A Dissertation Submitted to the Faculty of Science of the University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the Degree of Master of Science. / Impala are an ancient and successful specdes whose biogeography differs from other bovids. A detailed electrophoretic investigation of genetic variability within and between subpopulations found six polymorphic loci, CK-C"', GPl"', MPr\ PEP-Jr, PGM-2'" and PROT-2'" in a sample of 464 impala collected from 10 localities in southern Africa. Average gene diversity was 0,047. Between-population gene diversity was normal for bovid species. Allele frequency differences and genetic distances revealed low levels of subdivision into three broad regions. Wright's FST (0,035) revealed a significant yet low level of population subdivision. The distributions of single-locus heterozygosities and allele frequencies were significantly different to those predicted during mutation-drift equi librium, indicating that non-equilibrium conditions may prevail and that the population may be recovering from a recent bottleneck. / AC 2018

Bardet-Biedl syndrome (BBS) is an autosomal recessive disorder characterized by obesity, retinitis pigmentosa, polydactyly, mental retardation and hypogonadism. To date, twelve BBS genes have been identified, however, the precise functions of BBS genes are yet to be unraveled. To unravel the pathophysiology of BBS, we developed the zebrafish BBS model. In the zebrafish animal model system, we performed loss-of-function analysis using antisense morpholino oligonucleotides (MO). We observed strikingly overlapping phenotypes in the MO-injected embryos (morphants) including: Kupffer's vesicle (KV) disruption, a premature loss of KV cilia leading to alterations in left-right asymmetry and altered intracellular trafficking of organelles (melanosome). These phenotypes can be rescued by co-injection of in vitro synthesized wild-type RNA demonstrating the specificity of MO knockdown. In contrast to other bbs gene knockdowns, bbs1 knockdown resulted in an additional phenotype: pericardium effusion, which was rescued by co-injection with the bbs1 wild-type RNA and was significantly suppressed by the M390R mutant RNA. In addition, immunohistochemistry revealed that bbs1 wild-type protein localizes to the centrosome and cytoplasm, while bbs1 M390R mutant protein only has cytoplasmic localization. The BBS1 M390R missense mutation is the most common mutation in all BBS cases and the methionine at amino acid position 390 is evolutionarily conserved. Our data suggest that the BBS1 M390R allele is a hypomorphic allele and that the residual function of the mutant protein is necessary for embryonic development. Furthermore, we demonstrated the utility of the zebrafish BBS model in BBS candidate gene identification. Data from linkage analysis, expression profile correlation and mutation screen, suggested that TRIM32 is a BBS gene. Therefore, we identified the zebrafish trim32 gene and performed loss-of-function analysis. MO knockdown of zebrafish trim32 gene expression phenocopied the prototypic zebrafish BBS phenotypes: KV formation defect and a delay in intracellular transport. In addition, these phenotypes were rescued by co-injection of wild-type trim32 and LGMD2H mutant (D487N) RNA, but the RNA with the BBS-associated mutation (P130S) failed to rescue these phenotypes. The functional evidence, combined with data from linkage analysis, expression correlation and mutation screening demonstrate that TRIM32 is a BBS gene (BBS11).

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (p. 80-86).

Thesis (Ph. D.)--Ohio State University, 2003. / Title from first page of PDF file. Document formatted into pages; contains xii, 123 p.; also includes graphics. Includes abstract and vita. Advisor: Shili Lin, Dept. of Statistics. Includes bibliographical references (p. 119-123).

Thesis (Ph. D.)--York University, 2000. Graduate Programme in History. / Typescript. Includes bibliographical references. Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://wwwlib.umi.com/cr/yorku/fullcit?pNQ56247.

Thesis (Ph. D.)--York University, 1999. Graduate Programme in Sociology. / Typescript. Includes bibliographical references (leaves 316-336). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://wwwlib.umi.com/cr/yorku/fullcit?pNQ43440.

