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Masters Degrees (Biochemistry)

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    Antioxidative and antidiabetic activity and phytochemicals, analysis of some selected Sudanese traditional medicinal plants.
    (2021) Idris, Almahi Mohamed.; Islam, Shahidul.
    This study was conducted to evaluate the antioxidant and anti-diabetic properties of selected traditional Sudanese medicinal plants (Cyperus rotundus, Nauclea latifolia, and Hibiscus sabdariffa) using in vitro, ex vivo, and in silico experimental models. The crude extracts (ethyl acetate, ethanol, and aqueous) were screened in vitro for their antioxidant activities using ferricreducing antioxidant power (FRAP), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and nitric oxide radical (NO) scavenging activities, as well as their carbohydrate digesting enzyme inhibitory activities for antidiabetic evaluation. Subsequently, the extracts were subjected to Gas Chromatography-Mass Spectrometry (GC-MS) analysis to elucidate their possible bioactive compounds. Additionally, ex vivo studies was conducted to investigate their capability to promote muscle glucose uptake and suppress glucose absorption in the intestine as well as to analyze antioxidative effects in iron–induced oxidative stress in hepatic tissue. Molecular docking was carried out to determine the probable enzymes' inhibitory mode of action by ligands identified through GC-MS. This study indicates that these traditional Sudanese medicinal plants have remarkable antioxidant and antidiabetic activities, which may help to ameliorate oxidative stress and diabetes. Therefore, these plants may be considered a natural source of bioactive compounds beneficial for human health, particularly for managing diabetes and oxidative stress-related metabolic disorders.
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    Production and characterization of DNA ligases isolated from Kogelberg Biosphere metagenomics library.
    (2021) Zuma, Lindiwe Khumbuzile.; Pooe, Ofentse Jacob.
    Microbial enzymes have been described as an underutilized source of novel enzymes with potential economic advantages. Recently discovered enzymes such as DNA ligase from metagenomic studies, have been shown to achieve great potential in transforming the reagent market specifically in the African continent. Reagent proteins are frequently utilized in the research field widely and are prone to protein degradation and shelf-life reduction. Hence, this study sought to improve biological activity, shelf life and stability of the two DNA ligases identified from Kogelberg Biosphere metagenomics library. Two recombinant DNA ligases expression studies were done using E.coli BL21 and purification studies were done subsequently using affinity chromatography. Both recombinant DNA ligases (Ligsv081 & LigpET30) were successfully expressed and purified as homogenous proteins. In this study two approaches were used to enhance the biological DNA ligases, the first approach used was PEGylation. The purified proteins were conjugated to PEG using site-specific PEGylation and non-specific PEGylation. FTIR and UV-VIS spectroscopy were used to analyze the secondary structure of the PEG conjugated DNA ligases. Thermal stability assays were then employed to assess protein stability in the conjugation with PEG. Site-specific PEGylation enhanced ligase activity and reduced the formation of protein aggregates. The second approach involved DNA ligase co-expression in the presence of PfHsp70 or chimeric transcription factor, respectively. Protein co-expression and co-purification assays were conducted. The co-expression and co-purification assays of both proteins with chimeric transcription factor (cTF) were successful, followed by co-expression and co-purification of LigpET30-PfHsp70. Ligation assays were conducted to assess bioactivity of proteins. All DNA ligase complexes were functional and their melting point was increased. Taken together, site-specific PEGylation and protein co-expression with PfHsp70 potentially extended the shelf-life and stability of the proteins. PEGylation strategies and co-expression strategies can potentially be used to enhance reagents in diagnostic and therapeutic tools in molecular biology field.
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    Antioxidative and antidiabetic activity and phytochemicals analysis of some selected Sudanese traditional medicinal plants.
    (2021) Idris, Almahi Idris Mohamed.; Islam, Shahidul.
    This study was conducted to evaluate the antioxidant and anti-diabetic properties of selected traditional Sudanese medicinal plants (Cyperus rotundus, Nauclea latifolia, and Hibiscus sabdariffa) using in vitro, ex vivo, and in silico experimental models. The crude extracts (ethyl acetate, ethanol, and aqueous) were screened in vitro for their antioxidant activities using ferricreducing antioxidant power (FRAP), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and nitric oxide radical (NO) scavenging activities, as well as their carbohydrate digesting enzyme inhibitory activities for antidiabetic evaluation. Subsequently, the extracts were subjected to Gas Chromatography-Mass Spectrometry (GC-MS) analysis to elucidate their possible bioactive compounds. Additionally, ex vivo studies was conducted to investigate their capability to promote muscle glucose uptake and suppress glucose absorption in the intestine as well as to analyze antioxidative effects in iron–induced oxidative stress in hepatic tissue. Molecular docking was carried out to determine the probable enzymes' inhibitory mode of action by ligands identified through GC-MS. This study indicates that these traditional Sudanese medicinal plants have remarkable antioxidant and antidiabetic activities, which may help to ameliorate oxidative stress and diabetes. Therefore, these plants may be considered a natural source of bioactive compounds beneficial for human health, particularly for managing diabetes and oxidative stress-related metabolic disorders.
