<> "The repository administrator has not yet configured an RDF license."^^ . <> . . "Establishment of an in vitro platform for the characterization\r\nof proteins involved in the intestinal epithelial barrier\r\ndysfunction of Inflammatory Bowel Disease, with a focus on\r\nTNFα/TNR1A/TNR1B."^^ . "The gastrointestinal (GI) epithelium consists of a cellular monolayer that prevents the infiltration of\r\npathogens and antigens into the GI mucosa and concomitantly mediates the highly selective resorption\r\nand secretion of nutrients, solutes and water. An intact monolayer of epithelial cells is achieved by two\r\ntypes of intercellular junctional protein complexes that seal the paracellular space: the Adherens (AJs)\r\nand the Tight Junctions (TJs). Inflammatory Bowel Disease (IBD) is a complex pathological condition\r\noriginating from a disturbed homeostasis of the immune and epithelial barrier functions of the GI mucosa\r\nthat results in a perturbance of the tight balance in between pro- and anti-inflammatory cytokines, and\r\nconsequently in between the immune tolerance and the defensive inflammatory responses towards the\r\nmicrobiota and microbial antigens. The dysfunction of the GI epithelial barrier plays a major role in both\r\nthe pathogenesis and the chronic manifestation of IBD. Tumor Necrosis Factor alpha (TNFα) as a key\r\npro-inflammatory cytokine with pleiotropic functions is highly upregulated at the intestinal mucosa in\r\nIBD. The TNFα signalling cascade is initiated by two different ligands, the membrane (mTNFα) and the\r\nsoluble (sTNFα) forms of TNFα, and mediated by two different membrane receptors TNR1A\r\n(TNFRSF1A) and TNR1B (TNFRSF1B). The latter are not only activated in separate ways, but can\r\ndetermine divergent effects in a tissue- and cell-specific manner.\r\nIn my Ph.D. work, I aimed to: i) establish an in vitro platform for the characterization of candidate\r\ngenes/proteins with regards to the key mechanisms that regulate the paracellular permeability at the GI\r\nepithelium, ii) establish an inflammatory model to resemble the barrier dysfunction that takes place in\r\nIBD, iii) investigate the mechanisms of action of the key pro-inflammatory cytokines TNFα, Interferon\r\ngamma (IFNγ) and Interleukin1- beta (IL-1β), with a special focus on the dissection of the axis\r\nTNFα/TNR1A/TNR1B, in intestinal epithelial barrier function.\r\nAn in vitro model of the inflamed intestinal epithelium was generated by differentiating the colorectal\r\ncarcinoma cell lines T84 and CACO-2 on Transwell inserts and applying the pro-inflammatory cytokines\r\nsTNFα, IFNγ and IL-1β from the basolateral side at different concentrations and combinations. To assess\r\nthe consequences of these stimuli on the different permeability pathways of the paracellular route,\r\ndifferent assays were performed: measurement of the Trans Epithelial Electrical Resistance (TEER),\r\npermeability assays with fluorescently labelled molecular species, cell viability assays and assessment\r\nof the expression and subcellular localization of key tight junctional proteins (TJPs). Once defined the\r\noptimal pro-inflammatory input to mimic the IBD condition, the barrier function impairing effects of the\r\nsingle cytokines sTNFα and IFNγ were investigated in detail. Fully differentiated T84 monolayers were\r\nsubjected to a single cytokine induction in the presence of potent and selective blockers of both the\r\nligands and of the respective cognate membrane receptors to confirm the validity of the model. A part\r\nfrom applying the key functional and cell viability assays, the cross-regulatory mechanisms taking place\r\nin between both cytokines were investigated with transcriptional expression analyses. The results of\r\nthese experiments confirmed a synergism in between both cytokines based on not only a positive regulatory loop exerted by IFNγ on TNFRSF1A and TNFRSF1B, but also on a positive regulatory loop\r\nexerted by sTNFα on IFNGR1 and IFNGR2. Furthermore, a putative anti-necroptotic effect determined\r\nby IFNγ was observed to take place on these GI epithelial cells.\r\nIn the last part of my work, I applied potent and selective pharmacological modulators directed towards\r\ndifferent levels of the TNFα signalling. The colorectal carcinoma cell line T84 was fully differentiated\r\non Transwell inserts and different end-point experiments were run by applying combinations of sTNFα\r\n(natural agonist of TNR1A and marginal agonist of TNR1B), TROS (competitive, selective antagonist\r\nof TNR1A) and ADALIMUMAB (competitive, global antagonist of the sTNFα signalling). The\r\nconsequences of these modulations were analyzed with the different aforementioned functional assays,\r\nand a detailed image of the cellular pathways differentially regulated was obtained by a transcriptomic\r\nexpression analysis. The results of these experiments demonstrated that TNR1A mediates most of the\r\nbarrier function impairment determined by sTNFα. The complete dissection of the signalling, by future\r\ninvestigation of the role of TNR1B in this specific context, could provide the basis for the initiation of\r\nthe work towards the modification of the actual pharmacological therapies for IBD that are dominated\r\nby global TNFα inhibitors. A receptor-specific therapy could lead to a better outcome rather than the\r\nneutralization of the whole signalling pathway."^^ . "2022" . . . . . . . "Ignacio Andrés"^^ . "Vergara Dal Pont"^^ . "Ignacio Andrés Vergara Dal Pont"^^ . . . . . . "Establishment of an in vitro platform for the characterization\r\nof proteins involved in the intestinal epithelial barrier\r\ndysfunction of Inflammatory Bowel Disease, with a focus on\r\nTNFα/TNR1A/TNR1B. (PDF)"^^ . . . "THESIS SUBMITTED - Vergara Dal Pont.pdf"^^ . . . "Establishment of an in vitro platform for the characterization\r\nof proteins involved in the intestinal epithelial barrier\r\ndysfunction of Inflammatory Bowel Disease, with a focus on\r\nTNFα/TNR1A/TNR1B. (Other)"^^ . . . . . . "indexcodes.txt"^^ . . . "Establishment of an in vitro platform for the characterization\r\nof proteins involved in the intestinal epithelial barrier\r\ndysfunction of Inflammatory Bowel Disease, with a focus on\r\nTNFα/TNR1A/TNR1B. (Other)"^^ . . . . . . "lightbox.jpg"^^ . . . "Establishment of an in vitro platform for the characterization\r\nof proteins involved in the intestinal epithelial barrier\r\ndysfunction of Inflammatory Bowel Disease, with a focus on\r\nTNFα/TNR1A/TNR1B. (Other)"^^ . . . . . . "preview.jpg"^^ . . . "Establishment of an in vitro platform for the characterization\r\nof proteins involved in the intestinal epithelial barrier\r\ndysfunction of Inflammatory Bowel Disease, with a focus on\r\nTNFα/TNR1A/TNR1B. (Other)"^^ . . . . . . "medium.jpg"^^ . . . "Establishment of an in vitro platform for the characterization\r\nof proteins involved in the intestinal epithelial barrier\r\ndysfunction of Inflammatory Bowel Disease, with a focus on\r\nTNFα/TNR1A/TNR1B. (Other)"^^ . . . . . . "small.jpg"^^ . . "HTML Summary of #32402 \n\nEstablishment of an in vitro platform for the characterization \nof proteins involved in the intestinal epithelial barrier \ndysfunction of Inflammatory Bowel Disease, with a focus on \nTNFα/TNR1A/TNR1B.\n\n" . "text/html" . . . "570 Biowissenschaften, Biologie"@de . "570 Life sciences"@en . .