published_or_final_version / Microbiology / Master / Master of Medical Sciences

A sorghum gene SbLRR2, which is predicted to encode a simple extracellular leucine-rich repeat (LRR) protein, was previously isolated among a collection of fungal pathogen-induced sorghum cDNA clones generated by suppression subtractive hybridization. Phylogenetic analysis revealed that they are distinct from the simple extracellular LRR proteins reported previously. Subcellular localization analysis demonstrated that the SbLRR2-EYFP fusion protein was targeted to the extracellular space in tobacco leaf cells. Peptide N-Glycosidase F treatment revealed that the SbLRR2 is N-glycosylated with non-fucosylated oligosaccharides when transiently expressed in Nicotiana benthamiana leaves. Functional analysis was performed in SbLRR2 over-expression (OE) Arabidopsis plants which showed enhanced resistance against the necrotrophic pathogens Botrytis cinerea and Alternaria brassicicola. In addition, the OE lines were found to have elevated expression of several jasmonate acid (JA)-associated genes and higher endogenous JA contents. Hence, the SbLRR2-mediated defense responses in transgenic Arabidopsis are likely to be dependent on JA-signaling through increased JA production. On the other hand, the OE lines remained susceptible to Pseudomonas syringae pv. tomato as the wild type plants. Consistently, there was no up-regulation of salicylic acid (SA) defense marker gene expression or SA levels in the OE lines. Using yeast two-hybrid analysis, SbLRR2 was further shown to interact with Arabidopsis hypersensitive-induced response protein 1. Such interaction may suppress hypersensitive response which is known to enhance necrotrophic pathogen invasion. These data suggested a positive regulatory role of SbLRR2 in plant defense. Further phenotypic analysis of transgenic SbLRR2 revealed its novel role in enhancing lead [Pb(II)] tolerance in Arabidopsis. OE-lines were showed to alleviate Pb(II)-induced root inhibition, reduce the accumulation of Pb(II), and enhance transcription of AtPDR12 which was previously shown to function as a potential Pb(II) efflux pump contributing to Pb(II) detoxification. However, all the Pb(II) tolerance responses were abolished when SbLRR2 was transformed into the atpdr12 mutant. Meanwhile, the extracellular localization of SbLRR2 was shown to be essential for the enhanced Pb(II) tolerance in transgenic Arabidopsis. Together, these results indicated that SbLRR2-mediated Pb(II) tolerance was dependent on AtPDR12 via Pb(II) extrusion. Further investigations revealed the Pb(II)-induced transcriptional activation of SbLRR2 and several highly conserved AtPDR12 homologs in sorghum seedlings, suggesting the possibilities of a common molecular mechanism for Pb(II) tolerance in diverse plant species. Finally, an iTRAQ-based LC-MS/MS quantitative proteomics approach was used to investigate of lead responses in Arabidopsis. A total of 114 proteins showed significant changes in protein abundance with 58 up-regulated and 56 down-regulated proteins. Analysis of changes in the protein profile revealed that the photosynthesis, photorespiration and protein biosynthesis in Arabidopsis were inhibited under lead toxicity. On the other hand, abundances of proteins involved in the antioxidant system, glucosinolate-myrosinase system and JA biosynthesis pathway were elevated upon Pb(II) treatment. Further investigation revealed that Pb(II) stress induced a rapid increase of JA contents in Arabidopsis whereas a JA biosynthesis deficient mutant (AOS) showed hypersensitivity to Pb(II) toxicity, strongly implicating a significant role of JA in Pb(II) response. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

The protein phosphatase inhibitor okadaic acid (OA) previously has been shown to induce hyperphosphorylation of p53 protein both grossly and at specific tryptic peptide sites. However, the consequences of OA induced phosphorylation (and phosphorylation in general) on p53 function in vivo remain unclear. The focus of this study was to determine if hyperphosphorylation wrought by OA or expression of human p53 in protein phosphatase-deficient yeast strains could indeed regulate the interaction between p53 and a physiological downstream target, the cdk inhibitor, p21waf1. In S. pombe, one strain containing a mutant p53 (arg->his 175) and a type 1 protein phosphatase gene knockout was unable to grow whereas both parental strains were both able to thrive, indicating a possible gain of function related to p53 phosphorylation. Rat embryonic fibroblasts harboring a highly expressed mouse p53 transgene and a p53 null control cell line were treated with 50nM doses of OA. This treatment resulted in: (1) the formation and retention of acidic p53 protein isoforms, and, more specifically, phosphorylation of tryptic peptide sites in the transactivation domain, (2) an increase in p53 affinity for a p21waf1 promotor oligonucleotide, (3) an increase in cellular steady state levels of p21waf1 message, (4) an increase in p53-dependent transcriptional activity from a waf1 reporter construct, and (5) a G2/M cell cycle blockage that is associated with intact p53. These results demonstrate for the first time that hyperphosphorylation of p53 induced by OA may regulate a critical downstream affector of cell growth suppression in an intact cellular environment.