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    The use of zebrafish to assess water quality and remediation efforts.
    (2023) Zondi, Thandolwethu Beauty.; Hewer, Raymond.
    Although wastewater effluents continue to be significant polluters of aquatic ecosystems in developing countries with limited water resources, little is known about the ecotoxicity induced by these effluents on fish throughout their early life stages. Several wastewater treatment plants (WWTPs) in South Africa (SA) do not adequately meet the minimal wastewater treatment requirements established by the country's Department of Water and Sanitation (DWS). Moreover, contaminants of emerging concern (CECs) originating from synthetic or natural sources, are widely distributed in aquatic environments of SA. This includes a broad range of natural and chemical compounds, such as aspirin (44243 ng/L), Fluoroquinolones (27100 ng/L), Atenolol (25900 ng/L), Nalidixic acid (25234 ng/L) and Ciprofloxacin (20514 ng/L). In addition to chemical compounds, endocrine disrupting chemicals, pharmaceuticals and personal care products are also distributed in the water systems. In the process of wastewater treatment, agents such as flocculants, coagulants, chemical precipitants (e.g., calcium hydroxide or sodium hydroxide) and chlorine disinfectants are utilized in wastewater treatment settings. However, research to understand the adverse effects that can be caused by these agents on aquatic organisms is still ongoing in SA. In order to bridge this knowledge gap, advanced techniques could be employed to help reveal adverse effects of wastewater as well as any shortcomings of current water remediation techniques. Using an appropriate aquatic model organism with highly conserved physiological pathways present in higher vertebrates (including humans), a rich behavioural repertoire, and occurrence in a variety of habitats would be a novel approach. To this effect, this study employed zebrafish with the aim to monitor six distinct wastewater samples from various regions of SA and to assess the effectiveness of currently used water remediation techniques such as chlorination. Two wastewater effluents, namely, Southern Works Final Effluents (SWFE) and Jacob’s Incoming (JB) alerted potential toxicity during chemical characterization with suboptimal pH (SWFE = 9.02 ± 0.16 and JB = 5.65 ± 0.02) and total alkalinity of zero (0 mg/L) detected for both effluents. The lethal toxicity of these effluents was seen by the elevation of mortality rate up to 77 ± 2.89 % and 100 ± 0.00 %, respectively for SWFE and JB at 40 %, with corresponding LC50 values of 17.77 % and 16.46 %. The zebrafish jaw and face, heart, brain, fins, notochord, somite and tail were significantly deformed (p < 0.05) post-exposure to these effluents, as revealed by morphological scores upon the analysis of the zebrafish’s body structure. Moreover, there was a delay in development due to the aforementioned effluents, unsuccessful hatching, craniofacial abnormalities, pericardial and yolk sac oedema, notochord abnormality somite defects and spinal cord curvature. In addition, locomotor activity of zebrafish was inhibited following observation of distance travelled, frozen moments, acceleration rates, swimming trajectories and exploration rate. Surprisingly, safety of these wastewaters was restored by chemical precipitation revealing non-lethal pH ranges of 6.02 - 8.02 and 6.65 - 7.65 for SWFE and JB, reducing the mortality rate to non-significant levels (p > 0.05) compared to the control. Also, sodium bicarbonate (NaHCO3) at 120 mg/L was found effective at supplementing the wastewater total alkalinity. In contrast, Amanzimtoti water before and after chlorination (TB and TA), Incoming Badulla (IB) and Chatsworth Incoming (CI) exhibited no consistent lethality effects on zebrafish and induced no apparent stress as demonstrated by insignificant expression (p > 0.05) of the stress protein: heat shock protein 70 (HSP70). However, the insignificant mortality rate (p > 0.05) in the water tested before (TB) and after (TA) chlorination appeared to be the same (~25 %) indicating that chlorination is not enough at completely remediating wastewater. Our study is a pioneer in evaluating the ecotoxicological impact of wastewater effluents from localized regions of a developing country like South Africa in relation to the adjustment of water quality parameters for the neutralization of contaminants. To better understand emerging contaminants released as effluents in SA's water bodies and their interactions with aquatic organisms at the adult stage, more studies needs to be developed.
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    Quantitative detection and inactivation of Mycoplasma hyopneumoniae.
    (2023) Wei, Yanna.; Khoza, Thandeka Ntokozo.; Xiong, Qiyan.
    Abstract available in PDF.
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    Investigation of the interaction between antiretroviral drugs and the mucosal microbiome in African women.
