Philosophiae Doctor - PhD (Biochemistry)
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Item Investigation of socio-demographic, clinical and genetic factors associated with blood pressure and glycaemic control among indigenous South African adult patients(University of the Western Cape, 2021) Masilela, Charity Mandisa; Benjeddou, MongiAchieving blood pressure and glycaemic treatment targets remain a major public health challenge in individuals with hypertension and diabetes mellitus (DM). This research project was, therefore, designed to investigate the socio-demographic, clinical and genetic factors associated with blood pressure and glycaemic control among indigenous South African adult patients. The main aims of the project were as follows: (1) To assess the prevalence and socio-demographic factors associated with uncontrolled hypertension, in individuals receiving chronic care in primary healthcare facilities, based in the rural areas of Mkhondo Municipality (Study 1). (2) To investigate the association of nineteen single nucleotide polymorphisms (SNPs) with blood pressure control among adult patients treated with hydrochlorothiazide (Study 2). (3) To assess the level of association between twelve SNPs with uncontrolled blood pressure for adult patients treated with amlodipine (Study 3). (4) To examine the association of five SNPs in selected genes (ABO, VEGFA, BDKRB2, NOS3 and ADRB2) with blood pressure response to enalapril treatment, and further assess interaction patterns that influence blood pressure response (Study 4). (5) To determine the prevalence of poor glycaemic control and its influencing factors among adult patients from Mkhondo Municipality attending chronic care for DM (Study 5). (6) To evaluate the level of association between polymorphisms found in the SLC22A1, SP1, PRPF31, NBEA, SCNN1B, CPA6 and CAPN10 genes, and glycaemic response to metformin and Sulphonylureas (SU) combination therapy among South African adults with DM. Also, to investigate interaction patterns that influence glycaemic control in response to metformin and SU combination therapy (Study 6).Item Investigation of the intra-cellular localisation of Retinoblastoma Binding Protein 6 using immunofluorescence microscopy(University of the Western Cape, 2017) Szmyd-Potapczuk, Anna Victoria; Pugh, David JRHuman Retinoblastoma Binding Protein 6 (RBBP6) is a 200 kDa protein that has been implicated in a number of crucial cellular processes. It forms part of the mRNA 3'-end processing complex in both humans and yeast, and it contains an RS-like domain and interacts with core splicing proteins, suggesting multiple roles in mRNA processing. Through its RING finger domain it has been implicated in catalysing ubiquitination of the tumour suppressor p53, the oncogene Y-Box Binding Protein 1 (YB-1) and the DNA replication-associated protein zBTB38. It is one of only a few proteins known to bind to both p53 and pRb. At the N-terminus of the protein is the DWNN domain, an ubiquitin-like domain which is found only in this protein family. Four protein isoforms of RBBP6 have been identified in humans, all of which contain the DWNN domain: isoform 1 contains 1972 residues, isoform 2 contains 1758 residues and isoform 4 contains 952 residues. Isoform 3, which contains the first 101 residues of the full length protein (isoform 1), including the DWNN domain, followed by an unique 17-amino acid tail, is reported to be expressed independently of the other isoforms and to be down-regulated in a number of cancers.Item Metagenomic analysis and characterization of microbial diversity from hydrothermal samples of El Tatio geyser field, Chile(University of the Western Cape, 2012) Galada, Ncebakazi; Cowan, D. A.The El Tatio geyser field (ETGF) is the largest known geothermal field in Chile, forming part of a wide spectrum of extremophilic habitats in the country. The ETGF is NaCl rich, with high concentrations of toxic elements such as Li, As and Cs, which are contributed mainly by volcanic activities in the region. Most previous studies in the area have focused on the geology and geochemistry for mining purposes, as well as on the search for geothermal resources for power generation. Very little is currently known about the composition of the microbial communities of the ETGF, which makes the study reported here of particular novelty.A metagenomic approach, involving the amplification of 16S rRNA gene phylogenetic markers from metagenomic DNA was used to investigate seven different sites within the geyser field. The sample sites were characterized by high temperatures (80-85 °C) and a range of pH values (6.3-8). Various molecular methods, including clone library construction and PCR-DGGE analyses were used to target a wide range of microbial populations within the ETGF sites. Multivariate analysis was also applied to assess differences in the microbial diversity from different sites and to correlate microbial diversity with environmental conditions. Culture-dependent screening of novel nanoarchaeal species was also undertaken.These were coupled with PCR and other detection methods such as fluorescent in situ hybridization (FISH) to trace the presence of nanoarchaeal signals from enriched cultures.The results have shown that the ETGF encompasses a limited microbial diversity represented by only 30 dominant phylotypes, and most likely due to the toxic chemical content of the geyser field. The microbial representatives identified were assigned to OTUs from archaeal,nanoarchaeal and bacterial taxonomic groups. The dominant microbial taxa included members of the Proteobacteria, Firmicutes, Aquificae, Actinobacteria, Euryarchaeota(Halobacteriales, Archaeoglobales), Crenarchaeota (Thermoproteales, Desulfurococcales),together with uncultured representatives of the bacteria, archaea and nanoarchaeota. Notably,representatives of mesophilic, thermophilic and hyperthermophilic taxonomic groups were all detected in ETGF samples. This is attributed to various factors such as temperature gradients and dispersal mechanisms (e.g. natural forces such as rain and volcanic activities). Principal component analysis (PCA) showed significant differences (P < 0.05) in the microbial diversity of the ETGF