Analysis of the effect of novel PKC isozymes on autophagy and utilization of nanoparticles as a new therapeutic approach for gaucher disease
Yedier, Özlem (2017) Analysis of the effect of novel PKC isozymes on autophagy and utilization of nanoparticles as a new therapeutic approach for gaucher disease. [Thesis]
Autophagy is a mechanism that degrades long-lived proteins and non-functional organelles to sustain cellular homeostasis. It is implicated in many processes inside the cell ranging from regulation of energy and nutrient balance to the defence against pathogens. Defects or abnormalities in this mechanism are associated with various diseases including cancer and neurodegeneration. Protein Kinase C (PKC) isozymes are a large family of serine-threonine kinases that have many roles inside the cell. Each PKC isozyme might have unique and opposing functions in the same cellular pathway or same PKC isozyme can function differentially upon different stimuli. Several studies showed the relations of PKC isozymes in autophagy pathway mostly in the induction step. However, in most of the cases, complete mechanisms or the specific isozyme responsible from the particular effect was not identified. In this study, the effect of active PKC isozymes on autophagy was analyzed and candidate PKC isozymes, that are important for the modulation of autophagy by PMA were identified. In addition, the effect was shown as a conserved mechanism not only for human cancer cells but also mouse primary fibroblast cells. Moreover, lentiviral transduction method was established in order to use a lentiviral shRNA library for screening the target molecules functioning as effectors between PKC isozymes and autophagy. Optimum conditions for lentivirus production and transduction were investigated and the precise antibiotic concentration was identified for further screening. Defects in lysosomal enzymes are the main cause of malfunctional autophagy and thus lysosomal storage diseases. Gaucher Disease is the most common lysosomal storage disease around the world that results from mutations in GBA1 gene which expresses glucocerebrosidase enzyme. In this study, acidic nanoparticles were utilized as an alternative methodology to functionalize impaired autophagic flux and alkalinized lysosomes were corrected which eventually lead to decrease in the accumulation of glucosylceramide substrate in mutant cells.
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