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Identification of Mycobacterium tuberculosis DnaK substrates – towards inhibiting DnaK as a novel drug target for the treatment of tuberculosis

  • Authors: Tonui, Ronald
  • Date: 2024-04-05
  • Subjects: Uncatalogued
  • Language: English
  • Type: Academic theses , Doctoral theses , text
  • Identifier: http://hdl.handle.net/10962/435975 , vital:73217
  • Description: Access restricted. Expected release in 2026. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2024
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  • Date Issued: 2024-04-05

Elucidation of the roles of the carbonic anhydrase enzymes, CanA & CanB, in the physiology of Mycobacterium smegmatis

  • Authors: Jackson, Gabriella Teresa
  • Date: 2024-04-04
  • Subjects: Uncatalogued
  • Language: English
  • Type: Academic theses , Master's theses , text
  • Identifier: http://hdl.handle.net/10962/435299 , vital:73145
  • Description: The bacterial pathogen Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB) and one of the leading infectious causes of death globally. The success of Mtb as a pathogen depends on its ability to detect and respond to a variety of physical and chemical stresses it encounters during infection of its human host. These environmental stresses include shifts in temperature, oxygen concentration, osmolarity and nutrient availability. Mtb is, in addition, exposed to changes in pH and CO2 concentration in the intracellular and extracellular environments it inhabits, which the bacterium has to adapt to in order to ensure its growth, survival and/or persistence during infection. Carbonic anhydrases (CAs) are a widely distributed family of enzymes that catalyse the reversible hydration of carbon dioxide (CO2) to bicarbonate (HCO3−) in the reaction: CO2 + H2O ⇄ HCO3− + H+. In microbes, CA activity is important for the activity of enzymes involved in carbon fixation as well as for maintaining pH homeostasis. Mtb is known to express three CAs, encoded by the Rv3588c, Rv1284 and Rv3273 genes (canA, canB and canC, respectively). The role(s) of these CA enzymes in the physiology of Mtb and other mycobacterial species, such as Mycobacterium smegmatis (Msm), has not been elucidated to date. To gain insights into the function of the CanA and CanB enzymes in mycobacterial species, we generated both canA and canB knockdown (KD) and knockout (KO) mutants in the fast-growing mycobacterial species, Msm, and analysed their growth phenotypes under several growth conditions where CA activity is known to be required. Notably, Msm lacks the CanC homologue, which makes it an ideal surrogate to focus on CanA and CanB. The Msm canA KD mutant was found to display a growth defect following anhydrotetracycline (ATc)-mediated gene silencing at atmospheric (low) CO2 concentrations [~0.035% CO2 (v/v)]. The growth defect could be rescued by incubating cells at physiological (high) CO2 concentrations [~5% CO2 (v/v)] or by supplementing the growth media with either HCO3− or the metabolic end-products of certain HCO3−-dependent-carboxylase enzymes at low CO2 concentrations. The ability of these compounds to rescue the growth of the canA KD mutants was, however, dependent on the extent of ATc-mediated gene silencing, suggesting that the canA gene is required for Msm growth at both low and high CO2 concentrations. This was confirmed by our findings that canA could only be genetically inactivated when a second copy of the gene was provided on the chromosome in trans, regardless of the CO2 concentration used. In contrast to our observations for canA, no differences in the growth phenotypes of the Msm wild type (WT) and canB KD or knockout (KO) mutant strains were observed following silencing or inactivation of the canB gene at either low or high CO2 concentrations or different pH values. These observations suggest that, in contrast to canA, the canB gene is dispensable for the growth of Msm under standard laboratory growth conditions. The canB KO mutant strain, nevertheless, displayed a slight decrease in its ability to form biofilms when compared to the WT strain, which could be restored by genetic complementation. CanB activity may, therefore, be required to promote bacterial growth and/or survival under biofilm conditions where CO2 diffusion into cells is limited, a phenomenon that has recently been observed in other microbes. Further studies are required to confirm the role of CanB in biofilm formation and to determine how the different CA enzymes cooperate to promote the growth and survival of mycobacterial species in the various environments they are known to inhabit. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2024
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  • Date Issued: 2024-04-04

Towards a possible future solution against Multidrug Resistance: An in silico exploration of the Multidrug and Toxic compound Extrusion (MATE) transporter proteins as potential antimicrobial drug targets

  • Authors: Damji, Amira Mahamood
  • Date: 2024-04-04
  • Subjects: Multidrug resistance , Multidrug and toxic compound extrusion family, eukaryotic , Docking , Molecular dynamics , Drug development , Transmembrane protein
  • Language: English
  • Type: Academic theses , Master's theses , text
  • Identifier: http://hdl.handle.net/10962/435009 , vital:73123
  • Description: The rise of multidrug resistance (MDR) has become a pressing global issue, hindering the treatment of cancers and infectious diseases, and imposing a burden on healthcare systems and the economy. The Multidrug and Toxic compound Extrusion (MATE) superfamily of membrane efflux transporters is one of the key players contributing to MDR due to their ability to export a wide range of cationic and hydrophilic xenobiotics, including treatment drugs, from cells, diminishing their efficacy. Targeting MATE transporters holds great promise in achieving some cellular control over MDR, but first, a deeper understanding of their structure-function-dynamics link is required. This study aimed to explore the MATE transporters as potential antimicrobial drug targets using a two-fold in silico approach. First, virtual screening of compounds from the South African Natural Compounds Database (SANCDB) was performed to identify prospective lead inhibitory compounds against the MATE transporters using molecular docking, and top hits were selected based on their binding energy and interaction with the active site on the N-lobe of the protein. Second, to investigate the molecular-level dynamics of their extrusion mechanism, the MATE transporter structures were embedded in a POPC membrane bilayer using the CHARMM-GUI online tool and then subjected to MD simulations for 100 ns with the CHARMM 36m force field using GROMACS. The resulting trajectories were evaluated using three standard metrics – RMSD, RMSF, and Rg; significant global structural changes were observed and key functional regions in both membrane- and non-membrane transmembrane (TM) segments were identified, containing more dynamic and flexible residues than other regions. Furthermore, the MATE transporters showed more of a loosely-packed structure, providing flexibility to allow for conformational switching during their substrate-transport cycle, which is typical for proteins whose secondary structures are composed of all α-helices. The scope of this study lied in the preliminary stages of the computer-aided drug design process, and provided insights that can be used to guide the development of strategies aimed at regulating or inhibiting the function of the MATE transporters, offering a possible future solution to the growing challenge of MDR. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2024
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  • Date Issued: 2024-04-04

African population prevalent genetic variations of dihydropyrimidine dehydrogenase as the 5-flourouracil cancer drug metabolizing enzyme: computational approaches towards pharmacogenomics studies

