Electronic Theses and Dissertations (Masters)
Permanent URI for this collectionhttps://hdl.handle.net/10539/38018
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Item Characterising the Role of Cholesterol in Hypoxia-induced Epithelial- Mesenchymal Transition in Breast Cancer(University of the Witwatersrand, Johannesburg, 2022) Abdulla, Naaziyah; Kaur, MandeepThe 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 metastasisItem Antibacterial activity and susceptibility testing of bacterial isolates from nematodes (Cruznema spp.)(University of the Witwatersrand, Johannesburg, 2023-09) Mothapo, Maletjema Magdeline; Lephoto, Tiisetso E.Nematodes are unsegmented worms found in different niches associated with a diverse range of bacteria. Various types of nematodes exist including those that are parasitic to insects, known as entomopathogenic nematodes (EPNs). EPNS of genera Steinernema, Heterorhabditis and Oscheuis are symbiotically associated with Xenorhabdus, Photorhabdus and Serratia, respectively. The symbiotic bacteria of EPNs have been reported to produce a broad spectrum of antimicrobial compounds active against human pathogens. The aim of this study was to isolate and identify nematodes and their associated bacteria from soil samples collected from a vegetative farm in Lesotho and study their antimicrobial activity against four species of pathogenic bacteria (E. coli, S. aureus, E. faecalis and P. aeruginosa). An uncharacterized species of Cruznema was isolated and named Cruznema NTM-2021 (GenBank 18S rDNA accession number: OQ408141). Based on the BLASTN search incorporating the phylogenetic analysis of the 16S rDNA region, three genera of bacteria were identified as Alcaligenes sp., Enterobacter sp. and Elizabethkingia sp. The study revealed that all three bacterial isolates were pathogenic to Tenebrio molitor. Symbiosis tests, using lipid agar method demonstrated the ability of the host nematodes to develop and reproduce in the presence of their associated bacteria. Bacterial supernatants of Alcaligenes sp. and Enterobacter sp. showed some inhibitory activity against Escherichia coli and Enterococcus faecalis, by disk diffusion method. Staphylococcus aureus and Pseudomonas aeruginosa were the most resistant bacteria to supernatants of the three isolates. This study also showed that the Alcaligenes, Enterobacter, and Elizabethkingia species isolated from Cruznema NTM-2021 were resistant to ampicillin, amoxicillin, cefuroxime/sodium, vancomycin and cephalothin but susceptible to gentamicin.Item HIV-1 subtype C protease: enzyme kinetics, thermodynamics, and X-ray crystal structure(University of the Witwatersrand, Johannesburg, 2023-09) Dlamini, Nozinhle Precious; Sayed, YasienHuman immunodeficiency virus (HIV), a precursor for AIDS is still one of the most devastating pandemics in history. In 2021 alone there were 650 000 deaths associated with the virus and the number of people living with the infection was recorded to be 38.4 million globally. Sub-Saharan Africa suffers the most burden of the virus with approximately 8.3 million people living with virus, HIV-1 subtype C is the main driver of the disease in South Africa and accounts for 46% of global infections. Even with these alarming statistics this subtype is not the main focus point for the majority of HIV-1 research which mainly focuses on subtype B though it only accounts for 12% of infections globally. There is no vaccine or cure against HIV; however, great strides have been made in suppressing the virus. Viral suppression drugs have been developed to target different stages of viral replication such as those targeting the three important enzymes (protease, reverse transcriptase and integrase). In this study the focus will be on HIV-1 subtype C protease. This is a homodimeric aspartyl protease with 99 amino acids in each monomer. It plays a crucial role in the replication cycle of HIV-1 by producing mature infectious virions through cleavage of the Gag and Gag-Pol polyproteins. The subtype C protease differs from subtype B protease in that it has eight naturally occurring polymorphisms which are substitution mutations, some occurring in different regions of the protease with some in the fulcrum (T12S, I15V and L19I), others in the hinge region (M36I and R41K), with H69K and L89M found in the loops and I93L in the α helix. In this study, structural and functional characterisation of HIV-1 