School of Molecular & Cell Biology (ETDs)
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Item Metagenome sequencing of the lichen species Flavopunctelia flaventior and Parmotrema tinctorum from Gauteng, South Africa(University of the Witwatersrand, Johannesburg, 2024-06) Katane, Malebogo Dimpho; Botes, Angela; De Maayer, PieterLichens are defined as a mutualistic association between fungi (mycobiont) and an algal and/or cyanobacterial photobiont. Increasing evidence suggests that lichens comprise more diverse microorganisms than initially thought, where lichens represent an interaction between archaea, bacteria, filamentous fungi, green algae, yeasts, and viruses. Not many comprehensive studies have been done of South African lichen species. The present study employed metagenome sequencing to investigate the lichen microbiomes of Flavopunctelia flaventior and Parmotrema tinctorum sampled from Bryanston, Gauteng province, South Africa. Furthermore, the roles played by the members of the lichen microbiome within symbioses were also studied by functionally annotating the assembled metagenomes of the two lichen species. This study sets the groundwork for future studies on South African lichen species. In Chapter 1, an extensive literature review on lichens, their ecology, taxonomy and biology is discussed. Furthermore, it delves into the existence and shape of the microbiome beyond the mycobiont and the photobiont. Additionally, possible roles that the lichen microbiome may play in sustaining the lichen symbiosis is also discussed. In Chapter 2, the metagenomes of two lichen species were sequenced, the quality of the reads were assessed, and taxonomic classification was performed to elucidate the composition of microorganisms associated with each lichen species. Both microbiomes were dominated by bacteria, with limited fungi, viruses, and archaea. The majority of the identified phyla and genera were found to be common between the two lichen species. Similarities in the core microbiome was accounted for by the fact that F. flaventior and P. tinctorum were sampled from the same location and they are both members of the Parmeliaceae family. In Chapter 3, the metagenomic reads were assembled and functionally annotated using various bioinformatics tools. We demonstrate that the members of the lichen microbiome are involved in the cycling of nutrients such as carbon and nitrogen. We also found differences in carbon fixation pathways, which were attributed to the accessory microbiome. Finally, a summary highlights key results and recommendations on future work that could be undertaken to further provide insight into biological pathways essential to sustain the lichen symbiosis.Item Exploring the Structure, Function and Stability of Glutathione Transferases Engineered from Intra- and Inter-class Consensus Sequences: How Forgiving is Nature?(University of the Witwatersrand, Johannesburg, 2024-10) Mulenga, Thabelo; Achilonu, Ikechukwu; Sayed, YasienProtein folding is an enigmatic biochemical process that is foundational to the structural and functional requirements of a cell. The problem of protein folding, in a nutshell, concerns itself with the rate of protein folding as well as the conversion of amino acids from a linear sequence to a fully folded structure. This problem is partly answered by the existence of folding pathways. The folding funnel was conceptualised as a depiction of folding pathways, and it is a framework that illustrates that native proteins naturally favour the lowest energy state, encountering kinetic and thermodynamic barriers as they fold. Consensus protein design, based on this understanding, aims to: (1) enhance stability and (2) navigate the pitfalls of folding by modifying the folding funnel of a protein. This approach can also shed light on the significance of evolutionarily conserved residues. In this study, consensus protein mutants were generated for the Alpha and Mu glutathione transferases (GSTs) classes. The consensus proteins were then benchmarked against the parental proteins that were chosen (hGSTA1-1 and hGSTM1-1). The Alpha consensus mutant had 11 consensus mutations, including a notable M50L mutation, which affects the dynamic behaviour of helices α2 and α9, while the Mu consensus mutant had 13 unique mutations. Protein production and purification showed that the Mu consensus mutant had larger and purer yields. Data from far-UV circular dichroism studies and root-mean-squared-fluctuation (RMSF) from molecular dynamics (MD) simulations showed that the