  • Authors: Tendwa, Maureen Bilinga
  • Date: 2023-10-13
  • Subjects: Uncatalogued
  • Language: English
  • Type: Academic theses , Doctoral theses , text
  • Identifier: http://hdl.handle.net/10962/432263 , vital:72856 , DOI 10.21504/10962/432270
  • Description: In an era of newly emerging cases of non-communicable diseases such as cancer, research is vital for both the medical and economic well-being of humanity. Pharmacogenomics has laidthegroundworkfor the identification of potential genes in cancer progression and treatment outcome investigations. Researchers are increasingly discovering heterogeneity in the efficacy and toxicity responses of drugmetabolizing enzymes (DMEs) in diverse patient populations receiving anti-cancer therapy. DMEs comprise of Phase I (Cytochrome P450s) and Phase II (glutathione-S-transferases (GSTs), UDP-glucuronosyltransferases (UGTs), and dihydropyrimidine dehydrogenases (DPD)enzymes. The main cause of disparity in DME treatment outcomes is genetic variation,which causes missense mutations leading to structural and kinetic properties of the enzyme. These modifications have a deleterious impact on the pharmacodynamics and pharmacokinetics of drugs through multiple mechanisms. Presently, most cancer medicines are manufacturedin developed countries based on the genetic background of non-African subpopulations. Thus, these drugs may not be optimally effective or can cause adverse side effects. Even though heterogeneity in toxicity and efficacy of these drugs has been observed in African descent, the basis of this population variance remains partially understood. For instance,a deficiencyof DPD, the first-rate limiting metabolizing enzyme in the pyrimidinepathway, causes severe toxicity when exposed to 5-fluorouracil (5-FU) chemotherapy. However, minimum studies have been conducted to unravel itsmolecular mechanismwhich may unravel the observed drug treatment outcomes.The aim of this pharmacogenomics study was to determine the underlying mechanism by which DPD missense mutations, which are associated with an African ancestry subpopulation, provoke dysfunctional 5-FU metabolism, resulting in drug toxicity. This knowledge will be critical in designing drug modulators to aid in the restoration of DPD function, a hallmark of precision medicine. Therefore, in the first part of the research we identified and reviewed the general role of Phase I and Phase II cancer drug metabolizing enzymes. We then used World Health Organization (WHO) essential medicine and drug.com to authenticate the usage of 5-FU as an anti-cancer treatment agent. The 3D structure and chemical structure of the agent was then downloaded from the Drug bank. Subsequently, Human Mutation Analysis - Variant Analysis PORtal (HUMA) and Mendelian Inheritance in Man (OMIM) were used to obtain data on DPD non-synonymous genetic variants. Additionally, the aggregate information of DPD missense mutations and their relation to human health were extracted from ClinVar and Pharmacogenomics Knowledge Base (PharmKGB). This information, along with additional data from single nucleotide polymorphisms (dbSNP), 1000 Genomes Project and Exome Sequencing Project (ESP MAF) considering variants classified based on their minor allele frequency (MAF) of 0.001, as well as research articles, consolidated information on missense mutations associated with African subpopulations. Finally, the wild type (WT) and detected mutation sequences were obtained from the Universal Protein Resources database (UniProt). However, because the 3D structure of human DPD was missing, the dimeric wild type (WT) human 3-dimensional (3D) structure was modeled via MODELLER using the pig’s structure as a template. PRIMO, HHpred, and the Protein Data Bank (PDB) were all used to locate the suitable template. As a result, six clinical (C29R, M166V, Y186C, S534N, I543V, and D949V) and thirteen non-clinical (S201R, K259E, D342N, D432N, S492L, R592Q, A664S, G674D, A721T, V732G, T768K, R886C, and L993R) mutations were discovered. Using AMBER tools, we then determined accurate force field parameters for each monomer of DPD protein's Fe2+ centers. Following the creation of each mutation model structure in Discovery Studio, the resulting AMBER force field parameters were inferred. For each model structure, a drug free (inactive/open-conformation) and drug bound (active/closed-conformation) model structure was created (WT and mutations). The model structures were validated using the consensus of three validation programs, namely ERRAT, PROCHECK, and ProSA. Similarly, the impact on structural functionalities was predicted by consensus from Variant Analysis Porta (VAPOR) web server, which include three support vector machines (SVM)-based tools; PhD-SNP, MUpro, and I-Mutation. After protonation in the H++ web server, the six clinical and thirteen non-clinical (six active site and seven non-active site) mutations identified were then exposed to 600 ns molecular dynamic (MD) simulation. The non-clinical data was divided into two categories to better understand the impact of the mutation based on its position in the protein: six catalytic-domain (R592Q, A664S, G674D, A721T, V732G, and T768K) and seven remote (S201R, K259E, D342N, D432N, S492L, R886C, and L993R) missense mutations. The post-MD analysis was done using the typical existing computational global investigations [RMSD, all versus all RMSD, RMSF, RG, hydrogen bonds (H-bonds) and dynamic cross correlation (DCC)]. In addition, we used in silico tools newly developed within the Research Unit in Bioinformatics (RUBI) group, such as comparative essential dynamics (ED)-principal component analysis and dynamic residue network (DRN) multi-metric [betweenness centrality (BC), closeness centrality (CC), degree of centrality (DC), eigen-centrality (EC) and Katz centrality (KC)] analysis algorithms. From the analysis, it was observed that the loop regions of the mutation proteins had increased loop flexibility, particularly around the catalytic loop, which could account for the enhanced asymmetric behavior of the mutation’s monomers compared to the WT. Notably, the A664S mutant showed relatively lower fluctuations, deviating from the observed heightened flexibility in other mutants. A general decrease in hydrogen bonds was observed in the 5-FU binding environment of the mutations compared to the WT. In particular, 5-FU contact analysis of the WT versus the mutation revealed a reduction in contact between core 5-FU binding residues and catalytic residues Cys671 and Ser670, which form hydrogen bonds that initiate DPD catalytic action. Additionally, BC was used to quantify the importance of a protein residue based on how often it acted as a bridge along the shortest paths between other residues. It reflected the potential control or influence a residue may have over communication between different parts of a protein structure. DC assesses the number of connections or interactions a residue had with other residues in the protein, indicating its overall connectivity within the structure. In both drug free and drug bound state, DPD data from the active site hubs' BC and DC revealed a dimeric asymmetric communication pathway per monomer involving a cluster of newly introduced hubs ensemble along the oxidoreduction conduit from NADPH to 5-FU. The two BC communication pathways were located more on the interior of the oxidoreduction conduit, while the two DC communication pathways were located on the exterior. In both cases, one pathway dominated the other. Partially lost function reported in mutation systems could be credited to the compensation communication response to the catalytic site via the least compromised routes. Similar patterns were observed in allosteric communication pathways to the active site induced by remote mutations. Mutations may have destabilized the active-loop and 5-FU binding environment, resulting in a compensatory mechanism seen by the addition of new hubs to the communication network. Surprisingly, EC hubs in the WT were found within the catalytic site domain, indicating that the region is important in 5-FU metabolism. EC measured the importance of a residue by considering both its own degree of connectivity and the degrees of connectivity with its neighboring residues, highlighting its significance in information flow and communication. Herein, EC hubs in mutant systems were found to lose this importance, with active site domain mutations suffering the most. This could explain why non-clinical catalytic domain mutations R592Q, A664S, and G674D, as well as clinical catalytic domain mutations S534N and I543V, experienced drug exit in one of their monomers during simulation. In contrast, there was no 5-FU exit in the non-clinical remote domain. Additionally, aside from the active site, KC hubs were also found around the cofactors, indicating that these components were equally important in DPD overall function. KC combines the concepts of both degree centrality and eigen-centrality, it incorporated both direct and indirect interactions to evaluate the importance of a residue, assigning higher centrality to residues that have connections to other highly central residues. Hence, providing a more comprehensive measure of influence within the protein network. More importantly, CC is known to measure how efficiently a residue can interact with other residues in the protein, considering the shortest path lengths. It indicates the proximity of a residue to others, suggesting its potential for information transfer or functional integration. CC revealed that the majority of persistent hubs were found within the protein-cores known as cold-spots. Overall, this study highlighted the communication pathways triggered by active site domain mutations, as well as the allosteric communication pathways triggered by each remote mutation in both drug free and drug bound states of the DPD enzyme. Both clinical and non-clinical mutations revealed each protein's adaptive compensation mechanism, which results in partial function loss. In each case, the communication network of the different monomers changed from inactive to activated DPD protein. Cold-spot areas were discovered to contain key persistent residues involved in protein function and stability. These areas have been proposed as potential targets for new or repurposed pharmacological modulators that can restore enzyme function. In the pursuit of precision medicine, it also lays the groundwork for detecting and explaining the molecular mechanisms of other drug metabolizing enzymes related to the African-descent subpopulation. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2023
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  • Date Issued: 2023-10-13

