißt die Radiosendung der Universitäten Heidelberg, Mannheim, Karlsruhe und Freiburg. Die Reportagen über aktuelle Themen aus Forschung und Wissenschaft werden montags bis freitags jeweils um ca. 19.10h im Programm von Radio Regenbogen gesendet. (Empfang in Nordbaden: UKW 102,8. In Mittelbaden: 100,4 und in Südbaden: 101,1) Uni-Radio Baden: ein gemeinsames Projekt der Universitäten Freiburg, Heidelberg, Karlsruhe und Mannheim in Zusammenarbeit mit Radio Regenbogen – unterstützt von der Landesanstalt für Kommunikation. Sendung vom 18. April 2012
Selbstrezension Siegmund Günthers zu seinem Buch: Studien zur Geschichte der mathematischen und physikalischen Geographie. - Halle 1877-1879 1. Die Lehre von der Erdrundung und Erdbewegung im Mittelalter bei den Occidentalen 2. Die Lehre von der Erdrundung und Erdbewegung im Mittelalter bei den Arabern und Hebräern 3. Aeltere und neuere Hypothesen über die chronische Versetzung des Erdschwerpunktes durch Wassermassen 4. Analyse einiger kosmographischer Codices der Münchener Hof- und Staatsbibliothek 5. Johann Werner aus Nürnberg und seine Beziehungen zur Geschichte der mathematischen und physischen Erdkunde 6. Geschichte der loxodromischen Curve
Review of the article ''On the Equilibrium of Heterogeneous Substances'', which was published in ''Transactions of the Connecticut Academy of Arts and Sciences'', vol. 3 (1874-78), pp. 108-248 and 343-524.
W. Ostwald definierte 1904 in einem Wiener Vortrag das Glücksmaß mit Hilfe der - durchaus im physikalischen Sinn gemeinten - willensgemäß betätigten Energiemenge E und der widerwillig betätigten Energiemenge W als G = (E+W)(E-W) = E² - W² Boltzmann wendet sich gegen die willkürliche Formel - insbesondere bleibt die Wahl der 2. Potenz völlig unbegründet - als auch gegen die Reduktion auf die physikalische Energetik. Der Aufsatz Ostwalds befindet sich im HeiDOK-Archiv unter http://www.ub.uni-heidelberg.de/archiv/13200
W. Ostwald definiert das Glücksmaß mit Hilfe der - durchaus im physikalischen Sinn gemeinten - willensgemäß betätigten Energiemenge E und der widerwillig betätigten Energiemenge W als G = (E+W)(E-W) = E² - W²
Inhalt: * (30.10.1868) Zur Theorie der stationären Ströme in reibenden Flüssigkeiten * (12.02.1869) Physiologische Wirkung kurz dauernder elektrischer Schläge im Innern von ausgedehnten leitenden Massen * (30.04.1869) Elektrische Oszillationen * (25.06.1869) Schallschwingungen in der Schnecke des Ohres * (21.01.1870) Gesetze der inkonstanten elektrischen Ströme in körperlich ausgedehnten Leitern
Inhalt: * (30.06.1865) Stereoskopisches Sehen * (20.07.1866) Muskelton * (17.05.1867) Fortpflanzungsgeschwindigkeit der Nervenreizung * (09.08.1867) Mechanik der Gehörknöchelchen * (08.05.1868) Discontinuirliche Flüssigkeitsbewegungen * (22.05.1868) Grundlagen der Geometrie
Inhalt: * (27.02.1863) Über den Einfluss der Reibung in der Luft auf die Schallbewegung * (24.10.1862) Über die Form des Horopters, mathematisch bestimmt * (08.05.1863) Über die Bewegungen des menschlichen Auges * (04.12.1863) Über den Horopter * (27.05.1864) Ueber Muskelgeräusch * (25.11.1864) Ueber den Einfluss der Raddrehung der Augen auf die Projection der Retinabilder nach Aussen * (13.01.1865) Ueber die Augenbewegungen * (24.02.1865) Ueber Eigenschaften des Eises
Inhalt: * (11.11.1859) Über Farbenblindheit * (27.04.1860) Ueber die Contrasterscheinungen im Auge * (20.07.1860) Ueber Klangfarben * (23.11.1860) Über musikalische Temperatur * (26.07.1861) Zur Theorie der Zungenpfeifen * (08.12.1861) Über eine allgemeine Transformationsmethode der Probleme über elektrische Vertheilung * (24.01.1862) Über eine Arbeit des Herrn Professor v. Betzold in Jena * (31.05.1862) Über die arabisch-persische Tonleiter
