Doctoral Degrees (Biochemistry)
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Item Evaluating the effect of embryonic exposure to antiretroviral therapy (ART) on the development of type-2 diabetes mellitus (T2DM) in a zebrafish model.(2024) Shezi, Mlondi.; Niesler, Carola Ulrike.The use of combination antiretroviral therapy (cART) has significantly increased the quality of life and lifespan of people living with HIV (PLWH), however this new lease on life is threatened by a high risk of developing metabolic disorders such hyperglycemia and type 2 diabetes mellitus (T2DM). TLD, a fixed-dose combination antiretroviral drug comprising tenofovir (300 mg), lamivudine (300 mg) and dolutegravir (50 mg) is a first-line treatment regimen for all PLWH in South African and other mid-to-low income countries, as recommended by the World Health Organisation (WHO). In adults, TLD is however increasingly associated with adverse side effects such as hyperglycemia, bodyweight gain and T2DM. The mechanisms underlying these effects are not well understood and indicate the need to develop tools and models to better understand the development of cART-related T2DM. Of critical importance, TLD is also widely used for viral suppression in HIV-positive pregnant women as it crosses the placenta, conferring some level of protection in the developing fetus and thereby reducing the risk of mother-to-child transmission. However, whether in utero exposure to TLD predisposes the embryo to developing metabolic diseases such as T2DM later in life are not known. Studies on the short- and long-term effects of TLD exposure in utero are therefore required. In this context, the zebrafish (Danio rerio) is seen as an ideal animal model for many human diseases and several zebrafish models of T2DM exist. The aim of this study was therefore to utilise zebrafish to increase our understanding of the effects of embryonic TLD exposure on the development of T2DM in adult organisms, with post-natal bodyweight, fasting blood glucose, and the relative qPCR expression of hormones and enzymes central to glucose metabolism as indicator endpoints. In order to establish morphological (bodyweight), glycemic (fasting blood glucose) and relative qPCR genetic changes likely to occur in non-obese and obese diabetic zebrafish, we first developed adult zebrafish models of T2DM using glucose-immersion and overfeeding approaches. The mean bodyweight of the overfed group (0.73 g ± 0.05 g) was significantly higher than that of the control group (0.57 g ± 0.1 g), while there was no significant bodyweight change for the glucose model. The fasting blood glucose levels of the overfed (3.5 ± 0.9 mmol/L) and glucose-immersed (4.0 ± 0.7 mmol/L) zebrafish models were significantly higher than the controls indicating the progression to insulin resistance and T2DM. Both models revealed a lack of insulin and preproinsulin expression in some fish within these treatment groups relative to controls when assessed with qPCR (i.e. no target amplification). The toxicity of TLD and its individual components in vitro (on HEK293 cells) and in vivo (on one-day-old dechorionated embryos), and thereby the appropriate TLD concentration for treatment, were then determined. TLD did not induce toxicity in HEK293 cells even when used at the highest concentration (of 100 μM Tenofovir, 100 μM Lamivudine and 50 μM Dolutegravir), but did slow the rate of cell growth. TLD was also not toxic to zebrafish embryos after five days of exposure, but did slow their development by delaying the formation of the swim bladder and therefore the ability of developing embryos to maintain an upright posture. One-day-old dechorionated zebrafish embryos were then exposed to TLD for 5 days, followed by the evaluation of bodyweight and fasting blood glucose changes in adult zebrafish at 4 months. The bodyweight (0.26 g ± 0.12 g) and fasting blood glucose (3.5 ± 1.92 mmol/L) in response to TLD treatment was higher, but not significantly different to the controls (0.17 g ± 0.04 g and 2.0 ± 0.62 mmol/L) respectively. The relative expression of insulin (zins), preproinsulin (ppins) and phosphoenolpyruvate carboxykinase 1 & 2 (pck1 & pck2) (all referred to as candidate genes) was then assessed using qPCR. The qPCR expression of insulin (Δ𝐶𝑡 2.02 ± 1.0) and preproinsulin (Δ𝐶𝑡 1.87 ± 0.9) of pooled samples at 4 months after TLD treatment was variable but still higher than controls (Δ𝐶𝑡 1.0 ± 0.0). The expression of all the selected candidate genes at 5 days and at 1-month post treatment