Electronic Theses and Dissertations (Masters)

Permanent URI for this collectionhttps://hdl.handle.net/10539/38018

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Subject: search.filters.subject.SDG-4: Quality education

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    Unveiling the biochemical pathway between Type 2 Diabetes Mellitus and early Alzheimer’s disease
    (University of the Witwatersrand, Johannesburg, 2024-08) Tooray, Shweta; van der Merwe, Eloise
    Research related to Alzheimer's Disease (AD) remains a focal point in neurodegeneration studies. This is due to the severity of AD and the clear necessity for non-palliative treatment approaches, as underscored by the high prevalence of the disease. The combined formation of extracellular senile plaques and neurofibrillary tangles (NFTs) plays a crucial role in the development of the cognitive and behavioural symptoms observed in individuals with AD. Despite extensive research efforts, discovering a definitive cure for the disease remains a challenge. Therefore, it is imperative to explore new perspectives and identify the upstream molecular mechanisms that contribute to the onset of the disease. Metabolic disorders are widely recognized as a significant risk factor for AD. Specifically, the metabolic syndrome, Type 2 Diabetes Mellitus (T2DM), is connected to neurodegeneration by promoting the accumulation of neurotoxins, inducing neuronal stress, affecting synaptic communication, and leading to brain atrophy. Individuals with T2DM have an increased risk of developing dementia, with hyperglycaemia exacerbating the impact of AD by causing mitochondrial dysfunction and oxidative stress through reactive oxygen species (ROS) formation, which are also present in AD. Additionally, patients with T2DM exhibit shorter telomeres linked to cell death, which is an associated risk factor for developing AD. These key pathways involved in connecting T2DM and AD were explored in the current study to enhance the understanding of the early events that precede AD. Glucose uptake was measured and observed to decrease over time as a potentially protective response of the cell. Subsequently, mitochondrial activity, assessed using the Alamar blue assay, was found to be heightened as an initial protective mechanism of Aβ42. This was later overwhelmed by the elevated ROS detected through a Total ROS assay kit, induced by the hyperglycaemic state of T2DM. In turn causing the amount of Aβ42 to become toxic and leading to a decline in mitochondrial DNA (mtDNA) over time as measured through qPCR. Additionally, the increases in ROS induced by hyperglycaemia resulted in oxidative damage to telomeres. Simultaneously, Aβ42 physically hinders telomere-telomerase binding, leading to reduced telomerase activity and consequently, shorter telomeres. Furthermore, this study reveals, for the first time, that the novel glucose-lowering drug (GLD) caused an increase in Aβ42 production in the T2DM cell model, whilst effectively decreasing ROS production over a 24-hour period compared to the untreated cell model. The rise in Aβ42 levels caused by GLD could potentially be working to prevent the increase in hyperglycaemia-induced ROS through its metal chelating antioxidant properties by scavenging ROS, in the presence of oxidative stress associated with T2DM. These findings are indicative of an appealing function of GLD by reducing ROS and thereby impeding the progression towards AD. Hence making GLD an attractive therapeutic option for the treatment and/or prevention of AD.
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    Knockdown of long non-coding RNA PANDA improves the cytotoxic effects of cisplatin in oesophageal squamous cell carcinoma cell lines
    (University of the Witwatersrand, Johannesburg, 2024-11) Moonsamy, Sasha Sarasvathee Keshnee; Mavri-Damelin, Demetra; Jivan, Rupal