secondary structural components of the Alpha and Mu proteins remained largely the same, although the Alpha consensus mutant displayed a far lower molar residue ellipticity reading than its wildtype counterpart, indicating the disruption of secondary structural elements, likely caused by the M50L mutation. The ANS binding results showed that the M50L mutation in the Alpha consensus protein caused an increase in exposure of the surface area of the H-site, while the Mu consensus protein had a decrease in the solvent accessibility of its H-site. Thermal shift assay results indicated the consensus proteins had increased thermal stability. Enzyme kinetics results showed that the functionality of the proteins was severely diminished in the consensus mutants, particularly the Alpha consensus mutant. MD simulation results showed that there was an overall increase in the rigidity and compactness of the consensus mutant proteins, further affirming the improvement of thermal stability, while signalling the loss in functionality. The results produced herein have the potential to facilitate the proliferation of engineered GSTs for biotechnological applications that require proteins with an increased half-life and greater stability.Item The effect of cholesterol depletion on TGF-ß-induced epithelial-mesenchymal transition in pancreatic cancer cells(University of the Witwatersrand, Johannesburg, 2024-06) Breytenbach, Andrea; Kaur, MandeepPancreatic ductal adenocarcinoma (PDAC) is a highly metastatic cancer that relies on the epithelial to mesenchymal transition (EMT) program for its spread. EMT is a cell plasticity program that involves the reorganization of cell structure as cells transition from an epithelial to a mesenchymal phenotype. The dysregulated cholesterol metabolism resulting from metabolic reprogramming in PDAC is thought to play a role in EMT by affecting EMT-related signalling pathways. However, no publication has yet investigated the impact of EMT on cholesterol content in PDAC. To shed light on these dynamics, EMT was induced in PANC-1 cells using TGF-β1, thereafter the effect of cholesterol-depleting agents (KS-01 and methyl-β-cyclodextrin) alone or in combination with chemotherapeutic agents (Gemcitabine (GEM) and 5-Fluorouracil (5-FU)) on cholesterol content, EMT state, drug resistance, and invasion were investigated. Our results showed that mesenchymal cells rely on reduced membrane cholesterol levels, synthesis, and uptake, while storing more cholesterol and promoting efflux. EMT also promoted drug resistance via upregulation of ABCB1 expression and reduced hENT1 expression. Targeting cholesterol using cyclodextrins promoted a cholesterol compensatory mechanism, leading to a hybrid EMT state, drug resistance, and metastatic potential. Treating mesenchymal PANC-1 cells with GEM or 5-FU monotherapies were seen to promote EMT-transcription factors, as well as promote cholesterol efflux, synthesis, and import, an unexpected result as these chemotherapeutic agents are not known to affect cholesterol. When GEM was combined with KS-01, drug resistance, invasion, EMT-transcription factors, vimentin, and E-cadherin was promoted indicating the promotion of a hybrid EMT state. Interestingly however, combining KS-01 with 5-FU resulted in an interplay that was seen to mitigate the EMT-promoting effects typically associated with cholesterol depletion alone. The exact mechanism linking the cholesterol compensatory mechanism to EMT remains complex and unknown. Based on work presented in this dissertation, it is proposed that targeting cellular cholesterol should be continued to be investigated, particularly in understanding the repercussions of the use of cholesterol depleting agents for the treatment of other disorders in patients with PDAC.Item Investigating the DNA methylation status of the PXDN and PXDNL promoter regions in OSCC cell lines(University of the Witwatersrand, Johannesburg, 2024-06) Sebastian, Mistral; Mavri-Damelin, DemetraBackground: Oesophageal squamous cell carcinoma (OSCC) is the most prevalent form of oesophageal cancer in South Africa. Aberrant DNA methylation is a well-established epigenetic mechanism involved in various cancers, including OSCC. This study focuses on the DNA methylation status of the peroxidasin (PXDN) and perodixasin like (PXDNL) promoter regions and the expression of PXDN and PXDNL in OSCC cell lines. PXDN consolidates the basement membrane through collagen IV unit oligomerization, influences epithelial-mesenchymal transition and correlates with poor