Electronic Theses and Dissertations (Masters)

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

Browse

Search Results

Now showing 1 - 10 of 21
  • Thumbnail Image
    Item
    Antibacterial activity and susceptibility testing of bacterial isolates from nematodes (Cruznema spp.)
    (University of the Witwatersrand, Johannesburg, 2023-09) Mothapo, Maletjema Magdeline; Lephoto, Tiisetso E.
    Nematodes are unsegmented worms found in different niches associated with a diverse range of bacteria. Various types of nematodes exist including those that are parasitic to insects, known as entomopathogenic nematodes (EPNs). EPNS of genera Steinernema, Heterorhabditis and Oscheuis are symbiotically associated with Xenorhabdus, Photorhabdus and Serratia, respectively. The symbiotic bacteria of EPNs have been reported to produce a broad spectrum of antimicrobial compounds active against human pathogens. The aim of this study was to isolate and identify nematodes and their associated bacteria from soil samples collected from a vegetative farm in Lesotho and study their antimicrobial activity against four species of pathogenic bacteria (E. coli, S. aureus, E. faecalis and P. aeruginosa). An uncharacterized species of Cruznema was isolated and named Cruznema NTM-2021 (GenBank 18S rDNA accession number: OQ408141). Based on the BLASTN search incorporating the phylogenetic analysis of the 16S rDNA region, three genera of bacteria were identified as Alcaligenes sp., Enterobacter sp. and Elizabethkingia sp. The study revealed that all three bacterial isolates were pathogenic to Tenebrio molitor. Symbiosis tests, using lipid agar method demonstrated the ability of the host nematodes to develop and reproduce in the presence of their associated bacteria. Bacterial supernatants of Alcaligenes sp. and Enterobacter sp. showed some inhibitory activity against Escherichia coli and Enterococcus faecalis, by disk diffusion method. Staphylococcus aureus and Pseudomonas aeruginosa were the most resistant bacteria to supernatants of the three isolates. This study also showed that the Alcaligenes, Enterobacter, and Elizabethkingia species isolated from Cruznema NTM-2021 were resistant to ampicillin, amoxicillin, cefuroxime/sodium, vancomycin and cephalothin but susceptible to gentamicin.
  • Thumbnail Image
    Item
    HIV-1 subtype C protease: enzyme kinetics, thermodynamics, and X-ray crystal structure
    (University of the Witwatersrand, Johannesburg, 2023-09) Dlamini, Nozinhle Precious; Sayed, Yasien
    Human immunodeficiency virus (HIV), a precursor for AIDS is still one of the most devastating pandemics in history. In 2021 alone there were 650 000 deaths associated with the virus and the number of people living with the infection was recorded to be 38.4 million globally. Sub-Saharan Africa suffers the most burden of the virus with approximately 8.3 million people living with virus, HIV-1 subtype C is the main driver of the disease in South Africa and accounts for 46% of global infections. Even with these alarming statistics this subtype is not the main focus point for the majority of HIV-1 research which mainly focuses on subtype B though it only accounts for 12% of infections globally. There is no vaccine or cure against HIV; however, great strides have been made in suppressing the virus. Viral suppression drugs have been developed to target different stages of viral replication such as those targeting the three important enzymes (protease, reverse transcriptase and integrase). In this study the focus will be on HIV-1 subtype C protease. This is a homodimeric aspartyl protease with 99 amino acids in each monomer. It plays a crucial role in the replication cycle of HIV-1 by producing mature infectious virions through cleavage of the Gag and Gag-Pol polyproteins. The subtype C protease differs from subtype B protease in that it has eight naturally occurring polymorphisms which are substitution mutations, some occurring in different regions of the protease with some in the fulcrum (T12S, I15V and L19I), others in the hinge region (M36I and R41K), with H69K and L89M found in the loops and I93L in the α helix. In this study, structural and functional characterisation of HIV-1 subtype C protease was carried out. The secondary structure was characterised using far-UV CD, which is a technique that measures the difference in left and right circularly polarised light. The subtype C protease was estimated to be predominantly β-sheeted, with spectra showing a maximum at 195 nm and a minimum between 215-225 nm. Tertiary structure characterisation of protease was performed using fluorescence spectroscopy. The maximum emission at 347 nm close to that of water (350 nm), demonstrated that the tertiary conformation of the HIV-1 protease was conserved, and that the tryptophan