Faculty of Science (ETDs)

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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
A Phenotype Prediction Framework for Classifying Colorectal Cancer Patients’ Response to FOLFOX Treatment: An Integrated Approach
(University of the Witwatersrand, Johannesburg, 2024) Mashatola, Lebohang; Kaur, Mandeep
Colorectal cancer (CRC), characterised by its prevalence and heterogeneity, poses a significant challenge in understanding drug resistance, especially in the context of FOLFOX treatment. This study presents an innovative methodology that integrates diverse data analysis approaches to address the challenge of predicting the phenotype of CRC patients resistant or sensitive to FOLFOX. The initial analysis involved dierential and co-expression analyses, identifying pivotal hub genes crucial to drug resistance in CRC, regulating intricate molecular networks. Subsequent enrichment analysis revealed their significant roles in biological functions, particularly influencing DNA repair and nuclear division. To capture inherent topological characteristics within genetic expression data, a novel technique utilising topological data analysis (TDA) was employed. By applying persistence homology to generate persistence images, the Vietoris-Rips complex was constructed using the signed-topological overlap matrix, comprehensively capturing numerous topological features, including high-dimensional Betti-1 and Betti-2. This provided valuable insights into the structural patterns of gene expression between the hub genes. Furthermore, the integration of whole-slide images enhanced understanding of tissue anatomy, which is crucial for predicting cancer stages. Using a MobileNet architecture, a deep learning model classified cancer stages, contributing to a holistic understanding of colorectal tumor microenvironments. For predictive modelling of drug resistance, a multilayer perceptron applied topological summaries generated by TDA. The developed framework, GeTopology, exhibited remarkable performance metrics, achieving an overall 83% accuracy in predicting the FOLFOX response, demonstrating a 3% improvement over a previously published phenotype prediction framework (NSCLC ) that utilised similar data modes. Robust accuracies were consistently observed in independent datasets, classifying both cancer patients and healthy individuals. The results indicated an approximate 10% increase in model prediction accuracy compared to NSCLC, emphasising the potential clinical impact of this integrative approach. In conclusion, this study advances the understanding of drug resistance in CRC by proposing a novel approach that integrates topology with histopathological images, oering transformative insights into predictive modelling and precision medicine
An Assessment of Beauty Waste Management Practices: A Case Study of Rustenburg Beauty Salons
(University of the Witwatersrand, Johannesburg, 2024) Knight, Jasper; Knight, Jasper
The beauty salon industry is one of the fastest growing industries and is a significant waste generator in South Africa. Waste that results from beauty salons is a thorny environmental issue because it spans from different waste types and sources. Futhermore it requires waste treatment and different disposal methods. In view of this, this study seeks to assess waste management practices of beauty salons in Rustenburg, South Africa, in order to identify the types of waste salons generate and to identify recommendations that can assist beauty salons to be environmentally sustainable by improving their waste management practices. The aim of the research is to understand how beauty salon waste is discarded and to what degree beauty salon personnel understand the impacts of waste on the environment. The objectives of this research are to (1) determine the total amount of waste produced by selected beauty salons in Rustenburg, (2) identify the waste management practices undertaken by the beauty salons, (3) explore the challenges the beauty salons face in relation to waste management, and (4) identify recommendations of how beauty salons in South Africa can further improve their waste management practices. This study employed a mixed methods design through quantifying the amount of waste the selected beauty salons generate over a two month period, and by interviewing salon personell on their views on salon waste and waste management practices. Fifteen salons were surveyed. Results were analyzed using thematic analysis. The results show that the all the beauty salons combined produce a total annual estimated waste of 4732.2 kg. Through interviews, the study identified waste management practices of the beauty salons to be primarily premised on discarding waste in dustbins for municipal collection, burning waste, or dumping waste in unregulated dumping sites when waste service delivery poses challenges. These three practices are the most common modes of waste disposal in the Rustenburg beauty salons. Issues of waste management facing beauty salons includes lack of waste facilities and lack of knowledge about waste management. The major recommendation from beauty salons and civil (professional) organisations was for government to provide beauty salons with better infrastructure for waste sorting, recycling, pick-up and disposal. Promotion of good practice and awareness campaigns were cited as recommendations to improve waste management practices in beauty salons
An ethnobotanical study of indigenous knowledge of the medicinal plants used by traditional healers in the rural communities of Nkomazi Local Municipality, Mpumalanga province
(University of the Witwatersrand, Johannesburg, 2024) Khoza, Nompendulo; Dukhan, Shalini; Ramalepe, Phillemon; Risenga, Ida
Traditional medicine continues to significantly impact many people’s lives amid all the advancements in modern medicine. Many rural communities in Mpumalanga province depend on indigenous traditional medicines to manage various ailments. The available research on the traditional usage of medicinal plants among rural communities in Mpumalanga is highly fragmented and under-researched. The decline of medicinal plant populations has led stakeholders to take various initiatives to counteract over-exploitation, including cultivation as a viable conservation approach. However, the scientists' inadequate understanding of the acceptance of cultivated medicinal plants by traditional healers is one of the issues contributing to the failure of medicinal plant cultivation programs. Consequently, this study aimed to document medicinal plants utilised by the Nkomazi Local Municipality's traditional healers and assess opportunities and constraints for medicinal plant conservation in the Nkomazi Local Municipality. The ethnobotanical data was obtained through semi-structured questionnaires and guided field walks with traditional healers. Individual interviews were conducted with ten traditional healers from eight villages across Nkomazi during field visits between July 2021 and February 2022. The study employed qualitative and quantitative approaches to understand traditional healers' perspectives concerning the ethnobotanical significance and medicinal plant conservation. The study found that the indigenous knowledge of medicinal plants in the Nkomazi Local Municipality is diverse, encompassing 111 species from 59 different families employed to treat 70 ailments. Most of the reported medicinal plants for this study are of Least Concern. Additionally, the top fourteen most reported species in the Nkomazi Local Municipality included commercially valuable plants such as Psidium guajava, Ricinus communis, Sclerocarya birrea, Aloe ferox, Aloe maculata, Leonotis leonurus, and Moringa oleifera. Most of the Nkomazi Local Municipality's traditional healers did not know about protected plant species and the National Environmental Management Act (NEMA). Traditional healers were aware of the decline in wild populations of medicinal plants, which they attributed to various factors such as overharvesting. Diviner’s and herbalist perception of using cultivated plants did not differ significantly (𝜒2=0.4762, df=1, P= 0. 490). The study provided a comprehensive inventory of medicinal plants utilised by Nkomazi traditional healers and essential data for future assessments of the use local use of indigenous medicinal plants.