was not significantly different to controls. In conclusion, to study the implication of embryonic TLD exposure on the development of T2DM in adult organisms, we first established T2DM models in zebrafish in order to understand and establish the changes (physiological and genetic) reflective of a diabetic state. We demonstrate that changes in relative qPCR gene expression in adult zebrafish was variable in both models, with some fish displaying elevated levels while others lost the ability to express insulin and its precursor preproinsulin. Furthermore, our study suggests that exposure to TLD during embryonic development may contribute to weight gain and hyperglycemia (i.e. insulin resistance) later in life, even long after the drug is removed. The genetic effects of TLD were not clearly visible, however it is possible that dietary challenge in the adult fish is required to demonstrate the extent of the effect. These preliminary findings provide insight on the implications of embryonic TLD exposure in the context of the development of metabolic diseases such as T2DM in adults.Item Studies on the antioxidative and antidiabetic effects of Xylitol and Erythritol in type 2 diabetes: a comparative study.(2021) Msomi, Nontokozo Zimbili.; Islam, Shahidul.The present study comparatively investigated the antioxidative and antidiabetic effects of xylitol and erythritol using in vitro, ex vivo and in vivo experimental models. The free radical scavenging activities (DPPH, nitric oxide (NO) and ferric reducing power (FRAP) and inhibition of carbohydrate digesting enzymes of the increasing concentrations of xylitol and erythritol (90-720 mM) were determined in vitro. In ex vivo condition, xylitol and erythritol (360-2880 mM) were established for their effects on intestinal glucose absorption and muscle glucose uptake. Additionally, lipid peroxidation (LPO) and reduced glutathione (GSH) concentrations, and the activity of catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR) and glutathione peroxidase (GPx) enzymes were investigated in Fe2+ induced oxidative muscular tissue. Results from these experiments revealed that xylitol exhibited the best activities in terms of all the tested parameters. Xylitol and erythritol were further investigated for their antidiabetic and antioxidative effects in an intervention trial, using a fructose-streptozotocin induced type 2 diabetes rat model. The trial revealed the effects of these sugar alcohols at different concentrations (5%, 10% and 20%), on food and fluid intake, body weight modification, reducing blood glucose level and enhanced glucose tolerance capability. Upon euthanasia of the animals the curative capability on the complications associated with diabetes was determined on serum lipid profile, insulin resistance, pancreatic β-cell function, liver and muscle function. Oxidative stress biomarkers such as LPO, GSH, SOD and CAT were examined in the serum, pancreas, liver and muscle tissues. The in vivo experiment revealed that 10% xylitol dose displayed the most potent antioxidant and antidiabetic properties. Furthermore, data from the in vivo glucose absorption study revealed that xylitol (1 g/kg bw) exhibits better hypoglycemic and antidiabetic effects by delaying gastric emptying.Item Comparative antidiabetic effects and mechanisms of actions of five Chinese and South African indigenous teas.(2020) Xiao, Xin.; Islam, Shahidul.The present thesis assessed the in vitro, ex vivo and in vivo anti-oxidative and antidiabetic activities of five teas which are widely consumed in China or South Africa. Three of the selected five teas are from South Africa, namely red rooibos (Aspalathus linearis), green rooibos (Aspalathus linearis) and red honeybush (Cyclopia genistoides) tea. The remaining two from China are jasmine green (Camellia sinensis) and zhengshanxiaozhong (ZSXZ) black tea (Camellia sinensis). The different sequential solvent extracts following increasing polarity index (dichloromethane, ethyl acetate, ethanol, and water) and hot water extract of different teas were evaluated at in vitro and ex vivo conditions for their antioxidant properties, inhibitory potentials on α-glucosidase, α-amylase and pancreatic lipase, effects on ameliorating Fe2+- induced oxidative pancreatic or hepatic injury, as well as the glucose absorption inhibition in small intestine and the glucose uptake stimulation in isolated psoas muscle of rats. Possible bioactive components responsible for the activities of the extracts were identified by using Gas