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Fishes of the perciform suborder Notothenioidei are found in Antarctic and sub-Antarctic waters that are separated by the Antarctic Polar Front (APF), with some species being distributed on both sides of this front. In this wide latitudinal range, these fishes are exposed to different temperatures ranging from -2 °C in the High Antarctic regions to 12 °C in the sub-Antarctic regions. To survive in icy Antarctic waters, the Antarctic notothenioid species have evolved antifreeze glycoproteins (AFGPs) that prevent their body fluids from freezing. The findings of past research on the AFGP attributes of several notothenioid species inhabiting ice-free sub-Antarctic environments have presented a complex picture. Furthermore, previous taxonomic studies split widely distributed notothenioids into different species and/or subspecies, with other studies disagreeing with these splits. To understand the response of the sub-Antarctic notothenioids to warmer, ice-free environments, it is necessary to have a good understanding of their antifreeze biology and systematics. Therefore, this study aimed to determine the association, if any, between the antifreeze attributes of sub-Antarctic notothenioid fishes and their taxonomic status. And more...

Mycobacterium tuberculosis (M. tuberculosis) , the etiological agent of tuberculosis, is an intracellular bacterium which persists within macrophages. Successful control of tuberculosis depends on T-cell-mediated immunity. Immune protection involves the development of a Th1 response characterised by the secretion of cytokines such as IL-12, IFN-γ and TNF-α. The progression towards disease in humans and mice is often associated with a Th2 response characterised by the secretion of cytokines such as I L-4 and I L-10. Mycolic acids, the major cell wall lipid of M. tuberculosis, were previously shown to have a marginally protective effect on the development of disease in Balb/c mice when administered intravenously at an optimal dose of 25 µg one week before intravenous M. tuberculosis infection. Here it is shown that the protective effect is highly significant when infection is done intranasally. The protective effect of 25 µg mycolic acids against tuberculosis could not be explained by induction of a longer lasting Th1 response in Balb/c mice. This was determined by using semi-quantitative RT-PCR on the mRNA of cytokines characteristic of the different immune responses. It was observed that maximum sensitivity was obtained at the lowest possible PCR cycle and template concentrations for the samples. Mycolic acids were the first non-protein antigens shown to induce an immune response after presentation on CD1 membrane proteins. Balb/c mice predominantly generate a Th1 response during the first 3 - 4 weeks of M. tuberculosis infection, whereas they generate a Th2 response in the following weeks. Even though the protective effect of 25 µg mycolic acids could not be associated with a prolonged Th1 immune response in infected mice, it did induce IL-12 and IL-10 mRNA in uninfected mice. These cytokines are primarily. / Dissertation (MSc (Biochemistry))--University of Pretoria, 2005. / Biochemistry / unrestricted

