TY  - GEN
UR  - https://archiv.ub.uni-heidelberg.de/volltextserver/10349/
A1  - Medda, Rebecca
N2  - High resolution light microscopy is gaining in importance in the Life Sciences. This thesis reports on two applications of the STED (stimulated emission depletion) principle investigating alterations on the protein and lipid level occurring during aging.  One hallmark of age-related neurodegenerative diseases is the abnormal post-translationally occurring hyperphosphorylation of proteins. Here, the interplay of two main post-translational modifications of neurofilaments, phosphorylation and glycosylation, was investigated. The organization pattern of these modifications can not be resolved by conventional diffraction-limited light microscopy. However, with the about 10-times higher resolution of STED microscopy the organization for both modifications was revealed. Long term glucose deprivation of redifferentiated neuroblastomas results in axonal swellings and cell death, which could not be rescued by refeeding glucose. These severe alterations could be observed by confocal resolution (~250 nm). Taking advantage of the higher resolution of the STED microscope (20-30 nm) allowed the detection of earlier alterations in the pattern of both modifications, where cell death could be prevented by the refeeding of glucose. Two kinases were found to be involved in the phosphorylation events occurring upon glucose deprivation in redifferentiated SH-SY5Y: the mitogen activated protein kinase p38 MAPK and to a lower extent also the stress activated protein kinase JNK.   An effect occurring during aging is the change in the plasma membrane's fluidity. This thesis reports on the investigation of the diffusion characteristics of different phospholipids in dependency of the endogenous cholesterol content on a nanoscale. Fluorescent mono- and diacyl-phospholipid analogs were incorporated via BSA-complex formation into the plasma membrane of living cells. It was found that for best incorporation a molar ratio of 1:1 (fatty acid chains:BSA molecules) had to be used. Analogs of the phosphoglycerolipids and the sphingolipids, respectively, were the central study in this thesis. It was shown that sphingolipids, in contrast to phosphoglycerolipids, were trapped on spatial scales below 30 nm, which could not be examined by confocal microscopy. Upon changing the endogenous cholesterol level, enzymatically with cholesterol oxidase and by host-guest complex formation using the oligosaccharide ?-cyclodextrin, respectively, this hindered-diffusion could be reversibly abolished.
ID  - heidok10349
Y1  - 2009///
TI  - Investigation of age-related alterations occurring on the protein and the lipid level by using STED Microscopy
KW  - STED-Mikroskopie 
KW  -  humane Neuroblastoma 
KW  -  Zelllinie 
KW  -  posttranslationale ModifikationSTED microscopy 
KW  -  human neuroblastoma 
KW  -  cell line 
KW  -  posttranslational modification
AV  - public
ER  -