Chromatography-Mass Spectrometry (GC-MS) analysis or liquid chromatograph-mass spectrometry (LC-MS) analysis. In vitro and ex vivo tests presented promising antioxidant and antidiabetic activities of these five teas. The red honeybush, jasmine green and green rooibos teas, were further subjected to an in vivo intervention trial in a fructose-streptozotocin (STZ) induced T2D model of Sprague-Dawley rats. Assays were carried out to reveal the effects of these teas on lowering blood glucose level, improving oral glucose tolerance ability, stimulating insulin secretion and hepatic glycogen synthesis and ameliorating some diabetes related parameters such as serum lipid profile, hepatic and renal function tests and calculated insulin resistance (HOMA-IR), β-cell function (HOMA-β) from the blood glucose and serum insulin data. Furthermore, in vivo oxidative stress markers such as reduced glutathione, superoxide dismutase and catalase activity and lipid peroxidation were analysed in harvested organs (liver, kidney, heart and pancreas). The results of in vivo tests demonstrated that high dose of jasmine green tea showing the best activity followed by the high dose of red honeybush tea, low dose of jasmine green tea, high dose of green rooibos tea, low dose of red honeybush tea, when lowest activity was observed for the low dose green rooibos tea. The results of this study indicated promising anti-T2D properties of the above-mentioned teas. However, further clinical trials are needed to ascertain the results of these in vitro, ex vivo and in vivo studies.Item The development and use of in vitro and in silico beta-site amyloid precursor protein cleaving enzyme-1 protein-ligand binding assays.(2020) Chambers, Mark John.; Hewer, Raymond.Abstract available in PDF.Item Anti-c-myc cholesterol-based lipoplexes: development, characterisation and evaluation as Onconanotherapeutic agents in vitro.(2018) Habib, Saffiya.; Singh, Moganavelli.Strategies aimed at inhibiting the expression of the c-myc oncogene could provide the basis for alternative cancer treatment. In this regard, silencing c-myc expression using small interfering RNA (siRNA) is an attractive option. However, the development of a clinically viable, siRNAbased, c-myc silencing system is largely dependent upon the design of an appropriate siRNA carrier that can be easily prepared. Nanostructures formed by the electrostatic association of siRNA and cationic lipid vesicles represent uncomplicated, well-recognised siRNA delivery systems. Therefore, this study has focused on traditional cationic liposomes as the foundation for the development of a simple, but effective anti-c-myc onconanotherapeutic agent. Novel liposome formulations contained equimolar quantities of the cytofectin, N,Ndimethylaminopropylamidosuccinylcholesterylformylhydrazide (MS09), and cholesterol (Chol); with or without 2 mol % pegylation. Liposomes which contained dioleoylphosphatidylethanolamine (DOPE) as the co-lipid were included for comparative purposes. Pegylated and non-pegylated MS09/Chol (1:1) suspensions were reproducibly prepared by lipid film hydration to give unilamellar vesicles that were stable for at least 10 months at 4 ˚C. Liposomes successfully bound siRNA to form lipoplexes of less than 200 nm in size, with zeta potentials between -16 and -44 mV. These assumed globular and bilamellar structures in which siRNA was partially protected. Although all formulations were well tolerated at ≤14 nM siRNA, pegylation severely inhibited siRNA delivery in cancer cell lines, MCF-7 and HT-29, which overexpress c-myc. The non-pegylated MS09/Chol (1:1) lipoplex, at the MS09:siRNA (w /w) ratio of 16:1, was most effectively taken up by MCF-7 and HT-29 cells, with negligible effect in non-transformed cells when applied at 12 nM siRNA. Lipoplexes directed against the c-myc transcript (anti-c-myc siRNA), mediated a dramatic reduction in c-myc mRNA and protein levels. This was accompanied by a loss of migratory potential and apoptotic cell death. Moreover, oncogene knockdown and anti-cancer effects were superior to that of a commercially available transfection reagent, Lipofectamine™ 3000. Although the DOPE-containing counterpart performed with iii comparable efficacy under standard in vitro conditions, it was incapable of siRNA delivery at physiological serum concentration. Hence, the anti-c-myc MS09/Chol (1:1) lipoplex reported exemplifies a straightforward anti-cancer agent that warrants further investigation in vivo.Item An investigation into the mitochondrial toxicity of fusaric acid associated with aberrant energy metabolism and inflammatory responses.