Analysing RNA-sequence data for pancreatic ductal adenocarcinoma tissue samples to identify potential biomarkers
(University of the Witwatersrand, Johannesburg, 2023-09) Jamal, Khadija Sanober; Kaur, Mandeep
Pancreatic ductal adenocarcinoma (PDAC) accounts for approximately 90% of pancreatic cancer and is the fourth leading cause of death with a five-year survival rate of less than 10%. Patients are asymptomatic until detection is observed at a metastatic stage, hence contributing massively towards the high mortality rate. This study was conducted to explore PDAC and its two main subtypes, the classical and basal-like subtype, in an in-depth level via bioinformatic analysis. Bioinformatics is a computational approach to evaluate biological data by analysing omics data including genomic expression and proteomic sequences. A workflow consisting of programmes and web-tools was used to analyse PDAC RNA-sequence data. The sample sets were grouped according to tumour, stage, and subtype. The workflow began with quality control using FastQC and Trimmomatic. Alignment of sequencing files and counts were done through HISAT2 and HTSeq. The main component of this workflow was differential gene expression analysis to identify differentially expressed genes (DEGs), statistically significant genes, per compared conditions. WGCNA was used for co-expression analysis to identify the hub genes involved in regulating the biological network. Lastly, in-silico validation was done by using available web tools to support the findings of this workflow. The identified tumour genes included S100A11, PKM, GPRC5A, LAMC2 and ITGA2, which may represent as universal biomarkers as sample extraction was performed from data generated from individuals belonging to 8 different countries. KRT13 and IL6 were identified in the advanced stage and their role in cancer progression have been explored in this current study. The basal-like subtype had CAV1, DCVLD2 and TGFB2 genes that contribute to treatment resistance. The common dysregulated genes in the basal-like subtype and advanced stage were analysed to evaluate the link between subtype and stage which included WNT3A, TP63, KRT13 and IGF2BP. Coexpression analysis revealed hub genes for tumour (KIF4A, SPAG5, RRM2 and AURKA), basal-like subtype (BUB1, DEPDC1 and KIF14) and classical-subtype (PTPRN and CAMK2B). Through a machine learning model, recall, precision and accuracy scores per sample conditions for the DEGs were all above 94%. These potential biomarkers all have significant roles in promoting cancer progression, aggression and resistance. Hence, these may serve as a less invasive screening method for PDAC as DEGs were classified based on tissue or blood (extracellular vesicle) biomarkers. However, further wet laboratory validation is required for these biomarkers.
Are our Head Teachers okay? Decision-making processes during COVID-19 across South African independent schools
(University of the Witwatersrand, Johannesburg, 2023-10) Pahl, Julia; Evans, Mary
The COVID-19 pandemic significantly affected the education sphere globally and in South Africa. The impact on pupils and teachers has been investigated. However, the impact on Head Teachers in schools, particularly independent schools in South Africa, has received less attention. Head Teachers within independent South African Schools are the decision makers, leaders, and influencers who were placed in a key role during the COVID-19 pandemic. These Head Teachers were asked to lead, make decisions, and positively influence and manage those under their care while the educational landscape shifted. This shifting educational landscape placed Head Teachers within independent schools under increased pressure and stress. This study aimed to understand the technological and financial access and contexts of Head Teachers at independent schools within South Africa and explore their decision-making processes and the impact of their decisions on their well-being. An online survey was sent to independent Head Teachers, and four in-depth, one-on-one interviews were conducted. Head Teachers showed that their financial and technological contexts did not constrain their pandemic responses as the nature of their independent school allowed them to have access to adequate technological resources and alternative financial support sources. Key findings of this research were that Head Teachers were commonly influenced both positively and negatively by the complex communication networks and channels created and used during the pandemic and the complex decision-making drivers they had to navigate while leading during the pandemic. The decision drivers of well-being and shared moral purpose were more dominantly considered than the decision driver of National Government Directives, as Head Teachers had to consider their complex and multi-dimensional environments when deciding which decision driver was to be prioritized. Head Teachers had to navigate two main tensions: would they prioritize outcomes such as assessment and reports during the pandemic or would they prioritize awareness and well-being of staff and learners, and would they make their decisions from a compliance standpoint where full compliance was key or from a standpoint where complying as much as possible or enough for deniability was chosen. These results also associated increased communication networks during the pandemic with increased stress and burn-out levels and therefore a decreased well-being of Head Teachers, and increased numbers of Head Teachers leaving the profession. However, a positive outcome of the pandemic was that schools, staff, learners, and parents were forced to increase their device and/or internet access and technological skills to maintain education during the lockdown and the changing educational environment. When these results were studied across the geographical landscape of independent South African schools it was clear that the findings on access, well-being and decision-making were linked to the context of the Head Teacher and their school and not to their geographic location. Yet using complex, multi-faceted communication networks and the resultant stress was a country-wide experience and that National Government Directives was the country-wide main decision-driver for independent South African Head Teachers. Overall, this study identified six key lessons for education within any future South African pandemics: the need for clear and concise instructions within legislation, standard operating procedures need to consider access and context, blended education should become a norm in schools, consistency within education should be maintained throughout, personal boundaries should be maintained throughout and networking between leaders should be increased.