Analysis of the role and regulation of HOP1a and HOP1b splice variants in cancer biology

  • Authors: Schwarz, Kelly
  • Date: 2023-10-13
  • Subjects: Uncatalogued
  • Language: English
  • Type: Academic theses , Doctoral theses , text
  • Identifier: http://hdl.handle.net/10962/432240 , vital:72854
  • Description: Restricted access. Expected lease date in 2025. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2023
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  • Date Issued: 2023-10-13

Application of web design techniques and best practices in implementing web development, maintenance and enhancement of RUBi websites and web application systems

  • Authors: Tshabalalala, Thulani
  • Date: 2023-10-13
  • Subjects: Uncatalogued
  • Language: English
  • Type: Academic theses , Master's theses , text
  • Identifier: http://hdl.handle.net/10962/424688 , vital:72175
  • Description: The popularity of the web has seen various fields, such as the sciences taking advantage of this resource to further their scientific endeavours. This has seen science groups moving into developing websites and web applications, and such a group is the Research Unit in Bioinformative (RUBi). With the use of the web, the development and maintenance of whatever web-related tools become inevitable, given the continuous changes in the web space. This continuous evolution of web development and maintenance will come with techniques, principles and standards which will not only enable faster development of web entities but also ensure that modern hardware, fulfilment of the requirements to use such hardware and modern concepts are incorporated into forming web tools that enable such progression. Furthermore, introducing the previously mentioned progress of the web becomes an essential part of its development and maintenance. This paper did implement the processes of progressing the web using the technique of documentation and version control systems. The web development for the COVIDRUG website was done for the Covidrug-Africa Consortium (COVIDRUG) using the Django webdevelopment framework. The RUBi website and the MDM-Task we band the Job Management System (JMS) web applications were maintained for the maintenance aspect. Archives brought value regarding the traceability it provides of the various web-related aspects. The development showed a website’s potential value, particularly for research groups. The maintenance carried out showed how different techniques and approaches could be used in different maintenance prospects to achieve set objectives. The development and maintenance resulted in websites and web applications that have the features stated in their respective maintenance plans. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
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  • Date Issued: 2023-10-13

Arbuscular mycorrhizal fungi associated with maize in South Africa, under conventional and conservation agricultural cultivation

  • Authors: Maússe Sitoe, Sílvia Natal David
  • Date: 2023-10-13
  • Subjects: Uncatalogued
  • Language: English
  • Type: Academic theses , Doctoral theses , text
  • Identifier: http://hdl.handle.net/10962/431846 , vital:72808
  • Description: Access restricted. Expected release date in 2025. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2023
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  • Date Issued: 2023-10-13

Arbuscular mycorrhizal fungi associated with wheat under conventional and conservation agricultural cultivation

  • Authors: Dube, Makasithembe
  • Date: 2023-10-13
  • Subjects: Uncatalogued
  • Language: English
  • Type: Academic theses , Doctoral theses , text
  • Identifier: http://hdl.handle.net/10962/431688 , vital:72796
  • Description: Access restricted. Expected release date in 2024. , Thesis (PhD) -- Faculty of Science, Chemistry, 2023
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  • Date Issued: 2023-10-13

