Faculty of Science (ETDs)
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Item Identifying Markers of Differentiation in Monocyte-Derived-Macrophages(University of the Witwatersrand, Johannesburg, 2024-08) Gibson, Matthew Leo; Cronjé, Marianne; Gentle, NikkiThe importance of monocytes and monocyte-derived macrophages (MDMs) in both adaptive and innate immunity makes their study a topic of interest. Monocytes differentiate into macrophages through transcriptomic alterations, resulting in extensive changes in gene expression. Macrophage colony stimulating factor (M-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF) are the two primary cytokines that stimulate this differentiation, and are known to cause partial polarisation towards the M2 and M1 macrophage subtypes, respectively. However, the degree to which this polarisation takes place is not well-characterised. Therefore, this study aimed to use a computational approach to identify the differences and similarities in gene expression changes in macrophages induced with M-CSF and GM-CSF. RNA sequencing data for three human donors was obtained through EBI and used to quantify gene expression changes associated with M-CSF or GM-CSF treatment. Differential gene expression analysis was performed to identify the genes that were differentially expressed as a result of either treatment relative to the untreated monocytes. Over-representation analysis was used to determine the biological processes in which the differentially expressed genes (DEGs) were involved. Finally, transcription factors were identified within the lists of DEGs, as well as the genes encoding their known protein-protein interacting partners. Treatment with M-CSF and GM-CSF induced 4 072 and 4 399 DEGs, respectively, 2 734 of which were common. An examination of these DEGs revealed that the resultant macrophages lacked changes in expression of genes commonly associated with the M1 and M2 polarisation states. An investigation of the DEGs involved in myeloid cell differentiation and the regulation of inflammatory response revealed CCR2, IGF1 and INHBA to be inversely regulated by the two treatments. Furthermore, nine uniquely differentially expressed transcription factors involved in these biological processes were identified, each of which may be contributing to the lack of complete polarisation following differentiation. These results revealed that M-CSF and GM-CSF-induced macrophages, in the absence of activation, experience highly similar gene expression changes and lack changes in the expression of key polarisation marker genes.Item Synthesis and characterization of onion-like carbons for adsorption of tartrazine dye in water(University of the Witwatersrand, Johannesburg, 2024-08) Cwayi, Herbert Qaqambile; Maubane-Nkadimeng, Manoko S.; Coville, Neil J.; Maboya, Winny K.Industrial effluent often can contain a significant amount of synthetic dyes. The discharge of wastewater containing dyes into water streams without proper treatment consequently enters the soil and disturbs the aquatic and terrestrial life. Several wastewater treatment technologies have been proposed that can efficiently reduce the amount of synthetic dyes from the environment, in particular azo dyes. Among all the existing technologies for wastewater treatment, physical adsorption is a popular technology because it is inexpensive, simple, and efficient. The aim of this study is to synthesize, modify, and characterize onion-like carbons (OLCs) derived from four different waste oils for the adsorption of tartrazine dye in water. The OLCs derived from different carbon precursors (waste household oil, restaurant waste oil, engine waste oil, and paraffin oil bath waste) were synthesized using a flame pyrolysis method. The synthesized materials were doped with nitrogen using a chemical vapor deposition technique using 10% ammonia gas as a source of nitrogen. The N-doped OLCs were attached with hydroxyl groups through oxidation reactions to improve their solubility and adsorption efficacy. According to the high-resolution transmission electron microscopy and scanning electron microscopy images, the OLCs from all four-carbon precursor were quasi-spherical, agglomerated, and presented a chain-like structures of multi-layers. The distance between the graphitic layers was found to be 0.32 nm. The average particle size of OLCs was calculated to be 40.2 ± 2.5 nm. Adsorption studies revealed that the initial dye concentration, contact time, and pH of the dye solutions influenced the adsorption capacity of the tetrazine. Nitrogen doping of OLCs increased its capacity to adsorb the tartrazine dye. The nitrogen doped OLCs from household waste oil (H-N-OLCs) and engine waste oil (E-N- OLCs) were used in equilibrium adsorption studies in this work. For a concentration of 20 mg/L of tartrazine dye, an adsorption capacity of 28.9 mg/g was achieved using the N- doped OLCs from household waste oil. The adsorption process follows the pseudo second- order kinetic model. The adsorption isotherm is best fitted to the Freundlich mathematical model. The results obtained show that, the source of oil did not have major effect on the physicochemical properties of OLCs and that incorporation of nitrogen onto carbon matrix enhanced the