Biochemical and biophysical analysis of a hinge region protease variant in HIV-1 subtype C
(University of the Witwatersrand, Johannesburg, 2023-10) Mokhantso, Tshele; Achilonu, Ikechukwu; Sayed, Yasien
HIV-1 protease plays a crucial role in the maturation of the virus by cleaving gag and gag-pol polyproteins. Understanding the structural and functional consequences of mutations in this enzyme is essential for developing effective anti-HIV drugs, especially in the face of emerging drug-resistant variants. This study focused on the N37T↑V+10•D25A mutant, a novel HIV-1 subtype C protease variant harbouring an insertion (↑V) and a substitution (N→T) at position 37, along with 10 naturally occurring polymorphisms. Mutations occurring distal to the active site have long been thought to contribute to drug resistance, with this in mind the study aimed to assess the impact these mutations have on the structure, stability, dynamics and drug binding of HIV-1 protease. The N37T↑V+10•D25A mutant and wild-type (WT•D25A) HIV-1 protease were overexpressed and purified from inclusion bodies formed in Escherichia coli cells using ion-exchange chromatography. Far-UV CD and SE-HPLC analysis showed that N37T↑V+10•D25A exhibited a predominantly β-sheet secondary structure (218 nm trough) and had a homodimeric size of ~23 kDa, respectively, both similar to WT•D25A. Assessment of the local tertiary structure by intrinsic tryptophan fluorescence indicated that the protease retained its tertiary structure in the presence of mutations, with partial exposure of Trp residues (Trp 6, Trp 6', Trp 42/43, and Trp 42'/43’). Overall, the insertion and substitution mutations did not significantly alter the overall structure of HIV-1 protease. However, the conformational stability of N37T↑V+10•D25A was found to be reduced compared to WT•D25A as determined by urea-induced equilibrium unfolding and thermal unfolding experiments. When denatured using urea and temperature, the mutant exhibited a two-state mechanism of unfolding without stable intermediates during the folding and unfolding process. Thermal unfolding experiment determined the melting temperature of N37T↑V+10•D25A as 58 ± 1.2 °C, which is lower than that of the wild type of 62 ± 0.9 °C. This suggests a potential decrease in dimer stability due to the mutations present in N37T↑V+10•D25A. Isothermal titration calorimetry with acetyl pepstatin as a model inhibitor was employed to examine the impact of mutations on drug binding. The enthalpy (ΔH) for WT•D25A and N37T↑V+10•D25A were 35.94 and 36.02 kJ/mol, respectively. The entropy (ΔS) for WT•D25A and N37T↑V+10•D25A was found to be 256.7 and 261.6 J.mol.K, respectively. These differences in thermodynamic parameters between the WT•D25A and N37T↑V+10•D25A proteases, may indicate altered drug-protease interactions. Induced fit molecular docking predicted the binding strengths of both WT•D25A and N37T↑V+10•D25A with nine protease inhibitors (atazanavir, darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir and tipranavir), revealing that specific background mutations, such as P40S present in N37T↑V+10•D25A, significantly decreased the binding energy of the HIV-1 protease to these inhibitors. Molecular dynamics simulations provided insights into the structural dynamics of N37T↑V+10•D25A, showing reduced stability and increased dynamics, particularly in the flaps and hinges of the protease. An increase in flap tip curling and involvement of the cantilever tips were observed to be related to the flap opening mechanism of HIV-1 protease. Interactions between the HIV-1 protease and inhibitors were examined and the radius of gyration and solvent accessible surface area were calculated to evaluate protein compactness and solvent accessibility of the bound inhibitors, respectively. In general, the N37T↑V+10•D25A mutant exhibited decreased compactness when bound to inhibitors, which correlated with the increased solvent exposure of PIs when bound to N37T↑V+10•D25A. This may contribute to the drug resistance mechanisms of the protease, as inhibitors would have difficulty binding the active site and exhibit weaker binding. During the N37T↑V+10•D25A and PI interactions, there was a decrease in hydrogen interactions, which form the basis for protease inhibitor drug-design and these were replaced by water-bridge interactions, which are weaker and can be easily broken. In conclusion, this study provides a comprehensive characterisation of the N37T↑V+10•D25A mutant of HIV-1 protease, shedding light on its structural alterations, conformational stability, drug binding properties, and dynamic behaviour. These findings contribute to our understanding of drug resistance mechanisms in HIV-1 protease and offer valuable insights for the design of more effective inhibitors to combat HIV-1.
