Faculty of Science (ETDs)

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Subject: search.filters.subject.Drug Resistance

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    Characterising the Role of Cholesterol in Hypoxia-induced Epithelial- Mesenchymal Transition in Breast Cancer
    (University of the Witwatersrand, Johannesburg, 2022) Abdulla, Naaziyah; Kaur, Mandeep
    The cellular epithelial-mesenchymal transition (EMT) process is a complex labyrinth dependent on subversion of critical cellular signalling pathways, which crosstalk extensively to confer cancer cells with characteristics that mediate metastasis. Based on the pleotropic role of cholesterol in the cell, it is not surprising that cancer cells have evolved several mechanisms to facilitate cholesterol dyshomeostasis. In addition to meeting the increased metabolic demands of cancer cells, deregulated cholesterol metabolism also facilitates increased cellular cholesterol availability which is crucial to regulating the activity of protein intermediates in EMT-related signalling pathways. Despite evidence indicating that cholesterol directly regulates signalling pathways related to EMT, no publication to date has attempted to address the effect of EMT induction on cellular cholesterol levels in cancer. To shed light on the dynamics of cholesterol in the relationship between hypoxia and EMT, cholesterol content in MCF-7 cells pre- and post-hypoxia induced EMT was assessed. This dissertation presents findings indicating increased levels of free cholesterol, cholesteryl esters as well as lipid raft cholesterol in MCF-7 cells following hypoxia-induced EMT. Interestingly, MCF-7 cells post- EMT induction displayed increased sensitivity to treatment with cholesterol targeting agents and presented with reversion to an epithelial state as evidenced by the increased expression of epithelial markers, decreased expression of mesenchymal markers and also reduced invasive potential. Importantly, treatment with cholesterol targeting agents is also seen to abrogate the drug resistant potential following hypoxia-induced EMT. Based on these observations, it is proposed that targeting cellular cholesterol could be a promising area to invest in the search for novel therapeutics effective in combatting cancer metastasis
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    Insights into the Physiochemical Properties of the Interaction between the L38↑N↑L+4 HIV-1 Hinge Mutant Subtype C Protease and a Related Gag Cleavage Site
    (University of the Witwatersrand, Johannesburg, 2023-09) Adams, Taryn Racheal; Sayed, Yasien
    Frequent mutations in HIV protein drug targets such as the protease (PR), have led to a rise in resistance to clinically available treatments and inquiries into the associated biochemical mechanisms. In this study, the L38↑N↑L+4 PR (mutant) and related gag, were isolated from a PR inhibitor naïve child for further study. Wild-type and mutant PRs were successfully overexpressed and purified using ion exchange chromatography. Intrinsic fluorescence studies probing tryptophan residues located at the hinge revealed variations in tertiary structure. This coincided with significant differences in refolding efficiency (mutant PR recovery ~ 10% and wild-type PR recovery ~ 34%). Furthermore, differences in catalytic efficiency (mutant-specific activity ~ 7.4 µmol.min-1.mg-1, mutant kcat ~ 2.71 sec-1; wild-type specific activity ~ 31.6 µmol.min-1.mg-1, kcat ~ 11.60 sec-1). Thermal shift assays revealed reduced mutant PR structural stability (Tm ~ 67 ℃) compared to the wild-type PR (Tm ~ 64 ℃). Furthermore, reduced stability of inhibitor-mutant PR complexes (Tm ~ 73.5 ℃ Acetyl pepstatin (AP), 90 ℃ Darunavir (DRV), and 88 ℃ Saquinavir (SQV)) compared to complexes involving the wild-type PR (Tm ~ 70 ℃ (AP), 82 ℃ (DRV), and 82 ℃ (SQV)). Overall, the L38↑N↑L+4 PR mutations were found to influence the tertiary structure of the hinge, gag processing, and PI stability within the mutant PR active site. Computational docking studies highlighted the potential role of a gag single nucleotide polymorphism (SNP), located at the 4th amino acid (P4) position of the peptide-2-nucleocapsid (p2/NC) gag cleavage site, for future studies as a compensatory mutation aiding PR polymorphisms. Further studies should focus on the gag-PR functional pair to build a more accurate understanding of HIV drug resistance mechanisms.