Computational studies in human African trypanosomiasis

  • Authors: Muronzi, Tendai
  • Date: 2023-10-13
  • Subjects: African trypanosomiasis , Apolipoprotein L1 , Docking , Protein-protein interactions , Homology modeling , Tetrahydrofolate dehydrogenase , Pteridine reductase
  • Language: English
  • Type: Academic theses , Doctoral theses , text
  • Identifier: http://hdl.handle.net/10962/431883 , vital:72812 , DOI 10.21504/10962/431885
  • Description: Human African trypanosomiasis (HAT) is a neglected tropical disease (NTD) caused by two subspecies of the parasite, namely Trypanosoma brucei (Tb) gambiense (g-HAT) and rhodesiense (r-HAT). HAT is endemic in sub-Saharan countries, where the parasite transmission vectors, tsetse flies, breed. An estimated 70 million people remain at risk of contracting the disease, where the infection is classified as acute or chronic for g-HAT and r-HAT, respectively, with both forms ending in fatal meningoencephalitis when left untreated. Both g-HAT and r-HAT are responsible for widespread fatal epidemics throughout sub-Saharan African history, resulting from the complex molecular interplay between trypanosomes and humans through unique, innate immunity evasion mechanisms. Of interest, the Tbr subspecies expresses a serum resistance-associated protein (SRA), which binds to human serum lytic factor, apolipoprotein L1 (ApoL1), nullifying any trypanocidal activity. In response, ApoL1 (G1 and G2) variants found in humans of sub-Saharan African lineage have been cited for conferring resistance to the r-HAT infection in an interaction that is not fully elucidated In the event of successful infection, current HAT chemotherapeutics are plagued with complexity of administration, poor efficacy, toxicity, and potential drug resistance, highlighting a need for improved approaches. The parasite folate pathway provides a strategic target for alternative anti-trypanosomal drug development as trypanosomatids are folate auxotrophs, requiring host folate for growth and survival. Validated drug targets pteridine reductase (TbPTR1) and dihydrofolate reductase (TbDHFR) are essential for salvaging cofactors folate and folate biopterin crucial to parasite survival, making them viable targets for anti-folate investigation. The overall aims of this thesis were to a) provide insights into the molecular and dynamic basis of the SRA and ApoL1 interplay in HAT infection and b) identify safer and more efficient anti-folate anti-trypanosomal drug alternatives through in silico approaches. To achieve our first aim, in silico structure prediction was applied to generate 3D models of ApoL1 C-terminal variants G0, G1, G1G/M, G2 and G1G2, and four SRA variants retrieved from the NCBI database. The SRA and ApoL1 structures were inspected dynamically to identify the effect of the variants through molecular dynamics (MD) simulations. Dynamic residue network (DRN) analysis of MD trajectories was fundamental in identifying residues playing a vital role in the intramolecular communication of both proteins in the presence of mutations. Protein-protein docking was then applied to calculate plausible SRA-ApoL1 C-terminal wild-type complex structures to further elucidate the nature of SRA-mediated infection. Through MD simulations, twelve SRA-ApoL1 dimeric structures were narrowed down from five to two energetically sound complexes. The two feasible SRA-ApoL1 complexes (1 and 2) exhibited favourable communication observed through DRN analysis, including the retaining key communication residues identified in prior monomer DRN calculations. ApoL1 C-terminal variants were additionally incorporated into SRA-ApoL1 complexes 1 and 2 for further complex dynamics analysis This investigation into the nature of SRA-ApoL1 binding resulted in five primary outcomes: 1) highlighting the intramolecular effects ApoL1 variants have on the stability of the protein, 2) the identification of crucial SRA and ApoL1 communication residues in both monomeric or dimeric form, 3) the isolation of feasible SRA-ApoL1 complexes determined through global and local structural analyses 4) identification of residues crucial to the complex formation and maintenance of SRA-ApoL1, overlapping with those identified in (1), and 5) the minimal dissociative role of the G1 mutations in the complex, but compounding effect of the G2 deletion mutation. Computational modelling and drug repurposing were employed to achieve the thesis's second aim as they drastically cut down the costs involved in drug discovery and provide a more time-efficient screening method through numerous drug candidates. Using high throughput virtual screening, a subset of 2089 approved DrugBank compounds were screened against TbPTR1. The outputs were filtered to 24 viable compounds in 54 binding poses using binding energy and molecular interactions. Through subsequent MD simulations of 200ns, thirteen potential hit compounds were identified. The resultant hit compounds were subjected to further blind docking against TbDHFR and molecular dynamics to identify compounds with the potential for dual inhibition. The filtered subset was also tested in in vitro single concentration and dose-response bioassays to assess inhibitory properties against Trypanosoma brucei, complementing in silico findings. Post-molecular dynamics, four compounds exhibited high stabilities and molecular interactions with both TbPTR1 and TbDHFR, with two presenting favourable results in the in vitro assays. Three compounds additionally shared common structural moieties. In all, the in silico repurposing highlighted drugs characterised by favourable interactions and stabilities in TbPTR1, thus providing (1) a framework for further studies investigating anti-folate HAT compounds and (2) modulatory scaffolds based on identified moieties that can be used for the design of safe anti-folate trypanosomal drugs. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2023
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  • Date Issued: 2023-10-13

Falcipain 2 and 3 as malarial drug targets: deciphering the effects of missense mutations and identification of allosteric modulators via computational approaches

  • Authors: Okeke, Chiamaka Jessica
  • Date: 2023-10-13
  • Subjects: Antimalarials , Cysteine proteinases , Missense mutation , Allostery , Cysteine proteinase falcipain 2a , Cysteine proteinase falcipain 3
  • Language: English
  • Type: Academic theses , Doctoral theses , text
  • Identifier: http://hdl.handle.net/10962/432170 , vital:72848 , DOI 10.21504/10962/432170
  • Description: Malaria, caused by an obligate unicellular protozoan parasite of the genus Plasmodium, is a disease of global health importance that remains a major cause of morbidity and mortality in developing countries. The World Health Organization (WHO) reported nearly 247 million malaria cases in 2021, causing 619,000 deaths, the vast majority ascribed to pregnant women and young children in sub-Saharan Africa. A critical component of malaria mitigation and elimination efforts worldwide is antimalarial drugs. However, resistance to available antimalarial drugs jeopardizes the treatment, prevention, and eradication of the disease. The recent emergence and spread of resistance to artemisinin (ART), the currently recommended first-line antimalarial drug, emphasizes the need to understand the resistance mechanism and apply this knowledge in developing new drugs that are effective against malaria. An insight into ART's mechanism of action indicates that ferrous iron (Fe2+) or heme, released when hemoglobin is degraded, cleaves the endoperoxide bridge. As a result, free radicals are formed, which alkylate many intracellular targets and result in plasmodial proteopathy. Aside from the existing evidence that mutations in the Kelch 13 protein propeller domain affect ART sensitivity and clearance rate by Plasmodium falciparum (Pf) parasites, recent investigations raise the possibility that additional target loci may be involved, and these include a nonsense (S69stop) and four missense variants (K255R, N257E, T343P, and D345G) in falcipain 2 (FP-2) protein. FP-2 and falcipain 3 (FP-3) are cysteine proteases responsible for hydrolyzing hemoglobin in the host erythrocytic cycle, a key virulence factor for malaria parasite growth and metabolism. Due to the obligatory nature of the hemoglobin degradation process, both proteases have become potential antimalarial drug targets attracting attention in recent years for the development of blood-stage antimalarial drugs. The alteration of the expression profile of FP-2 and FP-3 through gene manipulation approaches (knockout) or compound inhibition assays, respectively, induced parasites with swollen food vacuoles due to the accumulation of undegraded hemoglobin. Furthermore, missense mutations in FP-2 confer parasites with decreased ART sensitivity, probably due to altered enzyme efficiency and momentary decreased hemoglobin degradation. Hence, understanding how these mutations affect FP-2 (including those implicated in ART resistance) and FP-3 is imperative to finding potentially effective inhibitors. The first aim of this thesis is to characterize the effects of missense mutations on the partial zymogen complex and the catalytic domain of FP-2 and FP-3 using a range of computational approaches and tools such as homology modeling, molecular dynamics (MD) simulations, comparative essential dynamics, dynamic residue network (DRN) analysis, weighted residue contact map analysis, amongst others. The Pf genomic resource database (PlasmoDB) identified 41 missense mutations located in the partial zymogen and catalytic domains of FP-2 and FP-3. Using structure-based tools, six putative allosteric pockets were identified in FP-2 and FP-3. The effect of mutations on the whole protein, the central core, binding pocket residues and allosteric pockets was evaluated. The accurate 3D homology models of the WT and mutants were calculated. MD simulations were performed on the various systems as a quick starting point. MD simulations have provided a cornerstone for establishing numerous computational tools for describing changes arising from mutations, ligand binding, and environmental changes such as pH and temperature. Post-MD analysis was performed in two stages viz global and local analysis. Global analysis via radius of gyration (Rg) and comparative essential dynamic analysis revealed the conformational variability associated with all mutations. In the catalytic domain of FP-2, the presence of M245I mutation triggered the formation of a cryptic pocket via an exclusive mechanism involving the fusion of pockets 2 and 6. This striking observation was also detected in the partial zymogen complex of FP-2 and induced by A159V, M245I and E249A mutations. A similar observation was uncovered in the presence of A422T mutation in the catalytic domain of FP-3. Local DRN and contact map analyses identified conserved inter-residue interaction changes on important communication networks. This study brings a novel understanding of the effects of missense mutations in FP-2 and FP-3 and provides important insight which may help discover new anti-hemoglobinase drugs. The second aim is the identification of potential allosteric ligands against the WT and mutant systems of FP-2 and FP-3 using various computational tools. Of the six potential allosteric pockets identified in FP-2 and FP-3, pocket 1 was evaluated by SiteMap as the most druggable in both proteins. This pipeline was implemented to screen pocket 1 of FP-2 and FP-3 against 2089 repositionable compounds obtained from the DrugBank database. In order to ensure selectivity and specificity to the Plasmodium protein, the human homologs (Cat K and Cat L) were screened, and compounds binding to these proteins were exempted from further analysis. Subsequently, eight compounds (DB00128, DB00312, DB00766, DB00951, DB02893, DB03754, DB13972, and DB14159) were identified as potential allosteric hits for FP-2 and five (DB00853, DB00951, DB01613, DB04173 and DB09419) for FP-3. These compounds were subjected to MD simulation and post-MD trajectory analysis to ascertain their stability in their respective protein structures. The effects of the stable compounds on the WT and mutant systems of FP-2 and FP-3 were then evaluated using DRN analysis. Attention has recently been drawn towards identifying novel allosteric compounds targeting FP-2 and FP-3; hence this study explores the potential allosteric inhibitory mechanisms in the presence and absence of mutations in FP-2 and FP-3. Overall, the results presented in this thesis provide (i) an understanding of the role mutations in the partial zymogen complex play in the activation of the active enzyme, (ii) an insight into the possible allosteric mechanisms induced by mutations on the active enzymes, and (iii) a computational pipeline for the development of novel allosteric modulators for malaria inhibition studies. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2023
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  • Date Issued: 2023-10-13