adsorption of the anionic tartrazine dye in aqueous solution.Item 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, EloiseResearch 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.Item Comparison of different bioassay methods for the assessment of dose-response relationships of entomopathogens and toxins against Helicoverpa armigera (Hübner, 1809) (Lepidoptera: Noctuidae)(University of the Witwatersrand, Johannesburg, 2024-11) Mogadingoane, Keitumetse Neo; Bouwer, GustavBioassays are an important tool for developing bioinsecticides against agricultural pests. The aim of this study was to compare two bioassay methods – diet overlay and droplet feeding – to identify the most suitable method for assessing dose-response relationships of entomopathogens and toxins against second instar larvae of Helicoverpa armigera. The toxins used were purified Bacillus thuringiensis Cry1A.105 and Cry2Ab2.820 proteins, the spore-crystal complex (SCC) of B. thuringiensis subspecies kurstaki strain HD-73, and the entomopathogen Helicoverpa armigera nucleopolyhedrovirus (HearNPV). Based on the heterogeneity factor, coefficient of variance (CV) and relative precision, the diet overlay bioassay was determined to be the best fit for use with HD-73 SCC and HearNPV. Suitable bioassay methods could not be determined for the purified B. thuringiensis proteins due to a poor probit model fit and low precision of estimated LC50s and LD50s. Validation of CV and relative precision across bioassays will ensure the most suitable methods are used for sustainable integrated pest management.Item In Silico Exploration of Endocannabinoid Receptor–CB1 and CB2–Interactions Comparing Cannabidiol and Cannabidiol Diacetate: A Comprehensive Computational Study(University of the Witwatersrand, Johannesburg, 2024-09) Soobben, Marushka; Achilonu, Ikechukwu Anthony; Sayed, YasienIn the rapidly evolving field of cannabinoid research, acetylated phytocannabinoids such as cannabidiol diacetate (CBDDA) have shown prominence due to its enhanced effects compared to its natural counterpart, cannabidiol (CBD). Despite the growing popularity in the consumption of acetylated phytocannabinoids, in-depth research on its pharmacological impact, especially on CB1 and CB2 receptors, remains scarce. With rising reports of adverse reactions to acetylated phytocannabinoids, a molecular understanding of their interaction with endocannabinoid receptors (CBRs) is imperative. This study aimed to fill this knowledge gap by analysing receptor interactions of CBDDA in comparison with receptor interactions of CBD. The study showed that CBDDA forms stronger interactions with CBRs than CBD. Recognised for its heightened potency, the potential of CBDDA as a biopharmaceutical product was examined. CBR interactions with known endocannabinoids, agonists and inverse agonists validated the computational models used to determine the difference in conformational dynamics upon ligand binding. In this work, bioinformatics, molecular docking, and molecular dynamics (MD) simulations were used to determine the structural differences of CBRs when bound to CBD/CBDDA. Simulations in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and water environment successfully mimicked physiological conditions. Subsequent high-throughput virtual screening (HTVS) was conducted using CBDDA as a reference where ligands 142730975 and 21568811 were identified as the top scoring hits for CB1 and CB2 receptors, respectively. The identification of these ligands via HTVS highlights the therapeutic potential of targeting CBRs and the biopharmaceutical potential of CBDDA. This study elucidates the specific interactions of CBD and CBDDA with CB1 and CB2 receptors, laying a foundation for assessing the safety and efficacy of acetylated phytocannabinoids. Overall, the differential interaction of CBDDA compared to CBD with CBRs suggests that acetylation changes the conformational dynamics of CBRs thereby potentially affecting signalling. The identification of ligands 142730975 and 21568811 as strong interactors with the receptors may provide valuable leads for the development of new cannabinoid-based therapies.Item Assessing the inter-annual and inter-seasonal climate-induced variation in caseload of respiratory diseases(University of the Witwatersrand, Johannesburg, 2024-06) Motlogeloa, OgoneIn South Africa, acute upper respiratory diseases pose a significant public health challenge, influenced heavily by climatic factors. Recognizing the critical need for detailed seasonal analysis. This thesis delves into the inter-annual and inter-seasonal impacts of climate on disease caseloads, offering four pivotal contributions to health biometeorology. The first contribution refines the understanding of the acute upper respiratory disease season in South Africa, previously recognized as the winter months of May to September. This research provides a more granular analysis by pinpointing specific onset timings and fluctuations within the season that are crucial for optimizing healthcare responses, particularly in vaccination schedules. The second contribution is an in-depth analysis of climatic variables affecting acute upper respiratory disease prevalence. Utilizing Spearman's correlation analyses and the Distributed