3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item Novel LRP/LR induced stem-cell-like cells to aid wound healing and regeneration(2019) Chigumba, StephanieThe 37/67kDa Laminin Receptor (LRP/LR) LR is a multifunctional cell surface receptor that maintains several survival processes. It has recently been found that there is a direct relationship between LRP/LR and the stem cell marker telomerase. Studies have shown that overexpression of FLAG tagged LRP increases hTERT levels and telomerase activity. Telomerase is a reverse transcriptase enzyme found in actively dividing cells whose core function involves telomere maintenance and elongation. The rate limiting component of telomerase, hTERT, is also often upregulated in rapidly dividing cells to aid stem cell renewal and cell survival and its ectopic expression can induce epithelial to mesenchymal transition (EMT) resulting in stem cell like characteristics. In adults, the primary role of stem cells is to repair and regenerate tissue. Hence, this study aims to determine whether overexpressing LRP::FLAG and the subsequent increase in hTERT levels induces stem-cell-like characteristics and promotes repair and regeneration in MRC5 lung fibroblasts and HEK293 embryonic kidney cells. Cells were stably transfected with the pCIneo-moLRP-FLAG plasmid in order to induce LRP::FLAG overexpression. Post-transfection, an increase in hTERT and phospho-TERT protein levels was observed in both cell lines which is crucial in maintaining the self-renewal capacity of stem cells. Additionally, an increase in the levels of pluripotency stem cell markers involved in cell reprogramming and alkaline phosphatase activity was also observed for HEK293 after transfection. However, in MRC5 cells there was an insufficient expression of reprogramming factors but, a Cadherin switch indicative of EMT. Moreover, HEK293 cells overexpressing LRP::FLAG showed no significant changes in the protein levels of the pro-inflammatory cytokine NF-κB1 and an increase in the anti-inflammatory cytokine TGFβ1 which modulates wound healing. In turn, it led to an increase in the adhesion and migratory capacity of HEK293 cells. This data suggests that overexpressing LRP::FLAG induces EMT similar to that observed during induced cell reprogramming and could possibly promote wound healing by upregulating TERT and TGFβ1 protein levels resulting in stem-cell-like characteristics.Item Targeting LRP/LR for the treatment of metastatic lung and colorectal cancer through impediment of telomerase activity(2018) Baichan, PavanThe 37 kDa/67 kDa laminin receptor (LRP/LR) plays a vital role in the malignancy of various cancer types contributing to invasion, adhesion, apoptosis evasion, proliferation and tumour angiogenesis. In addition, LRP/LR interacts with the catalytic reverse transcriptase subunit, TERT, of the ribonucleoprotein telomerase. Both LRP/LR and telomerase are implicated in cancer progression and knockdown of LRP/LR causes a decrease in telomerase activity in breast cancer cells. In the current study, LRP/LR was downregulated in lung adenocarcinoma (A549) and late-stage colorectal carcinoma (DLD-1) cells in an attempt to impede telomerase activity and ultimately impede cancer progression. Western blotting analysis showed a significant decrease in LRP/LR levels in HEK293 (Human Embryonic Kidney Cells) and A549 cells after siRNA mediated LRP/LR knockdown. To confirm LRP/LR knockdown confocal microscopy was performed; a reduction in LRP/LR protein levels was observed which also resulted in a subsequent decrease in hTERT mRNA levels with a corresponding decrease in hTERT levels in HEK293, A549, and DLD-1 cell lines. Furthermore, siRNA mediated knockdown of LRP/LR significantly decreased telomerase activity in HEK293, A549, and DLD-1 cells. The effect of LRP/LR downregulation on cellular viability was investigated via the MTT assay and a significant decrease in cell viability in A549 and DLD-1 cells was observed. Since downregulation of LRP/LR impedes telomerase activity and decreases cell viability, siRNAs directed against LRP mRNA acts as potential alternative therapeutic tools for treatment of lung adenocarcinoma and late-stage colorectal carcinoma.Item Molecular basis of metabolic reprogramming in innate immune cells: impact of drugs on the mitochondrial function(2016) Xelwa, Ntombikayise Hendrietta MarciaThis study focused on reprogramming of energy metabolism of cancer cells, since most cancer and proliferating cells have been shown to display a metabolic shift by displaying increased dependence on glycolysis and reduced oxidative phosphorylation (OXPHOS) for