Tuberculosis (TB) is an endemic health-crisis, particularly in sub-Saharan Africa. The rise of multiand extensively-drug resistant Mycobacterium tuberculosis (Mtb), the causative agent of TB, has led to further developments in understanding the physiology of Mtb during infection, as well as searching for novel drug targets, in order to combat the disease. Our understanding of cells, both eukaryotic and prokaryotic, has changed substantially in the last 50 years, incorporating the role of stable and transient protein-protein interactions which govern cell function and behaviour. Although there are many in vivo and in vitro methods for studying protein-protein interactions, they suffer from the lack of ability to distinguish physiological interactions from interactions that occur which are not physiologically relevant to the cell. Structure-based methods for determining protein interactions have the benefit of screening out false positives whilst simultaneously assessing the possible biological function of the protein complex in question. This study sought to assess different high-throughput methods for capturing stable, water soluble protein complexes from M. smegmatis (Msm), a close relative of Mtb, for structural characterisation by low-resolution transmission electron microscopy (EM). The use of partial biochemical fractionation was assessed, which produced low-resolution structures of glutamine synthetase I, bacterioferritin, and Encapsulin. These structures were unambiguously identified through a combination of fitting of homologous crystal structures into the low-resolution maps, and information obtained by liquid chromatography mass spectrometry (LC-MS/MS) of bands isolated from native- and SDS-PAGE gels. Since Encapsulin is likely to participate in the Msm oxidative stress response and functions to enclose the target proteins DyP-type peroxidase (DyP) and ferritin-family protein (BrfB), optimal conditions for cryo-EM were tested for further efforts to obtain a high-resolution structure. Furthermore, hypotheses were generated for the function of Mtb and Msm Encapsulin based on the Msm Encapsulin structure obtained with the aid of a crystal structure homologue; these related to the mode of cargo binding and pore selectivity. A single-step purification method was also assessed through grid blotting on blue native (BN) PAGE using GroEL as a test protein. The hydrophobicity and charge of the EM copper grid was tested to find the optimal grid property for particle transfer. This established that particles of GroEL could be transferred from BN-PAGE onto an EM copper grid and a successful negative stain reconstruction was obtained. In summary, the pipeline from purifying protein complexes to generating hypotheses based on structure was successfully investigated in Msm, which will aid in the production of novel drug targets for Mtb as well as in the application to other organisms.

Includes bibliographical references (p. 125-140). / Tobacco smoking is a major risk factor in the development of oesophageal squamous cell carcinoma (OSCC). Benzo[a]pyrene (BaP) is a tobacco smoke procarcinogen that is metabolically activated into the carcinogenic benzo[a]pyrene diol-epoxide (BPDE) by the CYP1 family of cytochrome P450 enzymes. BaP is a ligand for the aryl hydrocarbon receptor (AhR) which activates CYP1 gene transcription. Polymorphisms in these genes affect enzyme activity and therefore BaP bioactivation.

Bibliography: leaves 163-190. / Breast cancer is the most common cause of death amongst women, with the incidence of the disease varying between countries. Like all other cancers, breast cancer is a multigenic disorder with mutations in oncogenes and tumour suppressor genes playing an important role in cellular transformation and ultimately in tumour formation. In this study, 40 breast cancer patients from the Western Cape province in South Africa and 4 breast cancer cell lines were screened for mutations in the human Ha-ras oncogene and the p53 tumour suppressor gene.

Includes bibliographical references (leaves 71-80). / This thesis aimed to better define the co-receptor conformation by determining whether mutants of the CCR5 receptor stabilized in the active conformation were less able to mediate HIV fussion. Eight mutant receptors were generated by site-directed mutagenesis and screened for constitutive activity using intracellutal signaling assays.

Bibliography: leaves 95-104.

Bibliography leaves 70-97. / Type I collagen is the most abundant collagen molecule in vertebrate connective tissue and it consists of a heterotrimer of two alpha 1 (COL1A1) and one alpha 2 (COL1A2) chains. Reduced collagen gene expression is almost always correlated with pathological conditions and cellular transformation. Numerous studies have suggested that methylation of the cytosines in CpG dinucleotides is inversely correlated with transcriptional activity and plays a critical role in differential gene expression.

Includes bibliographical references (leaves 96-112). / The role of the dominant respiratory mucins (MUC5AC and MUC5B) and MUC2 has been investigated in chronic airway diseases as it is the mucin glycoprotein that confers upon mucus its biological, rheological and physicochemical properties. Within South Africa, specifically the Western Cape, TB has wreaked havoc especially amongst those of the lower socioeconomic groups. However, despite the prevalence of the disease in South Africa and the known morbidity and mortality associated with mucus and mucin hypersecretion in respiratory diseases, little is known of the association between respiratory mucins and TB. This is a novel study that investigated the association between respiratory mucins and TB at a biochemical and molecular level.

Includes bibliographical references.

Includes bibliographical references (leaves 74-83). / Oesophageal squamous cell carcinoma (OSCC) is a major cancer in South Africa, affecting mainly black males. Several risk factors for OSCC have been reported but this study focuses on the role of human papilloma virus (HPV) in the development of OSCC. HPV is a well-known risk factor for cervical cancer resulting in its classification into low- and high-risk HPV types. The role of the different HPV types in OSCC development is not known, but in cervical cancer the critical HPV transforming gene has been shown to be E6. In this project, the effects of HPV11 E6, a low-risk type, and HPV18 E6, a high-risk type, were investigated by transfecting HPV-negative cell lines (EPC2-hTERT, MCF12A and Rat1) with HPV11 and HPV18 E6.

Includes bibliographical references.