Viral infection of cells often requires many host cell factors. Despite the identification of numerous viral receptor molecules relatively little is known about the roles of these molecules in viral entry, and intracellular targeting. Major histocompatibility complex (MHC) class I proteins are components of the Simian Virus 40 (SV40) cell surface receptor. This interaction between virus and host cell is of interest since MHC class I proteins have typically been studied for their importance in cellular immune responses. MHC class I proteins acquire antigenic peptides in the endoplasmic reticulum (ER). SV40 is unique in its ability to target the ER from the cell surface. Thus, the possibility exists that surface MHC class I proteins may recycle and target the ER. A major question is do MHC class I proteins internalize and target SV40 to the ER. The goal of this thesis was to better characterize SV40 entry, and determine if MHC class I proteins are directly involved in viral internalization. Basic studies were therefore conducted to determine the fate of MHC class I proteins on fibroblasts. SV40 entry into cells was found to occur rather slowly. The majority of preadsorbed virions remain at the cell surface for up to 2.5 hours. Furthermore, three hours were required for preadsorbed SV40 to internalize and escape antiserum neutralization. This suggests that SV40 infectious entry is relatively slow. Cytosol acidification greatly reduced clathrin-dependent endocytosis, but had no inhibitory effect on SV40 infectious entry. Therefore, SV40 infectious entry does not occur by receptor-mediated endocytosis. Treatment of cells with PMA, nystatin, or filipin prevents internalization via caveolae. All three treatments prevented SV40 infectious entry. Thus, caveolae may be the sites of SV40 penetration into cells. To evaluate the role of MHC class I proteins in SV40 endocytosis, cell surface proteins were labeled with $\sp{125}$I. Protease treatment of cells was used to distinguish proteins that have internalized from those remaining at the cell surface. Protease resistant $\sp{125}$I-MHC class I proteins were not detected in the absence, or presence of SV40. Furthermore, a $\sp{125}$I-MHC class I protein-specific monoclonal antibody did not internalize into cells. These results suggest that surface MNC class I proteins are not internalized into cells, and SV40 does not induce their internalization. Truncated $\sp{125}$I-MHC class I proteins were detected in the media. 1,10-phenanthroline prevented accumulation of $\sp{125}$I-MHC class I proteins in the media. Thus, metalloprotease activity is involved in shedding of surface MHC class I proteins from CV-1 cells. The loss of the B$\sb2$-microglobulin subunit from the MHC class I heavy chain was found to precede metalloprotease cleavage of the heavy chain. Collectively, these results suggest that the fate of MHC class I proteins on fibroblast is shedding into the media, and these molecules are probably not directly involved in SV40 entry.

Programmed cell death (PCD) is an essential developmental process in all multicellular organisms. It serves multiple functions including selected removal of unneeded and/or deleterious cells, and regulation of cell numbers (reviewed in Milligan and Schwartz, 1996). The intersegmental muscles (ISMs) of the hawkmoth Manduca sexta provide an ideal model for studying PCD (reviewed in Schwartz, 1992). In response to a decline in the circulating titer of the steroid hormone 20-hydroxyecdysone (20-HE), these cells initiate a death program which includes both the up- and down-regulation of specific genes. Following eclosion, the ISMs undergo PCD that results in the complete destruction of the muscles during the subsequent 30 hours (Finlayson, 1956). This dissertation examines in detail the repression of actin and myosin heavy chain expression that occurs when the muscles become committed to die. At the protein level, actin expression was reduced by 84% at the time the muscles were committed to die, which presumably plays a role in the rapid dissolution of the muscles. When the ISMs became committed to die, there were dramatic increases in proteolytic activity that are correlated with an approximately eightfold increase in the absolute amounts of multicatalytic proteinase (MCP). At the time of commitment, four new MCP subunits were observed to be associated with the complex. Correlated with the addition of these new subunits was a dramatic increase in the levels of immunodetectable MCP throughout the cytoplasm and within the nuclei of dying muscles. These changes in MCP were regulated by the same hormonal signals that mediate cell death. Cells dying by PCD often display a characteristic set of features termed apoptosis. These features include chromatin condensation, DNA fragmentation, membrane blebbing and phagocytic removal of the dying cells. However, dying ISMs display few characteristics of apoptosis. Interestingly, apoptotic cell death does occur in Manduca embryogenesis. Evidently Manduca possesses the necessary biochemical machinery to undergo apoptosis and does so in specific developmental circumstances. These data suggest that more than one cell death mechanism is used during development.

