Includes abstract. / Includes bibliographical references. / Chemotherapeutic agents such as cisplatin, doxorubicin and 5-fluorouracil are currently used in the treatment of a variety of cancers, including oesophageal cancer. Although these agents have been part of our therapeutic repertoire for many years, there are several side effects that have been associated with their use in cancer therapy. Thus there is a need to develop novel chemotherapeutic agents with improved activity and less severe side effects. In this project, twelve compounds, consisting of 5 platinum dichloride complexes, four iminophosphine ligands and three gold (I) chloride complexes that were synthesised by a PhD student in our laboratory were tested for anticancer activity and the mode of action of these compounds was also explored.

Bibliography: leaves 171-191.

Includes abstract. / Includes bibliographical references (leaves 72-78). / The activation of Gonadotropin-releasing hormone receptor (GnRHR) by the GnRH ligand has been shown to mediate antiproliferative effects in extra-pituitary cells and in reproductive cancer cell lines. The GnRHR couples to Gαq in pituitary gonadotropes. However, the GnRHR expressed in reproductive cancer cell lines is thought to couple to Gαi. Recent evidence also suggests that the antiproliferative effects may be mediated via Gαq in these cells. Therefore our study involved determining the role of Gαi in the antiproliferative effects mediated by the GnRHR. The results suggest that the Gαi pathway could play a role in mediating the antiproliferative effects of GnRH.

Includes bibliographical references. / Cytochrome P450 (CYPs) are a superfamily of heme containing enzymes that catalyse a diverse range of biological reactions. They are responsible for over 80% of primary metabolism of currently available drugs and are therefore central to its medical importance. Investigating the effects of these enzymes on drugs by metabolite detection and kinetic studies is a step forward to the vision of personalised medicine. The enzyme family is known to be associated with the development of adverse drug reactions which are usually only discovered in late stages of drug development, therefore screening for potential adverse drug reactions earlier on would aid minimising such adverse events occurring. There is therefore a need to analyse the interaction profile of new drugs with CYPs in a cost effective and high throughput manner for early stage screening, since drug discovery efforts tend to utilise large compound libraries. Recently, a novel functional CYP microarray has been developed in the Blackburn laboratory at UCT to enable label-dependent analysis of metabolism of substrates by the major CYP3A4 isoform in a high throughput manner. This thesis describes efforts involved in expanding the functional CYP microarray format to the other major CYP isoforms namely, CYP2C9 and CYP2D6 and developing a new immobilisation-free technology with label-free mass spectrometric identification and quantitation of metabolites formed. The goals of expansion of functional CYP microarrays were achieved by using microarray or confocal fluorescence scanning in conjunction with atomic force microscopy to more accurately quantitate active CYP3A4, CYP2C9 and CYP2D6 protein levels for catalytic substrate-dependent turnover rates. Finally the label- and immobilisation-free CYP technology was evaluated using probe substrates and a complex drug, rifampicin. These two platforms are primed to be a useful tool in pre-clinical drug screening for use in the drug discovery field by the academic, pharmaceutical and biotechnology industries.

Infection by oncogenic human papillomavirus (HPV) is known to be the causative agent for the development of various anogenital cancers, including cervical cancer. Worldwide, the majority of cervical cancer cases occur in less developed regions, and while prophylactic vaccines exist to combat HPV infection, they are largely unattainable in these areas. Therefore, alternative preventative measures against HPV infection are needed to help eradicate cervical cancer over time. Since HPV employs multiple mechanisms to evade the host immune response, a proposed method for preventing infection may be by enhancing HPV recognition by the immune system. Surfactant proteins A and D (SP-A and SP-D) are innate immune proteins with a variety of functions including recognition and opsonisation of pathogens. They are primarily found in the lung, but have also been shown to be expressed at other sites of the body, including the female reproductive tract. It was hypothesised that SP-A and/or SP-D may enhance immune recognition of HPV, thereby preventing infection. To assess this hypothesis, co-immunoprecipitation and flow cytometry experiments were performed to determine whether SP-A and/or SP-D bind to HPV16 pseudovirions (HPV16- PsVs). SP-A was shown to bind to HPV16-PsVs as well as enhance viral uptake by RAW264.7 murine macrophages, while SP-D bound HPV16-PsVs weakly and had no effect on viral uptake. To confirm these observations and to assess whether SP-A had an effect on HPV16- PsVs infection in vivo, a well-established, but not yet available murine HPV16-PsVs cervicovaginal challenge model system was set up at UCT. It was determined that neither naïve nor C57BL/6 mice challenged with HPV16-PsVs expressed SP-A in the female genital tract. However, under the experimental conditions established herein, pre-incubation of HPV16-PsVs with purified SP-A at a 1:10 weight per weight ratio resulted in a reduction in infection. This study is the first to describe a biochemical and functional association of HPV16 virions with the innate immune molecule SP-A. In the long term, these observations may contribute to the development of topical microbicides incorporating recombinant fragments of SP-A to reduce the burden of new HPV infections.