samples, with principal components (based on the sequenced species from both 16S rRNA clone libraries and PCR-DGGE profiles) explaining up to 62.7% of variance. Furthermore, CCA showed that the differences in phylogenetic diversity were most influenced by temperature and salinity. This was also confirmed by the sequencing results,which showed that hyperthermophilic and haloarchaeal taxa were dominant in the ETGF sites. However, conductivity and pH were also found to contribute to variations in the microbial diversity of the experimental samples, with TDS (total dissolved solids) being a less influential factor. Attempts to generate nanoarchaeal-host co-cultures, and to recover sufficient nanoarchaeal genomic DNA for fosmid and/or large insert cloning for comparative genome analysis, were unsuccessful.This study is the first to employ metagenomic approaches to analyse the microbial diversity of sites in the ETGF, and has expanded our knowledge of microbiota present in this geyser field.Item Novel genomic approaches for the identification of virulence genes and drug targets in pathogenic bacteria(University of the Western Cape, 2001) Gamieldien, Junaid; Hide, Winston; Faculty of ScienceWhile the many completely sequenced genomes of bacterial pathogens contain all the determinants of the host-pathogen interaction, and also every possible drug target and recombinant vaccine candidate, computational tools for selecting suitable candidates for further experimental analyses are limited to date. The overall objective of my PhD project was to attempt to design reusable systems that employ the two most important features of bacterial evolution, horizontal gene transfer and adaptive mutation, for the identification of potentially novel virulence-associated factors and possible drug targets. In this dissertation, I report the development of two novel technologies that uncover novel virulence-associated factors and mechanisms employed by bacterial pathogens to effectively inhabit the host niche. More importantly, I illustrate that these technologies may present a reliable starting point for the development of screens for novel drug targets and vaccine candidates, significantly reducing the time for the development of novel therapeutic strategies. Our initial analyses of proteins predicted from the preliminary genomic sequences released by the Sanger Center indicated that a significant number appeared to be more similar to eukaryotic proteins than to their bacterial orthologs. In order determine whether acquisition of genetic material from eukaryotes has played a role in the evolution of pathogenic bacteria, we developed a system that detects genes in a bacterial genome that have been acquired by interkingdom horizontal gene transfer.. Initially, 19 eukaryotic genes were identified in the genome of Mycobacterium tuberculosis of which 2 were later found in the genome of Pseudomonas aeruginosa, along with two novel eukaryotic genes.Surprisingly, six of the M. tuberculosis genes and all four eukaryotic genes in P. aeruginosa may be involved in modulating the host immune response through altering the steroid balance and the production of pro-inflammatory lipids. We also compared the genome of the H37Rv M. tuberculosis strain to that of the CDC- 1551 strain that was sequenced by TIGR and found that the organisms were virtually identical with respect to their gene content, and hypothesized that the differences in virulence may be due to evolved differences in shared genes, rather than the absence/presence of unique genes. Using this observation as rationale, we developed a system that compares the orthologous gene complements of two strains of a bacterial species and mines for genes that have undergone adaptive evolution as a means to identify possibly novel virulence –associated genes. By applying this system to the genome sequences of two strains of Helicobacter pylori and Neisseria meningitidis, we identified 41 and 44 genes that are under positive selection in these organisms, respectively. As approximately 50% of the genes encode known or potential virulence factors, the remaining genes may also be implicated in virulence or pathoadaptation. Furthermore, 21 H. pylori genes, none of which are classic virulence factors or associated with a pathogenicity island, were tested for a role in colonization by gene knockout experiments. Of these, 61% were found to be either essential, or involved in effective stomach colonization in a mouse infection model. A significant amount of strong circumstantial and empirical evidence is thus presented that finding genes under positive selection is a reliable method of identifying novel virulence-associated genes and promising leads for drug targets.Item Physical and genetical investigation of the Xp11.3 region on the short arm of the human X-chromosome(University of the Western Cape, 2004) Wittwer, Pia Ethena; Gehring, Chris; Dept. of Biochemistry; Faculty of ScienceThe pattern of inactivation in the DXS8237E-UBE1-PCTK1 region is of particular interest, since the mechanisms of X chromosome inactivation and the escape from inactivation are, as yet, not fully understood. The inactivation status of the DXS8237E and PCTKl gene differ: the first undergoes normal inactivation and the second escapes this process. The status of the UBEl gene has been controversial, although it is widely excepted that it does escape X chromosome inactivation. Physical mapping of the region employing YACs and subsequently P ACs has been undertaken, but was restricted in scope by the high frequency of rearrangements occurring. DNA sequences between DXS8237E, UBE1, PCTKl and the distal gene, UHX1, have been investigated with regard to LINEI elements, which are thought to playa role in X-inactivation. The results obtained strongly suggest a link between LINE1 elements and X chromosome inactivation. Sequence analysis results also contributed to the understanding of difficulties with restriction mapping of the region. Further, this work includes the first reported establishment of the UBEl exonintron boundaries. Additionally, genomic sequence analysis showed that only 46kb separate DXS8237E from UHX1, which confirms that this region is extremely gene rich.