Biophysical studies of metal chelate binding by HSA: Towards an understanding of metallodrug transport
(University of the Witwatersrand, Johannesburg, 2023) Sookai, Sheldon; Munro, Orde
Human serum albumin (HSA) is the most abundant blood protein, transporting many exogenous compounds including clinically deployed and investigational drugs that are generally organic in nature. HSA may largely influence the pharmacokinetics and pharmacodynamics of these drugs. Therefore, studying their interactions with HSA is vital in progressing drug development. In this thesis we present work on the synthesis and characterisation of five Schiff base bis(pyrrolide-imine) ligands that were metalated with either Au(III) (Chapters 2 and 3) or Pt(II) (Chapters 4 and 5). One of the ligands H2L1 was further metalated with Ni(II) and Pd(II) (Chapter 6). In Chapters 2 and 3 focus on a patented class of anti-cancer bis(pyrrolide-imine) Au(III) Schiff base chelates. Three Au(III) chelates were synthesized in Chapter 2 and underwent National Cancer Institute (NCI)-60 cytotoxic screening. Among them, AuL1 and AuL3 underwent full-five dose testing and recorded GI50 values of 7.3 µM and 11.5 µM, and IC50 values of 15.7 µM and 30.9 µM, respectively. AuL1 was tested further and found to be an interfacial poison of topoisomerase II at 0.5–5 µM and a catalytic inhibitor at 50 µM. In Chapter 3, two chiral tetradentate cyclohexane-1,2-diamine-bridged bis(pyrrole-imine) Au(III) complexes were reported, both of which were found to be cytotoxic in the NCI-60 screen. The chiral Au(III) chelates had a different mode of action compared to AuL1. Hierarchical cluster analysis suggest that their mode of action is similar to that of taxol. All five Au(III) chelates bound to HSA with moderate affinity (104–105 M–1) and minimally perturbed the structure of the protein. This highlights the potential for the Au(III) complexes to be transported by the HSA-mediated pathway. Chapters 4 and 5 focused on the synthesis of novel and previously reported Pt(II) Schiff base chelates to spectroscopically and computationally study their interaction with HSA and elucidate if the chelates could act as theranostic agents. It was found that switching the linking bis(imine) carbon linkage altered the binding affinity of the complex. However, the Pt(II) ion ensured that all three Pt(II) chelates preferred binding to Sudlow’s site II of HSA. The data was corroborated by molecular docking simulations and ONIOM calculations. Only 2 was found to be cytotoxic when irradiated with UV light but was found to act as a photosensitizer rather than a theranostic agent. Chapter 6 investigated the influence of d8 metal ions (Ni(II), Pd(II) and Pt(II) within the same ligand scaffold (H2PrPyrr) binding to HAS, which was investigated by steady state fluorescence quenching. The affinity constants, Ka, ranged from -3.5 -103 M−1 to-1- 106 M–1 at 37 C, following the order Pd(PrPyrr) > Pt(PrPyrr) > Ni(PrPyrr) >H2PrPyrr. The Pd(II) chelate was prone to hydrolysis and had a unique binding mode which we attribute to the unusually high binding affinity. The complexes uptake is enthalpically driven, hinging mainly on London dispersion forces. In summation, twelve metal complexes were successfully synthesized, of which 11 bound to HSA with a moderate binding affinity. The Au(III) chelates preferred Sudlow’s site I, while the Pt(II) chelates preferred Sudlow’s site II. Overall, the metal complexes bound fully intact to HSA.
Computational modeling approaches to validate the druggability of the 26- and 28-kDa Schistosoma glutathione transferase enzymes using bromosulfophthalein as a benchmark ligand
(University of the Witwatersrand, Johannesburg, 2024) Valli, Akeel
PHARMACOPHORE MODELS are 3-D representations of the chemical and spatial features required for interaction with a drug target. These. models offer advantages in early-phase drug design by expediting screening experiments and enabling the sampling of highly specific chemical. space subsets, such as those containing quality drug-like candidates. The glutathione transferase enzyme of Schistosoma spp. (SGST) has been identified as an attractive drug target for the novel treatment of human schistosomiasis. We observed selective inhibition of SGST by bromosulfophthalein. Bromosulfophthalein was found to complex with SGST at a drug binding site in the target dimer interface, providing a suitable benchmark for the design of discriminative SGST pharmacophores. The aim of this research is to construct, deploy and evaluate pharmacophore models of the SGST drug binding site. The objectives are: to characterise the SGST drug binding site, to develop the pharmacophore models and finally to evaluate the drug-resolving ability of the models. We observed significant differences in the drug-binding character of SGST, compared to human glutathione transferase (hGST) counterparts, particularly that SGST supports binding of phenol and sulfonate moieties. Five- and four feature pharmacophores were developed for the respective 26- and 28 kDa SGST variants. Finally, the models demonstrated remarkable ability to retrieve candidates displaying drug-like qualities. In conclusion, we characterised and developed pharmacophore models of the drug binding domains from two major SGST variants. Assessment of drug-resolving power validates the capability of the models to sample drug-like chemicals. Altogether, these accomplishments enable efficient and reliable screening toward novel drug treatment for human schistosomiasis
Cranial orientation and the lateral semicircular canal in primates: implications for palaeobiological reconstructions and the evolution of locomotor repertoires
(University of the Witwatersrand, Johannesburg, 2023) Pestana, Christopher; Benoit, Julien; Beaudet, Amélie
The lateral semicircular canal and its predicted relation to head posture have been used in reconstructions of locomotion and posture of contemporary and extinct species, and in the evolution of bipedalism. Inferences of head posture in fossil species sometimes assume that the lateral semicircular canal is held near the earth’s horizontal when the head is at rest. Despite the physiological importance of the vestibular system, the relationship between head posture and lateral semicircular canal orientation in primates has not been explored on a statistically significant sample, using phylogenetically corrected methods. This study tests the hypothesised relationship between lateral semicircular canal orientation and head posture in primates, and investigates potential links to locomotor categories. This study finds that lateral canal orientation is not significantly correlated to positional repertoires. Significant differences in canal orientation are detected between terrestrial and arboreal species. Neutral head posture distinguishes several locomotor categories, and explains a moderate proportion of the variance in positional behaviour. Brain mass is found to correlate with positional behaviour when correcting for the effects of the phylogeny. The implications of the evolution of head posture in fossil species are discussed.