    (2022) Sibeko, Nomacusi Sibonganjalo Lindiwe.; Gumbi, Pamela Phumelele.; Coetzer, Theresa Helen Taillefer.
    Abstract available in PDF.
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    Recombinant expression and enzymatic characterisation of Trypanosoma vivax cathepsin L-like protease (TviCATL) for single chain variable fragment antibody production.
    (2022) Ramjeawon, Bhavana Roshenlal.; Coetzer, Theresa Helen Taillefer.
    Humans and animals in sub-Saharan Africa are at risk of African trypanosomiasis (AT), caused by tsetse fly-transmitted protozoan blood parasites of the Trypanosoma genus. Animal African trypanosomiasis (AAT), or nagana, is caused by T. brucei, T. congolense and T. vivax and negatively impacts livestock farming and consequently the economy of the continent. Since AAT occurs in rural areas, affordable rapid diagnostic tests (RDTs) and drugs are required. Diagnostic tests focus on antibody detection; however, antigen detection is more favorable since anti-trypanosome antibodies persist in blood for years following recovery. Due to the parasite’s defense by antigenic variation, development of a vaccine is unlikely. Molecules that are essential for parasite survival, such as peptidases, are currently being targeted for diagnosis and chemotherapy. A cathepsin-L-like cysteine protease from T. vivax, TviCATL, is released by dying parasites in the host bloodstream and was shown to be a diagnostic target for detecting host antibodies. To achieve diagnosis of current infections, detection of TviCATL is being explored. The overall aim of this study was to enzymatically characterise TviCATL; and to study the interaction of antibodies against the TviCATL antigen which could be used as a chemotherapeutic drug for the diagnosis of T. vivax infections. The protease, TviCATL, was recombinantly expressed in E. coli using the pET-28a expression vector and purified using a nickel chelate affinity column. The resulting 47 kDa protein was identified using western blot and was shown to hydrolyse H-D-Ala-Leu-Lys-AMC and was inhibited by bestatin and E-64 and had optimal activity between pH 6.5 and 7.5. The crossreactivity between TviCATL and antibodies produced against other Trypanosoma spp cysteine proteases was evaluated in western blots, and results confirmed cross-reactivity. In addition, chicken anti-TviCATL antibodies were able to detect TviCATL in TviCATL-spiked bovine serum. The production of antibodies using the Nkuku® phage library was employed as an alternative to the animal-based antibody production and single-chain variable fragment (scFvs) antibodies were selected by panning against the TviCATL antigen. After four rounds of panning, TviCATL-scFvs binders were enriched and four clones gave the highest signal when evaluated using a monospecific ELISA. Due to the low values obtained, optimisation of panning is necessary for improved results. Optimisation of recombinant expression and purification of the identified scFvs for use in a sandwich ELISA were explored to this end. This study showed that TviCATL is a promising chemotherapeutic and diagnostic target for African animal trypanosomiasis.
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    The role of MMP-14 and MMP-2 in mediating myoblast fusion.
    (2016) Nkosi, Mthokozisi Siphesihle.; Niesler, Carola Ulrike.
    Satellite cells are muscle precursor cells that have the ability to self-renew, proliferate and differentiate into myoblasts that eventually elongate and fuse to form myotubes which are vital for regeneration and repair of muscle. Satellite cells reside in a niche, between the sarcolemma of the muscle fiber and the basal lamina, which consists of mostly collagen IV, proteoglycans and laminin. Matrigel is a gelatinous protein mixture that consists primarily of collagen IV and laminin and therefore resembles the basal lamina. Matrix Metalloproteinases (MMPs) are zinc endopeptidases, proteolytic peptidases which break peptide bonds within their substrates. MMP-14 (membrane bound) also known as membrane-type 1 matrix metalloproteinase (MT1-MMP) is one of the major matrix metalloproteinases (MMPs) involved in muscle repair and regeneration, together with MMP-2 (secreted). MMP-2 is a secreted gelatinase A, which is activated by MMP-14. MMP-2 is also known to be activated by nitric oxide (NO), therefore allowing active MMP-2 to release growth factors such as Hepatocyte Growth Factor (HGF) from the extracellular matrix (ECM). There are two forms of MMP-2, intracellular MMP-2 and extracellular (secreted) MMP-2. Secreted MMP-2 contains a peptide signal that helps direct it outside the cell, while intracellular MMP-2 lacks this feature and is therefore retained within the cell. Intracellular MMP-2 activity is