Bibliography: leaves 88-104. / The current study was perfonned to give insights into the role of individual amino acid residues of both the GnRH receptor and the GnRH ligand in receptor function. A His7.36(3o5) residue on transmembrane helix seven of the GnRH receptor was investigated for its contribution to the overall function of the receptor.

The ADAMs (A Disintegrin And Metalloproteinase) is a family of transmembrane and secreted proteins essential in cellular fate determination, wound healing, cell migration, proliferation and angiogenesis. Previous studies have linked a range of ADAMs, which include ADAM10 to cancer development and progression. Research in our laboratory found endogenous ADAM10 levels to be higher in both oesophageal and cervical cancer cell lines. Reports in the literature have highlighted a correlation between high levels of ADAM10 expression with that of cancer cell biology; hence ADAM10 shows promise as an anti-cancer target. The aim of this study was to modulate ADAM10 activity in oesophageal and cervical cancer cell lines using the small molecule inhibitor GI254023X as well as previously undescribed two molecules generated en route to synthesizing GI254023X, namely SN-254 and SN-311. A CX₃CL1 ELISA functional assay as an indicator of ADAM10 activity showed a decrease in CX₃CL1 cleavage after treatment with GI254023X, SN-311 and SN-254 suggesting that all three compounds substantially inhibited ADAM10 activity. The effects of these compounds on the cell biology of WHCO5 oesophageal and HeLa cervical cancer cells were monitored. Our data shows that GI254023X, SN-254 and SN-311 inhibit oesophageal and cervical cancer cell proliferation, and cause cell death via apoptosis as observed by PARP cleavage, and elevated Caspase 3/7 activity. Drug treatment also resulted in an increase in cellular adhesion as well as a significant decrease in the invasion and migration of WHC05 and HeLa cells. The effect of ADAM10 inhibition on typical markers of the epithelial to mesenchymal transition state was also examined. An increase in epithelial cell markers (E-Cadherin, B-Catenin) and a decrease in mesenchymal marker expression (Vimentin) post treatment with the compounds tested strongly suggested that ADAM10 plays a role in mesenchymal cell transition. These results suggest that ADAM10 activity is necessary for the biological phenotypes that associate with cervical and oesophageal cancer cells and that targeting ADAM10 with inhibitors have potential as anticancer therapies.

Includes bibliographical references / Background: South Africa has the fourth highest prevalence of Human Immunodeficiency Virus (HIV) in the world, with over 340 000 new infections in 2013 alone. Implementation of Highly Active Antiretroviral Treatment (HAART) regimens, has improved the lives of many HIV-infected individuals, and has led to a decrease in the incidence of extreme forms of HIV-associated dementia. However, the extended lifespan afforded by HAART has also resulted in an increase in the prevalence of milder forms of HIV-associated Neurocognitive Disorders (HAND). Clinical diagnosis of such cognitive decline currently relies on neurocognitive assessments, as no definitive biochemical test exists. A point-of-care test designed for use with easily accessible samples (e.g. blood) would be of great utility in both HAND research and clinical diagnosis, prognostication, and provision of timeous therapy. A blood-based biomarker test would also be less subjective than neurocognitive testing, which can be adversely affected by the patient's level of education as well as operator competency. Recently, Professor Simon Lovestone (University of Oxford) developed a panel of blood-based biomarkers for the diagnosis of Alzheimer's disease. Due to pathological similarities between HAND and Alzheimer's disease, it is hypothesised that there may be applicability of this panel to diagnosis of HAND. The differential protein expression profile of patients with and without HAND is thus compared. Aims and Objectives: The aim of this study was to assess the applicability of a set of Alzheimer's disease biomarkers to the diagnosis of HAND. In order to achieve this, targeted proteomic assays were developed to measure the markers in human serum. This assay was then applied to defined patient cohorts. Methods: Targeted proteomic assays were developed in order to quantify candidate markers in patient blood serum. Parallel Reaction Monitoring assays were developed on a Thermo Q Exactive Quadrupole-Orbitrap mass spectrometer, for application to defined patient samples collected from Groote Schuur Hospital (Cape Town, South Africa). Patient samples were divided into groups according to HAND severity (based on the HIV Dementia Scale), namely: Normal (HIV+), Minor Neurocognitive Disorders and HIV-associated Dementia. Discovery proteomic analysis was performed on pooled patient samples in order to determine the optimal peptides for identification and quantitation of candidate proteins. Targeted methods were then developed and refined using the same pooled samples. Finally, patient samples (N=81) from the three classes were analysed in a statistically rigorous manner. Data were normalised to correct for possible loading inconsistencies by two independent methods. Quantitative differences were investigated using t-tests and non-parametric statistical tests where appropriate. Results: Statistical assessment of final data indicated no significant differences in proteins between patient classes. Discussion and Conclusions: It is likely that the inflammatory nature of HIV itself influences the levels of these markers in HIV-positive patient samples to a greater degree than neurocognitive effects. Alternatively, possible co-infection by TB may have confounded the results. Regardless, it was concluded that the candidate Alzheimer's biomarkers were not applicable to diagnosis of HAND. Further analysis on a larger sample cohort is recommended, utilising the assays optimised herein. Prospective studies to obtain viable biomarkers for definitive diagnosis may lie in proteomic analysis of model infection systems and cerebrospinal fluid.