Design and synthesis of chronic wound healing collagen peptide mimics
(University of the Witwatersrand, Johannesburg, 2024) Lesotho, Ntlama Francis
The South African wound care management market is expecting a compound annual growth rate (CAGR) of 6.75%. The numbers are expected to further increase because South Africa has the highest number (4.6 million) of people living with diabetes in Africa. Annually approximately 2% of patients with diabetes develop diabetic foot ulcers and hence chronic wounds. Many chronic wound patients must deal with the financial burden, as many current wound treatment options are expensive, ineffective, and inconvenient. Intervention in the form of synthetic collagen mimetic peptides has been limited due to cytotoxicity and susceptibility to protease degradation. These challenges have, for an ardent time affected the clinical and commercial development of synthetic wound healing peptides. The aim of the current study is to develop novel wound healing peptides by derivatizing bioactive peptides into selective and protease stable peptidomimetics. All the synthesized peptides are meant to mimic the function of collagen type I. Thus, the designed peptides comprise of the retro- integrin binding type I collagen motif, -GFOGER-, the DGD tripeptide for attraction of growth factors, the retro- tripeptides Thr-Thr-Lys (TTK), Gly-His-Lys (GHK), Gln-Pro-Arg (QPR) and Glu-Glu-Met (EEM) to stimulate collagen production. The importance of collagen is evidenced by the fact that it features in all four stages of wound healing. This therefore means, its inclusion in any biomaterial meant to curb chronicity in wound healing is indispensable. With this approach, the biomaterial would overcome the challenge of excess matrix metalloproteinases (MMPs), which degrade both viable and nonviable collagen used in the wound healing process. It would further provide a collagen-based wound scaffold that compensates for the loss of collagen required for proper tissue regeneration. The applications of collagens in wound healing are immense. Due to its material properties, and apparent effectiveness, collagen has the potential to be utilized as an unprecedented treatment protocol for chronic, slow-healing wounds. Sixteen palmitate and adamantane collagen mimetic peptides were designed and successfully synthesized using the solid-phase peptide synthesis strategy. Eight of the sixteen peptidescomprise of lipophilic moieties (adamantane and palmitic acid) for improved membrane permeability and different collagen inducing retro-tripeptides namely, TTK, GHK, QPR and EEM (retro-DGD-GG-GFOGER-GG-TTK-Adamantane (NL010)/palmitate (NL009), retro-DGD- GG-GFOGER-GG-GHK-Adamantane/palmitate, retro-DGD-GG-GFOGER-GG-QPR- Adamantane/palmitate and retro-DGD-GG-GFOGER-GG-EEM-Adamantane/palmitate). Another eight are control peptides without the retro-tripeptides (retro-DGRGOF- Adamantane/palmitate, retro-GOP-GFOGER-GOP-Adamantane/palmitate, retro-GG- GFOGER-GG-Adamantane/palmitate and retro-DGD-GG-GFOGER-GG-Adamantane (NL008)/palmitate). The tertiary structure and secondary features (folding patterns) of the peptides were determined using the Nuclear Magnetic Resonance (NMR) and Circular Dichroism (CD). From NMR experiments, medium-range couplings were detected for NL010 and NL009, suggesting a possibility of alpha helices. Temperature 1H NMR experiment for the peptide DGRGOF- Adamantane proved the presence of cis and trans geometric isomers. CD experiments revealed that NL009 mainly has α-helix while NL010 mainly consists of a parallel conformation. Synthesis of adamantane and palmitate peptides with enhanced integrin binding was accomplished by incorporation of para-fluorophenylalanine in place of phenylalanine in the peptide retro-GG-GFOGER-GG-Adamantane/palmitate. The peptides were obtained in low yields but with increased hydrophobicity. Structural features for the improvement of the stability of the peptides against protease degradation were accomplished by the synthesis of peptoids and N-methylated peptides. The peptoids were synthesized in low yields but with increased hydrophobicity. The efficacy of NL009 and NL010 in wound healing was tested both in vitro and in vivo. In the former, the efficiency of both NL009 and NL010 in inducing migration of cells in a scratch wound was accentuated by hyaluronic acid. In in vivo studies, NL010 performed better than NL009. However, NL010 was outperformed by a comparator, Puramatrix® The peptides have the ability to induce migration of cells and therefore have an ability to create an environment needed for proper wound healing. The peptides could be used in place of native collagen and bring about proper healing of wounds
Design and synthesis of triazine derivatives as non-nucleoside reverse transcriptase inhibitors