Lag Non-linear Model across Johannesburg, Cape Town, and Gqeberha, this study identifies negative correlations between temperature and disease cases, pinpointing significant risk thresholds most prevalent during the winter peak. The third contribution investigates the impact of extreme climate events (ECEs) over twelve years, elucidating how, while individual ECEs influence medical aid claims and disease incidence, it is the broader seasonal patterns that predominantly dictate acute upper respiratory disease prevalence. The fourth contribution offers a nuanced exploration of the climate-health nexus, demonstrating that routine weather variations play a more significant role in the peak transmission of acute upper respiratory viruses than extreme events. This thesis elucidates the substantial yet nuanced influence of climate on respiratory health in South Africa. By specifying the disease season with greater precision and clarifying the relationship between temperature variations and disease prevalence, the research provides essential data for health practitioners to plan targeted interventions. This study moves beyond the focus on extreme weather events to expose the subtler, yet more consistent, impact of seasonal climate shifts on health outcomes, enriching our understanding and serving as a vital reference for enhancing disease preparedness in an era marked by climatic uncertainty.Item Immunomodulation of the innate immune system: The role of vitamin D in the context of monocytes and macrophages(University of the Witwatersrand, Johannesburg, 2024-07) Mol, Bronwyn Ashleigh; Gentle, Nikki; Meyer, VanessaMacrophages are widely distributed cells of the innate immune system with essential roles in homeostasis and disease. Despite concerted efforts, several aspects of macrophage origin, biology, and functionality remain poorly understood. To gain a deeper understanding of these cells, a physiologically relevant, but practical model is required. In vitro, macrophages are principally generated from primary monocytes and monocyte-like cell lines through a natural process referred to as monocyte-to-macrophage differentiation. Monocyte-like cell lines have several practical advantages over the use of primary monocytes with the most commonly employed monocyte-like cell lines being THP-1 and U937 cells. Despite their frequent use, no standardised protocol is employed in the differentiation of monocyte-like cell lines to macrophages. Naturally, this results in large discrepancies and a lack of comparability between studies. Furthermore, many of these protocols are not physiologically relevant and produce macrophages that are not responsive to downstream stimuli. 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the biologically active form of vitamin D3, is a recognised immunomodulator that shows pronounced genomic and non-genomic effects in immune cells. It is also reported as an inducer of monocyte-to-macrophage differentiation, though heavily debated, and a potential macrophage polarisation agent. Despite this, there is relatively little information concerning the role of 1,25(OH)2D3 in monocyte-to-macrophage differentiation and macrophage biology. This study aimed to develop a more physiologically relevant differentiation protocol for the monocyte-like THP-1 and U937 cell lines. This model was then used to investigate the role of 1,25(OH)2D3 in monocyte-to-macrophage differentiation and macrophage biology. Assessment of morphological features and the macrophage markers, CD11b and CD14, indicated that in both THP-1 and U937 cells, differentiation induced using a combination of 5 nM of phorbol 12-myristate 13-acetate (PMA) and 10 nM 1,25(OH)2D3 over 96 hours produced the most mature macrophages. It was observed that 1,25(OH)2D3 alone was not capable of inducing differentiation, yet when combined with PMA, greatly enhanced macrophage features. THP-1 cells are the most widely employed monocyte-like cell line, and are proposed to be the most reflective of primary monocytes. In this study these cells were shown to be more responsive to the effects of 1,25(OH)2D3 than their U937 counterparts. As such, RNA-sequencing was used to explore the efficacy of the proposed differentiation protocols and the influence of 1,25(OH)2D3 on macrophage biology in THP-1 cells. Differential gene expression analysis confirmed that the most effective differentiation protocol was the combination of 5 nM PMA with 10 nM 1,25(OH)2D3 when considering macrophage associated features including transcription factor usage, adhesion, phagocytosis, and cytokine and cytokine receptor expression. This protocol also produced THP-1-derived macrophages that showed increased expression of genes considered to be primary macrophage markers. These results also suggested that THP-1 cells differentiated with neither PMA nor PMA with 1,25(OH)2D3 were likely to represent fully polarised macrophages. 