energy. Dichloroacetate (DCA) and Methyl pyruvate (MP) were used to attempt the reversal of the metabolic program of THP-1 cells. Flow cytometry was used to determine the mode of cell death and to analyse the changes in cell cycle. In this study, an overexpression of TLR4 was observed in THP-1 cells treated with 5ng/ml of lipopolysaccharides (LPS). Further analysis of cell death showed that MP and DCA-treated cells resulted to minimal induction of apoptotic cell death. This suggests that the 2 drugs (MP and DCA) cause cell death via apoptosis. Furthermore, LPS treated cells (infected cancer cells) showed an increase in glycolysis (Warburg effect). This study has shown that indeed treatment with drugs such as MP and DCA was effective in reversing the glycolytic phenotype of THP-1 cells, resulting in cell death via apoptosis by boosting OXPHOS.Item The biochemical functions of the Retinoblastoma binding protein 6 (RBBP 6) isoforms in metabolic reprogramming occurring during carcinogenesis(2018) Nsingwane, ZaneleABSTRACT The Retinoblastoma binding protein 6 is dysregulated in most cancers, indicating it may play a role in metabolic reprograming- a hallmark of carcinogenesis. Its human isoforms have been shown to play diverse roles in apoptosis. This study aimed to elucidate biochemical roles of RBBP6 isoforms in metabolic reprogramming during carcinogenesis. Drosophila melanogaster wild type and p53 null mutants were treated with drug permutations of irinotecan (DNA damaging agent) and exogenous pyruvate to perturb metabolism. Moreover, using RT-PCR and Western blot expression profiles of SNAMA (Drosophila Orthologue of RBBP6) isoforms were shown followed by survival studies to investigate the effects of these drugs. Furthermore, using bioinformatics the domains of RBBP6 isoforms in various species were shown. Results indicate that RBBP6 isoforms show contrasting expression patterns. Furthermore, exogenous pyruvate protects the wild type flies from irinotecan toxicity while killing p53 null mutants. RBBP6 proves to be a potential druggable target for chemotherapy.Item The integrative role of Glycogen Synthase Kinase 3B (GSK3B) in adhesion-originating signals human oesophageal squamous cell carcinoma cells(2017) Mahomed, SabeehaThe serine-threonine kinase glycogen synthase kinase 3β (GSK3β), has, in recent years, become established as hub for a myriad of intracellular signalling pathways. Many of these pathways have been implicated in cell cycle progression and proliferation in a multitude of carcinomas. GSK3β is ubiquitously expressed and considered to be constitutively active, and phosphorylation at the N-terminus serine 9 residue results in the inhibition. Interestingly, another prominent phosphorylation at tyrosine 216 in the activation loop has been reported to enhance GSK3β activity 200-fold. Its’ role in human oesophageal squamous cell carcinoma (HOSCC) migration, however is not well characterised. This study established that both active and inactive GSK3β are present in high abundance in HOSCC cells at rest. In order to characterise the influence of GSK3β on the migratory phenotype of HOSCC, focal adhesion kinase (FAK), a focal adhesion-associated protein known to be increasingly activated during cell migration (FAK(Tyr397)) was used as a marker for cell migration. The abundance of active GSK3β (pGSK3β(Tyr216)) was found to fluctuate during cell migration into a wound over 24 hours. Further investigation via the abrogation of GSK3β revealed that the observed variation was not a result of migration. Instead active GSK3β was found to differentially influence the migratory response observed in HOSCC cells by either 1.) promoting laemellipod extension and cell migration or 2.) partially-abrogate these processes. These findings however, did not produce the expected biochemical alterations with respect to the abundance of pFAK(Tyr397). Moreover, the effect of GSK3β-inhibition on HOSCC was shown to be dependent on the order in which wound initiation and GSK3β occurred, as abrogation of GSK3β prior to wound initiation was seen to either 1.) simply sustain the changes in the migratory response or 2.) produce little variation in the migratory response, suggesting the existence of “rescue-signalling”. These influences thus present GSK3β as a key regulator in HOSCC migration. Additionally culturing HOSCC cells on either collagen I of fibronectin, presented general decreases in the abundance of activated FAK, suggesting that varying suggesting that no one ECM-component, but rather the cell surface receptor distribution has become more diverse. This diversity may be a contributing