Inhalt: * Bericht über die Thätigkeit des Vereins im Jahre 1857 bis 1858 - Beitritt von Hermann Helmholtz * (15.03.1859) Über die Luftschwingungen in Röhren mit offenen Enden * Geschäftliche Mitteilung - Wahl Hermann Helmholtz' zum ersten Vorsitzenden am 14. Dezember 1858
a) Mathematische Instrumente Günther referierte über Astrolabien, Sextanten, Uhren und Globen. Ferner schildert er den aus der Universität Altdorf stammenden Bestand alter physikalischer Apparate. b) Quellbildung Günther geht insbesondere auf intermittierende kalte Naturspringbrunnen ohne Gasbeimischung ein. c) Neue Kartenserien: Eine meteorologische Kartenserie und eine Serie der Ostalpen werden vorgestellt. d) Geographieunterricht Günther fordert, die mathematische und physikalische Geographie als empirische Wissenschaft zu lehren und die Schüler zu eigenen Beobachtungen anzuhalten.
,,Heute ist die Paläontologie soweit ausgebildet, dass sie in den meisten Fällen sogar den Horizont anzugeben vermag, wohin ein irgendwie aufgefundener versteinerter Tier- und Pflanzenkörper gehört, und damit ist die Dynamik der Erdkruste in den Stand gesetzt, sich von den oft so abenteuerlichen Dislokationen und Schichtenstörungen kausale Rechenschaft zu geben, mit denen uns ein immer tiefer eindringendes Studium der Erdgebirge bekannt machte. Der Geophysiker stellt bloß die Frage der Altersfolge, der Paläontologe beantwortet ihm dieselbe, und im Übrigen gehen beide Disziplinen ihre gesonderte Bahnen. Damit es aber so weit kommen, damit auch hier die Arbeitsteilung im ausgedehntesten Maße Platz greifen konnte, mussten zuvor jene Zwischenstadien der Erkenntnis durchlaufen werden, auf welche dieser Vortrag das Augenmerk eines größeren Hörerkreises zurichten bestrebt war.'' (Résumé S. Günthers, S. 123) Dieser naturhistorische Aufsatz hat nur geringfügige Beziehungen zur Mathematik.
AUGUST HELLER wurde am 6. August 1843 in Budapest geboren und starb daselbst nach längerem Leiden am 4. September 1902. Nachdem er 1866 das Polytechnikum in Budapest absolviert und 1868 die Prüfung für das Lehramt bestanden hatte, wurde er Assistent am genannten Polytechnikum, studierte 1869 in Heidelberg, war 1870-1898 Professor der Mathematik und Physik an der Realschule seiner Geburtsstadt, zugleich 1872--1875 Privatdozent am Polytechnikum und seit 1894 Oberbibliothekar der Ungarischen Akademie der Wissenschaften. HELLER hat eine große Anzahl von Schriften vorzugsweise physikalischen und meteorologischen Inhalts veröffentlicht, von denen die meisten ungarisch geschrieben sind. Sein Hauptwerk ist die ,,Geschichte der Physik von Aristoteles bis auf die neueste Zeit'' in zwei Bänden (1882, 1884); die von ihm in Angriff genommene ,,Geschichte der Physik in Deutschland'', die den letzten Band der bekannten ,,Geschichte der Wissenschaften in Deutschland'' bilden würde, ist leider unvollendet geblieben. (Rezension von Gustaf Eneström (1852-1923) im Jahrbuch über die Fortschritte der Mathematik, Band 33.1903)
Die Erdkunde zählte im 15. Jahrhundert und noch lange danach zu den mathematischen Wissenschaften. Nürnberg war wegen seiner technischen Anstalten insbesondere für seine Buchdruckereien in dieser Zeit berühmt. Günther benennt u.a. folgende Personen, die wichtige Beiträge zur Geographie leisteten: Regiomontanus, Martin Behaim, Johannes Werner und Willibald Pirckheimer. In den folgenden Jahrhunderten entstanden in Nürnberg eine ganze Reihe bedeutender Karten. Die Seitenzählung des Originalaufsatzes ist am Seitenrand angegeben.