subtype C protease was carried out. The secondary structure was characterised using far-UV CD, which is a technique that measures the difference in left and right circularly polarised light. The subtype C protease was estimated to be predominantly β-sheeted, with spectra showing a maximum at 195 nm and a minimum between 215-225 nm. Tertiary structure characterisation of protease was performed using fluorescence spectroscopy. The maximum emission at 347 nm close to that of water (350 nm), demonstrated that the tertiary conformation of the HIV-1 protease was conserved, and that the tryptophan residues within the protease are solvent exposed. SE-HPLC was used to characterise the quaternary structure of the protease and the homodimeric size was determined to be approximately 22 kDa. Steady-state enzyme kinetics to assess the catalytic activity of the subtype C protease was performed using a fluorogenic substrate. The activity of the enzyme was confirmed, with the specific activity of 24.22±1.72 µmol. min-1.mg-1 and the binding of the substrate to the HIV-1 protease was demonstrated by the KM value of 79.546±6.491 µM. This correlates to literature indicating that the substrate was weakly bound and that a high substrate concentration will be required to reach the maximum velocity (Vmax), and Vmax was determined to be 0.036±0.003 µmol. min-1. Enzyme kinetics was coupled with displacement isothermal titration calorimetry for determination of thermodynamics parameters using second generation PIs (atazanavir, darunavir and lopinavir). Thermodynamic studies indicated that the HIV-1 protease has a high affinity for LPV (Kd = 1 nM), compared to ATV (Kd = 18.57nM) and DRV (Kd = 42.26 nM) and binding reactions were all spontaneous with ΔG values(ATV = -43.39 kJ/mol, DRV = -41.39 kJ/mol and LPV = -50.51 kJ/mol). The values also indicated that LPV complexed with HIV-1 is more a stable complex. Also, all the binding reactions were exothermic as indicated by the negative ΔH values of ATV = -45.54 kJ/mol, DRV = -55.62 kJ/mol and LPV = -54.71 kJ/mol. The entropy of all the reactions were determined to be unfavourable with the -T∆S of DRV = 14.23 kJ/mol followed by LPV: 4.2 kJ/mol and ATV: 2.15 kJ/mol. Overall this suggested that all the binding reactions were enthalpically driven. Furthermore, the three-dimensional structure of the HIV-1 subtype C protease was elucidated using X-ray crystallography. The three-dimensional structure the HIV-1 CSA (PDB ID: 8CI7) was solved at a 2.4 Å resolution which is better than the 2.7 Å (PDB ID: 3U71) initially solved in our lab. The high-resolution three-dimensional structure of the protease will provide precise information about the arrangement of atoms within the protease molecule, thus enabling the design and development of protease inhibitors that will be specific for the subtype C protease. This study emphasised the significance of investigating subtype C protease in the context of enzyme kinetics, thermodynamics and detailed X-ray crystallography.Item Establishing and characterizing organoid cultures from colon tissue of South African individuals(University of the Witwatersrand, Johannesburg, 2024) Du Plessis, Thea-Leonie; Kaur, MandeepColorectal cancer (CRC) has been poorly studied in South Africa, with limited studies on disease progression and development. Studies that have investigated CRC in South Africa have indicated that there is racial disparity between different racial groups that may be attributed to alternative developmental pathways, differences in genetic compositions or CRC initiators that result in these different clinical presentations. Furthermore, the lack of population-based studies substantiates the need for more intensive CRC research. A particular model used to study cancer in general is the use of two-dimensional (2D) cell cultures, which have provided novel insight into many cancers and their development processes. However, these models lack the complex biology observed in vivo. One such model that is gaining research interest is the use of three-dimensional (3D) organoid cultures. Organoids are derived from stem cells and are able to self-organize and mimic the corresponding organ from which they were derived. Research has indicated that organoids are able to maintain cell-type heterogeneity as well as gene expression levels that resemble the organ of origin. Therefore, this project aimed at standardizing a protocol