known to be a major cause of muscular atrophy. Secreted MMP-2 is known to degrade ECM components, facilitating satellite cell mobility and release of growth factors such as HGF, aiding in muscle regeneration. MMP-2 can cleave collagen IV due to the presence of a fibronectin-like domain within its catalytic domain; this is not the case with MMP-14. MMP-14 and MMP-2 together degrade collagens, fibronectin, laminin-2/4 and other adhesion molecules. This clears the path for the myoblast to align and fuse to form myotubes which then finally align to form mature muscle fibers. The levels of MMP-14 and MMP-2 must be regulated; low levels can cause muscular dystrophy. The current study analysed expression levels, activity and role of MMP-14 and MMP-2 in C2C12 myoblast differentiation. C2C12 myoblasts first proliferated (Day 0), then aligned and elongated (Days 1-2) and then finally fused into myotubes (Days 3-5) during differentiation. MMP-14 and MMP-2 protein levels were high during the elongation period and also during fusion of C2C12 myoblasts. MMP-14 was localised at the focal adhesions, where actin filaments terminate during myoblast proliferation and fusion. Inhibition of MMPs using BB94 (10 μM) was observed to significantly reduce C2C12 myoblasts fusion. Secreted MMP-2 seems to play a vital role in the C2C12 differentiation, as activity was seen during myogenesis; when neutralised with an antibody, an 18% decrease in fusion was observed. Matrigel promoted an increase of MMP-2 expression within the cell during fusion (day 5 of differentiation), while no intracellular MMP-2 protein was observed at day 2 of differentiation. Levels of secreted MMP-2 increased significantly from day 2 to day 5 of differentiation; however, the presence of Matrigel significantly reduced levels of secreted MMP-2 detected in conditioned media at day 5 compared to uncoated conditions. The decrease is, in part, due to the fact that MMP-2 was found to bind to Matrigel. In conclusion, MMP-14 and MMP-2 play an important role in C2C12 myoblast elongation and fusion. This study provides further insight into the role of MMPs in myogenesis and lays the foundation for future work.
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    Intercellular communication between fibroblast phenotypes, macrophages and myoblasts during cellular migration.
    (2022) Ramklowan, Dhamini Sanjay Hariduth.; Niesler, Carola Ulrike.
    Abstract available in PDF.
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    Identifying small molecule binding ligands for the pup-ligase (PafA) of Mycobacterium tuberculosis.
    (2022) Mthembu, Sandile Mehzi.; Hewer, Raymond.; Delport, Alexandre' Marie Chaplin.
    The rapid emergence of resistant TB strains (Mycobacterium tuberculosis (Mtb)) renders traditional treatment options ineffective and necessitates the generation of novel anti-TB drugs that possess innovative modes of action. The pup-ligase (PafA) of Mtb that solely mediates protein proteasomal removal via the pupylation cascade has recently been identified as a suitable target for TB drug development. A novel approach would be to recruit proteolysis targeting chimeras (PROTACs) technology as an alternative anti-TB treatment option by developing PROTAC-like molecules capable of recruiting the pupylation cascade. Therefore, the identification of novel PafA small-molecule binding ligands is an essential first step to establish possible new TB therapies. To this effect, PafA recombinant expression was successfully optimised in E. coli cells at 20°C for 20 h, where a 50-kDa protein was observed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Moreover, the identity of the protein was confirmed via immunoblotting with anti-His antibodies and PafA subsequently purified via immobilized metal affinity chromatography (IMAC) to high purity. A thermal shift assay (TSA) of PafA against 48 small-molecule compounds from a chemical library pre-screened for non-specific inhibition activity was conducted. Seven Hit compounds were detected significantly binding PafA (P < 0.05), all inducing a > 5 °C increase of PafA melting temperature (Tm) upon binding. Future research on these novel PafA binding ligands will be to ascertain whether they possess inhibitor qualities. Additionally, they will be used in the synthesis of heterobifunctional molecules to create the first PROTAC-like molecules for targeted proteasomal degradation of essential Mtb proteins – a novel type of anti-TB drug.
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    Investigation of intravaginal practices as a factor associated with a high prevalence of genital human papillomavirus infection in adolescent girls.
    (2021) Mntambo, Ntombenhle.; Gumbi, Pamela Phumelele.; Ngcapu, Sinaye.
