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Masters Thesis
Molecular techniques: HPgV and apoptosis, TMEM55B in MDCK cells
DETERMINING THE EFFECT OF HPGV INFECTION ON APOPTOTIC PROTEIN EXPRESSION IN JURKAT CELLS: The human pegivirus (HPgV) is a recently described Flavivirus which has been shown to be apathogenic to humans. It has been observed that HIV-1 positive patients coinfected with HPgV take longer to progress to AIDS, however the mechanisms are not fully understood. The Acquired Immunodeficiency Syndrome (AIDS) is acquired via the depletion of CD4+ T-cells, many of which are lost through apoptosis or programmed cell death. Two receptors involved in this pathway are FasR (Fas receptor) and TNFR (TNF receptor). It has been observed that HIV-infected patients who are coinfected with HPgV show decreased Fas receptor expression, suggesting that HPgV may hinder HIV-1 progression by protecting cells from apoptosis. While the relationship between HPgV and FasR expression has been studied, no significant investigations into the relationship between HPgV and TNFR expression have been done. It is possible that reduced TNFR expression is partially responsible for increased T-cell survival in HIV-1 patients coinfected with HPgV. The goal of this research is to determine if TNFR expression is indeed downregulated by HPgV infection. It is important to quantify increased expression levels of important proteins within the extrinsic apoptotic signaling pathway, such as TRADD, Caspase 8 and NF-κB to ensure that activation of the apoptotic pathway occurs through TNF activation. Upon conclusion of this research the relationship between HPgV infection and TNFR-mediated apoptosis would start to create a more distinct picture of the mechanism behind HPgV protection. The information gained will help us better understand this mysterious virus and aid in future studies into its interactions with HIV-1. CHARACTERIZATION OF TMEM55B EXPRESSION IN CANIS FAMILIARIS: TMEM55B, also known as phosphatidylinositol-4,5-bisphosphate 4-phosphatase, catalyzes the formation of phosphatidylinositol-5-phosphate via removal of its precursor’s 4’ phosphate. In conjunction with PI(5)P, TMEM55B is expressed in all cells and localizes to the lysosome and late endosome. TMEM55B has not been studied extensively, and its mechanisms are not well-understood. While its earliest function was discovered to be the production of PI(5)P, it has been implicated in several other cellular processes. Examples include participating in stress-induced apoptosis, regulation of cholesterol metabolism and direct involvement in lysosomal positioning. The implication of TMEM55B in these processes makes it an attractive target for cancer and diseaserelated studies. Efficient experimentation requires a biologically correct model, whether that be cellular or within the organism itself. The domestic dog, Canis familiaris, has been receiving more attention as a host for genomic disease-related studies. The dog’s genome shares more homology with our own than that of the mouse and dogs acquire many of the same diseases, at the same frequency as humans. In addition, dogs are divided into isolated genetic groups in the form of breeds, and that lends many advantages to genetic inquiries. Therefore, it would be beneficial to characterize TMEM55B in the domestic dog as a cellular model. This project shows that the TMEM55B transcript is indeed expressed in dog kidney cells (MDCK) and continuing research is aimed at creating an expression vector and observing its cellular localization.
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