Biographie des aus Mannheim stammenden Physikers und Mathematikers Philipp von Jolly, der von 1834 bis 1854 in Heidelberg lehrte. Die Originalseitenzählung ist jeweils am Rand in runden Klammern angegeben.
Nach der Ausgabe der Populären Schriften (1905) neu herausgegeben und mit einem Personenregister versehen von Gabriele Dörflinger, Universitätsbibliothek Heidelberg, 2005. Bericht Ludwig Boltzmanns (1844-1906) über seine Gastprofessur im Sommer 1905 an der Berkeley-Universität in Kalifornien.
Moritz Cantor schildert nicht den Künstler, sondern den Naturwissenschaftler Leonardo da Vinci. Er berichtet über den in Spiegelschrift verfassten Codex atlantico da Vincis und thematisiert die mathematisch-naturwissenschaftlichen Entdeckungen: - Messung des Reibungswiderstandes - Entdeckung der Kapillarität - Vorgänger des Kopernikus - Unmöglichkeit der Quadratur des Kreises - Unmöglichkeit des Perpetuum mobiles - Dampfschiff - Fallschirm - Wellenkreise in Wasser bewegen sich nicht horizontal, sondern vertikal - anatomische Abbildungen - das Auge arbeitet wie eine Camera obscura - Zentralperspektive
Nach der Ausgabe der Populären Schriften, 1905 neu herausgegeben und mit einem Personenregister versehen von Gabriele Dörflinger, Universitätsbibliothek Heidelberg, 2009. Biographie des Physikers und Mathematikers Gustav R. Kirchhoff (1824-1877), der von 1854 bis 1875 in Heidelberg lehrte und hier 1860 gemeinsam mit Bunsen die Spektralanalyse entdeckte.
We consider a mathematical model of a rigid body immersed in a viscous, compressible fluid moving with a velocity prescribed on the boundary of a large channel containing the body. We show continuity of the drag functional as well as domain shape stability of solutions in the incompressible limit, with the Mach number approaching zero.
Biographie des Technologen und Mathematikers Carl Christian von Langsdorf (1757-1834), der von 1806 bis 1834 an der Heidelberger Universität lehrte. Der Herausgeber der Badischen Biographien Friedrich von Weech (1837-1905) verfasste den Artikel.
Biographie des Physikers und Mathematikers Philipp von Jolly (1809-1884), der von 1834 bis 1854 an der Heidelberger Universität lehrte. 1854 folgte er einem Ruf an die Münchener Universität. Der Herausgeber der Badischen Biographien Friedrich von Weech (1837-1905) verfasste den Artikel.
The vice rector's address was delivered in the year 1862. It bears on a question often discussed, the comparative value of scientific and literary instruction. Translated by Charles Henry Schaible (1824-1899) German version: http://www.ub.uni-heidelberg.de/archiv/11487
Siegmund Günther (1848-1923) schildert, dass Cusanus (1401-1464) in astronomischer Hinsicht Kopernikus vorgriff, bereits das Trägheitsgesetz erkannte und eine Landkarte mit korrektem geometrischen Netz entwarf.