    Background: Human papillomavirus (HPV) is a common sexually transmitted infection (STI) in women which mainly infects the mucosal areas. Young women are disproportionately infected by HPV, and factors that may render adolescents or younger women more vulnerable to HPV acquisition than older women have not been fully elucidated. This study aimed to investigate the associations between the prevalence of HPV, the use of intravaginal products, the immune activation status of cervical T cells, and alterations of the composition and concentrations of antimicrobial peptides (AMPs) in vaginal fluid among adolescent females close to their sexual debut and older women in KwaZulu-Natal. Methodology: Genital specimens (cervical cytobrush and cervicovaginal swabs) were collected from 154 female participants aged 14-19 and 25-35 years. From cervicovaginal swabs, HPV genotyping was done using a deoxyribonucleic acid (DNA) Flow hybridization system. Flow cytometry was conducted from cervical cytobrush specimens (evaluating CD38+, HLA-DR+, andCCR5+ expressions) to assess T-cell immune activation status. Enzyme-linked Immunosorbent Assay (ELISA) kits were used to measure genital concentrations of human β- defensin (HBD-1, HBD-2), and psoriasin from cervicovaginal swabs. Statistical tests conducted were Robust Poisson regression models (RPRM), the Tukey multiple comparison adjustment, t-test, and Mann Whitney U test. 𝑃 values of <0.05 were statistically significant. Results: HPV prevalence was 85%, with high-risk genotypes being the most prevalent. The majority of the cohort was infected with multiple genotypes (76.62%). Genotypes associated with cancer and current Gardasil®9 HPV vaccine targets were more common (53.9%) than those associated with genital warts (14.9%). The risk of HPV in adolescent females was 15.9% higher than in adult females. The use of vaginal inserted products (VIPs) was associated with a 40% higher risk of contracting genotypes related to cancer (p=0.0503) compared to nonusers. The risk of HPV infection for adolescents using VIPs was 23% higher than that of adults using VIPs. However, these differences were not statistically significant at a 5% level of significance. Sexual debut after 18 years significantly reduced the risk of overall HPV infection (p=0.0040) and infection with genotypes associated with cancer (p=0.0024). When comparing HPV infected adolescents and adults, the proportion of activated CD4+ T cells was significantly higher in adolescents, particularly in CD4+ HLA-DR+ cells (p=0.0008). CD8+ T cells showed no difference. A significantly higher concentration of HBD-2 was observed in HPV+ adults compared to HPV- adults (p=0.0215) and HPV+ adolescents (p=0.0189). Conclusion: The overall HPV prevalence is higher than the previously reported prevalence in KwaZulu-Natal province. In addition, we demonstrate that the use of VIPs may be associated with some HPV infection risk, particularly in adolescent females. This finding suggests that young women should be warned about the potential risk of using VIPs. We also confirm that the age-phase, delay in sexual debut, and the number of sexual life partners have significant associations with HPV genotypes linked to cancer, highlighting the importance and the urgency of vaccinating young girls with Gardasil®9 HPV vaccine. Adolescents with HPV have significantly higher levels of activated CD4+ T cells in their cervical mucosa, suggesting the presence of reactive activated cells that lack efficiency in the clearing of HPV infection in this age group. HPV infection upregulates HBD-2 levels during HPV infection, notably significantly higher in adult females than in adolescents. This investigation has generated new insights into the risk factors for HPV acquisition in young women. These findings need to be confirmed further by larger cohort size studies, essential for HPV prevention.
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    Biochemical and structural characterization of ClpK from Klebsiella pneumoniae.
    (2022) Motiwala, Tehrim .; Khoza, Thandeka.
    Abstract available in PDF.
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    Enzymatic characterisation of a CATL-like protease from Trypanosoma brucei brucei and small-subunit rRNA sequence based phylogenetic analysis of freshwater fish trypanosomes.
    (2021) Mthethwa, Bongumusa Comfort.; Coetzer, Theresa Helen Taillefer.; Willows-Munro, Sandi.
    Abstract available in PDF.
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    Identification and analysis of Cryptosporidium Glutathione Transferase.
    (2021) Mfeka, Sizamile Mbalenhle.; Khoza., Thandeka Ntokozo.
    Cryptosporidiosis, caused by Cryptosporidium spp. is a gastrointestinal disease which gives rise to severe life threatening complications in immunecompromised patients. The disease causing parasite has a proficient defense system against xenobiotic compounds and substances that renders the only drug designed to treat the gastroenteritis infection inefficient in immune compromised patients. This defense system includes a phase II enzyme called Glutathione Transferase (GST) which detoxifies a wide range of oxidant based substrates. The overexpression of this protein in multi drug resistant cases and its presence in multiple stages of the parasites life cycle highlights the parasites dependence and utilization of the GST protein thus making it a suitable therapeutic target. This study was then set out to determine characteristic features of Cryptosporidium GSTs in comparison to well studied GSTs using molecular biology and bioinformatics tools. A genome wide search was performed across multiple protein databases to mine the Cryptosporidium GST. The 15 Cryptosporidium spp. found to possess full length proteins were compared amongst themselves within the species and against other species using phylogenetic analyses. This led to the discovery of three novel classes of Cryptosporidium GST based on amino acid sequence identity. The classes were named Gamma, Psi and Vega GSTs. The GSTs varied in amino acid length, and secondary structure characteristics determined through homology modeling. In comparison to preexisting GSTs, the Psi and Vega class GSTs did not have the typical active site Tyr7 found in most cytosolic GST, furthermore the Vega class GST also did not have the typical thioredoxin like fold conserved in the N-terminal region of all GSTs. The Gamma class GSTs were found to most resemble pre existing GSTs consisting of the typical thioredoxin fold and the active site Tyr7 and thus selected for expression and purification studies. pET, pCOLD1 and pCOLDTF vectors were used to determine a suitable vector to facilitate the expression of a soluble gamma class GST in Escherichia coli. pCOLDTF which utilizes cold shock proteins at low temperatures and a chaperone called trigger factor assisted in the recombinant expression of the gamma class GST resulting in a protein with the monomer size of ~50 kDa, which is double that of existing GSTs. This is owed to by the N-terminal and C-terminal extensions that the protein possesses. The protein was purified to homogeneity using affinity chromatography and size exclusion chromatography. The resulting protein was found to be dimeric under native conditions.