(University of the Witwatersrand, Johannesburg, 2024) Munetsi, Wendy; Bode, Moira; Ngwira, Kennedy
This research work was carried out to investigate the properties of different groups that can be used to modify the triazine core with the aim of designing a new library of possible HIV non- nucleoside reverse transcriptase inhibitors (NNRTIs). Triazine derivatives have been used extensively in the synthesis of numerous classes of drugs due to their significant biological activity. In this project, the specific focus was to synthesize 1,3,5-triazine derivatives by successive nucleophilic substitution reactions of the Cl atoms from cyanuric chloride. In the first step of the substitution reactions, 2,4,6-trichloro-1,3,5-triazine was reacted with various anilines, phenols and thiophenols which acted as nucleophiles to displace one of the Cl atoms upon reaction completion. The yields varied from 28% -90% with the best yields being observed when the anilines were used as a nucleophile and most of the substituents in this first step were anilines. The substituents used at each step of the substitution were vital in terms of determining the order of the reaction to enable a successful reaction. The introduction of different linkers to the triazine core such as -NH, -S, -O yielded compounds with different properties expected to provide significant interactions in the NNRTI binding pocket. We expected better binding properties from the -NH bearing compounds due to hydrogen bond formations with amino acid residues inside the allosteric binding pocket of the HIV-1 RT. The success of the second step of the substitution reactions was identified to be dependent on the substituent attached to the triazine ring from the first step. Some reactions were not successful when a stronger nucleophile was used in the first step and a weaker nucleophile was being used as the incoming nucleophile substituting the second Cl atom. Therefore, these reactions were repeated and the order of the reaction rearranged. Temperatures were increased and reaction times were increased at this stage as the reactivity of the triazine ring was reduced and therefore higher kinetic energy was required for successful reactions. In general, the synthesized triazine derivatives bearing two aromatic substituents exhibited the most significant presence of tautomers. The final stage in the synthesis of the trisubstituted triazine derivatives was relatively complex and required much higher temperatures and longer reaction times. The reactions were also performed at smaller scales and difficulties with the purification processes also contributed to the loss of product thereby resulting in lower yields, with one of the compounds giving a yield of 11%. The results obtained from the anti-HIV assay studies from the selected compounds tested, showed that antiviral activity was observed in triazine derivatives with electron withdrawing groups attached to the aromatic substituent as well as -NH and -O linkers at the right and left wing of the triazine core, respectively.
Development of a Commercial Manufacturing Process of 9-[(R)-2- (phosphonomethoxy)propyl] adenine (PMPA): A Key Intermediate for the Production of Tenofovir-based HIV Medicines
(University of the Witwatersrand, Johannesburg, 2023) Mbutho, Banele; Gohain, Mukut; De Koning, Charles
South Africa runs the largest antiretroviral (ARV) program in the world and yet 99% of the active pharmaceutical ingredients (APIs) used to make ARVs are imported from China. Dependence on imported APIs has major cost implications and influences the medication’s security of supply. This project was concerned with making it possible to produce the APIs tenofovir, a precursor for tenofovir disoproxil fumarate and tenofovir alafenamide locally and at a lower cost. A new synthetic route recently introduced by Medicines 4 All (M4ALL) was studied and used in this dissertation. The four-step process that produces an adenine derivative was optimized and scaled into a commercial industrial process producing tenofovir intermediates in repeatable yield and purity. This route was determined to be the most cost-effective since it utilized low cost and commercially available diaminomaleonitrile and triethyl orthoformate as starting materials—contrary to the synthetic routes currently used by the 17 largest tenofovir manufacturers. Key process improvements included a decrease in the number of solvents used and the minimization of by-product formation. Results showed that high yields of tenofovir intermediates were successfully synthesized using this new route. As such, the chemical company we conducted this research in, Chemical Process Technology Pharma will be able to employ this synthetic methodology to affordably produce the APIs used in the manufacturing of ARVs locally improving access to affordable medication.