Moritz Cantor (1829-1920) schildert Newtons Entdeckungen der Optik und des Gravitationsgesetzes, sowie die Erfindung der Differentialrechnung. 1696 übernahm Newton die Verwaltung der Königlichen Münze und beendete seine Forschungsarbeiten. Cantor skizziert die politischen Verhältnisse in England vom Ende des 17. Jahrhunderts bis zu Newtons Tod 1727.
Henry E. Roscoe was the favorite student of Robert W. Bunsen. His memoirs describe the Heidelberg university life in the second half of the 19th century. Extract from: Roscoe, Henry E.: The life & experiences of Sir Henry Enfield Roscoe / written by himself. - London ; New York : Macmillan, 1906. Chapter III. "Heidelberg du feine" Chapter IV. "Bunseniana" and Heidelberg Frieds
Auszug aus: Anna von Helmholtz : ein Lebensbild in Briefen / hrsg. von Ellen von Siemens-Helmholtz. - Berlin. - Band 1.1929 Hermann Helmholtz fuhr im Frühjahr 1864 vier Wochen nach England. Er hielt sich in London, Oxford, Glasgow und Manchester auf und besuchte u.a. Michael Faraday, Sir John Tyndall (der auch in Heidelberg tätig war), Sir Henry Enfield Roscoe (Lieblingsschüler Robert W. Bunsens) und seinen Freund Lord Kelvin (Sir William Thompson). Höhepunkt des Englandaufenthaltes waren Helmholtz' Vorlesungen in der Royal Society.
Auszug aus: Anna von Helmholtz : ein Lebensbild in Briefen / hrsg. von Ellen von Siemens-Helmholtz. - Berlin. - Band 1.1929 Hermann Helmholtz fuhr im April 1866 nach Paris. Dort lernte er Annas Onkel Julius von Mohl und ihre Tante Mary kennen, die in Paris einen bekannten Salon führte. Neben Wissenschaftlern wie Charles Hermite und Marie Henri Grandeau besuchte er Kunstmuseen und Konzerte.
Das Ziel der Naturwissenschaften ist nach Helmholtz, das Gesetz der Naturerscheinungen und damit ihre Ursachen zu erkennen. Er legt noch seine Erkenntnisse zum Gesetz der Energieerhaltung ("Erhaltung der Kraft") und seine physiologisch-physikalischen Anschauungen dar.
In der Besprechung des Inhaltes einer von Helmholtz hinterlassenen kurzen Aufzeichnung, welche wahrscheinlich eine Disposition zu der auf der Wiener Naturforscher-Versammlung im Jahre 1894 beabsichtigten Rede: "Über dauernde Bewegungsformen und scheinbare Substanzen werden sollte, wird vor allem festgestellt, daß mathematische Irrtümer, wie sie vielfach, vermutet worden, in der Tat in dieser Aufzeichnung nicht vorhanden sind, ferner die von philosophischer Seite wenig beachtete Auffassung der scheinbaren Substanzen in Übereinstimmung mit der von Kant gebracht, endlich werden einige Bemerkungen bezüglich einer Vertiefung des Begriffes der Substanz auf Grund einer durch Einführung kinetischer Potentiale höherer Ordnung mit beliebig vielen unabhängigen Variabeln erweiterten Mechanik hinzugefügt.
