Includes abstract. / Includes bibliographical references (leaves 122-157). / Stromal cells and the extracellular matrix (ECM) components provide the microenvironment that is pivotal for cell growth, motility, attachment and differentiation. Fibroblasts are some of the cells responsible for the synthesis of most of the extracellular matrix proteins. Type I collagen is the most abundant extracellular matrix protein in the human body and is found in tissues requiring high tensile strength. In this study we investigated the effect of a pre-formed fibroblast-derived extracellular matrix on the expression of type I collagen and associated matrix metalloproteinases in fibroblasts.

Includes abstract. Includes bibliographical references (leaves 96-113).

Includes bibliographical references. / Cancer is a leading cause of death worldwide and viral infections such as HBV/HCV and HPV have been known to be responsible for up to 20% of cancers in low- and middle-income countries. Approximately 500,000 of these deaths are due to oesophageal squamous cell carcinoma (OSSC) alone, one of the major cancers in Eastern and Southern Africa, Latin America and Asia. Previous studies have shown HPV DNA to be integrated in nearly 40% of oesophageal tumours whereas it was present in only 3% of normal healthy asymptomatic individuals, implicating it as a possible risk factor. The aim of this study was to compare the roles and effects of the E6 gene from the low risk HPV11 and high risk HPV18 on the cellular gene expression profile in order to identify genes required for the initiation of cellular transformation and also to identify genomic alterations associated with oesophageal squamous cell carcinoma. Cancer is a leading cause of death worldwide and viral infections such as HBV/HCV and HPV have been known to be responsible for up to 20% of cancers in low- and middle-income countries. Approximately 500,000 of these deaths are due to oesophageal squamous cell carcinoma (OSSC) alone, one of the major cancers in Eastern and Southern Africa, Latin America and Asia. Previous studies have shown HPV DNA to be integrated in nearly 40% of oesophageal tumours whereas it was present in only 3% of normal healthy asymptomatic individuals, implicating it as a possible risk factor. The aim of this study was to compare the roles and effects of the E6 gene from the low risk HPV11 and high risk HPV18 on the cellular gene expression profile in order to identify genes required for the initiation of cellular transformation and also to identify genomic alterations associated with oesophageal squamous cell carcinoma.

Includes bibliographical references / Angiotensin-1 converting enzyme (ACE) is a zinc metallopeptidase that consists of two homologous catalytic domains (N and C) with different substrate specificities. ACE is a central component of the intrinsic brain renin angiotensin-aldosterone system (BRAAS), well renowned as the regulator of blood pressure. The BRAAS has alternate functions that extend beyond fluid and blood pressure homeostasis into areas such as neurological function. As a result, it is implicated in many neurodegenerative diseases including Alzheimer's disease (AD). ACE's specific mechanistic role in AD is not entirely clear and is somewhat controversial. However, it has been shown that ACE hydrolyses the amyloid beta (Aβ) peptide, the putative causative agent of AD. This study aimed to investigate the molecular basis of ACE hydrolysis of Aβ by determining : 1) the kinetic parameters of five different forms of human ACE with various N-terminal amyloid beta (Aβ) substrates; 2) the specific active site determinants of Aβ-domain selectivity; and 3) the high-resolution crystal structures of the N-domain of ACE in complex with Aβ(1-16), Aβ(10-16), Aβ(4-10), the FRET Aβ(4-10)Y and Aβ(35-42) peptides. For the physiological Aβ(1-16) peptide, a novel ACE cleavage site was found at His14/Gln15. Furthermore, Aβ(1-16 ) was preferentially cleaved by the truncated N-domain; however, the presence of an inactive C-domain in full-length ACE greatly reduced enzyme activity and affected domain-selectivity. Two fluorogenic substrates, designed specifically to assess ACE's mechanism of Aβ hydrolysis Aβ(4-10)Q and Aβ(4-10)Y, underwent endoproteolytic cleavage at the Asp7/Ser8 bond. The Aβ(4-10)Q peptide was a poor substrate of ACE but was N-selective, with a selectivity driven largely by interactions with the domain-specific residues of the S2 and S2' pockets. The selectivity of the S2' residues were confirmed with a similar, more physiological, fluorogenic Aβ(4-10)Y peptide. This work provides further understanding towards the substrate determinants of N-selectivity, highlighting the importance of the S2' Ser357. ACE C-domain hydrolysed Aβ(4-10)Y with modest efficiency compared to the other substrates, where hydrolysis under the same conditions did not occur. Moreover, Aβ(4-10)Y also displayed N-domain selectivity. In contrast to Aβ(1-16) and Aβ(410)Q, both sACE and the double C-domain (CC-sACE) construct showed positive domain cooperativity towards Aβ(4-10)Y. The high-resolution crystal structures of the N-domain in complex with five Aβ peptide fragments provided an overlapping, conserved, molecular mechanism of peptide binding and evidence of the enzyme's broad exoprotease activity. In addition to the kinetic and structural studies, ACE's signalling response to the N-selective Aβ(1-16) and Aβ(1-42) was investigated using immunodetection and mass spectrometry. Similar to the ACE inhibitor lisinopril, the Aβ peptides elicited ACE signalling by phosphorylation of the cytoplasmic Ser1270 residue and JNK activation. The signalling response of ACE was coupled to increased ACE activity an d expression on treatment with Aβ(1-42). These studies allowed us to rationalise the increased ACE activity and expression found in AD, may arise through direct interactions with Aβ. This work provides a kinetic, structural and mechanistic understanding of the selective cleavage of Aβ by the N and C catalytic sites of ACE. Due to the broad substrate specificity of the two domains of ACE, and the overarching N- selectivity of Aβ hydrolysis, these findings provide rationale for further in vivo pharmacological studies on the mechanism of action C- domain-selective inhibitors, in the context of AD.

