Successful antiretroviral (ARV) treatment is associated with suppression of HIV viral load and the reduction of clinical disease progression. Despite marked improvements in ARV medication, side effects from long-term treatment, such as loss of muscle mass do occur. The mechanism by which ARVs affect muscle mass is unclear, however, published in vitro data suggests a negative effect on myoblast fusion during differentiation. The objective of this study was therefore to determine the effect of ARVs on processes required for successful myogenesis; these included proliferation, migration during wound repair, and differentiation. C2C12 mouse skeletal myoblasts and human primary culture skeletal (HSk) myoblasts were incubated with Zidovudine (nucleoside reverse transcriptase inhibitor-NRTI), Tenofovir (nucleotide reverse transcriptase inhibitor-NtRTI) or Ritonavir (protease inhibitor-PI) at a concentration range of 0.01 μM to 10 μM. Proliferation was determined using crystal violet and migration was analyzed using a 2D wound healing assay. The commitment of myoblasts into the myogenic lineage was assessed via the expression of the transcription factor Pax7. Differentiation was measured by assessing the fusion index of multinucleated myotubes. C2C12 myoblast proliferation was observed to increase significantly in response to Tenofovir (1 μM and 10 μM). In HSk cells however, proliferation was observed to decrease significantly in response to Tenofovir (1 μM). Zidovudine had no consistent effect on C2C12 proliferation at any dose tested, but caused a decrease in HSk myoblast proliferation (0.01 μM and 0.1 μM); however this was statistically non-significant. A small dose-dependent increase in C2C12 and HSk cell number, although not significant, was seen in response to Ritonavir. Wound closure results revealed both dose-dependent and time-dependent effects of Tenofovir and Zidovudine on human myoblast migration, with significant decreases in the rate of wound closure (4-7 hours) noted at 0.1 μM and 0.01 μM doses respectively. Zidovudine had no significant effect on migration while Ritonavir (0.01 μM) was observed to significantly increase percentage wound closure of human myoblasts, suggesting an increased ability to migrate during wound repair. Differentiation results indicated a decrease in myoblast fusion in response to all three ARVs. However only Ritonavir was shown to negatively affect myosin heavy chain expression. Further research into the exact mechanism of decreased fusion is required. To our knowledge, this study is the first to suggest that selected ARVs may significantly influence myoblast regeneration capabilities by modulating myoblast proliferation, migration, differentiation and fusion, and thereby decrease their myogenic capability. Extended human myoblast studies on differentiation could confirm this hypothesis. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2013.

Several diseases, including cancer, have been associated with high membrane type-1 matrix metalloproteinase (MT1-MMP) expression levels. MT1-MMP together with a non-membrane-bound, soluble MMP, MMP-2, also associated with many other biological functions, have been implicated in breast cancer progression, invasion and metastasis, and poor prognosis. Researchers who ran early clinical trials that employed broad-spectrum MMP inhibitors (MMPIs) lacked understanding of the intricate physiological and patho-physiological roles that MMPs play in tissues. In addition, structural similarities between MMPs hamper selective inhibition. Selective inhibition of MT1-MMP is of particular interest as MT1-MMP is overexpressed in target cancer cells relative to normal cells and is key in signalling for invasion. The inhibition profiles of two structurally similar synthetic pyrimidine-class MMPIs, with increased bioavailability and stability compared to hydroxamates tested in earlier clinical trials, TF 17-2 and TF 22d, were assessed. TF 17-2 and TF 22d were applied to a normal MCF-10A breast epithelial cell line and its premalignant H-ras(V12)-transfected MCF-10AneoT derivative to assess their efficacy for inhibiting MT1-MMP-mediated normal and premalignant cell migration, and indirectly, invasion. Both inhibitors form a co-ordination complex with the zinc ion of the catalytic site of MT1-MMP and MMP-2 with greater affinity for MT1-MMP. The computational molecular docking package, AutoDock Vina, was used for in silico predictions of binding affinities that could potentially substitute for in vitro kinetic assays when assessing inhibitor potential for inhibiting target MMPs. The binding of the two MMPIs was assessed using AutoDock Vina and compared to established kinetic data. The AutoDock Vina program was found to be an unreliable predictor for assessing relative efficacy of inhibition. During in vitro applications, analysis of the induction of apoptosis and metabolic effects were assessed using flow cytometry and the MTS assay, respectively. These showed no significant toxicity. Effects of inhibitors on collective and single cell migration in the normal and premalignant cell model, assessed using time lapse live cell imaging, cell morphology and labelling for vinculin and F-actin (for focal adhesions, FAs) showed that the TF 17-2 and TF 22d inhibitors reduced the collective cell migration of MCF-10A cells in scratch assays. Live-cell analysis of single cell migration, however, showed that TF 22d increased cell migration rates, and reduced the size of FAs and actin stability in MCF-10AneoT cells, resulting in a predominently rounded cell morphology in the premalignant cell line. TF 17-2, on the other hand was seen to be a relatively selective inhibitor of premalignant cell migration and resulted in MCF-10AneoT cells re-establishing larger focal - v - adhesions due to more stable F-actin networks resembling those of the non-transfected MCF-10A cell line, but reduced MCF-10AneoT cell migration most markedly. FA size and velocity of movement seemed inversely related in the normal and premalignant cells. The results of the current study suggest that, TF 17-2 seemed to have the greater therapeutic potential than TF 22d for inducing phenotype reversion, inhibition of dissemination, invasion and metastasis. Three promising selective pharmacological actions of TF 17-2 on the premalignant MCF10AneoT cell line include the suppression of proliferation, induction of increased in metabolic activity (possibly indicating cell stress) and a decrease in premalignant cell migration. A lack of cytotoxicity, however, suggests that TF 17-2 would need to be administered with an ancillary chemotherapeutic agent. This study showed that MMPIs directed against MT1-MMP, may still represent an effective strategy for inhibiting the migration of premalignant cells expressing high levels of MT1-MMP, and suggests further studies on this topic may be profitable. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