"Campus-Report" heißt die Radiosendung der Universitäten Heidelberg, Mannheim, Karlsruhe und Freiburg. Die Reportagen über aktuelle Themen aus Forschung und Wissenschaft werden montags bis freitags jeweils um ca. 19.10h im Programm von Radio Regenbogen gesendet. (Empfang in Nordbaden: UKW 102,8. In Mittelbaden: 100,4 und in Südbaden: 101,1) Uni-Radio Baden: ein gemeinsames Projekt der Universitäten Freiburg, Heidelberg, Karlsruhe und Mannheim in Zusammenarbeit mit Radio Regenbogen – unterstützt von der Landesanstalt für Kommunikation. Sendung vom 15. Juni 2011
"Campus-Report" heißt die Radiosendung der Universitäten Heidelberg, Mannheim, Karlsruhe und Freiburg. Die Reportagen über aktuelle Themen aus Forschung und Wissenschaft werden montags bis freitags jeweils um ca. 19.10h im Programm von Radio Regenbogen gesendet. (Empfang in Nordbaden: UKW 102,8. In Mittelbaden: 100,4 und in Südbaden: 101,1) Uni-Radio Baden: ein gemeinsames Projekt der Universitäten Freiburg, Heidelberg, Karlsruhe und Mannheim in Zusammenarbeit mit Radio Regenbogen – unterstützt von der Landesanstalt für Kommunikation. Sendung vom 12. August 2009
Das Ziel dieser Arbeit war die biochemische, molekulare und genetische Charakterisierung des Drosophila Gens frühstart. In früheren Studien konnte bereits gezeigt werden, daß Frs als mitotischer Inhibitor wirkt, der spezifisch der Funktion der Protein-Phosphatase String entgegenwirkt, dadurch die Mitosen in den Zellen der Ventralfurche verzögert und so diesen Zellen erlaubt, die für die Ventralfurchenbildung erforderlichen morphogenetischen Prozesse ohne Störungen durch mitotische Zellteilungen zu durchlaufen. Darüber hinaus wurde gezeigt, daß frs ausreichend und zum Teil notwendig ist, um die schnellen syncytialen Kernteilungen nach der 13. Teilung und während der nachfolgenden Zellularisierung auszusetzen. Die vorliegende Arbeit beschreibt verschiedene weitergehende und bisher unbekannte physiologische und biochemische Eigenschaften von Frs. So wird gezeigt, daß durch die ektopische Expression von Frs in späteren embryonalen Entwicklungsstadien auch der reguläre Zellzyklus inhibiert werden kann, diese Inhibition durch Blockierung der M-Phase verursacht wird und der damit verbundene direkte Übergang von der G2 zur G1 Phase zu Endoreplikationen in den betroffenen Zellen führt. Dieser durch Frs-Überexpression verursachte Phänotyp ist konsistent mit den Phänotypen von Cdk1 und CycA Mutanten. Um eine molekulare Verbindung zwischen Cdk1 und Frs herstellen zu können, wurde in dieser Arbeit nach mit Frs interagierenden Faktoren gesucht, deren Interaktionen mit Hilfe vielfältiger biochemischer Methoden analysiert wurden. Frs interagiert hauptsächlich mit zwei verschiedenen Arten von Proteinen: Nukleoporinen und Cyclinen. Durch molekulare in vitro Analyse konnte gezeigt werden, daß die Aminosäuresequenz von Frs neben einer Leuzinreichen Region (putatives NES), die für die Bildung des Frs-Nup50-Komplexes notwendig ist, zwei Haupt-Phosphorylierungsstellen (T22 and T48) und ein KxL-Motiv enthält, das essentiell für die direkte Interaktion von Frs mit dem hydrophoben Patch von Cyclinen ist. Die physiologische Funktion dieser Motive wurde in Rescue-Experimenten überprüft. So konnte gezeigt werden, daß das KxL-Motiv essentiell für die Frs-Funktion im Embryo ist. Die beiden Phosphorylierungsstellen tragen in vivo teilweise, das putative NES-Motiv hingegen überhaupt nicht zur antimitotischen Aktivität von Frs bei. Mit Hilfe von surface-plasmon-resonance-Analyse wurde zudem gezeigt, daß Frs bevorzugt an mitotische Cycline bindet und eine viel höhere Affinität für das mitotische Cyclin A als für das G1/S spezifische Cyclin E aufweist. Mit der gleichen Methode konnte keinerlei Interaktion von Frs mit der Cdk-Untereinheit nachgewiesen werden. Somit unterscheidet sich das Bindungsverhalten von Frs von dem bereits bekannter Mitglieder der Cyklin-abhängigen Kinase-Inhibitor-Familien INK4 und CIP/KIP, was darauf schließen läßt, daß die Funktion von Frs auf einem neuen Mechanismus der Cdk-Inhibierung basiert. Zusammenfassend bleibt festzustellen, daß die Bindung von Frs an den hydrophoben Patch ausreichend ist, um den Eintritt in Mitose 14 zu inhibieren und das der hydrophobe Patch somit eine wichtige Rolle in der Zellzyklusregulation während des Drosophila- Midblastula-Übergangs spielt.