(2019) Sheik-Abdul, Naeem.; Chuturgoon, Anil Amichund.; Nagiah, Savania.No abstract available.Item An investigation into the analytical, cytotoxicity and immunotoxicity of mycotoxins found in commercially available pelleted pet foods in Durban, South Africa.(2018) Singh, Sanil Duleep.; Chuturgoon, Anil Amichund.Introduction: Dry pelleted dog food in the South African market is available via supermarket, pet stores (standard brands - SB) and veterinary channels (premium brands-PB). Similarly, cat food were viewed in two market segments. Methodology: Representative feeds from both categories were analysed for four main mycotoxins viz. aflatoxins (AF), fumonisin (FB), ochratoxin A (OTA), and zearalenone (ZEA) using standard well-described extraction, characterisation and quantitation processes. Results: All foods showed contamination with fungi (mainly Aspergillus flavus, Aspergillus fumigatus and Aspergillus parasiticus) and mycotoxins (the most prevalent being aflatoxins and fumonisins), irrespective of the brand. This study determined the immunotoxicity of extracts from pelleted dog and cat feed for mycotoxins. Isolated dog peripheral blood mononuclear cells (PBMCs) were treated with feed extracts to determine mitochondrial function, oxidative stress, and markers of cell death using luminometry and flow cytometry. Glutathione was significantly depleted by SB extracts. Markers of apoptosis and necrosis were elevated by both SB and PB feeds when compared to controls, with SB extracts being significantly higher than PB. ATP levels decreased with increased mitochondrial depolarization in cells that were exposed to both feed extracts with SB showing the greatest differences when compared to the control. Cat peripheral blood mononuclear cells (PBMCs) were isolated and treated with various feed extracts to determine oxidative stress (TBARS and GSH assay), mitochondrial integrity and cell death (Luminometry and Flow cytometry). Both PB and SB extracts showed significantly decreased ATP levels and increased mitochondrial depolarization except for the PB acid fraction. Lipid peroxidation was significantly increased in both PB and SB extracts with a concomitant decrease in GSH levels. Phosphatidylserine externalization and necrosis levels were increased in both PB and SB extracts when compared to the control. Executioner caspases-3/7 was also elevated following extract exposure except for the PB acid fraction. Conclusion: There were high levels of fungal contamination and mycotoxins in both categories of feed, regardless of the notion that higher priced PB’s were of a higher quality.Item Developing in vitro multi co-culture models and analysis tools for muscle regeneration.(2019) Venter, Colin.; Niesler, Carola Ulrike.Skeletal muscle regeneration represents a complex process mediated by non-myogenic cell types. These cells, such as macrophages and fibroblasts, display a range of interactions with muscle stem cells (myoblasts) during myogenesis (the differentiation and fusion of myoblasts into muscle fibres). Our knowledge of these interactions has been elucidated using in vivo and in vitro skeletal muscle models. Although in vivo models are more physiologically relevant, in vitro models, such as co-culture, offer a simpler and cost-effective means to study muscle regeneration. We therefore developed a novel and inexpensive co-culture method using three different cells types, which closely resembled the in vivo microenvironment by permitting a range of cellular interactions. Once this method was established, cellular behaviour in response to various experimental conditions could be evaluated. A second challenge we encountered was that the strategies available to us for assessing myogenesis in vitro were suboptimal in terms of speed and accuracy. We therefore sought to optimize image processing methods to rapidly and accurately quantify cellular numbers (proliferation), wound area (migration) and orientation (alignment) in our co-culture model. We then used these methods to evaluate the roles of macrophages and/or fibroblasts during the early (proliferation and migration) and late (alignment and fusion) stages of myogenesis. We observed a significant increase in myoblast proliferation and migration in response to coculture with either unstimulated macrophages or fibroblasts. In triple co-culture, macrophages continued to promote myoblast proliferation in the presence of