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    Recombinant cathepsin L-like cysteine proteases for species-specific diagnosis of animal African trypanosomiasis.
    (2019) Mbhele, Ziphezinhle Elaine.; Coetzer, Theresa Helen Taillefer.
    African trypanosomiasis is a disease caused by protozoan parasites i.e. Trypanosoma spp. in livestock and humans and affects 37 sub-Saharan countries. Animal African trypanosomiasis (AAT) is known as nagana and African human trypanosomiasis (HAT) as sleeping sickness. Trypanosoma congolense, T. vivax and T. brucei brucei cause AAT which is an economic burden and hampers agricultural development in Africa. The parasite escapes the host’s immune response by switching the genes coding for the variable surface glycoproteins, resulting in new variable antigen types. This has made it unlikely to develop a vaccine against the disease and therefore many studies now focus on non-variant trypanosome antigens as potential diagnostic and drug targets. Trypanosomal cysteine proteases, such as the cathepsins B and L, have been identified and validated as potential diagnostic targets. They are expressed throughout the parasite life cycle and are essential for the survival of the parasite. The mammalian host produces an antibody response against trypanosomal cysteine proteases which do not affect the survival of the parasite, however, the antibodies are believed to play a role in trypanotolerance by neutralising the effects of the enzyme. Antigen-based ELISA is a good tool for accurate diagnosis of AAT, but also relies on good antibodies. The overall aim of this study was to produce antibodies against the cathepsin-L-like protease of T. b. brucei (TbbCATL) which can be used for specific diagnosis of T. b. brucei infections. This included polyclonal antibodies as well as single chain variable fragments (scFvs) using phage display. The protease, TbbCATL, was recombinantly expressed in E. coli for the first time as a 61 kDa protease (including the GST tag) using the pGEX-4T expression vector. The homologues from T. congolense (TcoCATL) (29 kDa) and T. vivax (TviCATL) (28 kDa and 32 kDa) were also recombinantly expressed using the P. pastoris yeast expression system and were shown to hydrolyse the Z-Phe-Arg-AMC substrate and to be inhibited by E-64. Antibodies against whole TbbCATL were produced in chickens and together with anti-TcoCATL peptide antibodies and anti-TviCATL antibodies, were evaluated in a western blot to determine possible cross-reactivity. Whereas the anti-TbbCATL antibodies were specific for TbbCATL, the anti-TcoCATL peptide and anti-TviCATL antibodies cross-reacted with TbbCATL. The production of scFvs was optimised using TviCATL as the panning antigen against the Nkuku® phage display library. The TviCATL-specific phages were enriched after the fourth round of panning and a total of seven clones gave high signals when analysed by monospecific ELISA. Future work will include recombinant expression and purification of the selected TviCATL-specific scFvs for testing in diagnostic assays as well as panning with the TbbCATL antigen. This study laid the groundwork for evaluating TbbCATL as a diagnostic target for AAT
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    Molecular cloning, recombinant expression and characterisation of serine and cysteine protease inhibitors from Trichinella zimbabwensis.
    (2019) Maseko, Thando Glory.; Coetzer, Theresa Helen Taillefer.
    Trichinellosis is a disease caused by parasitic helminths of the genus Trichinella. Infection occurs by ingestion of meat contaminated with infective Trichinella larvae. Cases of Trichinella zimbabwensis, a non-encapsulating species of Trichinella infecting mammals and reptiles, have been reported in Ethiopia, Mozambique and South Africa. The parasite life cycle alternates between the enteric and skeletal muscle phases of infection. Trichinella species release various excretory-secretory products that enable successful parasitism. These include cysteine protease inhibitors, cystatins and serine protease inhibitors. To illustrate, cystatins have roles in cellular invasion and immune evasion while serpins inhibit blood coagulation, resist host protease damage and interfere with host immunoregulatory signals. The potential roles of endogenous parasite cysteine and serine protease inhibitors in T. zimbabwensis make these inhibitors attractive targets for the development of novel antiparasitic interventions. The genes encoding a cysteine protease inhibitor, cystatin B, and a Kazal-type serine protease inhibitor, SPINK4, were identified in the T. zimbabwensis genome. Following the synthesis of cDNA from nematode extracted mRNA, the respective genes were amplified and cloned into E. coli expression vectors. The recombinantly expressed proteins, rTzcystatin B and rTzSPINK4, were purified using immobilised metal affinity chromatography, their inhibitory activity evaluated and antibodies were produced in chickens. The antibodies were used for the detection of the recombinant proteins on western blots and ELISA. Recombinant Tzcystatin B inhibited the activity of the catalytic domain of the cathepsin L-like peptidase from Trypanosoma congolense (TcoCATL), the homologues from T. vivax (TviCATL) and Theileria parva (ThpCATL) as well as cathepsin B from T. zimbabwensis (TzCATB). Conversely, rTzSPINK4 was unable to inhibit either host serine proteases, chymotrypsin or trypsin. Future studies will be aimed at exploring the effect of the T. zimbabwensis protease inhibitors on host proteases involved in antigen processing. This may indicate a possible role for the nematode protease inhibitors in host immunoregulation.
