Department of Veterinary Medicine Theses and Dissertations

Permanent URI for this collectionhttp://hdl.handle.net/1903/2762

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FUNCTIONAL CHARACTERIZATION OF THE VIRAL FLICE INHIBITORY PROTEIN OF RHESUS MONKEY RHADINOVIRUS
(2011) Ritthipichai, Krit; Zhang, Yanjin; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Rhesus monkey rhadinovirus (RRV) is a gamma-2 herpesvirus closely related to Kaposi's sarcoma-associated herpesvirus (KSHV). KSHV is associated with several malignant diseases, including Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman's disease. Here we found that RRV viral FLICE inhibitory protein (vFLIP) inhibits apoptosis. In HeLa cells with vFLIP expression, cleavage of poly [ADP-ribose] polymerase 1 (PARP-1) and activities of caspase 3, 7, and 9 were much lower than controls. RRV vFLIP was able to enhance cell survival under starved condition or apoptosis induction. After apoptosis induction, autophagosome formation was enhanced in cells with vFLIP expression and when autophagy was inhibited, these cells underwent apoptosis. Moreover, RRV latent infection of BJAB B-lymphoblastoid cells protects the cells against apoptosis. Knockdown of vFLIP expression in the RRV-infected BJAB cells with siRNA abolished the protection against apoptosis. These findings indicate that RRV vFLIP protects cells against apoptosis by enhancing autophagosome formation.
REGULATION OF MACROAUTOPHAGY BY VITAMIN A/ RETINOIDS
(2013) Rajawat, Yogendra Singh; Bossis, Ioannis; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Retinoic acids (RAs) have diverse biologic effects and regulate several cellular functions. Here, we investigated the role of RA on autophagy by studying its effects on autophagosome (AUT) maturation, as well as on upstream regulators of autophagosome biogenesis. Our studies, based on the use of pH-sensitive fluorescent reporter markers, suggest that RA promotes AUT acidification and maturation. By using competitive inhibitors and specific agonists, we demonstrated that this effect is not mediated by the classic Retinoic Acid Receptor (RAR) and Retinoid X Receptors (RXR). RA did not affect the protein expression levels of upstream regulators of autophagy, such as Beclin-1, phospho-mTOR, and phospho-Akt1, but induced redistribution of both endogenous cation-independent mannose-6-phosphate receptor CI-MPR and transiently transfected GFP and RFP full-length CI-MPR fusion proteins from the trans-Golgi region to acidified AUT structures. Those structures were found to be amphisomes (acidified AUTs) and not autophagolysosomes. The critical role of CI-MPR in AUT maturation was further demonstrated by siRNA-mediated silencing of endogenous CI-MPR. Transient CI-MPR knockdown resulted in remarkable accumulation of nonacidified AUTs, a process that could not be reversed with RA.These results suggest that RA induces AUT acidification and maturation by regulating CI-MPR subcellular location, a process critical in the cellular autophagic mechanism.