In developmental neurobiology, it is a fundamental topic but it is not still well investigated how newborn neurons elaborate axonal and dendrite processes to navigate complicated pathways and travel long distances before they reach their target. Recent studies have suggested that Rho family GTP-binding proteins are important components of the signalling pathways that link the reception of extracellular cues to the cytoskeleton. Rho family GTP-binding proteins regulate many different aspects of the actin cytoskeleton in a wide variety of organisms. Small GTPases of the Rho family have been suggested to be involved in the regulation of formation of neurites and their differentiation into axons and dendrites, but the function of Rho GTPases is not still clear in terms of axonal and dendritic growth during mammalian development. There are numerous data suggesting the important role of Rho GTPases in axonal guidance in vitro, however, there has been little direct evidence of these proteins in the in vivo context in mammals. To modulate the activity of Rho during early nervous system development, we expressed either a RhoA dominant negative (N19-RhoA) mutant, a constitutively active (V14-RhoA) mutant, or a natural inhibitor, C3 transferase from Clostridium botulinum, in neuborn neurons under the control of the tau gene. Their protein expression in neurons can be activated by application of Cre recombinase. The tau gene was used because it is known to drive the high expression of genes specifically in neurons (Binder et al., 1995). We used this transgenic strategy to analyze the effects of Rho family GTP-binding proteins on axonal outgrowth in early nervous system and the effect of long-term inhibition of Rho function in the adult brain. The recombinant protein of N19-RhoA was expressed in the postnatal mouse brain, and we found that the somatosensory cortex in the adult mouse brain contained more severe involutions and aggregations of the cells in specific area of somatosensory cortex in brain, particularly in layer IV. Also, the barrel-like discontinuous pattern was more extended toward the posterior part of the brain in the mice that have expressed a dominant negative RhoA.
In dieser Arbeit wird das dreidimensionale System der gasdynamischen Gleichungen in allgemein krummlinig-orthogonalen Koordinaten formuliert und auf zwei Raumdimensionen reduziert, indem nur Probleme mit bestimmten Symmetrien betrachtet werden. Zur Lösung der so erhaltenen hyperbolischen Erhaltungsgleichungen wird ein neues numerisches Verfahren für krummlinige Gitter entwickelt. Umfangreiche Tests belegen, dass die mit dieser Methode gewonnenen Resultate eine im Vergleich mit anderen Verfahren mindestens ebenso gute Approximation für die tatsächlichen Lösungen darstellen. Mit Hilfe des neuen numerischen Verfahrens wird die Dynamik der strahlungsgetriebenen Scheibenwinde untersucht. Es werden stationäre Lösungen für bestimmte Einflussrandbedingungen gefunden. Außerdem wird untersucht, inwiefern die Resultate von der künstlichen Viskosität des Verfahrens abhängen. Berechnungen auf sphärischen und zylindrischen Gittern ergeben kleine Unterschiede in den Lösungen in Abhängigkeit von der jeweiligen Geometrie.