residues within the protease are solvent exposed. SE-HPLC was used to characterise the quaternary structure of the protease and the homodimeric size was determined to be approximately 22 kDa. Steady-state enzyme kinetics to assess the catalytic activity of the subtype C protease was performed using a fluorogenic substrate. The activity of the enzyme was confirmed, with the specific activity of 24.22±1.72 µmol. min-1.mg-1 and the binding of the substrate to the HIV-1 protease was demonstrated by the KM value of 79.546±6.491 µM. This correlates to literature indicating that the substrate was weakly bound and that a high substrate concentration will be required to reach the maximum velocity (Vmax), and Vmax was determined to be 0.036±0.003 µmol. min-1. Enzyme kinetics was coupled with displacement isothermal titration calorimetry for determination of thermodynamics parameters using second generation PIs (atazanavir, darunavir and lopinavir). Thermodynamic studies indicated that the HIV-1 protease has a high affinity for LPV (Kd = 1 nM), compared to ATV (Kd = 18.57nM) and DRV (Kd = 42.26 nM) and binding reactions were all spontaneous with ΔG values(ATV = -43.39 kJ/mol, DRV = -41.39 kJ/mol and LPV = -50.51 kJ/mol). The values also indicated that LPV complexed with HIV-1 is more a stable complex. Also, all the binding reactions were exothermic as indicated by the negative ΔH values of ATV = -45.54 kJ/mol, DRV = -55.62 kJ/mol and LPV = -54.71 kJ/mol. The entropy of all the reactions were determined to be unfavourable with the -T∆S of DRV = 14.23 kJ/mol followed by LPV: 4.2 kJ/mol and ATV: 2.15 kJ/mol. Overall this suggested that all the binding reactions were enthalpically driven. Furthermore, the three-dimensional structure of the HIV-1 subtype C protease was elucidated using X-ray crystallography. The three-dimensional structure the HIV-1 CSA (PDB ID: 8CI7) was solved at a 2.4 Å resolution which is better than the 2.7 Å (PDB ID: 3U71) initially solved in our lab. The high-resolution three-dimensional structure of the protease will provide precise information about the arrangement of atoms within the protease molecule, thus enabling the design and development of protease inhibitors that will be specific for the subtype C protease. This study emphasised the significance of investigating subtype C protease in the context of enzyme kinetics, thermodynamics and detailed X-ray crystallography.
  • Thumbnail Image
    Item
    Comparison of Saccharomyces cerevisiae and the novel wild yeast used in beer fermentation and their future in industrial biotechnology
    (University of the Witwatersrand, Johannesburg, 2024) Zviuya, Patience; Moodley, Sanchia; Rumbold, Karl
    The alcohol industry has grown over centuries due to the increase in alcohol demand by customers. Beer consumers now understand the brewing process and they well understand the role of yeast in fermentation. The alcohol industry now has a lot of customers because of the growth that this sector has made, and this has resulted in an increased demand for new beer styles and high flavor profile beers. Due to this demand, research has been conducted on unconventional wild yeasts that can be employed in making beer, demonstrating the variety of fermentation yeasts that are available and capable of enhancing beer quality and producing a wide range of new beer varieties. The commercial S. cerevisiae yeast and eight wild yeasts (Samson’s Saison, Ragnarok, Dark knight, B. brusc, Neipa, The Proletariat, La Trappist and B. clauss) were used to ferment pale ale in different fermentation vessels. The commercial yeast was the control of this research because this yeast has been used for generations in the brewing industry. The wild yeast strains used were identified using sanger sequencing and seven of these yeasts were S. cerevisiae wild species with similarity index of more than 80% and one was B. bruxellensis strain similarity index more than 95% before and after fermentation. The research outcomes demonstrated that most of the wildyeast performed the same as the commercial yeast in terms of physical and chemical parameters however most of the wild yeast produced more volatiles and esters as compared to the commercial brewing yeast. Commercial S. cerevisiae produced the highest alcohol content 4.5% and the average alcohol content for wild yeast was 3.4% because they are challenging to regulate during fermentation and have low alcohol tolerance unlike the commercial yeast that has been harvested and used for generations. Overall, the utilization of unconventional wild yeast to make beer was identified as a promising alternative to produce beers with exotic flavours and alcohol-free beers. Future work identifying specific yeast that suit different fermentation processes and beer types are recommended.