to establish and characterise colorectal organoid cultures from South African patient-derived tissues. Patient samples were obtained from individual patients with informed consent and were processed to generate organoids. The morphology of the organoids was monitored across several days and across passages. Once the organoids had reached maturity and were at passage 2, characterization was performed using real-time quantitative polymerase chain reaction (RT-qPCR) and immunofluorescence which indicated that the genetic composition and spatial localization of cell types of interest in non-cancerous tissue was recapitulated in the organoids. Based on these observations, it is proposed that organoids could be a promising model to investigate CRC disease development and progression and potentially search for novel therapeutics. This project has established the protocols for growing and characterizing organoids from African samples and provides baseline data, and outlines the complexities and issues involved in growing organoid cultures for the future studiesItem A Clot to Uncover: FOXP3 and SARS-CoV-2 Nucleocapsid Interactions and Their Effect on DNA Binding(University of the Witwatersrand, Johannesburg, 2024) Mcinnes, Keiran; Fanucchi, SylviaDuring COVID-19, systemic coagulopathy, which can lead to strokes and embolisms, is often observed in COVID-19 patients and may also contribute to long COVID. This coagulopathy is the result of overactivated platelets in circulation that leads to inappropriate clot formation. FOXP3 is a transcription factor involved in platelet development and loss of FOXP3 function leads to platelets that resemble those seen during COVID-19. Thus, FOXP3 may be dysregulated in COVID-19. The SARS-CoV- 2 nucleocapsid (NC) is a multifunctional protein typically associated with viral genome packaging and virion assembly. However, it is also capable of binding DNA and is potentially able to alter regulation of host protein expression. Here, potential interactions between the DNA-binding forkhead domain (FHD) of FOXP3 and the SARS-CoV-2 NC were investigated. Identification of a novel interaction between FOXP3 and SARS CoV-2 NC may provide new clues as to the pathophysiology of COVID-19. To address this aim, both proteins were overexpressed in T7 E. coli, purified via immobilised metal affinity chromatography, and monitored for potential interactions in the absence and presence of DNA using pull-down assays and fluorescence anisotropy. A direct interaction was identified between the two proteins in the absence of DNA. Additionally, it was found that both proteins are capable of binding to DNA at the same time, but excess NC was found to cause FHD dissociation from the FHD- NC-DNA complex. This result implicates NC in FOXP3 dysfunction which may be associated with the coagulopathy and other symptoms seen during COVID-19. Additionally, NC DNA binding does not appear to be driven by the FOXP3 consensus sequence, indicating that FOXP3 may not be the only transcription factor potentially dysregulated by NCItem Effects of Mg2+, Ni2+ and Ca2+ on ATP binding kinetics of nicotinate nucleotide adenylyltransferase from Klebsiella pneumoniae and Enterococcus faecium: insights from empirical and computational studies(University of the Witwatersrand, Johannesburg, 2023-07) Van Deventer, Ruan; Achilonu, Ikechukwu AnthonyNNAT is an attractive target for drug development due to its crucial role in NAD+ synthesis. However, its characterisation in ESKAPE species, such as Klebsiella pneumoniae and Enterococcus faecium, remains limited. This study aimed to elucidate the binding mechanism of ATP, a pivotal substrate, to these NNAT species, focusing on the role of divalent metal ion cofactors. KpNNAT and EfNNAT enzymes were overexpressed and purified, yielding approximately 2 mg/ml for both. Various techniques were employed to investigate their properties, including far-UV CD, extrinsic ANS fluorescence, stopped-flow kinetic analyses, and MD simulations. The results revealed that KpNNAT could bind ATP independently of divalent metal ions, but catalytic activity required the presence of Mg2+. The kinetic analysis showed ka values of 5.99 μM-1 .sec-1 without divalent metal ions and 5.72 μM-1 .sec-1 in the presence of Mg2+. The "pseudo"-specific activity values were 0.005 μmol/min/mg without divalent metal ions and 0.374 μmol/min/mg in the presence of Mg2+. Conversely, recombinant