We identify anthropometric parameter for models of human beings and the corresponding macroscopic movement. The models are based on rigid--body formalisms and formulated as mechanical DAEs. We use the Generalized Gauß--Newton method based on multiple shooting discretization to estimate the parameters of this dynamic nonlinear parameter estimation problem using measurement data taken from motion capturing. We adapt modelling, integration and optimization independently and in combination. The modelling based on Natural Coordinates is modified to efficiently evaluate the right-hand side of the DAE in linear time w.r.t. the number of bodies. The formulation does not introduce additional redundant constraints and we developed constraint partitioning to treat inherent singularities of the physical model. Furthermore, we include additional biomechanical elements like passive muscles and wobbling masses. As Natural Coordinates lead to a set of redundant coordinates, we have to relax to treat inconsistent variables during the optimization process. To efficiently generate the sensitivity information needed for the optimization algorithm, we apply reduced methods to evaluate a minimal number of directional derivatives and exploit the structure of the model equations to calculate each of them. Finally, we present parameter estimation results for a complex, full three-dimensional biomechanical model of the human body with 82 kinematic degrees of freedom. This is implemented in the object-oriented modelling tool MBSNAT and the parameter estimation package Parfit++.
Climate on earth strongly depends on the radiative balance of its atmosphere, and, thus, on the abundance of the radiatively active greenhouse gases. Largely due to human activities since the Industrial Revolution, the atmospheric burden of many greenhouse gases has increased dramatically. Direct measurements during the last decades and analysis of ancient air trapped in ice from polar regions allow to quantify the change of these trace gas concentrations in the atmosphere. From a presumably "undisturbed" pre-industrial situation several hundred years ago until today, the CO2 mixing ratio increased by almost 30%. In the last decades this increase was nearly exponential, leading to a global mean CO2 mixing ratio of almost 370 ppm by the turn of the millenium. The atmospheric abundance of CO2 the main greenhouse gas containing carbon, is strongly controlled by exchange with the organic and inorganic carbon reservoirs. The world oceans are definitely the most important carbon reservoir, with a buffering capacity for atmospheric CO2 largest on time scales of centuries and longer. In contrast, the buffering capacity of the terrestrial biosphere is largest on shorter time scales from decades to centuries. Although today equally important, the role of the terrestrial biosphere as a sink of anthropogenic CO2 emissions is still poorly understood. Any prediction of future climate strongly relies on an accurate knowledge of the greenhouse gas concentrations in the present day atmosphere, and of their development in the future. This implies the need to quantitatively understand their natural geophysical and biochemical cycles including the important perturbations by man's impact. In attempting to disentangle the complexity of these cycles, Radiocarbon observations have played a crucial role as an experimental tool enlightening the spatial and temporal variability of carbon sources and sinks. Studies of the “undisturbed” natural carbon cycle profit from the radioactive decay of 14C in using it as a dating tracer, e.g. to determine the turnover time of soil organic matter or to study internal mixing rates of the global oceans. Moreover, the anthropogenic disturbance of 14C through atmospheric bomb tests has served as an invaluable tracer to get insight into the global carbon cycle on the decadal time scale.
About 4900 values of 14CO2 activity have been measured on stratospheric air samples collected between 1953 and 1975 when the major nuclear weapon tests injected large amounts of 14C into the atmosphere. However, the validity of these data published in the Health and Safety Laboratory reports where repeatedly criticized and their relevance is thus usually denied in model studies tracing the global carbon cycle with bomb 14CO2. To oppose this criticism, we perform here a comprehensive analysis of the measurements and calculate stratospheric bomb 14CO2 inventories for the period in question. We find out that the recognized weakness of the survey do not justify a general discrimination against the 14CO2 observations. Our 14CO2 inventories determined using numerical methods to interpolate the observations widely confirm more "hand-made" results from a former study from Telegadas (1971) except in the northern poleward stratosphere. We are also able to clear away the reasons commonly advanced to call into question the stratospheric bomb 14CO2 inventories by up to 20%. These findings rehabilitate the most extensive data set of stratospheric 14CO2 observations and establish them, together with our corresponding bomb 14CO2 inventories, as a valuable observational constraint which should be seriously accounted for in global carbon cycle models and in other studies relying on an accurate simulation of air mass transport in the atmosphere.