HOP abundance affects nuclear pore components and the export of protein and RNA cargo

  • Authors: Oladipo, Hannah Oluwakemi
  • Date: 2023-10-13
  • Subjects: Uncatalogued
  • Language: English
  • Type: Academic theses , Doctoral theses , text
  • Identifier: http://hdl.handle.net/10962/432192 , vital:72850
  • Description: Access restricted. Expected release date 2025. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2023
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  • Date Issued: 2023-10-13

Identification of novel Arf1 GTPase inhibitors for cancer target validation

  • Authors: Mqwathi, Nomxolisi Vuyokasi
  • Date: 2023-10-13
  • Subjects: ARF1 , GTPase , Guanosine triphosphatase , Cancer Treatment
  • Language: English
  • Type: Academic theses , Master's theses , text
  • Identifier: http://hdl.handle.net/10962/424666 , vital:72173
  • Description: The key regulators of both anterograde and retrograde vesicular traffic, adenosine diphosphate-ribosylation factors (Arfs), also coordinate various signalling pathways and regulate cellular processes required for cell survival and function. In addition to its role in mediating secretory trafficking in the Golgi apparatus, the involvement of Arf1 in signalling pathways that contribute to the formation and progression of cancer has become apparent, and the overexpression and deregulation of Arf1 activity has been associated with cancer cell invasion, proliferation and metastasis. As with other small GTPases, Arf1 must cycle back and forth between an inactive (GDP-bound) and active (GTP-bound) conformation to carry out its function. However, the cycle of Arf1 inactivation and activation is controlled by Arf GTPase activating proteins (Arf-GAPs) that stimulate Arf1 to hydrolyse the bound GTP to GDP and Arf guanine nucleotide exchange factors (Arf-GEFs) that facilitate GDP for GTP exchange on Arf1, respectively. The identification of Arf1 inhibitors that indirectly disrupt Arf1 function by blocking its interaction with Arf-GAPs or Arf-GEFs has generated interest in their use as possible anti-cancer agents. The suppression of Arf1 activation (by targeting Arf-GEFs) has been investigated as a potential cancer therapeutic target and resulted in inhibitor compounds that have micromolar-range activity against cancer cells and targets and promising results in mouse models, but experience problems with bioavailability when used in vivo. This motivates the search for novel Arf1 inhibitors for validation purposes to question whether Arf1 is a viable target for cancer therapy. The purpose of the study was to employ a recently developed colourimetric screening assay to identify inhibitors of Arf1 activation (Arf-GEF inhibitors) and deactivation (Arf-GAP inhibitors), with a focus on evaluating the potential of Arf1 deactivation as an entirely novel anti-cancer target. The proteins required for the assay (Arf1, Arf-GEF and -GAP domains and a reporter protein, GST-GGA3) were expressed in E. coli. and purified using affinity chromatography. The assay could detect the activation of Arf1 by the catalytic Sec7 domain of the three Arf-GEFs chosen for this study, but reproducibility was compromised by the occasional spontaneous activation of Arf1 in the absence of the Arf-GEFs. By contrast, the assay could reproducibly detect Arf1 deactivation by an Arf-GAP domain (Arf-GAP1GAP) and was subsequently used to screen a library of α-helix mimetics. Thirteen hit compounds with IC50 values ranging from 0.53 to 20.95 μM were found to inhibit Arf-GAP1GAP-mediated stimulation of GTP hydrolysis by Arf1-GTP in this assay format, however, they did not effectively suppress the proliferation of three tested cell lines (HeLa, MCF-7 and MCF-12A). Interestingly, the results obtained from fluorescence microscopy studies suggested that the compounds disrupt Golgi structure and Arf1 localisation, presumably by keeping Arf1 in its active conformation by blocking Arf-GAP1 function. This suggests that the compounds affect Arf1 function in cells, and may be used to explore the feasibility of targeting Arf1 deactivation for anti-cancer purposes in a wider range of cell lines and experiments. It has been reported that Arf-GAP1 inhibition is associated with the suppression of cell migration, and the potential of the compounds as metastasis inhibitors may also be explored. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
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  • Date Issued: 2023-10-13

Identification of novel therapeutic agents targeting Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication

  • Authors: Okpara, Michael Obinna
  • Date: 2023-10-13
  • Subjects: Uncatalogued
  • Language: English
  • Type: Academic theses , Doctoral theses , text
  • Identifier: http://hdl.handle.net/10962/432181 , vital:72849
  • Description: Access restricted. Expected release date 2025. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2023
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  • Date Issued: 2023-10-13

In silico characterization of missense mutations in infectious diseases: case studies of tuberculosis and COVID-19

