Electronic Theses and Dissertations (PhDs)

Permanent URI for this collectionhttps://hdl.handle.net/10539/37932

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Start date: 2024Subject: search.filters.subject.UCTD

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Now showing 1 - 6 of 6
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    Identification of (Novel) Immune Targets with Potential Roles in the Progression of Pancreatic Ductal Adenocarcinoma (PDAC)
    (University of the Witwatersrand, Johannesburg, 2024) Nsingwane, Zanele; Nweke, Ekene
    Background: Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with a growing incidence and mortality despite novel therapeutic strategies. Its aggressiveness and difficulty to treat suggest the need for a better understanding of associated molecular mechanisms which could be targeted for treatment. The complement signalling pathway may play diverse roles in PDAC by eliciting an immune response, inducing inflammatory responses, and may elevate pathways linked to chemoresistance. However, their role in the progression of PDAC is not fully understood. This study aimed to identify potential immune response-related targets in a group of patients. Methods: In this study, 30 tissue samples (tumours and corresponding normal tissues) were obtained from 15 PDAC patients, 34 plasma samples were obtained from 25 PDAC patients, 6 patients with chronic pancreatitis, and 3 healthy control participants. Targeted pathway-specific PCR analysis was conducted to determine the gene expression profiles of immune-response-related genes. The circulating levels of complement proteins C3 and C5 were further investigated. Pharmacological inhibition of the complement pathway in MIA PaCa-2 pancreatic cancer cell lines was performed and the effect on cells was assessed by cell proliferation, cell migration, and cell cycle assays. Finally, SWATH-mass spectrometry was performed to identify potential molecular mechanisms during inhibition. Results: The results identified C3 to be overly expressed in early PDAC compared to later stages in plasma (p=0.047). Pharmacological inhibition of the complement pathway led to increased cell growth (p<0.0001), proliferation (p=0.001) and migration (p=0.002) in vitro. Proteomic analysis implicated several proteins such as the mitochondrial and histone proteins, that could play a role in inducing this phenotype. Conclusion: Both Complement C3 and C5 are elevated in PDAC samples compared to healthy ones. Furthermore, the inhibition of the complement pathway was shown in vitro to result in a more aggressive phenotype by stimulating cellular growth, proliferation, and migration, indicating the involvement of complement C3 and C5 in tumour progression. This study helps to further delineate the role of the complement pathway in PDAC progression.
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    An in-silico analysis of the glycosylation inhibitors Brefeldin A and Tunicamycin C in colorectal cancer; characterization of novel targets
    (University of the Witwatersrand, Johannesburg, 2024) Naidoo, Vivash
    Colorectal cancer (CRC), a prevalent malignancy in South Africa, is significantly influenced by posttranslational modifications such as glycosylation. This study investigates the complex interactions between genes, signalling pathways, and cellular processes involved in CRC progression and glycosylation. The glycosylation inhibitors, Tunicamycin and Brefeldin A, are known to hinder colon cancer cell proliferation, migration, and invasion, making them potential therapeutic agents. We used Swiss Target Prediction Software to identify target proteins for both compounds and revealed that Protein Kinase C Alpha (PRKCA), Peroxisome Proliferator- Activated Receptor Gamma (PPARG), and Mitogen-Activated Protein Kinase 1 (MAP2K1) are specific for Brefeldin A, and TK1 and PRKCA for Tunicamycin, respectively. These proteins were selected based on their potential role in the glycosylation process and their role in CRC-related pathways. Further, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis disclosed significantly enriched pathways, including Epstein-Barr virus infection, cellular senescence, and cancer pathways. The 3D-crystallographic structures of PrKC1 (PDB ID 6ar4), TK1 (PDB ID 1w4r), PrCK1 (PDB ID: 6ar4) and MAPK (PDB ID: 3eqc) were retrieved from RCSB Protein Data Bank. The compounds BSP and EGCG were downloaded from PubChem. All non-relevant co-crystallized molecules, including ions, crystallographic water, and others, were removed. Missing residues in the proteins were filled in using the MODELLER algorithm on the UCSF Chimera Graphic User Interface. Molecular docking of Tunicamycin C and Brefeldin A was performed with UCSF Chimera, and the docked conformations were visualised in Maestro and Chimera. The complexes with the top docking scores were selected and prepared for molecular dynamics simulation studies to offer structural and dynamic perspectives on the inhibitory potential of the compounds against the target proteins. The Origin Lab software tool was used to post- analyze the docking conformations. Molecular Dynamics simulation was conducted using Graphics Processing Units version of the Particle Mesh Ewald Molecular Dynamics engine in the AMBER18 suite. Our investigation into the dynamic events leading to the proximal binding of Tunicamycin at the pockets of TK1 and PrKC1 suggested that the binding of Tunicamycin induced a conformational perturbation of the 3D structures of these proteins, resulting in a structural deviation that inhibited their activity. Tunicamycin's time-based dynamics indicated a stable pattern, leading to optimal interaction and maximal stabilization in the hydrophobic pockets of TK1 and PrKC1. Binding energy calculations showed a high-affinity interaction of Tunicamycin with these proteins. Similarly, the structural investigation revealed that the binding of Brefeldin A to Mitogen- Activated Protein Kinase (MAPK) and Protein Kinase C (PrKC1) inhibited their activity. A detailed analysis of active site residues revealed crucial residues that contributed to the binding stabilization of Brefeldin A. It was noted that the Brefeldin A/MAPK complex produced a binding energy of -22.18±4.50Kcal/mol while the Brefeldin A/PrCK1 complex produced a binding energy of -23.90±5.36Kcal/mol. These findings provide crucial insights into designing novel inhibitors of TK1 and PrKC1, potentially blocking glycosylation progression in cancer treatment. This study underscores the potential for exploiting glycosylation inhibition as a therapeutic strategy against CRC, opening avenues to mitigate cancer progression