Differential expression analysis of PMA and 1,25(OH)2D3-induced monocyte-to-macrophage differentiation in THP-1 cells
(University of the Witwatersrand, Johannesburg, 2023-09) Perumal, Kelda Chloe; Meyer, Vanessa; Gentle, Nikki
The process of monocyte-to-macrophage differentiation is studied in vitro through the use of promonocytic model cell lines, such as the THP-1 cell line, where commonly used differentiation inducing agents include phorbol-12-myristate-13-acetate (PMA) and the active metabolite of vitamin D3, (1,25(OH)2D3; VD3). While both induce differentiation, differences in their mechanisms of action, as well as how the end states of the differentiation process differ, are not well understood. Therefore, this study used computational approaches to compare the effects of PMA and VD3 on the differentiation of monocytes into macrophages, using the promonocytic THP-1 cell line. Through the use of RNA-sequencing, gene expression was quantified in differentiated and undifferentiated THP-1 cells, treated with both PMA and VD3. Differential gene expression analysis was performed to determine genes that were differentially expressed as a result of either treatment relative to the untreated cells. This was followed by over-representation analysis to determine the pathways and processes in which the differentially expressed genes (DEGs) were involved. PMA treatment (3 989 DEGs) resulted in more changes in expression relative to VD3 treatment, where only 384 genes were found to be differentially expressed in response to treatment with VD3. Only TFE3, KIT and TRIB1 were observed to be crucial to the process of differentiation, irrespective of treatment. Apart from this, the treatments were observed to largely involve different biological pathways, resulting in cells that were phenotypically distinct from each other at the transcriptional level. This included changes observed in the expression of genes encoding transcription factors known to be involved in the differentiation process, such as CEBPA, GATA2, IRF8 and PU.1, as well as those encoding surface markers representative of monocytes and macrophages, such as CD14, CD64 and CD11b. The expression patterns observed here indicate that, at least at the concentrations and time points included in this study, PMA and VD3 induce macrophage-like cells that are at different stages of differentiation and are not comparable to either each other or primary macrophages. Furthermore, key differences observed in the expression of genes encoding pathogen recognition receptors and cytokines suggest that which differentiation inducing agent is used may have important implications for these cells’ capacity to recognise pathogens and produce cytokines. The findings of this study therefore emphasise that it is crucial to carefully consider the choice of differentiation-inducing agent when using THP-1 cells as an experimental system for studying monocyte-to-macrophage differentiation.
Differential Gene Expression Analysis of PMA Treated Pro-monocytic Cell Lines
(University of the Witwatersrand, Johannesburg, 2023) Kama, Asavela Olona; Meyer, Vanessa; Gentle, Nikki
HL-60, THP-1, and U937 are model cell lines that can undergo myeloid differentiation in vitro, allowing the study of myeloid cell function in drug metabolism, cytotoxicity, and the aetiology of infections. However, the differentiated end-state of these cells is not well characterised. Moreover, cell line-specific differences in the level of gene expression may confound results obtained from such studies. The aim of this study was thus to compare changes in gene expression between HL-60, THP-1, and U937 cells in response to the differentiation agent, phorbol 12-myristate 13-acetate (PMA), 48 hours after treatment. Gene expression profiles were compared across all three cell lines prior to and post-PMA treatment. Differential gene expression analysis between treated and untreated cells was performed using DESeq2 (v 4.2). Gene over-representation analysis was performed using cluster Profiler (v 4.0). HL-60, THP-1, and U937 cells had similar expression profiles prior to PMA treatment, but different sets of genes were significantly differentially expressed in these cell lines 48 h after treatment with PMA. A total of 475 genes were consistently differentially expressed across all cell lines. These genes were found to be involved in phagosome formation and cell cycle transition. HL-60, THP-1, and U937 cells had 944, 1231, and 624 uniquely differentially expressed genes, respectively. These genes were predominantly involved in energy metabolism and pathogen recognition. Overall, THP-1 cells showed greater potential to detect viruses, while U937 cells showed greater potential to detect bacteria. From this, it can be concluded that while all three cell lines did indeed undergo myeloid differentiation, the macrophage-like cell state produced in each case differed between cell lines.
Effects of acidification on the survival of pathogens in reconstituted infant formula
(University of the Witwatersrand, Johannesburg, 2023-10) Nemakonde, Mufunwa; De Maayer, Pieter
Infants are at a high risk of developing food-borne illnesses due to the consumption of powdered infant formulas (PIFs) contaminated by pathogenic microorganisms. These pathogens may be introduced during PIF production, transportation or during preparation and storage due to poor hygiene practices. Therefore, it is important to improve the microbiological safety of PIFs to reduce illness. Manufacturers have added further measures to ensure continued control of pathogenic microorganisms during the reconstitution and consumption, including the addition of prebiotics and probiotics, as well as acidification of the PIFs, which negatively affect the growth of pathogens. Different organic acids have been biologically or chemically added to commercial PIFs, however, little is known about the efficacy of the different acids in controlling pathogen growth post-reconstitution. The present study aimed to evaluate and compare the effects of two acids, namely citrate and lactate, integrated in commercial PIFs from two manufacturers on pathogen growth within the products. In Chapter 1, we present a review of the current literature pertaining to PIF production and consumption, its microbiological safety and key problem pathogens, as well as measures to control these pathogens (with specific emphasis on PIF acidification). In Chapter 2, the methodology of the present study is discussed. The effects of lactate and citrate incorporated in commercial PIFs against eight key PIF pathogens was evaluated using plate-based assays. Furthermore, the efficacy of PIF acidification at different storage temperatures and over prolonged storage periods was determined. Finally, to validate the effect of acidity on controlling pathogen growth, a spectrophotometric approach was used. Chapter 3 presents and discusses the key findings of the study. PIF acidification was found to negatively affect the growth of pathogens in comparison to the non-acidified infant formula. Acidification and storage at suboptimal temperatures resulted in little to no microbial growth. Lactate acidification of PIFs demonstrated greater inhibitory effects against most pathogens compared to citrate acidification. Further, lactate was found to have a bactericidal effect on the growth of some pathogens. Similarly, Luria Broth acidification resulted in the reduction of microbial growth. This validated that the acids and not other constituents of the infant formula were responsible for the inhibitory effect. Finally, Chapter 4 provides a summary of the key findings as well as recommendations and guidance on future studies which could be undertaken to enhance the effective control of pathogens in powdered infant formulas, thereby ensuring a safe and nutritious food source for infants.