  • Thumbnail Image
    Item
    Using ChIP-seq and Gene Expression Microarray data to explore transcriptional dysregulation of PXDN and PXDNL in cardiovascular diseases
    (University of the Witwatersrand, Johannesburg, 2024) Naidoo, Shiven; Gentle, Nikki; Mavri-Damelin, Demetra
    Background: Cardiovascular diseases (CVDs) remain one of the leading causes of death globally. The genes PXDN and PXDNL are both expressed in the cardiovascular system, and their dysregulation has been linked to various disorders, including CVDs, but little is known of their transcriptional regulation in the cardiovascular system or their roles in CVD pathogenesis. Methods: This study developed two custom bioinformatics pipelines in R to mine and analyse ChIP-seq data from ChIP-Atlas and gene expression microarray data from the Gene Expression Omnibus (GEO). The first pipeline used ChIPseeker to identify regulatory transcription factors (TFs) of PXDN and PXDNL in cardiovascular cells and tissues. ChIP-seq data from 400 experiments across 63 TFs was filtered to isolate TFs with high confidence binding peaks in the promoter and first intron of PXDN and PXDNL. The second pipeline used R Bioconductor packages to explore the expression profiles of PXDN, PXDNL, and their TFs in seven microarray datasets across three CVD-related contexts: cardiomyopathies, heart failure and TNF-α stimulation. Results and discussion: This study identified 27 TFs binding to PXDN and 18 TFs binding to PXDNL in cardiovascular cells. Sixteen of these TFs were shared by both PXDN and PXDNL, suggesting potential coregulatory mechanisms in cardiovascular cells where they are both expressed. Unique TFs were also identified for PXDN (11) and PXDNL (2). Differential gene expression analysis revealed no significant change in expression (log2FC > 0.5; p.adj < 0.05) for PXDN, PXDNL and many of their identified TFs in the CVD-related conditions investigated, suggesting that changes at the transcript level may not contribute to the progression of these conditions. Conclusions: This study advances our understanding of the transcriptional regulation of PXDN and PXDNL in healthy cardiovascular cells as well as their expression levels in the investigated CVD-related contexts. This study also contributes a bioinformatics pipeline which can be further developed and applied to analysing data from ChIP-Atlas and GEO. Future research can elucidate the roles of each TF in regulating PXDN and PXDNL in healthy and diseased cell lines
  • Thumbnail Image
    Item
    Isolation and characterisation of entomopathogenic fungi
    (University of the Witwatersrand, Johannesburg, 2024) Kwinda, Fhatani; Lephoto, Tiisetso E.
    The purpose of the study was to isolate and identify fungal isolates in soil samples, followed by virulence characterisation to study their effectiveness in controlling insect pests using Tenebrio molitor as our model organism. Lastly, a combination study was conducted to evaluate the effectiveness of the joint use of two entomopathogenic microorganisms. For isolation, T. molitor was used as bait then the isolated fungal isolates were identified using molecular and morphological characterisation. Morphological characterisation included macroscopic (fungal cultures) and microscopic (conidia shape and size) analysis while molecular characterisations included extraction of DNA, amplification of the internal transcribed spacer region and sequence alignment. Once identification was done, virulence was assessed through in-vitro virulence parameter like vegetative growth and in-vivo assessment where bioassays were done against T. molitor. Lastly, entomopathogenic fungi were combined with Cruznema sp. NTM-2021 in a soil assay. From the study, two of the five isolates were identified as entomopathogenic fungi, Metarhizium anisopliae ARSEF 7487. M. anisopliae had the slowest vegetative growth but was the highest in virulence. When used for a single application in a soil environment it reaches 97.8% mortality and its combination with Cruznema sp. NTM-2021 resulted in a 57.8% mortality and an additive interaction. In conclusion, M. anisopliae used alone was effective in its control of T. molitor
  • Thumbnail Image
    Item
    Establishing and characterizing organoid cultures from colon tissue of South African individuals
    (University of the Witwatersrand, Johannesburg, 2024) Du Plessis, Thea-Leonie; Kaur, Mandeep
    Colorectal cancer (CRC) has been poorly studied in South Africa, with limited studies on disease progression and development. Studies that have investigated CRC in South Africa have indicated that there is racial disparity between different racial groups that may be attributed to alternative developmental pathways, differences in genetic compositions or CRC initiators that result in these different clinical presentations. Furthermore, the lack of population-based studies substantiates the need for more intensive CRC research. A particular model used to study cancer in general is the use of two-dimensional (2D) cell cultures, which have provided novel insight into many cancers and their development processes. However, these models lack the complex biology observed in vivo. One such model that is gaining research interest is the use of three-dimensional (3D) organoid cultures. Organoids are derived from stem cells and are able to self-organize and mimic the corresponding organ from which they were derived. Research has indicated that organoids are able to maintain cell-type heterogeneity as well as gene expression levels that resemble the organ of origin. Therefore, this