The baculovirus Autographa californica nucleopolyhedrovirus (AcNPV) has two viral forms, budded virus (BV) and occlusion derived virus (ODV). The envelopment of these two viral forms occurs at different locations: BV acquires envelopes at the plasma membrane while ODV acquires envelopes in the nucleus. The two viral forms carry out different functions in the viral life cycle. The purpose of this study is to investigate how viral envelope proteins sort/traffic to the nucleus. Of particular interest is BV/ODV E26 (E26). E26 is an envelope protein of both BV and ODV (Braunagel and Summers, 1994); therefore it must traffic to the plasma membrane and the nucleus during infection. Thus, E26 is a bi-directional trafficking protein, which interacts with membranes in both locations of the cell. As such it has been shown that there are several immunoreactive forms of E26 (Beniya, Braunagel, and Summers, 1998). The da26 gene produces at least 2 protein products of 26 and 28 kDa with different functions respectively, which correlate with localization, solubility, membrane association, and temporal requirements. The 28 kDa form is likely a soluble protein that interacts with transcriptional activators and DNA in the nucleus in the early stages of infection. A part of the 26 kDa population is a membrane bound form interacting with an integral membrane protein in the ER and likely functions as an INM sorting factor. The 26 kDa membrane bond form is also found in the inner nuclear membrane, intra-nuclear microvesicles, ODV envelopes, and ODV in the nucleus.

Enterovirus 71 (EV71) infection is one of epidemic disease in children commonly in Taiwan. The clinical manifestation of EV71 infection may include acute respiratory disease, hand foot and mouth disease, herpangina, myocarditis, aseptic meningitis, acute flaccid paralysis, brainstem or cerebellar encephalitis. EV71 infection usually occurs through the fecal¡Voral route, leading to viremia and invasion of the skin and mucosa. Infection is initiated by attachment to putative receptor, which induces conformational changes in the virus that facilitate translocation of the viral RNA into the cytoplasm. Some cell surface molecules have been primarily identified for enterovirus which like poliovirus receptor (CD155), coxsackievirus and adenovirus receptor, Decay accelerating factor (CD55) belong Echoviruses but no EV71 receptor has yet been found. Rhabdomyosarcoma cells were used as a model for EV71 infection. We use two-dimensional gel electrophoresis to analyse membrane fraction from rhabdomyosarcoma cells infected with EV71 at 6 h post infection. Twenty-eight differentially expressed protein spots were identified. Some lipid-associated protein slightly change after EV71 infection that may indicate EV71 infection will change membrane structure of rhabdomyosarcoma cells. And some O-linked glycosylation proteins were also upregulated after EV71 infection. It is interesting to reveal the role of these proteins in early EV71 infection and cell response.

Thesis (M.Sc.)--York University, 2006. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 62-69). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR29595

Thesis (Ph. D.)--Ohio State University, 2004. / Title from first page of PDF file. Document formatted into pages; contains xiv, 201 p.; also includes graphics (some col.). Includes abstract and vita. Advisor: Stephen A. Osmani, Dept. of Molecular Genetics. Includes bibliographical references (p. 184-201).

Thesis--M.D., University of Hong Kong, 1982. / 6 leaves of curriculum vitai in pocket.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 92-112).

Oxidative damage by reactive oxygen species (ROS) is a major cause of sperm dysfunction. Excessive ROS generation reduces fertilization and enhances DNA damage of spermatozoa. In mammals, including humans, oviduct functions as a sperm reservoir which is created by the binding of spermatozoa to the epithelial lining in the oviduct. Interaction between sperm and oviductal epithelial cells improves the fertilizing ability of and reduces chromatin damage in spermatozoa. However, the mechanism(s) by which spermatozoa-oviduct interaction producing these beneficial effects is unknown. One possibility is that oviduct protects spermatozoa from oxidative stress. The hypothesis of this project was that oviductal cell membrane proteins interact with spermatozoa to protect them from oxidative damage. Due to the limited availability of human oviductal tissue for research, an immortalized human oviductal epithelial cell line, OE-E6/E7, was used as a study model. The first objective examined the effect of OE-E6/E7 membrane proteins on human spermatozoa. The extracted OE-E6/E7 membrane proteins bound to sperm head and preferentially to uncapacitated sperm. Pretreatment with OE-E6/E7 membrane proteins significantly suppressed ROS-induced adverse effects in sperm motility, membrane integrity, DNA integrity, and intracellular ROS level. OE-E6/E7 membrane proteins also increased the endogenous enzyme activities of sperm superoxide dismutase (SOD) and glutathione peroxidase (GPx). Sperm fucosyltransferase-5 (sFUT5) is a membrane carbohydrate-binding protein on human sperm. The second objective investigated the involvement of sFUT5 in sperm-oviduct interaction. Purified sFUT5 bound to OE-E6/E7 cells and anti-FUT5 antibody inhibited this interaction. Pre-absorption of OE-E6/E7 membrane proteins with purified sFUT5 or blocking of sFUT5 on sperm with anti-FUT5 antibody significantly inhibited the protective effects of OE-E6/E7 membrane proteins against ROS-induced damages in spermatozoa. Asialofetuin, a reported sFUT5 substrate, can partly mimic the protective effect of OE-E6/E7 membrane proteins. Sperm processing in assisted reproductive technology (ART) treatment, including centrifugation and cryopreservation, has shown to induce ROS production and oxidative damage in sperm. The third objective investigated the possible use of OE-E6/E7 membrane proteins or asialofetuin as an antioxidant supplement during centrifugation and cryopreservation. No adverse effect on sperm functions was detected by centrifugation using our centrifugation protocols. OE-E6/E7 membrane proteins or asialofetuin pretreatment suppressed the cryopreservation-induced damage on sperm in terms of motility and DNA fragmentation. The fourth objective aimed to identify the sFUT5-interacting proteins from OE-E6/E7 membrane extracts. By using immuno-affinity chromatography and mass spectrometry analysis, cell adhesion molecule 4 (CADM4) was identified as a potential sFUT5-interacting protein. This result was further supported by co-immunoprecipitation, immunofluorescent staining and immunohistochemistry. CADM4 expression level was shown to be higher at follicular phase when compared to luteal phase of the menstrual cycle. In conclusion, this thesis demonstrated that oviductal epithelial cell membrane proteins bind to the human spermatozoa and protect them from ROS-induced damages in terms of motility, membrane integrity, and DNA integrity. sFUT5 mediates the spermatozoon-oviductal epithelial cell interaction and the beneficial effects of such interaction on the fertilizing ability of spermatozoa. Results from this study provide the potential use of sFUT5-interacting proteins to enhance the fertilization ability of human spermatozoa by protecting them from oxidative stress. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

Mitochondria are essential organelles in the cell. They are enveloped by a two membrane system, an inner and outer membrane. Residing in the outer membrane is the voltage-dependant anion-selective channel (VDAC) which is in very high abundance in the outer membrane. Previously, when VDAC was deleted in N. crassa the mitochondria developed unusual morphology and the fungus became temperature sensitive. This was suspected to be caused by alterations in the membrane architecture. It was found that deletion of VDAC causes a reduction in mitochondrial outer membrane fluidity leading to a fragile outer membrane. In the current study it was also found that loss of VDAC significantly impacts the mitochondrial proteome. These results taken together suggest that VDAC is not only a channel for traversing the outer membrane but appears to be important for the structural integrity of the mitochondria. / October 2014

published_or_final_version / Medicine / Master / Doctor of Medicine

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