  • Authors: Barozi, Victor
  • Date: 2023-10-13
  • Subjects: Microbial mutation , COVID-19 (Disease) , Drug resistance in microorganisms , Antitubercular agents , Tuberculosis , Molecular dynamics , Single nucleotide polymorphisms
  • Language: English
  • Type: Academic theses , Doctoral theses , text
  • Identifier: http://hdl.handle.net/10962/431626 , vital:72791 , DOI 10.21504/10962/431626
  • Description: One of the greatest challenges facing modern medicine and the global public health today is antimicrobial drug resistance (AMR). This “silent pandemic,” as coined by the world health organization (WHO), is steadily increasing with an estimated 4.95 million mortalities attributed to AMR in 2019, 1.27 million of which were directly linked to AMR. Some of the contributors to AMR include self-prescription, drug overuse, sub-optimal drug prescriptions by health workers, and inaccessibility to drugs, especially in remote areas, which leads to poor adherence. The situation is aggravated by the upsurge of new zoonotic infections like the coronavirus disease 2019, which present unique challenges and take the bulk of resources hence stunting the fight against AMR. Quite alarming still is our current antimicrobial arsenal, which hasn’t had any novel antimicrobial drug discovery/addition, of a new class, since the 1980s. This puts a burden on the existing broad-spectrum antimicrobial drugs which are already struggling against multi-drug resistant strains like multi-drug resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB). Besides the search for new antimicrobial agents, the other avenue for addressing AMR is studying drug resistance mechanisms, especially single nucleotide polymorphisms (SNPs), that change drug target characteristics. With the advancement of computational power and data storage resources, computational approaches can be applied in mutational studies to provide insight into the drug resistance mechanisms with an aim to inform future drug design and development. Therefore, in the first part of this thesis, we employ integrative in silico approaches, including 3D structure modeling, molecular dynamic (MD) simulations, comparative essential dynamics (ED), and protein network analysis approaches i.e., dynamic residue network (DRN) analysis to decipher drug resistance mechanisms in tuberculosis (TB). This involved an investigation of the drug resistance mutations in the catalase-peroxidase (KatG) and pyrazinamidase (MtPncA) enzymes which are responsible for activation of TB first-line drugs; Isoniazid (INH) and Pyrazinamide (PZA), respectively. In the case of KatG, eleven high confidence (HC) KatG mutations associated with a high prevalence of phenotypic INH resistance were identified and their 3D structures modeled before subjecting them to MD simulations. Global analysis showed an unstable KatG structure and active site environment in the mutants compared to the wildtype. Active site dynamics in the mutants compromised cofactor (heme) interactions resulting in less bonds/interactions compared to the wildtype. Given the importance of the heme, reduced interactions affect enzyme function. Trajectory analysis also showed asymmetric protomer behavior both in the wildtype and mutant systems. DRN analysis identified the KatG dimerization domain and C-terminal domain as functionally important and influential in the enzyme function as per betweenness centrality and eigenvector centrality distribution. In the case of the MtPncA enzyme, our main focus was on understanding the MtPncA binding ability of Nicotinamide (an analogue of PZA) in comparison to PZA, especially in the presence of 82 resistance conferring MtPncA mutations. Like in KatG, the mutant structures were modeled and subjected to MD simulations and analysis. Interestingly, more MtPncA mutants favored NAM interactions compared to PZA i.e., 34 MtPncA mutants steadily coordinated NAM compared to 21 in the case of PZA. Trajectory and ligand interaction analysis showed how increased active site lid loop dynamics affect the NAM binding, especially in the systems with the active site mutations i.e., H51Y, W68R, C72R, L82R, K96N, L159N, and L159R. This led to fewer protein-ligand interactions and eventually ligand ejection. Network analysis further identified the protein core, metal binding site (MBS), and substrate binding site as the most important regions of the enzyme. Furthermore, the degree of centrality analysis showed how specific MtPncA mutations i.e., C14H, F17D, and T412P, interrupt intra-protein communication from the MtPncA core to the MBS, affecting enzyme activity. The analysis of KatG and MtPncA enzyme mutations not only identified the effects of mutations on enzyme behaviour and communication, but also established a framework of computational approaches that can be used for mutational studies in any protein. Besides AMR, the continued encroachment of wildlife habitats due to population growth has exposed humans to wildlife pathogens leading to zoonotic diseases, a recent example being coronavirus disease 2019 (COVID-19). In the second part of the thesis, the established computational approaches in Part 1, were employed to investigate the changes in inter-protein interactions and communication patterns between the severe acute respiratory coronavirus 2 (SARS-CoV-2) with the human host receptor protein (ACE2: angiotensin-converting enzyme 2) consequent to mutations in the SARS-CoV-2 receptor binding domain (RBD). Here, the focus was on RBD mutations of the Omicron sub-lineages. We identified four Omicron-sub lineages with RBD mutations i.e., BA.1, BA.2, BA.3 and BA.4. Each sub-lineage mutations were modeled into RBD structure in complex with the hACE2. MD analysis of the RBD-hACE2 complex highlighted how the RBD mutations change the conformational flexibility of both the RBD and hACE2 compared to the wildtype (WT). Furthermore, DRN analysis identified novel allosteric paths composed of residues with high betweenness and eigenvector centralities linking the RBD to the hACE2 in both the wildtype and mutant systems. Interestingly, these paths were modified with the progression of Omicron sub-lineages, highlighting how the virus evolution affects protein interaction. Lastly, the effect of mutations on S RBD and hACE2 interaction was investigated from the hACE2 perspective by focusing on mutations in the hACE2 protein. Here, naturally occurring hACE2 polymorphisms in African populations i.e., S19P, K26R, M82I, K341R, N546D, and D597Q, were identified and their effects on RBD-hACE2 interactions investigated in presence of the Omicron BA.4/5 RBD mutations. The hACE2 polymorphisms subtly affected the complex dynamics; however, RBD-hACE2 interaction analysis showed that hACE2 mutations effect the complex formation and interaction. Here, the K26R mutation favored RBD-hACE2 interactions, whereas S19P resulted in fewer inter-protein interactions than the reference system. The M82I mutation resulted in a higher RBD-hACE2 binding energy compared to the wildtype meaning that the mutation might not favor RBD binding to the hACE2. On the other hand, K341R had the most RBD-hACE2 interactions suggesting that it probably favors RBD binding to the hACE2. N546D and D597Q had diminutive differences to the reference system. Interestingly, the network of high betweenness centrality residues linking the two proteins, as seen in the previous paragraph, were maintained/modified in presence of hACE2 mutations. HACE2 mutations also changed the enzyme network patterns resulting in a concentration of high eigenvector centrality residues around the zinc-binding and active site region, ultimately influencing the enzyme functionality. Altogether, the thesis highlights fundamental structural and network changes consequent to mutations both in TB and COVID-19 proteins of interest using in silico approaches. These approaches not only provide a new context on impact of mutations in TB and COVID target proteins, but also presents a framework that be implemented in other protein mutation studies. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2023
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  • Date Issued: 2023-10-13