project aimed at standardizing a protocol to establish and characterise colorectal organoid cultures from South African patient-derived tissues. Patient samples were obtained from individual patients with informed consent and were processed to generate organoids. The morphology of the organoids was monitored across several days and across passages. Once the organoids had reached maturity and were at passage 2, characterization was performed using real-time quantitative polymerase chain reaction (RT-qPCR) and immunofluorescence which indicated that the genetic composition and spatial localization of cell types of interest in non-cancerous tissue was recapitulated in the organoids. Based on these observations, it is proposed that organoids could be a promising model to investigate CRC disease development and progression and potentially search for novel therapeutics. This project has established the protocols for growing and characterizing organoids from African samples and provides baseline data, and outlines the complexities and issues involved in growing organoid cultures for the future studies
  • Thumbnail Image
    Item
    A Clot to Uncover: FOXP3 and SARS-CoV-2 Nucleocapsid Interactions and Their Effect on DNA Binding
    (University of the Witwatersrand, Johannesburg, 2024) Mcinnes, Keiran; Fanucchi, Sylvia
    During COVID-19, systemic coagulopathy, which can lead to strokes and embolisms, is often observed in COVID-19 patients and may also contribute to long COVID. This coagulopathy is the result of overactivated platelets in circulation that leads to inappropriate clot formation. FOXP3 is a transcription factor involved in platelet development and loss of FOXP3 function leads to platelets that resemble those seen during COVID-19. Thus, FOXP3 may be dysregulated in COVID-19. The SARS-CoV- 2 nucleocapsid (NC) is a multifunctional protein typically associated with viral genome packaging and virion assembly. However, it is also capable of binding DNA and is potentially able to alter regulation of host protein expression. Here, potential interactions between the DNA-binding forkhead domain (FHD) of FOXP3 and the SARS-CoV-2 NC were investigated. Identification of a novel interaction between FOXP3 and SARS CoV-2 NC may provide new clues as to the pathophysiology of COVID-19. To address this aim, both proteins were overexpressed in T7 E. coli, purified via immobilised metal affinity chromatography, and monitored for potential interactions in the absence and presence of DNA using pull-down assays and fluorescence anisotropy. A direct interaction was identified between the two proteins in the absence of DNA. Additionally, it was found that both proteins are capable of binding to DNA at the same time, but excess NC was found to cause FHD dissociation from the FHD- NC-DNA complex. This result implicates NC in FOXP3 dysfunction which may be associated with the coagulopathy and other symptoms seen during COVID-19. Additionally, NC DNA binding does not appear to be driven by the FOXP3 consensus sequence, indicating that FOXP3 may not be the only transcription factor potentially dysregulated by NC
  • Thumbnail Image
    Item
    Metabolic Engineering of Streptomyces
    (University of the Witwatersrand, Johannesburg, 2023-09) Nosarka, Zainab; Moodley, Sanchia; Rumbold, Karl
    The Streptomyces genus demonstrates remarkable potential as a source and host for the production and discovery of industrially relevant secondary metabolites. The genus natively produces approximately 75% of antibiotics and several other compounds encoded by biosynthetic gene clusters (BCGs). However, most BCGs are poorly expressed or dormant under laboratory conditions and thus require metabolic engineering. Several technologies have been developed for this purpose but the pCRISPomyces-2 plasmid system, which employs Cas9 engineering, exhibits the most promising efficacy. This dissertation outlined foundational research to determine the capacity to introduce pCRISPomyces-2 plasmids into Streptomyces albulus BCRC11814 that produces high quantities of ɛ-poly-L-lysine, an antimicrobial and anti-phage compound. In addition, the strain has several other industrially relevant BCGs that have not been studied but possess engineering potential. To achieve the outlined aim, pCRISPomyces-2 plasmids were Sanger sequenced to ensure structural integrity and related functionality. A ClustalW alignment referenced against the plasmid’s nucleotide sequence verified a sequence identity > 98%. Subsequently, an intergeneric conjugation system was established by transforming pCRISPomyces-2 plasmids into Escherichia coli donor cells with an average transformation efficiency (TE) of 1.49 × 105 cfu/µg. Attempts to optimise TE were hindered by the plasmids’ large size and inherent Cas9 toxicity. Thereafter, the transformed donor cells were conjugated with S. albulus BCRC11814 and a comparative model strain Streptomyces coelicolor A3(2). Successful exconjugants were only obtained with S. coelicolor A3(2). The absence of conjugal mating with S. albulus BCRC11814, despite optimisation attempts, was hypothesised to result from the presence of a methyl-specific restriction modification system. This was confirmed by comparative electro-transformation with methylated and non-methylated DNA that demonstrated specificity to dam and dcm methylated DNA. Furthermore, spontaneous resistance to the selectable marker apramycin was confirmed in both Streptomyces strains. Additional efforts are required to effectively introduce pCRISPomyces-2 plasmids into S. albulus BCRC11814.