Bibliography: pages 183-200. / This thesis comprises a report of investigations into the interaction of the volatile anaesthetic agents, fluroxene, 2,2,2-trifluoroethyl ethyl ether(TFEE), methoxyflurane and enflurane, with hepatic drug metabolizing enzymes in vivo and in vitro. Each of the anaesthetic agents interacts with the type P-450 cytochromes of hepatic microsomes in vitro resulting in the appearance of a type I difference spectrum, enhancement of NADPH oxidation and production of potentially toxic metabolites, 2,2,2-trifluoroethanol (TFE) (from fluroxene and TFEE) and free fluoride ion (from methoxyflurane and enflurane).

The identification of novel cancer-associated biomarkers against which drugs can be developed is anticipated to be beneficial in multiple ways; including their use as monotherapies and in combination with current chemotherapeutic agents for improved anti-cancer treatment outcome. Recently, research in our own laboratory and others have reported elevated expression of the nuclear transporter Kpnβ1 in multiple cancers. Using the cervical cancer model, we showed that its inhibition using small-interfering RNA (siRNA) resulted in cancer cell death via apoptosis while sparing normal cells, suggesting it has potential as a target for anti-cancer therapy. An in silico screen for Kpnβ1 inhibitors identified several small molecules that showed inhibitory effects on nuclear import as well as cancer killing activity. In this study, we aimed to examine the potential of one such small molecule, the Inhibitor of Nuclear Import-43 (INI-43) as a lead compound with anti-cancer activities using multiple cancer models. Through culture-based in vitro assays, we demonstrated that INI-43 inhibited the proliferation of cancer cells grown anchorage-dependently and independently. These effects were similarly observed in Kpnβ1 knock-down cells, and Kpnβ1 over-expression was able to partially reverse these effects, suggesting that the anti-cancer effects of INI-43 is mediated through interference of the Kpnβ1 function. Toxicology studies and liver microsomal assay showed that INI-43 has an acceptable toxicity profile in nude mice and is metabolically stable, allowing its use in in vivo testing. Intraperitoneal administration of INI-43 significantly reduced the growth of subcutaneously xenografted cervical and oesophageal tumour cells in nude mice, supporting its anti-cancer activity in vivo. To examine the potential of using INI-43 in combination therapy, we examined the effects of the combined treatment of INI-43 and Cisplatin (CDDP), a first-line chemotherapeutic agent used in the treatment of many cancers. INI-43 treatment at sub-lethal concentrations enhanced cancer cells' sensitivity to CDDP, which was similarly observed in Kpnβ1 knock-down cells. Using an ovarian cancer model, we demonstrated that CDDP treatment led to elevated expression and nuclear localization of Kpnβ1, suggesting that Kpnβ1 is involved in CDDP-induced stress response. INI-43 treatment impeded the CDDP-induced nuclear accumulation of Kpnβ1 which correlated with increased cell death, suggesting that nuclear localization of Kpnβ1 may be important for ovarian cancer cell survival when challenged with genotoxins such as CDDP. Using the cervical cancer model, we demonstrated that INI-43 enhanced CDDP-induced cell death synergistically, and that the enhanced cell death is mediated through stabilizing p53 protein. This associated with decreased levels of Myeloid Cell Leukemia 1 (Mcl-1), an anti-apoptotic factor negatively regulated by p53. Furthermore, INI-43 treatment reduced the nuclear import of NFκB, a stress-regulated response known to promote cancer cell survival. Decreased levels of various downstream pro-survival and DNA-repair targets of NFκB were observed, including cyclinD1, c-Myc and X-Linked Inhibitor of Apoptosis Protein (XIAP), which correlated with increased DNA damage and apoptosis. Taken together, we show that nuclear import inhibition using small molecules could have therapeutic benefits in the treatment of cancer, and that INI-43 is a promising candidate for further development to be used in anti-cancer monotherapy or combination chemotherapy.