EfNNAT exhibited limited ATP association, and the reasons for this remain unclear. Overall, this study shed light on the structural dynamics and functional kinetics of ATP association in both NNAT species. The findings contribute to our understanding of this druggable target and provide insights into the inactivity of EfNNAT, which warrants further investigation.Item 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, YasienFrequent 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.Item Differential Gene Expression Analysis of PMA Treated Pro-monocytic Cell Lines(University of the Witwatersrand, Johannesburg, 2023) Kama, Asavela Olona; Meyer, Vanessa; Gentle, NikkiHL-60, THP-1, and U937 are model cell lines that can undergo myeloid differentiation in vitro, allowing the study of myeloid cell function in drug metabolism, cytotoxicity, and the aetiology of infections. However, the differentiated end-state of these cells is not well characterised. Moreover, cell line-specific differences in the level of gene expression may confound results obtained from such studies. The aim of this study was thus to compare changes in gene expression between HL-60, THP-1, and U937 cells in response to the differentiation agent, phorbol 12-myristate 13-acetate (PMA), 48 hours after treatment. Gene expression profiles were compared across all three cell lines prior to and post-PMA treatment. Differential gene expression analysis between treated and untreated cells was performed using DESeq2 (v 4.2). Gene over-representation analysis was performed using cluster Profiler (v 4.0). HL-60, THP-1, and U937 cells had similar expression profiles prior to PMA treatment, but different sets of genes were significantly differentially expressed in these cell lines 48 h after treatment with PMA. A total of 475 genes were consistently differentially expressed across all cell lines. These genes were found to be involved in phagosome formation and cell cycle transition. HL-60, THP-1, and U937 cells had 944, 1231, and 624 uniquely differentially expressed genes, respectively. These genes were predominantly involved in energy metabolism and pathogen recognition. Overall, THP-1 cells showed greater potential to detect viruses, while U937 cells showed greater potential to detect bacteria. From this, it can be concluded that while all three cell lines did indeed undergo myeloid differentiation, the macrophage-like cell state produced in each case differed between cell lines.Item Expression and Methylation of Peroxidasin in Breast Cancer Cell Lines(University of the Witwatersrand, Johannesburg, 2023-07) Falkov, Jemma Lilian; Mavri-Damelin, DemetraPeroxidasin (PXDN) is a haem-containing extracellular matrix peroxidase protein which forms hypohalous acids in the presence of hydrogen peroxide (H2O2). The predominant role of PXDN is that of a collagen IV crosslinker within the basement membrane. Increased collagen IV deposition has been linked to tissue invasion and metastasis in breast cancer and PXDN has also been shown to assist in the process of epithelial-mesenchymal transition (EMT) in cancer. Various cancer types display dysregulated levels of PXDN expression including breast cancer and this dysregulation has been associated with poor prognosis. This study aimed to investigate whether DNA methylation of the PXDN promoter may be a mechanism through which changes in PXDN expression observed in breast cancer are regulated. Non-invasive MCF-7 and invasive MDA-MB-231 cells were used as models for luminal A and triple negative breast cancer (TNBC) respectively. The HEK-293 cell line was used as a non-cancerous control cell line. DNA methylation levels of the PXDN promoter and PXDN protein expression was investigated in these cell lines through the methods of methylation sensitive PCR (MS PCR) and immunofluorescence microscopy. Relative levels of PXDN expression were determined through immunofluorescence microscopy. Corrected total cell fluorescence (CTCF) analysis of these images revealed the highest PXDN levels to be found within the invasive MDA-MB-231 cell line, which was double that of the MCF-7 cell line. All cell lines were treated with 10 nM β-Oestradiol, which caused an increase in PXDN expression within the MCF-7 and HEK-293 cell lines and a decrease in expression within the MDA-MB-231 cell line to half its untreated value. PXDN was found to be localised in the ECM in all three cell