fibroblasts. However, the presence of macrophages abrogated the positive effect of fibroblasts on myoblast migration; qualitative analysis also suggested a decrease in fibroblast number. Following analysis of later differentiation, we found that macrophages significantly promoted alignment, but prevented fusion, in a cell density-dependent manner. Fibroblasts, on the other hand, had no significant effect on myoblast alignment, but either promoted (at low fibroblast numbers) or inhibited (at higher fibroblast numbers) fusion. In triple co-culture, the effect of macrophages on myoblast alignment and fusion was unaltered by the additional presence of fibroblasts. In order to determine whether pro-macrophages have a direct quantitative effect on fibroblast number, M1 macrophages were generated following incubation with LPS and then cocultured with a fibroblast population. The latter population was characterised as containing both fibroblasts and their differentiated counterpart, myofibroblasts. A significant decrease in the size of this population (potentially as a result of cell death) was observed in response to M1 macrophages; this decrease was prevented by the addition of LY294002, a phosphoinositide 3-kinase (PI3K) inhibitor. Subsequent analysis demonstrated that LY294002 decreases macrophage numbers, suggesting a potential mechanism for the rescue of the fibroblast population by this inhibitor. Dexamethasone, on the other hand, caused the fibroblast population to acquire a rounder myofibroblast morphology, but the implications of this morphological change requires further investigation. In this thesis, we presented optimized and novel methods which were used to study skeletal muscle regeneration in vitro. The findings provided new insights into the temporal regulation of myogenesis by non-myogenic cells. During the early stages of myogenesis, macrophages need to increase in number to promote myoblast proliferation, but subsequently resolve with an increase in fibroblast numbers to promote myoblast migration into the wound. During the later stages of myogenesis, macrophage and fibroblast numbers need to subside to promote myoblast alignment and fusion, respectively. The communication between these nonmyogenic cells and the phenotypes they acquire can also indirectly influence myogenesis. The fibroblast population is important for promoting myoblast fusion, but macrophages with an M1 phenotype resulted in death of myofibroblasts. This makes it imperative that the population of M1 macrophages timeously subsides. However, M1 macrophage-mediated death of myofibroblasts was prevented by inhibition of the PI3K pathway which resulted in macrophage, but not myofibroblast, death. This suggests a potential therapeutic target for the treatment of muscle diseases, such as myositis, caused by the dysregulated presence of macrophages.Item Evaluation of the anti-diabetic potentials of some African medicinal plants: a multimode study.(2018) Olajumoke, Arinola Oyebode.; Islam, Mohammad Shahidul.The present study investigated the effects of five African medicinal plants (Alstonia boonei, Acalypha wilkesiana, Boerhaavia diffusa, Bridelia ferruginea, and Crassocephalum rubens) for their antioxidative and antidiabetic potentials by using several experimental protocols. The crude extracts (ethyl acetate, ethanol and aqueous) of the different parts (leaves, stem bark, root bark or aerial parts when applicable) were initially investigated for their detailed antioxidant and antidiabetic activity using in vitro, ex vivo and in silico experimental models. Then the most active crude extract from each plant was chosen for further fractionation with the solvents of increasing polarity. The solvent obtained fractions were then subjected to screening in terms of their antioxidant, -glucosidase, -amylase and lipase inhibitory activity in vitro and intestinal glucose absorption and muscle glucose uptake ex vivo. Results from these assays revealed that the butanol and aqueous fractions of A. boonei, butanol fraction of B. ferruginea, ethanol extract of A. wilkesiana and B. diffusa and the aqueous extracts of C. rubens showed the best activities in terms of all the tested models. The most active crude extracts and fractions were consequently subjected to GC-MS and LC-MS analyses in order to identify their bioactive components. Then the structures of the most bioactive components were docked with the tested enzymes using in silico modelling. The anti-diabetic effect of the butanol fractions of A. boonei