Cancer is a leading cause of death in the modern world. Chronic inflammation facilitates tumourigenesis and cancer progression by providing an environment conducive to cancer. Dysregulation of the immune response, and particularly inflammation, is an important part of this process. Garlic (Allium sativum) has been used for centuries as both a prophylactic and therapeutic medicinal agent, more recent epidemiological and experimental evidence shows that garlic has both cancer-preventative and immune system-enhancing effects. While garlic contains many bioactive compounds, garlic organosulfur compounds (OSCs) have been most widely studied for their anti-cancer properties. In this study, we hypothesize that garlic OSCs modulate the inflammatory immune response by downregulating pro-inflammatory while stimulating anti-inflammatory responses, preventing the formation of a cancer-friendly chronic inflammatory environment. To test this hypothesis we established and optimised an in vitro inflammatory model using lipopolysaccharide-stimulated RAW264.7 murine macrophages. Expression analysis of selected inflammatory genes was performed by qPCR on RNA harvested 4 h and 8 h post treatment, while protein expression was analysed by ELISA using cell culture supernatant samples harvested 8 h and 24 h post treatment. These experiments were complemented by gene and protein arrays. Results showed that allicin had a more pronounced upregulatory effect on LPS-induced gene expression 4 h post-LPS treatment. In contrast, Z-ajoene generally had mild downregulatory effects on the expression of LPS-induced genes. Conversely, Z-ajoene had pronounced downregulatory effects on LPS-induced inflammatory proteins after 24 h, while allicin showed mild up- or downregulatory effects. Overall, we found that allicin induced an initial pro-inflammatory gene response, while Z-ajoene induced a longer-lasting anti-inflammatory response at a protein level. Finally, as many of the inflammatory genes investigated are regulated by the transcription factor STAT3, we investigated the effects of allicin and Z-ajoene on STAT3 phosphorylation and hence activation. Western blot analyses showed that allicin increased LPS-induced STAT3 phosphorylation (2-8 h), while Z-ajoene was found to decrease the phosphorylation of STAT3 after 4 h. These effects on STAT3 phosphorylation are in agreement with the early pro-inflammatory effect of allicin and the later anti-inflammatory effect of Z-ajoene on LPS-induced gene and protein expression. Further, using Western blotting we showed that E/Z-ajoene directly interacts with and reversibly alkylates STAT3 via a thiol-disulfide reaction with a cysteine thiolate on STAT3.

Transmitted founders (TF) might carry motifs that provide a phenotypic advantage that enables human immunodeficiency virus type-1 (HIV-1) to overcome immune barriers within the female genital tract. One study compared over 5000 subtype B TF and mismatched chronic infection envelope (env) sequences and identified two putative transmission motifs: Histidine at position 12 of the signal peptide (His12) and a potential N-glycan site (PNG) at position 413-415. Although, His12 was shown to be important for subtype B Env expression and viral infectivity, in our own sequence analysis subtype C variants did not carry the transmission motifs and the aim of this study was to determine whether His12 and PNG413 was important for subtype C Env expression, processing, function and viral replication. Mutagenesis of a subtype C Env clone indicated that His12 decreased pseudovirion (PSV) entry efficiency without influencing Env expression, secretion and cleavage with no changes in the N-glycosylation profile. This suggested that His12 had a fitness cost and was thus selected against. However, His12 significantly enhanced the entry efficiency of infectious molecular clones (IMCs), suggesting that it might be beneficial for in vivo replication. The variation between the PSV and IMC entry of TZM-bl cells could be due to differences in assay conditions. On the other hand deletion of PNG413 enhanced Env expression, secretion, cleavage and PSV and IMC entry efficiency of TZM-bl cells. This would suggest that subtype C TFs carrying a PNG at 413-413 would have lower viral replicative capacity due to poor expression and processing of Env. The benefit of this phenotype on HIV-1 subtype C transmission needs to be further investigated. Unfortunately, PSV and IMC entry of TZM-bl cells could not be confirmed by IMC replication in peripheral blood monocytes because the clones could not replicate to measurable levels in these cells over the culture period. Overall, this study has shown that amino acid residues at positions 12 and 415 do play a role in modulating Env processing and function however the actual mechanism by which these polymorphisms impact viral fitness most likely differ to that of subtype B, explaining why His12 is absent and PNG413 is present in subtype C TFs.