During muscle differentiation in vertebrates, myoblasts initially form in somites and then migrate to proper locations in the trunk and limbs. Once there, these cells are faced with one of three choices: differentiate into myotubes, arrest as satellite cells, or initiate apoptosis and die. The molecular mechanisms that regulate the decision of myoblasts to die are poorly understood. To gain insight into this process, we have cloned death-associated genes from the intersegmental muscles of the moth Manduca sexta, a model system for developmentally regulated muscle cell death. One of the genes isolated in this screen was DALP (Death Associated LIM Only Protein), a protein that shares 52% similarity at the protein level with mammalian Hic-5. Ectopic expression of DALP in the skeletal muscles of the fruit fly Drosophila caused atrophy and disorganization of the contractile apparatus. To determine the role of DALP/Hic-5 in mammalian myogenesis, we took advantage of the mouse myoblast cell line C2C12. Ectopic expression of either DALP or Hic-5 blocked the ability of myoblasts to differentiate following serum withdrawal. These cells failed to express muscle differentiation markers such as MyoD or myosin heavy chain. In addition, these cultures displayed greatly enhanced rates of cell death. Hic-5 expression is restricted to mononucleated and apoptotic C2C12 cells in serum-depleted medium. The effects of ectopic DALP or Hic-5 expression could be prevented by contact with wild type C2C12 cells or by ectopic expression of MyoD. Gene profiling experiment demonstrated that the ectopic expression of Hic-5 results in enhanced expression of pro-apoptotic Bcl-2 family members and of polyubiquitin. Taken together, these data suggest that Hic-5 acts upstream of MyoD and functions as a negative-regulator of myoblast differentiation and may facilitate the initiation of apoptosis. In separate studies, the functional roles of another death-associated molecule, m56, were studied in Ratl fibroblasts. Our data strongly support the hypothesis that m56 is a proteasome subunit and misexpressing m56 can sensitize Ratl cells to apoptotic stimuli.

The pituitary gland is the major endocrine gland in the forebrain. The hormones secreted from this gland regulate vital processes such as reproduction, growth and stress response. Distinct endocrine cells arise from pituitary precursors cells. The endocrine cells are spatially organized along the anterior-posterior axis within the anterior lobe of the pituitary, the adenohypophysis. Several signaling molecules have been shown to play roles in the development of this endocrine gland. Previous work in our lab showed that Sonic Hedgehog (Shh) is required for induction and patterning of the adenohypophysis. Hedgehog (Hh) signaling is involved in many developmental processes including induction, patterning and differentiation of many tissues. In addition, independent studies show that Fibroblast growth factor (Fgf) signaling also plays a role in the development of zebrafish adenohypophysis. ^ One of the main aims of my dissertation was to determine how the Hh and Fgf signaling pathways specify the functional patterning of the adenohypophysis. Using small molecule inhibitors I show that high levels of Hh signaling are required for the formation of the anterior adenohypophysis, the pars distalis (PD) and high levels of Fgf signaling are required for the formation of the posterior adenohypophysis, the pars intermedia (PI). My dissertation work also shows that high Hh levels are required for differentiation of the endocrine cells in the PD, and in contrast high Fgf levels are required for differentiation of the endocrine cells in the PI. Using live-imaging of a transgenic zebrafish line, I show that the PD and PI originate from distinct regions. My analyses has revealed that graded Hh and Fgf signaling help pattern the adenohypophysis along anterior-posterior axis by guiding endocrine cell differentiation in a dose dependent manner. ^ A related aim of my research was to analyze the role of Hh signaling in zebrafish neural tube patterning. The transcriptional response to varying Hh levels is well characterized in chick and mouse neural tube, and this transcriptional response has been partially described in zebrafish. The analysis of the Hh transcriptional response in wild type, Hh mutant and Hh over-expressing embryos show that there is a conserved transcriptional response to Hh signaling in the zebrafish neural tube. My comprehensive analyses of the Hh transcriptional response in the zebrafish neural tube provides a useful tool for the characterization of Hh signaling in zebrafish. ^

A-kinase anchoring protein AKAP95 is implicated in mitotic chromosome condensation by recruiting the condensin complex. Here, we report a differential regulation of condensation of maternal and paternal chromosomes mediated by AKAP95 and chromatin composition in mitotic mouse zygotes. AKAP95 is synthesized upon oocyte activation, targeted to the female pronucleus and specifically associates with maternal chromosomes at mitosis. Peptide competition and rescue experiments show that AKAP95 is required for recruitment of the mCAP-D2 condensin subunit to, and condensation of, maternal chromosomes. In contrast, AKAP95 is dispensable for mCAP-D2 targeting and condensation of paternal chromosomes. In vitro nuclear reconstitution and disassembly assays indicate that human hCAP-D2 targets protamine-containing chromatin independently of AKAP95, but requires AKAP95 for association with histone-containing chromosomes. We propose a concept whereby (1) recruitment of condensins to chromatin is affected by chromatin composition and (2) AKAP95 renders histone-containing chromatin permissive to condensin targeting.