prognosis in various cancers. PXDNL modulates the extracellular matrix (ECM) by antagonising PXDN. Since PXDNL shares domains with PXDN, that allow PXDN to interact with the ECM, it is speculated that PXDNL may possess other ECM modulation roles that require further elucidation. Dysregulated PXDNL expression also correlates with poor cancer prognosis. To date, within the context of South African derived OSCC cell lines, no studies pertaining to the DNA methylation status of the PXDN and PXDNL promoter regions and the expression of PXDN and PXDNL have been carried out. Aim: The aim of this project was to investigate the DNA methylation status of the PXDN and PXDNL promoter regions and observe PXDN and PXDNL expression in the SNO and WHCO5 OSCC cell lines. Methods: PXDN and PXDNL localisation was observed using immunofluorescence microscopy; expression of PXDN and PXDNL was quantified using western blotting and the DNA methylation status of the PXDN and PXDNL promoters was assessed using methylation specific PCR and bisulfite sequencing, respectively. Results: Immunofluorescence microscopy results indicated that both cell lines show varying degrees of PXDN and PXDNL expression. In addition, these results also showed that PXDN and PXDNL localise in the ECM. The western blotting results established that these cell lines express the canonical version of PXDN and possibly a PXDNL isoform (146kDa). Methylation specific PCR has shown that the promoter region of PXDN is differentially methylated across both cell lines. The sequencing results of the bisulfite converted PXDNL promoter region were unsuccessful. Hence, bisulfite sequencing requires further optimisation before the DNA methylation status of the PXDNL promoter region can be determined. Conclusion: This study is the first to show the novel finding that PXDN and PXDNL are expressed in South African derived OSCC cell lines. Within the context of OSCC, further investigation is warranted in order to elucidate the underlying mechanisms that these proteins play a role in. In addition, further study may determine whether a correlation exists between PXDN and PXDNL promoter methylation, protein expression as well as prognosis and whether these aspects should serve as novel markers for diagnosis and therapy. This may subsequently lead to increased OSCC patient survival rates by contributing to early diagnosis of OSCC and efficacious targeted therapeutic intervention.Item Diversity and Abundance of Arthropods on Conventional Sugarcane under Field Conditions in South Africa(University of the Witwatersrand, Johannesburg, 2024-09) Smith, Roshay; Malinga, Lawrence; Bouwer, GustavInsect diversity and abundance are often the base for formulating strategies that involve the appropriate application of pest control methods, considering the ecosystem services provided by insects. Therefore, the aim of this study was to provide recent baseline data on the diversity and abundance of insects in conventional sugarcane based on two sugarcane fields in KwaZulu-Natal. Three sampling methods, namely pitfall, sticky and water pan traps, were used to sample insects in rain-fed and irrigated sugarcane in Gingindlovu and Pongola from March to October 2022. This study collected 12 493 insects belonging to 14 insect orders and 88 families in rain-fed sugarcane and 22 309 insects belonging to 14 orders and 94 families in irrigated sugarcane. Significant differences in the diversity indices were found between the sampling methods and the sampling periods. This study provides recent baseline data on the diversity and abundance of insects in sugarcane.Item Biophysical evaluation of the kinetics, thermodynamics, and structure-stability relationship of Wuchereria bancrofti glutathione transferase in comparison with human µ and π glutathione transferases(University of the Witwatersrand, Johannesburg, 2024-06) Oyiogu, Blessing Oluebube; Achilonu, Ikechukwu AnthonyLymphatic filariasis is an endemic disease caused mainly by the Wuchereria bancrofti parasite and has been classified as a major neglected tropical disease. The emergence of drug-resistant strains of W. bancrofti and the limited efficacy of the available drugs on adult worms threatens the eradication of the disease. W. bancrofti glutathione S-transferase (WbGST) is a homodimeric enzyme central to detoxifying electrophilic compounds in the parasite due to its lack of cytochrome P-450. Therefore, WbGST is a potential therapeutic target for lymphatic filariasis. Bromosulphophthalein (BSP) and epigallocatechin gallate (EGCG) were previously shown to inhibit glutathione S-transferase activity. In this study, the interaction of WbGST with BSP and EGCG in comparison with human glutathione S-transferase P1-1 (hGSTP1-1) and human glutathione S-transferase M1-1 (hGSTM1-1) isoforms was investigated. Soluble WbGST, hGSTP1-1 and hGSTM1-1 were recombinantly produced and purified successfully to homogeneity. Glutathione and 1-chloro-2,4-dinitrobenzene conjugation assay was employed to analyse the enzyme activity, kinetics and inhibitory potency of the compounds. Spectroscopic studies were employed to investigate the functional and structural impact of ligand binding to the enzymes. Both thermal and chemical stability studies were performed, and binding energetics were analysed using isothermal titration calorimetry. The activity of WbGST was predominantly inhibited, with IC50 values of 5 μM for BSP and 12 μM for EGCG. The EGCG displayed uncompetitive and mixed modes of inhibition towards WbGST with respect to glutathione and hydrophobic binding sites, respectively. Whereas BSP showed a mixed type of inhibition for both active sites of WbGST. Ligands reduced the turnover rates (kcat) and the catalytic efficiencies (kcat/KM) of the enzymes. Upon ligand binding, 8-anilino-1-napthalene sulphonate was displaced from WbGST and hGSTM1-1 by 67%(BSP), 24%(EGCG) and 72%(BSP), 5%(EGCG), respectively; suggesting that the ligands bind to the 8-anilino-1-napthalene sulphonate binding site. Stability studies indicate that WbGST is the least stable of the three enzymes and that glutathione increases its stability. Isothermal titration calorimetry showed that BSP binds to multiple sites in WbGST with binding at site-1 (S1) and site-2 (S2), which are entropically and enthalpically driven, respectively. S1 showed a higher affinity for BSP than S2. EGCG binding to WbGST was entropically driven. BSP had a higher affinity for the enzymes than EGCG. All the results indicated that the ligands significantly impact WbGST more than the human GSTs. Further investigations, such as crystallography and molecular dynamics simulations, will shed more light on the ligan-protein interactions on a molecular level. Overall, this study suggests that BSP and EGCG are efficient inhibitors of WbGST that probably bind to both L and H-sites of WbGST, altering catalytic activity of the enzyme. The unique properties of the L-site are particularly suitable for rational drug design. Therefore, both ligands can be repurposed as new-generation therapeutics against filariasis.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 Exploring temporal changes in the malting barley seed microbiome with meta-omics to understand nitrogen content effects(University of the Witwatersrand, Johannesburg, 2024-10) Tshisekedi, Kalonji Abondance; De Maayer, Pieter; Botes, AngelaBarley (Hordeum vulgare L.) is a critical cereal crop, particularly in beer production, where it plays a significant role in the economy, especially in South Africa. Despite its importance, the barley seed microbiome, which affects seed storage and quality, is not well understood. This research addresses two key questions: (1) how microbial composition and function evolve during storage and (2) how the inherent nitrogen content of the grain affects these dynamics. Using metagenomic and metaproteomic approaches, eight barley samples from the Kadie cultivar, stored for various durations (harvest, three, six, and nine months) with high and low nitrogen content, were analysed. Metagenomic sequencing revealed a predominance of bacterial sequences and minimal fungal presence, with storage time having a greater impact on microbial diversity than nitrogen content. However, specific bacterial genera such as Erwinia, Pantoea, Pseudomonas, and Stenotrophomonas showed nitrogen-dependent prevalence. Metagenome-assembled genomes (MAGs) were reconstructed, representing 26 bacterial genera, with minimal shared orthologues, highlighting taxonomic diversity. Functional analysis identified key metabolic pathways and carbohydrate-active enzymes (CAZymes) essential for microbial adaptation during storage. Metaproteomic analysis further showed the active expression of proteins related to nutrient transport and stress response, indicating functional changes over storage time. Overall, this research enhances the understanding of the barley seed microbiome, providing valuable insights into storage practices that could improve brewing quality and agricultural sustainability.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.