3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item The molecular basis of glycopeptide Resistence in two Clinical Isolates: Bacillus Lentus RSA1208 and Paenibacillus Thiaminolyticus RSA 1221(2006-10-25T08:10:34Z) Moodley, ArshneeThe molecular mechanisms of glycopeptide resistance in two Gram-positive clinical isolates, Bacillus lentus RSA1208 and Paenibacillus thiaminolyticus RSA1221 were investigated. The glycopeptide resistance genotypes were determined by PCR. If van genes were detected, recombinant DNA techniques and sequencing were used to determine the gene sequence. The location of the resistance determinant was investigated using Southern hybridization techniques. To determine the 5’ and 3’ ends flanking the resistance operon, sub-genomic libraries were constructed. Transmission electron microscopy was used to assess possible structural changes of the B. lentus RSA1208 cell wall. B. lentus RSA1208 exhibits inducible, high-level resistance to both glycopeptides, but does not possess any known van resistance genes. Electron micrographs showed a visible increase in cell wall thickness in B. lentus RSA1208 grown in vancomycin compared to the isolate grown in vancomycin-free media. However, it remains to be confirmed as to whether this resistance is solely responsible for the high-level resistance phenotype. P. thiaminolyticus RSA1221 exhibits constitutive, high-level resistance to vancomycin only. It was found to possess a chromosomally-borne, vanA gene cassette. The vanA gene showed the highest amino acid identity to the vanA-like D-ala: D-lac gene found in P. thiaminolyticus PT-2B1 and Enterococcus faecium BM4147. All five genes of the vanA gene cluster (vanR, vanS, vanH, vanX, vanY) were amplified and sequenced. No vanZ gene was detected. The vanA operon in P. thiaminolyticus RSA1221 was found not to be associated with any known mobile elements. The observed constitutive expression of resistance maybe due to a two amino acid insertion in the VanSBpt1221 protein. vItem Characterization of the novel domain with no name gene in colon cancer(2006-03-23) Rupnarain, CharleenNormal colonic epithelium bombarded by a range of molecular changes, affecting cell proliferation and apoptosis, result in the initiation of an adenoma and consequently an invasive carcinoma, which is usually lethal. One of the main characteristics of tumour progression is the loss of regulation between the cell cycle and apoptosis. Under normal circumstances, these processes are strictly controlled by a number of regulators and inhibitors. Previous studies have implicated the novel Domain With No Name gene in apoptosis. This study aimed to characterize the expression patterns and levels of the gene in colon cancer and to determine its role in apoptosis. In situ hybridisation, immunocytochemistry and quantitative PCR localised the gene and its products in cancerous and normal colon tissue. Combined with apoptosis detection studies, proliferation assays and Bcl-2 assays, the results suggest that the gene is involved in promoting apoptosis in cancerous cells i.e. the targeting of undesirable cells. Helicobacter pylorus was implicated in the progression of noninvasive colon cancer to the invasive state. From this study DWNN is proposed to be a pro-apoptotic participant in programmed cell death and classification studies such as these allow for potential manipulation of the apoptotic system to serve as a therapeutic corridor.Item Characterization of a novel cell death related gene, DWNN, in cervical cancer(2006-03-13) Ledwaba, Ramatsobane JohannaDWNN-deficient Chinese Hamster Ovary cells have been found to be resistant to staurosporine-induced apoptosis. The human DWNN gene is located on chromosome 16p21, with 18 exons and is 36 kb long. It is alternatively spliced at exon 16 and makes two major mRNA transcripts, 1.1 and 6.1 kb, encoding 13 kDa and 200 kDa proteins respectively. The purpose of the study was to elucidate the possible role of DWNN in cervical cancer and apoptosis, to establish tissue distribution and expression levels of DWNN at protein and mRNA levels in cervical cancer. In situ hybridization studies showed elevated levels of the three mRNA transcripts in cervical cancer as compared to the normal tissues. The transcripts were localized in the nuclei of invaded stroma, moderately differentiated islands of tumours, dysplastic epithelium and some infiltrating lymphocytes. Immunocytochemistry showed that DWNN proteins were highly expressed in the dysplastic epithelium, dysplastic endocervical glands, moderately and well differentiated islands of tumours and the invaded stroma. Image analysis indicated elevated expression levels in the islands of tumours. Apoptosis detection by TUNEL revealed high apoptotic levels in the invaded stroma and moderately differentiated islands of tumours and this significantly correlated with DWNN localization. Proliferation assay using Ki67 antibody was found to be indirectly directly proportional to DWNN expression. Antiapoptotic Bcl-2 expression levels were found to be inversely proportional to the expression levels of DWNN. The up-regulated levels of DWNN in cervical cancers in contrast to normal tissues suggest DWNN to be proapoptotic, as there were elevated levels of apoptosis in the same sites where there were high levels of DWNN expression and Bcl-2 was down-regulated in the same sites. DWNN expression significantly correlated with apoptotic levels and was indirectly proportional to ki67 in human cervical cancers. Real Time PCR also confirmed the up-regulation in levels of DWNN in cervical cancer. This study suggests that the DWNN gene may be involved in apoptosis. Further characterization of this gene could lead to its manipulation as a diagnostic marker and a potential therapeutic target for cancer treatment.