Radiocarbon produced naturally in the upper atmosphere or artificially during nuclear weapons testing is the main tracer used to validate models of oceanic carbon cycling, in particular the exchange of carbon dioxide with the atmosphere and the mixing parameters within the ocean itself. Here we test the overall consistency of exchange fluxes between all relevant compartments in a simple model of the global carbon cycle, using measurements of the long-term tropospheric CO2 concentration and radiocarbon composition, the bomb 14C inventory in the stratosphere and a compilation of bomb detonation dates and strengths. We find that to balance the budget, we must invoke an extra source to account for 25% of the generally accepted uptake of bomb 14C by the oceans. The strength of this source decreases from 1970 onwards, with a characteristic timescale similar to that of the ocean uptake. Significant radiocarbon transport from the remote high stratosphere and significantly reduced uptake of bomb 14C by the biosphere can both be ruled out by observational constraints. We therefore conclude that the global oceanic bomb 14C inventory should be revised downwards. A smaller oceanic bomb 14C inventory also implies a smaller oceanic radiocarbon penetration depth, which in turn implies that the oceans take up 25% less anthropogenic CO2 than had previously been believed.
This paper has been written to make an enormous threat known to the public. We all know about the forest fires that raged in Indonesia and parts of Malaysia in 1997, and about the noxious haze covering the whole region, but only a few experts are aware of the threat of extinction of the last rain forest areas in Southeast Asia. As this paper will show, droughts have always been a part of these ecosystems, which normally receive plenty of rain. Mild droughts can be regarded as slight or medium disturbances, which can even have a stimulating effect according to the Intermediate Disturbance Hypothesis. However, severe droughts and fires have a destructive effect, which will only be overcome in successional stages. Logging and forest fragmentation reduce the ability of forests to overcome severe droughts. Forest fires rage in selectively logged forests, whereas they are extremely rare in undisturbed rain forests. Forest regeneration in fragmented forests is disturbed, so that we have to expect a total degradation of even effectively protected forest areas. Due to drought and fire, the remnant rain forest areas are being roasted like slices of toast . New research is also revealing that rain forests under drought stress consume more oxygen than they produce. The “green lung” and carbon sink are thus out of function. Furthermore, severe ENSO-(El Niño/Southern Oscillation)-droughts have been increasing in recent years. The consequences both for development of the region and for the whole atmosphere are imminent. Forest fires during ENSO droughts have caused extensive damage to forests and plantations, and noxious haze clouds in the whole region. Awareness about ENSO and droughts exists and is growing within the local population. Strict regulations, drought and fire alerts, and law enforcement can reduce at least the effects of drought. A logging ban, also including selective logging, may have to be considered. While this paper covers the whole of tropical Asia, it focuses on Sumatra, Borneo and the Malay Peninsula, which are the main evergreen rain forest areas. Other areas in the region have either seasonal vegetation, which is more drought adapted, or only small pockets of evergreen rain forest depending on local rain exposure – or the lowland forests ha ve disappeared. The paper concentrates on Western Indonesia, Malaysia and the very south of Thailand.
Das Heidelberger Life-Science-Lab will Schülerinnen und Schüler für die Naturwissenschaften begeistern. Dazu geht man neue Wege und hat ein gemeinsames Bildungsangebot von Schulen, Wissenschaft und Technik unter dem Dach der Technologiepark Heidelberg GmbH ins Leben gerufen. Der Leiter des Projekts, Dr. Thomas Schutz, hat das Projekt initiiert, weil seiner Erfahrung nach gerade die Naturwissenschaften heute im Bildungsleben zu kurz kommen. Das Angebot umfasst Labortage etwa im Deutschen Krebsforschungszentrum oder im Europäischen Labor für Molekularbiologie, aber auch Vorträge von Wissenschaftlern und Praktikern. Die jungen Teilnehmer haben gerade eine Portugalreise hinter sich und zeigen sich begeistert über die vielen Möglichkeiten, die Wissenschaft und Forschung heute bieten.