In-silico investigation of the effects of genetic mutations on the structural dynamics of thiopurine s-methyltransferase and their implications on the metabolism of 6-mercaptopurine

  • Authors: Mwaniki, Rehema Mukami
  • Date: 2023-10-13
  • Subjects: Mutation , Thiopurine S-methyltransferase , Mercaptopurine , Molecular dynamics , Protein structure , Structural dynamics
  • Language: English
  • Type: Academic theses , Master's theses , text
  • Identifier: http://hdl.handle.net/10962/432553 , vital:72880
  • Description: Thiopurine S-methyltransferase (TPMT) is a cytosolic enzyme that catalyzes the S-methylation of aromatic and heterocyclic sulfhydryl compounds such as 6-mercaptopurine (6MP), 6-thioguanine (6TG) and azathioprine (AZA) which is first converted to 6MP through reduction by glutathione S- transferases (GST). The compounds, generally referred to as thiopurines, are immunosuppressants used to treat childhood acute lymphoblastic leukemia (ALL), autoimmune disorders and transplant rejection. Thiopurines are prodrugs which require metabolic activation to give thioguanine nucleotides that exert their cytotoxic effects by incorporation into DNA or inhibiting purine synthesis. The methylation reaction by TPMT utilizing S-adenosylmethionine (SAM) as the methyl donor prevents their conversion to these toxic compounds. The catalytic activity of TPMT in metabolising these compounds has been associated with occurrence of genetic variations. The variations that result to missense mutations cause amino-acid changes and in turn alter the polypeptide sequence of the protein. This could alter functionality and structural dynamics of the enzyme. This study sought to understand the underlying mechanism by which 7 specially selected mutations impede metabolic activity of the enzyme on 6-MP using in silico techniques. VAPOR and PredictSNP were used to predict the effects of single nucleotide polymorphisms (SNPs) on the stability and function of the enzyme. Of the 7 mutations, only H227Q was predicted to be functionally benign while the rest (L49S, L69V, A80P, R163H, R163C and R163P) were predicted to be deleterious or associated with disease. All the SNPs were predicted to destabilize the enzyme. Molecular dynamics (MD) simulations were preformed to mimic the behaviour of the apo, holo and drug-bound WT and mutant enzymes in vivo. This was followed by post-MD analysis to identify changes in the local and global motions of the protein in the presence of mutations and changes in intra-protein communication networks through contact map and centrality metrics calculations. RMSD and Rg analyses were performed to assess changes in global motions and compactness of the enzyme in the apo, holo and drug-bound states and in the presence of mutations. These revealed that binding of the ligand had a stabilizing effect on the WT enzyme evident from more steady trends from the analyses across trajectories in the holo and drug-bound enzymes compared to the apoenzyme. The occurrence of mutations had an effect on the global motions and compactness of the enzyme across the trajectories. Most mutations resulted in destabilized systems and less compact structures shown by unsteady RMSD and Rg across trajectories respectively. The drugbound systems appeared to be more stable in most of the systems meaning that the binding of 6MP stabilized the enzyme regardless of the presence of a mutation. RMSF analysis recorded local changes in residue flexibility due to the presence of mutations in all the systems. All the drug-bound mutant systems lost flexibility on the αAhelix which caps the active site. This could have an effect on drug binding and result to defective drug metabolism. The A80P mutation resulted to a more rigid structure from both global and local motions compared to the WT enzyme which could be associated with its nearly loss of function in vivo and in vitro. Dynamic cross correlation calculations were performed to assess how the atoms moved together. Correlated, anti-correlated and areas of no correlations were recorded in all the systems and in similar places when compared to each other. This meant that occurrence of mutations had no effect on how the atoms moved together. Contact map analysis showed that occurrence of mutations caused changes in interactions around the positions where the mutations occurred, which could have an effect on protein structural dynamics. The A80P substitution which occurred on the surface away from the binding site was identified as an allosteric mutation that resulted to changes in the catalytic site. Contact maps for the drug-cofactor complex in the mutant systems in comparison with the WT protein revealed changes that could suggest reorientation of the drug at the catalytic site. This could be an implication to altered drug metabolism. Eigenvector centrality (EC) and betweenness centrality (BC) for the most equilibrated portions of the trajectories were calculated for all the studied systems to identify residues connected to the most important residues and those that were spanned the most in shortest paths connecting other residues. Areas that scored highest in these metrics where mostly found in regions surrounding the catalytic site. Top 5% centrality hubs calculations showed loss of major hubs due to mutations with gaining of new ones. This means that mutations affected communication networks within the protein. The gained hubs were in areas close-by the lost ones which could have been an attempt of the protein to accommodate the mutations. Persistent top 5% BC hubs were identified at positions 90 and 151 while one persistent top 5% EC hub was identified at position 70. This positions play important roles in shaping the catalytic site and are in direct contact with the ligands. It was concluded that in silico techniques and analysis applied in this study revealed possible mechanisms in which genetic variations affected the structural dynamics of TMPT enzyme an affecte 6MP metabolism. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
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  • Date Issued: 2023-10-13

Screening isolation and biological characterization of antibacterial secondary metabolites from macrofauna endemic to the southern African coast

  • Authors: Njanje, Idris
  • Date: 2023-10-13
  • Subjects: Uncatalogued
  • Language: English
  • Type: Academic theses , Doctoral theses , text
  • Identifier: http://hdl.handle.net/10962/431944 , vital:72817
  • Description: Access restricted. Expected release date in 2025. , Thesis (PhD) -- Faculty of Science, Chemistry, 2023
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  • Date Issued: 2023-10-13

The development and op timisation of a Theiler’s murine encephalomyelitis virus antiviral assay