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    Gene expression patterns of signalling pathways in PDAC: towards inhibiting metastases
    (University of the Witwatersrand, Johannesburg, 2024) Xelwa, Ntombikayise Hendrietta Marcia
    PDAC has a poor prognosis, with its prevalence varying by geographical location. In South Africa, PDAC ranked seventh among all cancer-related deaths in 2020 for both sexes. Specifically, it was the seventh leading cause of cancer death among males and the sixth among females. In 2020, an estimated 1,982 cancer deaths in South Africa were attributed to pancreatic cancer, with 1,006 occurring in males and 976 in females. However, the annual reported number of PDAC deaths in South Africa varies. This study aimed to identify potential novel therapeutic targets for PDAC in patients from the African population. Following ethical approval, tissue from fifteen patients (15 tumour and 15 corresponding normal tissues) were obtained during Whipple procedures from PDAC patients who consented to the study. Despite the development of new treatment strategies, patient outcomes have not significantly improved underscoring the necessity for extensive research to identify novel treatment options. A discovery study was conducted to determine the gene expression profile of signalling pathway-related genes using PDAC tissue samples. Top upregulated pathways included those involved in cytokine signalling, receptor kinase signalling, and PI3/Akt signalling. SPP1 was one of the most highly expressed genes identified in PDAC patients compared to normal corresponding tissues suggesting its potential role in the progression of the disease. To investigate SPP1's role in PDAC, RNA interference was employed to knockdown SPP1 in a PDAC cell line, MIA PaCa-2. Knockdown was confirmed by a significant reduction in SPP1 expression at the mRNA level. Combining SPP1 knockdown with conventional chemotherapy used for PDAC, gemcitabine, resulted in a synergistic effect, leading to an enhanced early apoptotic response. The study also examined the migratory and invasive capabilities of MIA PaCa-2 cells, revealing a noticeable decline in these abilities upon reduction in SPP1 expression with gemcitabine treatment. Furthermore, proteomic analyses uncovered the complex network of cellular processes influenced by the downregulation of SPP1 and the synergistic effects of combination therapy. Altogether, the findings from this study demonstrate the role of SPP1 in PDAC indicating that it could serve as a promising therapeutic target. The synergistic effects observed when SPP1 knockdown was combined with gemcitabine treatment suggest a potential avenue for developing more effective treatments for PDAC while exploring tumour cell adaptation for survival.
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    Evaluation of the postgraduate family medicine decentralised training programme at the university of Witwatersrand, South Africa, using the logic model
    (University of the Witwatersrand, Johannesburg, 2024) Erumeda, Neetha Joe; George, Ann Zeta
    Postgraduate family medicine decentralised training programmes were implemented in South Africa about 15 years ago, but the University of the Witwatersrand’s programme has not been comprehensively evaluated. This study evaluated the programme using a complex programme evaluation logic model based on linearity theory. This theory assumes ideal inputs and processes produce good programmatic outputs and outcomes. Resources and support were evaluated as inputs, postgraduate supervision and workplace-based learning as processes, supervisory feedback as outputs and workplace-based assessments as outcomes. A parallel convergent mixed-methods instrumental case study was conducted with purposively-sampled family physicians (n=11) and trainees (n=11) from five decentralised training sites. Semi-structured interviews were audio recorded, transcribed verbatim, and analysed inductively using MAXQDA 2020 software. Descriptive statistical analysis was conducted on components of registrars’ learning portfolios (scores, supervisory feedback, and skills competence) and examination results using Stata 14.2 software. An integrative analysis involving transforming the quantitative results to qualitative findings and drawing meta-inferences was conducted. The integrated findings were used to modify the initial logic model and identify key recommendations to optimise the programme. The integrative analysis identified the need for more material and human resources, university and district management support, and standardised resources, supervision, and learning practices. Supervisors’ knowledge, skills, and behaviours varied across sites and their feedback was insufficient regarding soft skills like clinical reasoning and patient negotiation. Workplace-based assessments did not meet the required standards across training years and districts. Interpersonal interactions with patients, peers, supervisors and other professionals, engagement in district activities, promoted learning. Registrars’ professionalism and self-learning need improvement. The key recommendations include more explicit national guidelines, sufficient support from the provincial department, university, and district management, well maintained infrastructure, sufficient skilled supervisors, more professional development training for supervisors, protected time for registrar learning, and better use of self-learning and reflection. Emulating successful contextual adaptations while addressing challenges across sites contributes to thriving decentralised training programmes in district health systems. An improved understanding of the concepts and their interrelationships in training programmes could be translated to similar decentralised training platforms across medical disciplines of sub-Saharan Africa or low-middle income countries