and B. ferruginea together with the aqueous extract of C. rubens were investigated in an in vivo intervention trial using a type 2 diabetes rat model. The in vivo experiment revealed that the fractions and extract exhibited potent in vivo hypoglycaemic activity. Interestingly, these fractions were also able to alleviate T2D-associated complications involving oxidative stress. Analysis of in vivo oxidative stress markers such as superoxide dismutase, catalase, glutathione and thiobarbituric acid reactive substances, in the serum, liver, kidney, heart and pancreas of the animals also suggested their strong antioxidative effects. The results of this study suggest that the different extracts/fractions of the above-mentioned plants have promising anti-diabetic potentials; however further clinical trials are required in order to justify the usefulness of these plants for the development of potent and cost effective anti-diabetic drugs.Item Antidiabetic and toxicological properties of some African medicinal plants used in the treatment of diabetes and its complications.(2018) Erukainure, Ochuko Lucky.; Islam, Mohammad Shahidul.This study investigated the antioxidant, antidiabetic and toxicity properties of antidiabetic medicinal plants comprising of Vernonia amygdalina, Cola nitida, Raffia palm (Raphia hookeri) wine, Phaseolus lunatus, Dacryodes edulis, and Clerodendrum volubile using in vitro, ex vivo, in silico and in vivo models. The leaves of V. amygdalina and D. edulis, as well as C. volubile flower were sequentially extracted with solvents of increasing polarity to yield ethyl acetate, ethanol and aqueous extracts. Cola nitida and V. amygdalina were infused in hot water to yield infusion extracts. Phaseolus lunatus was subjected to aqueous extraction to yield aqueous extract, while Raffia palm wine was concentrated to yield the concentrate. The extracts and concentrate were screened for their in vitro and ex vivo antioxidant activities, as well as their inhibitory effect on α-glucosidase, α-amylase and pancreatic lipase activities, and their ability to stimulate muscle glucose uptake and inhibit intestinal glucose absorption in vitro. The ethanol extracts of D. edulis, C. volubile and V. amygdalina were subjected to GC-MS analysis, while the aqueous extract of P. lunatus, palm wine concentrate and the infusions were analyzed with LC-MS to elucidate the active compounds that may be responsible for their bioactivities. The ethanol extracts of C. volubile and D. edulis were further subjected to liquid-liquid fractionation to yield the hexane, dichloromethane, ethyl acetate, butanol and aqueous fractions. These fractions were also assayed for their antioxidant and antidiabetic properties in vitro and ex vivo. The dichloromethane, ethyl acetate and butanol fractions were subjected to GC-MS analysis to elucidate their active compounds. The identified compounds were molecularly docked with the test enzymes in silico to further validate their bioactivities. The antidiabetic properties of palm wine concentrate, C. nitida infusion, and D. edulis butanol fraction were investigated in a type 2 diabetes rat model. The in vivo study revealed a potent hypoglycemic activity, with concomitant amelioration of oxidative stress in the serum, pancreas, testes and brain. This was further substantiated by the downregulation of Nrf2 expressions in the pancreas and brain. These results further validate the use and safety of these plants in diabetes management.Item A comparative investigation of transgene expression and gene silencing of non-functionalized layered double hydroxides versus amino-acid functionalized hydrotalcites.(2017) Nundkumar, Nirasha.; Singh, Moganavelli.; Singh, Sooboo.Abstract available in PDF file.Item Molecular characterisation of metacaspase 5 and the production of oligopeptidase b-specific single chain variable fragment antibodies for potential animal African trypanosomosis chemotherapies and diagnostics.