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    The stabilisation of the cysteine protease of Carica papaya (papain) and the catalytic domain of the Cathepsin L-like cysteine protease of Trypanosoma congolense (TcoCATL)
    (2019) Chetty, Ryan.; Hewer, Raymond.; Coetzer, Theresa Helen Taillefer.
    Proteases play an intricate role in the numerous functions of a living organism. Proteases are responsible for the cleavage of proteins into smaller fragments by catalysing the hydrolysis of peptide bonds. The class of cysteine proteases have a cysteine thiol group in their active site have been found in lower and higher organisms. They have been investigated as promising drug targets for various diseases due to their fundamental functions in catabolism and protein processing. The thermal stability of a protease is a key characteristic feature that is largely dependent on its amino acid sequence and composition and quantified through the determination of its melting temperature (TM). Papain is the most well characterised cysteine protease and is commonly used as a model for other cysteine proteases. Congopain is the major cysteine protease of Trypanosoma congolense which has been identified as the main causative agent of trypanosomiasis in livestock. The thermal stability for papain and congopain were investigated in this study via the thermal shift assay. Papain was purchased and the catalytic domain of congopain was expressed using the Pichia pastoris yeast expression system. The thermal stability of the proteases were determined under neutral pH conditions and the effect of pH and ligand binding were evaluated to determine if the proteases could be further stabilised. The most stable forms of papain and the catalytic domain of congopain in its monomeric form was observed at pH 5.0 with 50 μM chymostatin. The thermal stability of both cysteine proteases was successfully evaluated via the thermal shift assay and conditions to further stabilise papain and the catalytic domain of congopain were determined. The thermal shift assay has been proven to be a reliable technique in identifying factors which increase the stability of a protein. More specifically, the technique serves as a simple and primary diagnostic tool to screen potential inhibitors of a protein and detect changes in the TM of a protein.
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    Transferrin receptor-mediated gene delivery using functionalised gold nanoparticles.
    (2018) Padayachee, Jananee.; Singh, Moganavelli.
    Gene therapy strategies have shown their potential in treating numerous central nervous system (CNS) disorders, including highly aggressive brain cancers. Gold nanoparticles (AuNPs) are popular vectors for gene delivery, due to their low toxicity, and ease of synthesis and functionalisation. However, the in vivo efficacy of these vectors is dependent on their ability to cross the blood-brain barrier (BBB), a specialised capillary network preventing the movement of compounds into the CNS. Passage across the BBB is often facilitated through targeting of the transferrin (Tf) receptor, leading to uptake by receptor-mediated transcytosis. This study aimed to develop untargeted and Tf-targeted functionalised AuNP (FAuNP) vectors and assess their potential as gene delivery vectors. AuNPs were prepared through citrate reduction and functionalised with chitosan (CS) and poly(ethylene) glycol 2000 (PEG2000) in two weight ratios [2% and 5% (ww⁄)] to produce untargeted FAuNPs. The holo-transferrin protein was conjugated to both PEGylated and unPEGylated FAuNPS to produce the Tf-targeted FAuNPs (TfAuNPs). The physicochemical characteristics of FAuNPs were evaluated using UV spectroscopy, Fourier-transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). TEM revealed AuNP to be spherical and relatively monodisperse. FAuNPs displayed hydrodynamic diameters ranging from 94.7 – 196.4 nm with good colloidal stability, as evidenced by NTA. Binding studies viz. band shift and ethidium bromide intercalation assays showed that all FAuNPs were able to fully complex and efficiently condense pCMV-luc plasmid DNA, with PEGylated and targeted FAuNPs being capable of partially protecting DNA from nuclease degradation, as determined in nuclease protection assays. In vitro studies were conducted in the HEK293, Caco-2, and the Tf receptor-positive HeLa cell lines. Cytotoxicity was assessed using the MTT cytotoxicity assay, which revealed FAuNPs to be relatively non-toxic to HeLa and HEK293 cells. Notably, TfAuNPs displayed low cytotoxicities, and generally exhibited increased cell viabilities compared to the untargeted FAuNPs. The luciferase gene reporter assay was conducted to assess the transfection efficiency of the FAuNPs. Transfection levels were highest in Caco-2 cells, with PEGylated FAuNPs observed to produce reduced transfection compared to the unPEGylated FAuNPs. TfAuNPs displayed favourable transfection in HeLa cells; with the competition binding assays confirming receptor-mediated uptake for AuCSTf and AuCSTf-5% PEG FAuNPs only, suggesting that a grafting density of the 2% (ww⁄) PEG interfered with receptor binding. These Tf-targeted FAuNPs show the potential to be utilised as vectors for brain delivery; however further optimisation and investigations in an in vivo system are required.