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    The effects of indigenous South African plant extracts (cotyledon c. orbiculata and tulbaghia. violacea) on triple negative breast cancer cells
    (University of the Witwatersrand, Johannesburg, 2024) Alaouna, Mohammed; Dlamini, Zodwa
    This dissertation explored the potential therapeutic applications of water and methanol extracts of C. orbiculata and Tulbaghia violacea, indigenous to Southern Africa, targeting triple-negative breast cancer (TNBC). TNBC, a significant subset of breast cancer cases, is notably challenging because of the absence of key hormone receptors, often leading to less favourable patient outcomes and a high relapse rate within five years. The research approach was both thorough and meticulous, utilising two cell lines: one representing normal breast tissue and the other representing TNBC. Extensive cytotoxicity assays were conducted to determine the IC50 values for TNBC cells, which is critical for understanding how plant extracts affect cellular activities such as migration, invasion, adhesion, cell cycle regulation, and apoptosis induction. Additionally, the antioxidant properties of these extracts were examined, which showed significant effects, especially in the aqueous extract of Tulbaghia violacea, on TNBC cellular dynamics. This study employed a comprehensive array of analytical techniques, including Fourier transform infrared spectroscopy (FTIR), gas chromatography-mass spectrometry (GC-MS), and nuclear magnetic resonance (NMR) spectroscopy, to identify the specific molecular constituents of these extracts. Computational docking studies have focused on the interactions between these molecules and the anti-apoptotic protein, COX2. Whole transcriptome sequencing of RNA from both TNBC and normal breast cells treated with T. violacea extract provided valuable insights into the affected signaling pathways. An antibody array assay further elucidated protein changes in the receptor tyrosine kinase (RTK) pathway. The half-maximal inhibitory concentration (IC50) values were determined for the aqueous and methanol extracts of T. violacea at 400 μg/mL and 820 μg/mL, respectively, and for C. orbiculata at 830 μg/mL and 700 μg/mL, respectively. Exposure to the water-soluble extract of T. violacea resulted in a marked increase in apoptosis in TNBC cells, with approximately 82% undergoing programmed cell death, compared to 32% in normal breast cells. Chemical profiling identified a range of compounds, including 36 distinct compounds identified through GC-MS and 61 identified through NMR, many of which bear structural similarities to known anti-cancer agents. Notably, five compounds demonstrated a high affinity forbinding to COX2, with d-glycero-d-galacto-heptose achieving an impressive docking score, surpassing several established COX2 inhibitors. This study highlights the therapeutic potential of T. violacea compounds and lays the groundwork for further exploration of their mechanisms of action and potential applications in cancer treatment. This emphasises the importance of investigating natural plant extracts as a source for the development of new and effective treatments for TNBC, which is an area of urgent need in oncology
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    An ethico-legal analysis of broad consent for biobank research in South Africa: Towards an enabling framework
    (University of the Witwatersrand, Johannesburg, 2024) Maseme, Mantombi Rebecca
    Biobanks preserve collections of human biological material and data for the benefit of medical research. Using and transferring human biological data and materials both inside and outside of South Africa is often a requirement of biobank research. Broad consent is allowed by the South African National Department of Health Ethics Guidelines but appears to be prohibited by section 13(1) of the Protection of Personal Information Act 4 of 2013. Additionally, the Act mandates that all personal data (including biobank sample data) be collected for legitimate, definite, and clearly stated purposes. There is room for several interpretations of the Act because of this discord between the two instruments. Given the connection between the transfer of samples and data, the long-term nature of biobanking, which makes it impractical to provide too much or adequate information because it is simply not available at the time of sample collection, and the various ways that the Protection of Personal Information Act 4 of 2013 have been interpreted, I aim to respond to the following question: How should South Africa’s current regulatory framework appropriately permit broad consent use for biobank research where the transfer of samples and their associated data are contemplated? The research question is addressed by applying ethical principles and theories, as well as analysing and evaluating relevant ethico-legal frameworks and literature. The study involves no research participants and no collection or analysis of any new data. Arguments for and against using broad consent for biobank research are discussed by demonstrating the potential for biobank research to do a great deal of good for humanity; the ambiguity in the current regulatory framework regarding whether broad consent is permissible for personal information/data; and the ethical justifiability of broad consent. In summary, the proposed regulatory framework amendments are those that would be required to allow for ethically justifiable biobank research broad consent use. These include removing regulatory ambiguity regarding broad consent use, ensuring adequate safeguards for research participants by specifying rules for data access and personal information processing, and incorporating consent form information requirements into the national Consent Template as specified in the National Department of Health Ethics Guidelines