  • Authors: Naidoo, Urisha Tirah
  • Date: 2023-10-13
  • Subjects: Theiler's encephalomyelitis virus , Picornaviruses , Antiviral agents , Immunofluorescence , Western immunoblotting , Drug development
  • Language: English
  • Type: Academic theses , Master's theses , text
  • Identifier: http://hdl.handle.net/10962/424677 , vital:72174
  • Description: Picornaviruses belong to the Picornaviridae family which are one of the largest and most diverse family of RNA viruses that cause a broad spectrum of infections in both humans and animals. These diseases range from severe infections such as poliomyelitis, meningitis, myocarditis to mild illnesses such as the common cold. Picornavirus outbreaks are a worldwide threat as they are continuously occurring. A recent outbreak of foot-and-mouth disease caused by a picornavirus occurred in South Africa, resulting in a temporary ban on the movement of cattle. Currently, the FDA has not approved any antiviral drugs against this virus, increasing the urgency for identifying effective antivirals. Picornaviruses have similar genomes and capsid organisation as such, those that are non-hazardous to humans can be used as a model system. A Theiler’s murine encephalomyelitis virus (TMEV) strain GDVII and Baby Hamster Kidney fibroblasts (BHK-21 cells) was used as a replication system to develop and optimise a medium-throughput antiviral screening assay. The TMEV GDVII replication system in BHK-21 cells was validated, and preliminary experiments were performed that were necessary for the development of the TMEV GDVII antiviral assay. This was achieved by conducting a CPE assay to visually monitor the onset and development of CPE induced by TMEV GDVII. Plaque assays accurately quantified the number of infectious virus particles required for calculating the MOI in downstream experiments. Lastly, indirect immunofluorescence and Western blot analysis detected the expression of viral proteins using previously generated antibodies against the TMEV GDVII VP1 capsid and 2C protein, thereby confirming infection in BHK-21 cells. The development of robust and reproducible assays is an essential component in antiviral drug discovery. Therefore, the confirmed replication system was then used as a foundation to develop a medium-throughput CPE-based TMEV GDVII antiviral assay whereby the parameters were optimised to produce one of high quality. Firstly, the quantitation of viral-induced CPE was examined and confirmed in a 96-well plate using resazurin as a cell viability indicator. Each parameter was tested at varying conditions, and the optimal was concluded as 2 % FBS in the assay media, a 15 000 cells/well seeding density, infecting the cells with TMEV GDVII at an MOI of 0.00625 and measuring resazurin at an endpoint of 72 hpi. Furthermore, the parameters were ii validated by calculating the Z’- factor, which consistently produced scores above 0.5, indicative of a reliable, robust, reproducible antiviral assay. Currently, there are no inhibitors against TMEV GDVII that have been reported or confirmed in cell lines, animal models or clinical trials. Therefore, once the optimal assay parameters were selected, it presented an opportunity to assess whether potential compounds, including itraconazole (ITZ) and dipyridamole (DIP), possessed antiviral activity that could firstly, be utilised as a control inhibitor when screening compounds against TMEV GDVII and secondly, contribute to research on this virus. Additionally, the previously produced anti-TMEV GDVII capsid antibody was shown to neutralise viral infection and was also included as a potential control. The sensitivity of the cells towards DMSO, a solution in which the compounds were solubilised, was first investigated. It was found that concentrations above 1 % are toxic to the cells; as such, the final DMSO concentrations were always kept below 1 % when screening compounds. Lastly, the generation of dose-response curves aided in the conclusion that the antibody was the most suitable control inhibitor as it displayed potent antiviral activity and no cytotoxicity towards the cells. In contrast, ITZ and DIP did not possess effective antiviral action and were toxic to cells at high concentrations. Finally, after all the components of the medium-throughput TMEV GDVII antiviral assay were identified, it was possible to screen 24 compounds from a coumarin and marine natural product library for cell cytotoxicity and antiviral activity. After generating dose-response curves, it was concluded that no compound effectively inhibited virus-induced CPE, and most were toxic to cells at relatively high concentrations. In conclusion, this is the first study that describes the development and optimisation of a robust medium-throughput CPE-based antiviral assay that has immense potential to screen other libraries of compounds for antiviral activity against TMEV GDVII. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
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  • Date Issued: 2023-10-13

The development of a plate-based assay to detect the activation status of ARF1 GTPase in Plasmodium falciparum parasites

  • Authors: Du Toit, Skye Carol
  • Date: 2023-10-13
  • Subjects: ARF1 , GTPase , Plasmodium falciparum , Malaria , Drug resistance , Drug targeting , Enzyme-linked immunosorbent assay , Proteins
  • Language: English
  • Type: Academic theses , Master's theses , text
  • Identifier: http://hdl.handle.net/10962/424654 , vital:72172
  • Description: The exponential rise in antimalarial drug resistance in the most infectious malaria species, Plasmodium falciparum, has emphasised the urgency to identify and validate novel drug targets that decrease parasite viability upon inhibition. In addition to several publications indicating that the regulation of human Arf1 GTPase activity (mediated by ArfGEFs and ArfGAPs) serves as a pertinent drug target for cancer research, the identification of Arf1 and its regulatory proteins in Plasmodium falciparum led to the question whether these protein homologs could be exploited as drug targets for anti-malarial drug therapies. To investigate this prospect, the establishment of a novel in vitro colorimetric ELISA-based assay was needed to be able to detect changes in the activation status of P. falciparum Arf1 (PfArf1) in parasite cultures exposed to potential Arf1 inhibitors. By exploiting the selective protein interaction that occurs between active GTP-bound Arf1 and its downstream effector, GGA3, an assay protocol was established that could be used to detect the activation status of purified, truncated PfArf1 obtained from E. coli and endogenous PfArf1 sourced from parasite lysates. The assay relies on the use of anti-Arf1 antibodies to detect the binding of active PfArf1 in the lysates of inhibitor-exposed cultured parasites to GST-GGA3 immobilised in glutathione-coated plates. The results from chemical validation experiments conducted using the novel assay developed in this study, using the known ArfGEF inhibitor brefeldin A (BFA) and ArfGAP inhibitors Chem1099 and Chem3050, yielded the anticipated results: decrease in active PfArf1 after parasite incubation with the ArfGEF inhibitor, and increased active PfArf1 after ArfGAP inhibition. The results confirmed PfArf1 as a potential anti-malarial drug target and encourages the further development of this assay format for the identification of subsequent inhibitors in library screening campaigns. Additional pilot experiments were conducted to further explore whether the assay could detect the activation status of human Arf1 using HeLa cell lysates and to provide further evidence that the assay could be exploited as a tool in the identification of Arf1 GTPase inhibitors with BFA and the known ArfGAP inhibitor, QS11. The results suggested that, while the assay can detect the increase in active cellular Arf1 due to the inhibition of human ArfGEF following BFA treatment, subsequent treatment with QS11 showed no evidence of a reduction in active human Arf1 due to ArfGAP inhibition. Further experimentation is required to investigate the ability the assay to confirm inhibition of human Arf1 deactivation by ArfGAP inhibitors and develop the assay as a useful tool to support cancer drug discovery, in addition to antimalarial drug discovery projects aimed at Arf1. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
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  • Date Issued: 2023-10-13

Phytoplankton communities provide insight into ecosystem functioning of the Agulhas Current system

  • Authors: Gibb, Ross-Lynne Alida
  • Date: 2023-03-31
  • Subjects: Uncatalogued
  • Language: English
  • Type: Academic theses , Doctoral theses , text
  • Identifier: http://hdl.handle.net/10962/422646 , vital:71965
  • Description: Access restricted. Embargoed until 2025. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2023
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  • Date Issued: 2023-03-31

South African supratidal microbialites: prokaryote communities, metabolic capabilities, and biogeochemical processes

  • Authors: Isemonger, Eric William
  • Date: 2023-03-31
  • Subjects: Uncatalogued
  • Language: English
  • Type: Academic theses , Doctoral theses , text
  • Identifier: http://hdl.handle.net/10962/422641 , vital:71964
  • Description: Access restricted. Embargoed until 2025. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2023
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  • Date Issued: 2023-03-31
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