  • Thumbnail Image
    Item
    Effects of Mg2+, Ni2+ and Ca2+ on ATP binding kinetics of nicotinate nucleotide adenylyltransferase from Klebsiella pneumoniae and Enterococcus faecium: insights from empirical and computational studies
    (University of the Witwatersrand, Johannesburg, 2023-07) Van Deventer, Ruan; Achilonu, Ikechukwu Anthony
    NNAT is an attractive target for drug development due to its crucial role in NAD+ synthesis. However, its characterisation in ESKAPE species, such as Klebsiella pneumoniae and Enterococcus faecium, remains limited. This study aimed to elucidate the binding mechanism of ATP, a pivotal substrate, to these NNAT species, focusing on the role of divalent metal ion cofactors. KpNNAT and EfNNAT enzymes were overexpressed and purified, yielding approximately 2 mg/ml for both. Various techniques were employed to investigate their properties, including far-UV CD, extrinsic ANS fluorescence, stopped-flow kinetic analyses, and MD simulations. The results revealed that KpNNAT could bind ATP independently of divalent metal ions, but catalytic activity required the presence of Mg2+. The kinetic analysis showed ka values of 5.99 μM-1 .sec-1 without divalent metal ions and 5.72 μM-1 .sec-1 in the presence of Mg2+. The "pseudo"-specific activity values were 0.005 μmol/min/mg without divalent metal ions and 0.374 μmol/min/mg in the presence of Mg2+. Conversely, recombinant EfNNAT exhibited limited ATP association, and the reasons for this remain unclear. Overall, this study shed light on the structural dynamics and functional kinetics of ATP association in both NNAT species. The findings contribute to our understanding of this druggable target and provide insights into the inactivity of EfNNAT, which warrants further investigation.
  • Thumbnail Image
    Item
    Insights into the Physiochemical Properties of the Interaction between the L38↑N↑L+4 HIV-1 Hinge Mutant Subtype C Protease and a Related Gag Cleavage Site
    (University of the Witwatersrand, Johannesburg, 2023-09) Adams, Taryn Racheal; Sayed, Yasien
    Frequent mutations in HIV protein drug targets such as the protease (PR), have led to a rise in resistance to clinically available treatments and inquiries into the associated biochemical mechanisms. In this study, the L38↑N↑L+4 PR (mutant) and related gag, were isolated from a PR inhibitor naïve child for further study. Wild-type and mutant PRs were successfully overexpressed and purified using ion exchange chromatography. Intrinsic fluorescence studies probing tryptophan residues located at the hinge revealed variations in tertiary structure. This coincided with significant differences in refolding efficiency (mutant PR recovery ~ 10% and wild-type PR recovery ~ 34%). Furthermore, differences in catalytic efficiency (mutant-specific activity ~ 7.4 µmol.min-1.mg-1, mutant kcat ~ 2.71 sec-1; wild-type specific activity ~ 31.6 µmol.min-1.mg-1, kcat ~ 11.60 sec-1). Thermal shift assays revealed reduced mutant PR structural stability (Tm ~ 67 ℃) compared to the wild-type PR (Tm ~ 64 ℃). Furthermore, reduced stability of inhibitor-mutant PR complexes (Tm ~ 73.5 ℃ Acetyl pepstatin (AP), 90 ℃ Darunavir (DRV), and 88 ℃ Saquinavir (SQV)) compared to complexes involving the wild-type PR (Tm ~ 70 ℃ (AP), 82 ℃ (DRV), and 82 ℃ (SQV)). Overall, the L38↑N↑L+4 PR mutations were found to influence the tertiary structure of the hinge, gag processing, and PI stability within the mutant PR active site. Computational docking studies highlighted the potential role of a gag single nucleotide polymorphism (SNP), located at the 4th amino acid (P4) position of the peptide-2-nucleocapsid (p2/NC) gag cleavage site, for future studies as a compensatory mutation aiding PR polymorphisms. Further studies should focus on the gag-PR functional pair to build a more accurate understanding of HIV drug resistance mechanisms.