1,25(OH)2D3 treatment of THP-1 monocytes and THP-1-derived macrophages produced distinct gene expression profiles with considerably less overlap than expected. Though 1,25(OH)2D3 treatment often affected similar biological processes in both cell types, the genes within these processes found to be differentially expressed in each cell line were often distinct. For example, in THP-1- derived macrophages, but not THP-1 monocytes, 1,25(OH)2D3 treatment resulted in the increased expression of genes encoding numerous antibacterial peptides, several small GTPases and their regulators. Additionally, several type I interferon response related proteins showed decreased expression, while expression of cytokines and cytokine receptors was variable. This, taken together with the morphological work, indicates two potential roles for 1,25(OH)2D3 in macrophages. Firstly, a protective role as it suggests the potential to prime an antibacterial response, while still balancing inflammatory responses and protecting against autoinflammation induced by aberrant type I interferon response. Secondly, a potential role in determining the morphological features, clearly demonstrated through microscopy, and further suggested by the differential expression of a variety of small GTPases and their regulators.Item Detecting Disease in Citrus Trees using Multispectral UAV Data and Deep Learning Algorithm(University of the Witwatersrand, Johannesburg, 2024-06) Woolfson, Logan Stefan; Adam, ElhadiThere is a high prevalence, in South Africa, of fruit tree related diseases infesting lemon trees, subsequently affecting overall crop yield and quality. Ultimately, the income for the farmers is significantly diminished and limits the supply of nutritional food crops for the South African population, who already suffer from a high incidence of malnutrition. Currently, there are various methods utilized to detect diseases in fruit trees, however they pose limitations in terms of efficiency and accuracy. By employing the use of drones and machine learning methods, fruit tree diseases could be detected at an earlier stage of development and with a much higher level of accuracy. Consequently, the chances of remedying the trees before the disease spreads is greatly improved, and the supply of nutritious fruit within South Africa is increased. This research report’s aim is to investigate the effectiveness of a deep learning algorithm for detecting and classifying diseases in lemon orchards using multispectral drone imagery. This entails assessing the performance of a pretrained ResNet-101 model, fine-tuned with additional sample images, in accurately identifying and classifying diseased lemon trees, specifically those affected by Phytophthora root rot. The methodology involves the utilization of a pretrained ResNet-101 model, a deep learning architecture, and the retraining of its layers with an augmented dataset from multispectral aerial drone images of a lemon orchard. The model is fine-tuned to enhance its ability to discern subtle spectral variations indicative of disease presence. The selection of ResNet-101 is grounded in its proven success in image recognition tasks and transfer learning capabilities. The results obtained demonstrated an impressive accuracy of 80%. The deep learning algorithm exhibited notable performance in distinguishing root rot-affected lemon trees from their healthy counterparts. The findings indicate the promise of utilizing advanced deep learning methods for timely and effective disease detection in agricultural farmlands, facilitating orchard management.Item 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, RupalOesophageal 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.Item Investigating 2-hydroxypropyl-β-cyclodextrin (HPβCD) as a novel therapeutic agent for breast cancer(University of the Witwatersrand, Johannesburg, 2019) Saha, Sourav Taru; Kaur, MandeepCancer cells have an increased need for cholesterol, which is required for cell membrane integrity. Cholesterol accumulation has been described in various malignancies including breast cancer. Cholesterol has also been known to be the precursor of estrogen and vitamin D, both of which play a key role in the histology of breast cancer. Elevated cholesterol levels have been linked to breast cancer therefore depleting cholesterol levels in cancer cells can be a viable strategy for treatment. 2-hydroxypropyl-β-cyclodextrin (HPβCD) is a cholesterol depleting compound which is a cyclic amylose oligomer composed of glucose units. It solubilizes cholesterol and is proven to be toxicologically benign in humans. This led us to hypothesise that it might deplete cholesterol from cancer cells and may prove to be a clinically useful compound. Our work provides experimental evidences to support this hypothesis. We identified the potency of HPβCD in vitro against two breast cancer cell lines: MCF7 (Estrogen positive, ER+), MDA-MB-231 [Triple negative breast cancer (TNBC)], and compared the results against two normal cell lines: MRC-5 (Normal Human Lung Fibroblasts) and HEK-293 (Human embryonic kidney) using cytotoxic, apoptosis and cholesterol based assays. HPβCD treatment reduced intracellular cholesterol resulting in significant breast cancer cell growth inhibition through apoptosis. The results hold true for both ER+ and TNBC. We have also tested HPβCD in vivo in MF-1 mice xenograft model and obtained 73.9%, 94% and 100% reduction in tumour size for late, intermediate and early stage TNBC. These data suggest that HPβCD can prevent cholesterol accumulation in breast cancer cells and is a promising anti- cancer agent