Phylogeographic mitochondrial DNA (mtDNA) population structure was determined for Aethomys namaquensis and A. ineptus from southern Africa and Swaziland. It was evident from the study that A. namaquensis reflected a pattern of phylogenetic discontinuity with and without spatial separation between populations. Previously documented mtDNA phylogeographic patterns recorded in the rock hyrax, Procavia capensis and the red rock rabbit species, Pronolagus rupestris and P. randensis, coincided with the phylogeographic break that was detected in one of the mtDNA lineages (C) within A. namaquensis. Similar vicariant events may have been responsible for shaping evolutionary processes in the independent Procavia, Pronolagus and Aethomys lineages. In contrast, A. ineptus showed a pattern of shallow phylogeographic structuring. The marked genetic differences detected in A. namaquensis and A. ineptus may reflect the influences of habitat specificity, its fragmentation and the effects of life history on mtDNA gene flow. The study also revealed three genetically well-supported lineages within A. namaquensis: a lineage (A) found in the Limpopo valley, a lineage (B) widely distributed across the Karoo and a lineage (C) found across the grasslands of the North-West, Limpopo and Mpumalanga Provinces. These spatial distributions broadly coincided with the ranges of four previously proposed morphologically defined subspecies. From the present sample sizes, there is only good support, from a molecular point of view, for the subspecies A. n. lehocla (lineage B). In addition to the three well-supported lineages, six geographically restricted lineages were identified that could not be assigned to any of the four previously proposed subspecies, A. n. namaquensis, A. n. monticularis, A. n. alborarius and A. n. lehocla. Molecular techniques, specifically the analysis of the mtDNA cytochrome b gene, have been useful in the identification of sibling species. This technique has also proved to be useful in the identification of two cryptic species, A. chrysophilus and A. ineptus in this study. Phylogenetic analysis revealed two maternal groups corresponding to A. chrysophilus and A. ineptus. Distributional data of these two species, suggest that A. chrysophilus occupies the low elevations of the Limpopo River drainage, while A. ineptus occupies the remainder of South Africa at higher elevations, but expands into lower elevations in the southern portion of its range. Phylogenetic relationships among four southern African species of Aethomys suggest the presence of two clades that included: 1) A. chrysophilus and A. ineptus and 2) A. namaquensis and A. granti. This study, however, revealed that Aethomys may be paraphyletic, suggesting that the allocation of A. namaquensis and A. granti to the subgenus Micaelamys needs to be investigated further. / Dissertation (MSc (Genetics))--University of Pretoria, 2006. / Genetics / unrestricted

The experiments presented were designed to test the hypothesis that the well-known carcinogen, benzo[a]pyrene has epigenetic effects, specifically the ability to alter cytosine methylation patterns. MCF-7 and MDA-MB-231 human breast cancer cells were treated for a period of sixty days with 100 nM benzo[a]pyrene. The methylation status of two genes, Ecadherin and GSTP 1 were examined using methyl-specific PCR and Southern blot analysis. After sixty days, no detectable change in methylation was observed. Evidence exists that de novo methylation is a consequence of transcriptional inactivity. Benzo[a]pyrene can contribute to transcriptional repression by sequestering the transcription factor, Spl. To test this hypothesis in our system, MCF-7 cells were transiently transfected with a reporter construct containing Sp 1 sites. These experiments demonstrated an 8.4 fold increase in reporter gene activity over a promoterless control plasmid; however, a difference could not be established between benzo[a]pyrene-treated and untreated cells.

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The cellular slime mold, <i><b>Dictyostelium discoideum</i></b>, has two developmentally regulated forms of the enzyme glycogen phosphorylase, which are encoded by two distinct, but related genes (Rutherford, et. aI., 1991). A complementary DNA (cDNA) encoding glycogen phosphorylase-}, gp-l, was isolated from a λgtll expression library made from amoebae stage mRNA. The 5' upstream region of the gp-l gene was cloned by inverted polymerase chain reaction (IPCR) and partial genomic DNA library screening. The gp-l gene was found as one copy or low copy number gene in the <i><b>Dictyostelium</i></b> genome, and an adjacent 22 kilobase pair region was physically mapped. The deduced amino acid sequencing analysis revealed that there were 862 amino acid residues encoded by the gp-1 mRNA of 2729 nucleotides. It was also found that most regulatory and catalytic domains were similar to those in other glycogen phosphorylases. One intron of 139 bp was verified beginning after the 40th amino acid codon. The transcriptional start site was determined at 134 nucleotides upstream of the ATG initiation codon. Gel retardation assays demonstrated that there were at least two nuclear DNA binding proteins from vegetative amoebae (V 1 and V2 factors) and two from developing cells (D 1 and D2 factors). Experiments with a luciferase reporter gene suggested that a basal expression of the gp-l gene can be conferred by the 5' region containing 363 bp upstream of the ATG codon and the entire regulatory region is located at 157 to 700 bp upstream of the ATG site. It was also demonstrated that the 363 bp deletion fragment did not support cyclic AMP (cAMP) responsiveness of the gp-l gene. DNase I footprinting mapped two regions that were protected by nuclear DNA binding proteins and one of them was a palindromic sequence: CAAGTCGCTIG. / Ph. D.