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Abstract

Cell polarity describes the asymmetric distribution of proteins, RNA, organelles and lipids. One of the best studied models of polarity are epithelial cells. They form the barriers that separate and protect the organism from the outside environment. Establishment and maintenance of polarity is ensured through a highly complex network of proteins and lipids that mediate asymmetry through trafficking processes and reorganization of the cytoskeleton. One of the key players in polarity are phosphoinositides, phosphorylated forms of the lipid phosphatidylinositol. In this thesis I investigated the role of one such phosphoinositide, PI(3)P, on cellular polarity and immunity in human intestinal epithelial cells. In my work I used established methods to evaluate the role of PI(3)P in human intestinal epithelial cells. These three methods were (1) the use of specific inhibitors targeting VPS34 (2) the development of cell lines depleted of kinases and phosphatases needed for PI(3)P biogenesis and (3) on demand depletion of PI(3)P from endosomes using a chemical dimerizer system. Each of these methods were evaluated both for their ability to modulate PI(3)P levels and for their impact on cellular polarity and immune response. While I could demonstrate that all three methods successfully modulated PI(3)P levels the chemical dimerizer (AP21967) interferes with polarity and immunity itself and we require a new dimerizer before conclusions can be made using this method. Interestingly, I determined that reduced PI(3)P levels, either by chemical inhibition or knock-down of the kinase VPS34, impaired cellular polarity in T84 cells indicating a critical role of PI(3)P in the polarity program. Additionally, knock-down of the PI(3)P metabolizing enzymes VPS34 and MTM1 showed interesting results for IFNλ production upon reovirus infection. IFNλ production in MTM1 and VPS34 knock-down cells is increased and decreased respectively. This could be due to the previously reported PI(3)P dependent TLR3 sorting adaptor WDFY1 and should be further investigated. In parallel I also created a novel viral vector system in our lab. This viral vector is based on the BacMam system which are baculovirus based vectors with a large cargo capacity. The BacMam was initially modified with an S/MAR sequence to allow for its persistence in cells. The vector was then further modified to allow expression of reporter genes. Both vectors were shown to be fully functional and will provide a valuable tool for future projects.