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    Expression and evaluation of a 297-amino acid fragment of the blood stage protein PfC0760c from Plasmodium falciparum.
    (2019) Baig, Zainab.; Goldring, James Philip Dean.
    Abstract available in pdf.
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    Evaluating the effect of nitric oxide on myoblast proliferation, migration and differentiation.
    (2018) Sibisi, Ntethelelo Charles.; Niesler, Carola Ulrike.; Myburgh, Kathryn Helen.
    Wound healing is the process of restoring tissue integrity in response to injury. This process involves four major phases namely; haemostasis, inflammation, regeneration and remodelling. These phases are regulated by various growth factors and cytokines that are released at the site of injury to facilitate wound repair. Cells involved in wound healing include neutrophils, macrophages, lymphocytes, fibroblasts and stem cells. Satellite cells are mesenchymal stem cells which facilitate skeletal muscle regeneration through a process known as myogenesis. These cells are quiescently located underneath the sarcolemma of the muscle fiber and are activated upon injury to enter the growth phase of cell cycle. They then proliferate and migrate to the injury site to differentiate and fuse with existing fibers to form multinucleated muscle cells.Growth factors and signalling molecules, such as hepatocyte growth factor (HGF) and nitric oxide (NO), induce satellite cell activation by altering the expression of transcription factors such as paired box transcription factor 7 (Pax7), myogenic regulatory factor 5 (Myf5), myogenic differentiation antigen (MyoD) and Myogenin. The role of NO in the subsequent process of myoblast proliferation, migration and differentiation is however unclear. The present study therefore evaluated the effect of nitric oxide on myoblast proliferation, migration and terminal differentiation. C2C12 myoblasts were cultured in standard growth media and subsequently plated for analysis in serum free media. Proliferation or differentiation was induced via the addition of either 2 ng/ml HGF or 2% horse serum respectively, while migration was stimulated using the standard in vitro wound healing assay. L-NAME (a NOS inhibitor; 100 µM and 200 µM) and SIN-1 (a NO donor; 10 µM or 25 µM) were utilized to modify NO levels in vitro, while NO levels were assessed using a nitric oxide colorimetric assay kit. Proliferation was assessed via cell counts, migration by assessing the percentage wound closure and differentiation determined by calculating myoblast alignment and subsequent fusion into multinucleated myotubes. There was no significant change in nitric oxide generated by myoblasts during proliferation and migration studies. However, NO levels increased significantly in response to differentiation, L-NAME significantly prevented this NO increase at day of differentiation. L-NAME also significantly decreased myoblast terminal differentiation by inhibiting myoblast alignment and fusion at day 5 of differentiation. L-NAME also significantly reduced the proliferative effect of HGF on myoblasts at 24 hours, and significantly reduced percentage wound closure at 16 hours post-injury. NO release by C2C12 myoblast was observed to increase in response to SIN-1 in a dose dependent manner. NO levels significantly increased from 0.58 nmol in a control up to 1.35 nmol and 1.9 nmol at 1 hour in response to 10 µM and 25 µM SIN-1 respectively. These levels increased until they reached 2.5 nmol in response to 25 µM SIN-1 at 16 hours. SIN-1 showed no significant effect on myoblast proliferation, however, it significantly promoted myoblast migration in a dose dependent manner by increasing the percentage wound closure to 42% and 45% at 7 hours for 10 µM and 25 µM respectively compared to 38% of the control. SIN-1 also significantly stimulated myoblast fusion with myofiber area of 26% as compared to 18.6% of the control at day 5 of differentiation. In conclusion, nitric oxide levels increase significantly during myoblast differentiation, but not during proliferation and migration. Despite this, inhibition of nitric oxide synthase significantly affects all these processes. In contrast NOS-independent elevation of NO (through incubation with SIN-1) significantly increased myoblast migration and fusion, but not proliferation. This suggests a central role for NO in regulating myogenesis; however, this role requires further investigation.