Includes abstract.~Includes bibliographical references. / Crushed garlic contains organosulfur compounds (OSC), which are reported to have cancer chemotherapeutic properties both in vitro and in vivo. A library of 15 organosulfur analogues were obtained as mechanistic probes in WHCO1 oesophageal cancer cells. Structure-activity studies showed a positive correlation between the anti-proliferative-IC50 of disulfides and the relative stability of their anion leaving groups, as assessed through resonance and quantified by predictive pKa-values.

Includes abstract. Includes bibliographical references.

Understanding HIV transmission mechanisms is essential for the design and development of an efficacious, broadly acting vaccine that targets features common to transmitted viruses. However, there is a lack of consensus amongst current HIV studies characterising transmitted founders (TFs). When investigating the methods employed across studies, it becomes clear that methodologies are highly variable and thus, could be impacting research outcomes. This study therefore aimed to determine whether Envelope (Env) expression and processing affects function and whether cell type and/or expression system were responsible for these differences. Our data suggest that even though we did not observe differential expression of recombinant Env clones across cell types, when pseudovirus and infectious molecular clone (IMC) backbones were introduced, expression of Env decreased. We also found differences in processing in the form of cleavage, N-glycosylation and incorporation of Env across cell types. We conclude from this that methods used to study Env characteristics are highly sensitive to cell type and HIV backbone which suggests that a more standardised system is required to make meaningful comparisons between studies. The results of our functional Env analysis revealed high variation depending on the methodology used. We found that entry of TZM-bl cells by pseudovirus (PSV) is dependent on the cell line used to produce the viral particles. Unfortunately, due to low IMC titre, we had to expand the virus in PBMCs, negating the effect that cell type might have had on IMC expression. We could thus not directly compare PSVs to IMCs. However, PSV and IMC entry as well as IMC replication in PBMCs suggested that CHO cells were not suitable for robust viral production and better suited for recombinant Env expression. Overall, the findings in this project support previous findings that PSVs and IMCs are not directly comparable due to multiple factors that influence Env expression and virus production. We suggest that researchers who focus on HIV functional analysis, particularly Env, with the end-point of informing vaccine design, need regulated methods across laboratories, similar to the way that neutralisation assays were standardised.

A significant proportion of human immunodeficiency virus type 1 (HIV)-positive individuals are affected by the cognitive, motor and behavioural dysfunction that characterises HIV associated neurocognitive disorders. While the molecular aetiology of this important HIV complication remains largely uncharacterised, HIV transactivator of transcription (HIV-Tat) has been identified as a plausible aetiological cause. Here we have used mass spectrometry-based discovery proteomics to identify the quantitative, cell-wide changes that occur when non-transformed, differentiated human neurons are treated with HIV-Tat over time, as a novel cell culture model representing the initial progression of HIV associated neurocognitive disorders, and as a means to identify putative biomarkers for the illness. We found that our stem cell-based model system displayed morphological and functional neuronal properties and using a Q-Exactive mass spectrometer, we identified over 4000 protein groups (FDR < 0.01) in this system with 131,118 and 45 protein groups differentially expressed at 6, 24 and 48 hours post treatment, respectively. We found changes to the gene expression machinery (nucleic acid binding proteins), which suggests that HIV-Tat is involved in preparing the host cell for altered transcriptional and translational activity. We also found cytoskeletal dysregulation in response to HIV-Tat treatment. The 24-hour time point of the time course experiment was largely corroborated with a repeat experiment. A repeat of the entire time course experiment at a lower cell confluence showed that the effect of HIV-Tat treatment to the gene expression machinery was unchanged by cell confluence, while the effect to cytoskeletal proteins upon HIV-Tat treatment was present, but less prominent, in lower cell confluence samples. We hypothesise that the gene-expression-machinery effect may be a biphasic response. We further hypothesise that cytoskeletal dysregulation may form part of the molecular mechanism responsible for synaptic injury - as the cytoskeleton is crucial for synapse development and maintenance - and may contribute to memory impairment in HIV associated neurocognitive disorder patients.