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.
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
Evaluating the in vitro anti-metastatic effects of silver(I) phosphine complexes on malignant breast cancer cell lines
(University of the Witwatersrand, Johannesburg, 2023-08) Ferreira, Mizan; Engelbrecht, Zelinda; Cronje, Marianne Jacqueline
Breast cancer is the most diagnosed cancer type among females worldwide. Metastasis, the spread of cancer cells from the primary tumour and establishment of macroscopic secondary tumours, is regarded as the most dangerous characteristic of cancer cells as it is responsible for over 90% of cancer-related deaths. Globally there is a lack of drugs available to specifically target or prevent either the dissemination of cells from the primary tumour or the establishment of distant metastases. The purpose of this study was to ascertain whether a series of silver(I) phosphine complexes, which have previously been shown to display anti-cancer properties in vitro, are also effective as anti-metastatic compounds. The migration, invasion and adhesive abilities of two malignant breast cancer cell lines, MCF-7 and MDA-MB-231, in response to silver(I) phosphine treatment were evaluated. In addition, the colony-forming abilities of cells under both anchorage-dependent and -independent conditions were investigated. Furthermore, the effects of silver(I) phosphine treatment on the expression and activities of key metastatic proteins, matrix metalloproteinases (MMPs), were studied. Of the nine complexes evaluated, all of them showed the ability to reduce one or more metastatic steps namely cell migration, invasion through collagen towards a chemoattractant or adhesion to collagen. In addition, a selected number of complexes reduced the colony-forming abilities of MCF-7 and/or MDA-MB-231 cells in culture plates as well as in soft agar. Moreover, three of these complexes increased the in vitro invasion and colony formation of breast cancer cells. Further investigation into complexes showing anti-metastatic abilities revealed that, apart from one complex on MDA-MB-231 cells, anti-metastatic effects were not achieved through a reduction in MMP levels or activities. The findings presented here show the potential for silver(I) phosphine complexes to reduce the in vitro metastatic abilities of breast cancer cells, warranting further investigations into these complexes for their use as anti-metastatic drugs.
Expression and Methylation of Peroxidasin in Breast Cancer Cell Lines
(University of the Witwatersrand, Johannesburg, 2023-07) Falkov, Jemma Lilian; Mavri-Damelin, Demetra
Peroxidasin (PXDN) is a haem-containing extracellular matrix peroxidase protein which forms hypohalous acids in the presence of hydrogen peroxide (H2O2). The predominant role of PXDN is that of a collagen IV crosslinker within the basement membrane. Increased collagen IV deposition has been linked to tissue invasion and metastasis in breast cancer and PXDN has also been shown to assist in the process of epithelial-mesenchymal transition (EMT) in cancer. Various cancer types display dysregulated levels of PXDN expression including breast cancer and this dysregulation has been associated with poor prognosis. This study aimed to investigate whether DNA methylation of the PXDN promoter may be a mechanism through which changes in PXDN expression observed in breast cancer are regulated. Non-invasive MCF-7 and invasive MDA-MB-231 cells were used as models for luminal A and triple negative breast cancer (TNBC) respectively. The HEK-293 cell line was used as a non-cancerous control cell line. DNA methylation levels of the PXDN promoter and PXDN protein expression was investigated in these cell lines through the methods of methylation sensitive PCR (MS PCR) and immunofluorescence microscopy. Relative levels of PXDN expression were determined through immunofluorescence microscopy. Corrected total cell fluorescence (CTCF) analysis of these images revealed the highest PXDN levels to be found within the invasive MDA-MB-231 cell line, which was double that of the MCF-7 cell line. All cell lines were treated with 10 nM β-Oestradiol, which caused an increase in PXDN expression within the MCF-7 and HEK-293 cell lines and a decrease in expression within the MDA-MB-231 cell line to half its untreated value. PXDN was found to be localised in the ECM in all three cell lines. To elucidate the role of DNA methylation, methylation sensitive PCR (MS PCR) was performed on all three cell lines, with four primer pairs spanning a region of 1305 base pairs (bp) within the PXDN promoter. A region of differential methylation was found between the MDA-MB-231 and HEK-293 cell lines between 524 bp and 53 bp upstream of the transcription start site (TSS). This region was unmethylated within the MDA-MB-231 cell line and methylated within the HEK-293 cell line, which correlates with expression differences between these two cell lines and suggests this region could be of regulatory significance. The four primer pairs designed to amplify the PXDN promoter were unable to amplify this region within the MCF-7 cell line. A heterochromatic DNA conformation or a point mutation increasing CpG content creating a thermodynamically ultra-fastened (TUF) region could be the explanation behind this phenomenon, however further research is required to elucidate the mechanism responsible. In conclusion, PXDN shows higher expression in TNBC cells than in luminal A subtype cells. The oestrogen receptor is involved in regulating PXDN expression, however, different mechanisms seem to be at play between the two cell lines. The contribution of CpG methylation to this change in PXDN expression remains unknown, as does the nature of the interaction between the oestrogen receptors and the gene. Further research is required to clarify the mechanisms involved.

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