(2018) Eyssen, Lauren Elizabeth-Ann.; Coetzer, Theresa Helen Taillefer.African trypanosomosis (AT) is a major obstacle in the establishment of agriculture and economic sustainability in Africa. Animal AT is responsible for large numbers of livestock succumbing to the tsetse transmitted kinetoplastid parasites, Trypanosoma congolense and T. vivax, and as a result, losses in further downstream sectors are experienced. Due to the ability of the trypanosomal parasites to undergo antigenic variation, vaccine candidates are highly unlikely. Peptidases have been identified as virulence factors and are the focus of the development of novel chemotherapies and diagnostics. The metacaspases (MCAs) are a prime example of a chemotherapeutic target and oligopeptidase B (OPB), that of a diagnostic target. Towards the validation of a chemotherapeutic target, recombinant expression was used to obtain an active peptidase which could be enzymatically characterised. Various inhibitors were investigated and their effect on the parasite, analysed. Current diagnostics are based on antibody detection, but an antigen detection format would be preferable as it could differentiate between active and cured infections as anti-trypanosomal antibodies can persist for years. Given the rural, resource-poor locations in the areas of AT incidence, an ideal rapid diagnostic test (RDT) would be robust, affordable, sensitive and specific and requiring minimal training, such as a dipstick test. The MCAs are cysteine peptidases which are found in all kingdoms other than the metazoa, and share a secondary structure fold and catalytic dyad with the metazoan caspases. Since the caspases play a role in apoptosis, it is thought that the MCAs may function in a similar manner. The single copy MCAs of Trypanosoma spp. and Leishmania spp. differ from the multicopy MCAs in that they possess a Pro-, Gln-, Tyr-rich C-terminal domain which is thought to mediate protein-protein interactions. The activity of the single copy MCAs from T. cruzi and L. major has been implicated in the cell cycle of the kinetoplastid parasite. The aim of the project was to express, purify and enzymatically characterise the recombinant and native MCA5 from T. congolense and T. vivax. Using the 3D structures, solved by X-ray diffraction, of MCA2 from T. b. brucei, molecular docking studies were used to validate the inhibition potential of a published library of inhibitors, designed based on the, then, hypothetical structure of TbbMCA2. Since the elucidation of the 3D structure of TbbMCA2 by X-ray diffraction, the inhibitory power of the library of inhibitors against TbbMCA2 and the MCA5s was investigated. The serine peptidase, OPB, has been shown to be released into the host bloodstream by dead and dying parasites. The use of phage displayed scFv (single chain fragment variable) antibodies for the detection of OPB in serum from infected cattle is reported, towards the development of a RDT. Recombinantly expressed TcoMCA5 was shown to autoprocess and over autoprocess when purified using nickel affinity chromatography. Mutagenesis of the catalytic dyad residues reduced the over autoprocessing and the mutated form was enzymatically active at a pH between 6 and 9. This active mutant and purified TcoMCA5 showed a prefence for Arg over Lys at the P1 substrate position and were able to hydrolyse gelatin. Possible novel inhibitors of TbbMCA2 and the MCA5s of T. congolense and T. vivax were identified using a library of ligands (Berg library) based on the P1 specificity of TbbMCA2 and molecular docking. Commercial fluorogenic peptide substrates and inhibitors reported in literature for the characterisation of various MCAs, revealed interactions with the MCAs which should be taken into consideration when modifying the Berg ligands to achieve higher affinity for the MCAs. The application of scFv antibodies, derived from the Nkuku® phagemid library, for the diagnosis of current AAT infections by the detection of OPB, released in the bloodstream of the infected mammalian host, was investigated. After the successful isolation and production of OPB-specific scFv, MCA-specific scFv antibodies can be pursued using the Nkuku® phagemid library. The resulting OPB-specific scFv identified a conserved peptide between T. congolense and T. vivax and was able to detect native OPB in a western blot format. It was predicted that the scFv interacted with OPB in such a way that it would restrict the hinge motion between the C-terminal catalytic and N-terminal regulatory domains of the enzyme and limit access to the active site pocket. The ability of scFv and rabbit-anti-OPB polyclonal antibody in an antigen detection ELISA with sera from T. congolense infected cattle indicated that detection of OPB fluctuated with parasitaemia.Item Functionalised gold nanoparticle delivery for c-MYC siRNA in cancer gene therapy.(2018) Daniels, Aliscia Nicole.; Singh, Moganavelli.; Singh, Sooboo.Abstract available in PDF file.Item Starburst poly-amidoamine dendrimer grafted gold nanoparticles as scaffolds for targeted gene delivery in vitro.