Pollen tube elongation is a polarized cell growth process that directionally transports the male gametes from the stigma to the ovary for fertilization inside the ovules. Actin cytoskeleton is known to support this growth process and Rac-like GTPases have been shown recently to be important to regulate actin organization in elongating pollen tubes. Actin depolymerizing factor/cofilins (ADF/cofilins) are actinbinding proteins that increase actin dynamics by enhancing actin depolymerization. They are also responsible to regulate actin organization in Rac-mediated signaling. My thesis research focuses on establishing a signaling pathway from Rac GTPase to the actin cytoskeleton via the regulation of ADF/cofilins in tobacco pollen tubes. I have isolated and characterized cDNAs for tobacco pollen ADF, NtADFs, to study their function in pollen tube growth. First, I showed the activity of Rac-like GTPase is essential for pollen germination. Tobacco pollen germination and early pollen tube growth stimulates the activation of these small GTPases, the phosphorylation of NtADFs and an increase in the ratio of F- to G-actin. Moreover, over-production of a pollen-expressed Rac-like GTPase, NtRac1 from tobacco, induces increased ADF phosphorylation in transformed pollen and diminished the binding of GFP-tagged NtADF1 (GFP-NtADF1) to actin filaments in growing pollen tubes. These observations are consistent with the presence of a signaling pathway in pollen whereby Rac-like GTPase are stimulated by germination to activate a phosphorylation cascade that down regulates the activity of ADFs. This ultimately affects actin dynamics and growth characteristics in pollen tubes. Second, I also showed that NtADF activity is important for actin organization in pollen tube growth. When expressed in a moderate level in pollen tubes, GFP-NtADF1 associated prominently with a sub-apical actin mesh comprised of short dynamic actin filaments and with long dynamic actin cables in the shank. Over-producing NtADF1 resulted in the reduction of fine, axially oriented actin cables in transformed pollen tubes. Pollen tube growth was also inhibited by over-expressed NtADF1 in a dosage-dependent manner, suggesting proper regulation of actin turnover by NtADF1 is critical for the pollen tube growth process. In addition, NtADF1 activity is regulated by phosphorylation and pH. By creating mutants on the serine 6 residue on NtADF1, the result showed that the charge characteristics on serine 6 is important for NtADF1 interaction with actin and for its activity on pollen tube growth. By an in vitro depolymerization assay, recombinant NtADF1 depolymerizes actin more efficiently at pH 8 than pH6. This and localization of a NtADF1-rich actin mesh in the sub-apical region of elongating pollen tube, which is known to have a more alkaline cytoplasmic condition relative to the apex, suggest that interaction between NtADF1 and actin in this vicinity maybe critical for the pollen tube growth process. Finally, to examine a signaling pathway from Rac to ADF, I showed that overexpression of NtADF1 suppressed Rac-induced isotropic pollen tube growth. These observations demonstrating biologically that pollen ADFs mediate signaling activated by Rac-like GTPase to the actin cytoskeleton in pollen tube growth.* *This dissertation is a compound document (contains both a paper copy and a CD as part of the dissertation). The CD requires the following system requirements: QuickTime.

The Testis Specific Serine Kinase (Tssk) family of proteins is a large group of kinases that present high level of conservation within paralogs, as well as within species. In addition, in all reported cases as well as in the analysis of expressed sequence tags available in databases, this family of proteins presents a very strict pattern of either testicular or male-gonadal expression. This high level of conservation prompted the postulate that these kinases ought to be important for either testicular function or fertilization. In this work we attempt a biochemical characterization of one family member (Tssk6) in the mouse. We also analyze the male infertility phenotype presented by mice null for Tssk6 revealing its requirement for actin dynamics and the relocalization of proteins necessary for gamete fusion. In this analysis we described Tssk6 as the second protein known to date to be necessary in the sperm for gamete fusion to take place. We also examined a novel member of the Tssk family in the mouse as well as ortholog proteins in two invertebrates (C. elegans and D. melanogaster ). Although our understanding of the function, activity and regulation of these kinases remains small, this work constitutes a significant advance towards the understanding of the identity of the Tssk family. The results that follow have far reaching effects that surpass the realm of the Tssk family. They influence the study of sperm biological processes like the changes in sperm cytoskeletal structures and the acrosome reaction. They also influence the field of developmental biology and scientist working in the molecular characterization of the process of gamete fusion and zygote formation. Lastly, the work here presented influences as well evolutionary developmental biology through the study of a highly conserved family of proteins that is essential for reproduction and could play a role in the process of speciation.