factor to the varied influence of active GSKβ on the migratory response observed in HOSCC.Item Investigating telomere dynamics in oesophageal squamous carcinoma cells using standard and gold nanoparticle-based assays(2017) Bernert, MartinCancer is characterised by abnormal cell proliferation and is one of the leading causes of death in first world countries and the second leading cause in developing countries. In 2012 alone, over 14 million cases were reported and over 8 million deaths were attributed to cancer worldwide, with sub-Saharan Africa, especially South Africa having one of the highest oesophageal cancer rates in the world. An important aspect of cancer is the telomeres, which are 10-15kbp of TTAGGG DNA repeats in humans at the ends of chromosomes. These repeats are maintained by the enzyme telomerase. Up to 90% of all cancers show increased telomerase activity to overcome the "end-replication" problem in which the telomeres shorten after each cell division. This eventually leads to cellular senescence. Due to the high number of cancers relying on increased telomerase activity to bypass senescence, telomerase could be a viable target for anti-cancer therapies. The limiting factor of the multi-subunit telomerase enzyme is its telomerase reverse transcriptase component (hTERT). hTERT has also been shown to migrate to the mitochondria during times of high oxidative stress caused by reactive oxygen species (ROS). Here it confers protection to the mitochondria against ROS, potentially preventing the cell form undergoing apoptosis and reaching senescence. This can potentially be detrimental, as cells become damaged by the ROS and continue dividing. This could lead to further genetic damage. Metformin, a drug used for the treatment of type-2 diabetes, has been linked to lower incidences of cancer. The mode of action of metformin is not yet fully understood, however it is known that it affects the mitochondria. Since hTERT and metformin could co-localise, the drug may influence hTERT and potentially telomerase activity. This makes metformin an anticancer candidate to be used in conjunction with traditional anticancer therapies. To determine telomerase activity in metformin treated oesophageal carcinoma cells, qPCR based telomerase activity assays must be used. These assays can be very expensive and time consuming, so a faster and cheaper alternative would be beneficial. Therefore, the aim of this project was to alter and improve a nanoparticle based detection method for telomerase activity, by decreasing the time required to prepare the DNA functionalised nanoparticles as well as determining a more rapid method of data measurement, and compare it to conventional qPCR based techniques (TRAPeze RT Telomerase Activity Kit – Merck). Thereafter the effects of the metformin treatment on telomere dynamics, such as telomere length, telomerase activity and hTERT mRNA expression, in oesophageal squamous carcinoma cells were determined. Gold nanoparticles were synthesised and functionalised with thiolated-DNA (telomerase substrate). These functionalised particles were characterised using transmission electron microscopy. To assess telomerase activity the extracted protein was added to the functionalised nanoparticle solution and allowed to elongate the coupled DNA. A characteristic of gold nanoparticles is that the size of the particles as well as their proximity to one another determines the colour of the nanoparticle solution. Due to the steric hindrance caused by the now elongated DNA, a distinct colour change was observable. The change in absorption spectra of the nanoparticle solution was recorded after the enzyme elongated the substrate. This nanoparticle based assay was then compared to TRAPeze RT Telomerase detection kit (Merck-Millipore) as a positive control. Using the conventional qPCR based telomerase activity assay, it was found that metformin significantly decreased telomerase activity in oesophageal cancer cell lines, however this was not seen using the nanoparticle assay. A colour change was observed with the nanoparticle assay compared to the negative control reflecting detection of telomerase activity. However, no significant decrease in telomerase activity could be detected due to metformin treatment. More optimisation is required, however this technique has great potential, as nanoparticle based assays are also known for their high sensitivity. This technique is also far more rapid and significantly cheaper that the qPCR based method. The gold nanoparticle based telomerase activity assay could become an alternative to conventional qPCR based techniques.