(2017) Mbatha, Londiwe Simphiwe.; Singh, Moganavelli.Abstract available in PDF file.Item Studies on the anti-hyperglycemic potentials and possible mode of actions of some commonly used sugar alcohols.(2016) Chukwuma, Chika Ifeanyi.; Islam, Mohammad Shahidul.Abstract available in PDF file.Item Factors governing seed recalcitrance in two species of contrasting storage longevity.(2017) Moothoo-Padayachie, Anushka.; Naidoo, Sershen.; Varghese, Boby.; Varghese, Dalia.; Pammenter, Norman William.; Govender, Patrick.; Berjak, Patricia.Abstract available in PDF file.Item Mitochondrial localisation and cellular uptake in vitro using novel ‘mitochondriotropic’ liposomes.(2016) Narainpersad, Nicolisha.; Ariatti, Mario.; Masola, Bubuya.; Singh, Moganavelli.Mitochondrial research has made tremendous strides since the 1980/90s when mitochondrial DNA mutations were first identified as a primary cause for human diseases and the organelle’s role in apoptosis was elucidated. These mutations of the mitochondrial genome have been implicated in a spectrum of clinical disorders especially involving the muscle and central nervous system. This makes the mitochondrion a prime candidate for organelle-specific delivery of exogenous materials such as therapeutic DNA and drugs, for therapy of diseases caused by mitochondrial dysfunction. However, reports of mitochondrial targeted delivery systems are limited. Hence vector design and development is of paramount importance. The success of liposomes viz. cationic liposomes, in chromosomal gene therapy make them potential vectors for mitochondrial gene targeting. In this investigation novel ‘mitochondriotropic’ liposomes were synthesised to evaluate their cellular uptake and mitochondrial localisation activity in vitro using four different mammalian cell culture models. Cationic cholesterol derivative, 3β [N-(N’,N’-dimethylaminopropane)-carbamoyl] cholesterol (CHOL-T) was formulated with dioleoylphosphatidylethanolamine (DOPE) to produce cationic liposomes, to which a mitochondrial targeting sequence (MTS) and octaarginine (R8) peptides were attached via two different novel cholesterol-derived cross-linking agents. Size, zeta potential, shape and lamellarity of liposomes and corresponding lipoplexes were assessed by the innovative technique, Nanoparticle Tracking Analysis (NTA) and cryogenic transmission electron microscopy. Their ability to bind, condense and protect plasmid DNA (pCMV-luc), was determined using the band shift, dye displacement and nuclease protection assays repectively. In vitro cytotoxicity and mechanism of cell death prompted by these novel liposomal preparations was determined using the MTT, AlamarBlue® and acridine orange and ethidium bromide (AO/EB) dual staining assays respectively, in the hepatocyte-derived human cell line (HepG2), human embryonic kidney cells (HEK293), the human intestinal cell line (Caco-2) and human cervical carcinoma (HeLa-Tat luc) cells. Fluorescently labelled DNA was used to determine cellular uptake and mitochondrial targeting and localisation ability of these cationic mitochondriotropic liposomal formulations in the target organelles, mitochondria using fluorescence microscopy and the quantitative evaluation of fluorescence in the mitochondrial fraction of cell homogenate cocktails. These mitochondriotropic liposomes successfully bind, condense and protect plasmid DNA in the presence of serum, are fairly well tolerated by all cell lines tested in culture with cell death observed to be apoptotic and not necrotic in nature. The liposomes were shown to successfully enhance cellular uptake in all cell culture models tested. Furthermore, results demonstrate positive mitochondrial targeting and localisation activity facilitated by the presence of MTS peptide and a combination of MTS and R8 peptides on the liposomal surface for all four of these novel liposomal nanovectors.Item Expression of monocyte heat shock protein 70 (HSP70) during malaria fever in the presence of antimalarial, anti-inflammatory drugs and β-haematin.(2017) Fowdar, Kajal.; Goldring, James Philip Dean.Abstract available in PDF file.Item Antidiabetic properties of centella asiatica in type II diabetic rats.(2016) Oyenihi, Ayodeji Babatunde.; Masola, Bubuya.; Mukaratirwa, Samson.Abstract available in PDF file.Item Synthesis, characterization and evaluation of antibacterial, antidiabetic and toxicological profiles of newly-derived therapeutic agents.(2016) Gannimani, Ramesh.; Govender, Patrick.; Pillay, Karen.Abstract available in PDF file.