lines. To elucidate the role of DNA methylation, methylation sensitive PCR (MS PCR) was performed on all three cell lines, with four primer pairs spanning a region of 1305 base pairs (bp) within the PXDN promoter. A region of differential methylation was found between the MDA-MB-231 and HEK-293 cell lines between 524 bp and 53 bp upstream of the transcription start site (TSS). This region was unmethylated within the MDA-MB-231 cell line and methylated within the HEK-293 cell line, which correlates with expression differences between these two cell lines and suggests this region could be of regulatory significance. The four primer pairs designed to amplify the PXDN promoter were unable to amplify this region within the MCF-7 cell line. A heterochromatic DNA conformation or a point mutation increasing CpG content creating a thermodynamically ultra-fastened (TUF) region could be the explanation behind this phenomenon, however further research is required to elucidate the mechanism responsible. In conclusion, PXDN shows higher expression in TNBC cells than in luminal A subtype cells. The oestrogen receptor is involved in regulating PXDN expression, however, different mechanisms seem to be at play between the two cell lines. The contribution of CpG methylation to this change in PXDN expression remains unknown, as does the nature of the interaction between the oestrogen receptors and the gene. Further research is required to clarify the mechanisms involved.Item Differential expression analysis of PMA and 1,25(OH)2D3-induced monocyte-to-macrophage differentiation in THP-1 cells(University of the Witwatersrand, Johannesburg, 2023-09) Perumal, Kelda Chloe; Meyer, Vanessa; Gentle, NikkiThe process of monocyte-to-macrophage differentiation is studied in vitro through the use of promonocytic model cell lines, such as the THP-1 cell line, where commonly used differentiation inducing agents include phorbol-12-myristate-13-acetate (PMA) and the active metabolite of vitamin D3, (1,25(OH)2D3; VD3). While both induce differentiation, differences in their mechanisms of action, as well as how the end states of the differentiation process differ, are not well understood. Therefore, this study used computational approaches to compare the effects of PMA and VD3 on the differentiation of monocytes into macrophages, using the promonocytic THP-1 cell line. Through the use of RNA-sequencing, gene expression was quantified in differentiated and undifferentiated THP-1 cells, treated with both PMA and VD3. Differential gene expression analysis was performed to determine genes that were differentially expressed as a result of either treatment relative to the untreated cells. This was followed by over-representation analysis to determine the pathways and processes in which the differentially expressed genes (DEGs) were involved. PMA treatment (3 989 DEGs) resulted in more changes in expression relative to VD3 treatment, where only 384 genes were found to be differentially expressed in response to treatment with VD3. Only TFE3, KIT and TRIB1 were observed to be crucial to the process of differentiation, irrespective of treatment. Apart from this, the treatments were observed to largely involve different biological pathways, resulting in cells that were phenotypically distinct from each other at the transcriptional level. This included changes observed in the expression of genes encoding transcription factors known to be involved in the differentiation process, such as CEBPA, GATA2, IRF8 and PU.1, as well as those encoding surface markers representative of monocytes and macrophages, such as CD14, CD64 and CD11b. The expression patterns observed here indicate that, at least at the concentrations and time points included in this study, PMA and VD3 induce macrophage-like cells that are at different stages of differentiation and are not comparable to either each other or primary macrophages. Furthermore, key differences observed in the expression of genes encoding pathogen recognition receptors and cytokines suggest that which differentiation inducing agent is used may have important implications for these cells’ capacity to recognise pathogens and produce cytokines. The findings of this study therefore emphasise that it is crucial to carefully consider the choice of differentiation-inducing agent when using THP-1 cells as an experimental system for studying monocyte-to-macrophage differentiation.