Bibliography: leaves 60-72. / The stress response protein Hsp 12 is induced in S. cerevisiae cells upon exposure to salt stress, heat shock, ethanol, and upon entry to stationary phase (Mtwisha et aI., 1998). In this study, the occurrence of proteins related to Hsp12 was investigated in a number of yeasts (namely, Saccharomyces cerevisiae S288C, Schizosaccharomyces pombe, Debaromyces hansenii, Lipomyces starkeyi Y-2024, Saccharomyces cerevisiae IFO 23X7 (Kaokai), Zygosaccharomyces rouxii and Pichia sorbitophila. This was performed by selective protein extraction followed by SDS-P AGE and western blotting using a S. cerevisiae anti-Hsp 12 antibody. The results showed that almost all the yeasts investigated possessed a protein that had an identical migration to that of Hsp 12 with the exception of S. pombe, which contained a 9 kDa protein. Western blotting using the antiHsp 12 antibody cross-reacted only with the two S. cerevisiae species in addition to the 12 kDa protein from Lipomyces starkeyi of all the species investigated. MALDI-TOF peptide mass analysis after tryptic digestion of the L. starkeyi 12 kDa protein showed that a close sequence similarity existed to that of S. cerevisiae Hsp 12 and none to rest of the 12 kDa proteins isolated from all the other species investigated. In order to determine the sequence of the Hsp 12 protein, the L. starkeyi Hsp 12 gene was amplified using S. cerevisiae Hsp 12 primers. Gene sequencing of both S. cerevisiae and L. starkeyi Hsp 12 genes revealed three nucleotide differences existed between them. L. starkeyi Hsp 12 was found to be present in relatively small amounts during early growth stages but increased during log phase with a slight further increase during stationary phase. Increasing the salt concentration in the growth medium was found to induce Hsp 12. Increased levels of Hsp 12 appeared to confer a degree of protection during desiccation and subsequent rehydration of both L. starkeyi and S. cerevisiae.

No description available.

Microtubule-disrupting agents have been studied for decades for their potential anticancer activity and resulted in discovery of an endogenous 17β-estradiol derivative, 2-methoxyestradiol (2ME2). Since 2ME2 possesses low bioavailability, several analogues with improved efficacy was in silico-designed to target tumourigenic cells. This study investigated the influence of an 17β-estradiol analogue, (8R, 13S, 14S, 17S)-2-ethyl-13-methyl-7, 8, 9, 11, 12,13, 14, 15, 16, 17-decahydro-6H-cyclopenta[a]phenanthrane-3, 17-diyl bis(sulphamate) (EMBS) on cell growth, cytotoxicity, metabolism, morphology, cell cycle progression, reactive oxygen species generation and induction of cell death via apoptosis in two adenocarcinoma cell lines (MCF-7 and MDA-MB-231) and the non-tumourigenic epithelial breast cell line (MCF-12A). Crystal violet staining and the real-time xCELLigence approach indicated statistically significant antiproliferative activity in an estrogen-independent manner (0.4 μM; 24 h) in all three cell lines. Influence on morphological demonstrated several apoptotic hallmarks including compromised cell density, apoptotic bodies, shrunken cells, hypercondensed chromatin and several cells trapped in metaphase culminating in apoptosis. Cell cycle progression studies revealed apoptosis induction and cells blocked in the G2M phase. Apoptosis induction was verified by means of Annexin V-FITC.EMBS-treated cells demonstrated a reduced mitochondrial membrane potential. Furthermore, autophagy characteristics were observed including vacuoles and autophagosomes. Mitotic indices demonstrated an increase in cells possessing abnormal morphology associated with apoptosis and the number of cells trapped in metaphase culminating in apoptosis. This was confirmed by cell cycle progression studies that revealed apoptosis induction and a G2M block. Apoptosis induction was verified by means of Annexin V-FITC and additional flow cytometry studies indicated EMBS-treated cells demonstrated a reduced mitochondrial membrane potential. Fluorescent microscopy exhibited increased lysosomal staining suggesting autophagy induction which was verified by conducting flow cytometry employing LC3B conjugated to DyLight 488. Flow cytometry studies also demonstrated that EMBS exposure resulted in statistically significant increased hydrogen peroxide and superoxide production. EMBS exposure resulted in a statistically significant increase in p53 protein expression, decreased Bcl-2 expression and a decrease in pBcl-2(s70) phosphorylation supporting the notion that EMBS utilises crosstalk pathways to induce both autophagy and apoptosis. These results were observed in all three cell lines with caspase 6 and 8 activation being more prominent in the tumourigenic cell lines and cell growth recovering after 24 h exposure in the non-tumourigenic MCF-12A cell line. Further research will focus on the molecular signal transduction utilized by EMBS and an in-depth analysis of specific anticancer targets identified in vitro and subsequent in vivo investigation. Thus this study contributes to the discovery of targets for cancer therapies that will aid in the design of microtubule disrupting agents. / Thesis(PhD)--University of Pretoria, 2014. / Physiology / PhD / unrestricted