    Oesophageal cancer is one of the leading causes of cancer death worldwide, of which oesophageal squamous cell carcinoma (OSCC) is the major subtype in southern and eastern Africa. Cisplatin is a well-established drug used to treat multiple cancers, including OSCC. Drug resistance is a major impediment to continued cisplatin therapy in numerous cancers. LncRNA P21-associated non-coding RNA DNA damaged activated RNA (PANDA) is known to function in cell cycle regulation in response to DNA damage and is upregulated in OSCC. We aim to determine lncRNA PANDA expression in South African-derived OSCC cells and establish whether down-regulation of this lncRNA can be used to supplement cisplatin therapy. In this study, MTT assays were performed to determine the EC50 concentrations of cisplatin in OSCC (WHCO1, WHCO5, and SNO) cells and HEK293 cells as a non-cancer control. The cytotoxic effects of cisplatin were exerted in all cell lines, with WHCO5 and SNO appearing more responsive to cisplatin than WHCO1 and HEK293. RT-PCR was used to detect if lncRNA PANDA is expressed in untreated and cisplatin-treated cells and was detected in all cell lines. Knockdown of lncRNA PANDA by siRNA was assessed with RT-PCR. Phase contrast microscopy was used to assess whether siRNA reagents altered cell morphology at 5, 24, and 48 hours post treatment. No significant alterations in cell morphology were observed in WHCO1, WHCO5, SNO, and HEK293 cells. MTT assay evaluation after 48 hours of cisplatin exposure, with or without siRNA for lncRNA PANDA, showed a significant reduction in EC50 concentrations in WHCO5, SNO, and HEK293 cell lines, suggesting that knockdown of lncRNA PANDA may improve cisplatin cytotoxicity in some cell lines. However, the EC50 values were higher with lncRNA PANDA knockdown in the WHCO1 cell line, suggesting that not all OSCC cell types may be responsive to this approach. In conclusion, lncRNA PANDA is expressed in response to cisplatin-induced DNA damage, and the down regulation of lncRNA PANDA improves the cytotoxic effects of cisplatin; however, further investigations are warranted in OSCC.
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    Using ChIP-seq and Gene Expression Microarray data to explore transcriptional dysregulation of PXDN and PXDNL in cardiovascular diseases
    (University of the Witwatersrand, Johannesburg, 2024) Naidoo, Shiven; Gentle, Nikki; Mavri-Damelin, Demetra
    Background: Cardiovascular diseases (CVDs) remain one of the leading causes of death globally. The genes PXDN and PXDNL are both expressed in the cardiovascular system, and their dysregulation has been linked to various disorders, including CVDs, but little is known of their transcriptional regulation in the cardiovascular system or their roles in CVD pathogenesis. Methods: This study developed two custom bioinformatics pipelines in R to mine and analyse ChIP-seq data from ChIP-Atlas and gene expression microarray data from the Gene Expression Omnibus (GEO). The first pipeline used ChIPseeker to identify regulatory transcription factors (TFs) of PXDN and PXDNL in cardiovascular cells and tissues. ChIP-seq data from 400 experiments across 63 TFs was filtered to isolate TFs with high confidence binding peaks in the promoter and first intron of PXDN and PXDNL. The second pipeline used R Bioconductor packages to explore the expression profiles of PXDN, PXDNL, and their TFs in seven microarray datasets across three CVD-related contexts: cardiomyopathies, heart failure and TNF-α stimulation. Results and discussion: This study identified 27 TFs binding to PXDN and 18 TFs binding to PXDNL in cardiovascular cells. Sixteen of these TFs were shared by both PXDN and PXDNL, suggesting potential coregulatory mechanisms in cardiovascular cells where they are both expressed. Unique TFs were also identified for PXDN (11) and PXDNL (2). Differential gene expression analysis revealed no significant change in expression (log2FC > 0.5; p.adj < 0.05) for PXDN, PXDNL and many of their identified TFs in the CVD-related conditions investigated, suggesting that changes at the transcript level may not contribute to the progression of these conditions. Conclusions: This study advances our understanding of the transcriptional regulation of PXDN and PXDNL in healthy cardiovascular cells as well as their expression levels in the investigated CVD-related contexts. This study also contributes a bioinformatics pipeline which can be further developed and applied to analysing data from ChIP-Atlas and GEO. Future research can elucidate the roles of each TF in regulating PXDN and PXDNL in healthy and diseased cell lines