In traditional forage breeding programmes, breeders have spent decades improving the agronomic characteristics of grasses, such as herbage yield, persistence and resistance to diseases, without considering the nutrient requirements of the grazing animal. In an attempt to improve the nutritive value of Italian ryegrass, which is widely utilised for intensive dairy, lamb and beef production in South Africa, Enhancer ryegrass was developed from predominantly Italian types of Lolium multiflorum, with a minor Westerwolds component, by selecting for a higher concentration of total nonstructural carbohydrate (TNC) and lower moisture content than that currently available in commercial cultivars. The nutritional value of Enhancer was compared with Midmar ryegrass in a controlled environment study and in a grazing trial with weaned lambs; and with Dargle ryegrass in a grazing trial with Holstein dairy cows. Neutral detergent fibre, acid detergent fibre, lignin, nitrogenous compounds, mineral content and in vitro digestibility were also investigated as parameters of nutritive value. The anatomical features of Enhancer and Midmar were studied to determine possible structural differences. Weaned lambs grazed Enhancer and Midmar in an eight-paddock rotational grazing system, with 3.5 days spent in each paddock, allowing a 24.5 day regrowth period for the pastures. Holstein dairy cows grazed Enhancer and Dargle which were established on 16 and 19 hectare pastures, respectively. The n-alkane technique was used to estimate dry matter intake (DMI) in both grazing trials. Results from the controlled environment study suggest that the differences in the dry matter and TNC concentration of Enhancer are not positively linked to anti-quality factors associated with forage species, but can be attributed to genetic differences between the two grasses. Despite the significantly higher (P < 0.01) DMI of weaned lambs grazing Midmar compared with Enhancer, the lambs on Enhancer outperformed those on Midmar in terms of liveweight gain and carcass quality. The superior animal performance on Enhancer is likely due to an improvement in the readily digestible energy to protein ratio as a result of its significantly higher (P < 0.001) concentration of TNC compared with Midmar. Milk yield for cows grazing Enhancer in period 1 of the cross-over study was significantly higher (P < 0.05) than for cows grazing Dargle, despite the significantly lower (P < 0.05) DMI of animals on Enhancer. The higher TNC concentration relative to the true protein content of Enhancer would suggest that the protein metabolism in the rumen can be enhanced. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2003.

The work presented here represents research done on mycotoxins and plant toxins by the author and his postgraduate students over a period from 1964 to date. The first phase, which ends at 1980, mainly addresses the biosynthesis of the aflatoxins. The involvement of anthraquinone derivatives in this process was investigated and the role of versicolorin A and its derivatives was partially elucidated. Novel active enzymes systems were derived from protoplasts and used in these studies. The period lasting from 1980 to 1992 concentrates on the occurrence of mycotoxins in agricultural commodities and effects on animals and their systems. Over 7000 samples were analysed using a multimycotoxin analytical method and a fungal screen. The most common mycotoxin found was aflatoxin B₁ and prevalent fungus was Fusarium moniliforme. Later work is indicating that fumonisin B₁ is the most commonly occurring mycotoxin. As this was only discovered in 1988, its presence was only looked from 1995 onwards. It was also found that rumen fluid could metabolise trichothecenes. During this period (1980-1992) further work on aflatoxin metabolism was done and a novel dehydrogenase involved in aflatoxin B₁ was isolated and characterised. An Elisa assay was developed for atractyloside, a toxin found in a plant (Callilepis laureola) used in tradition medicine. The site of atractyloside storage was found to be in the plant vacuole. The final period covers 1992 to the present, where the occurrence and effects of mycotoxins in human disease were studied. The major and most important finding is that fumonisin B₁ is present in the blood and tissues of many of the Black population examined in Kwazulu Natal. This includes, oesophageal cancer patients, eclamptic patients, school children and members of the rural population. A similar circumstance also appertains for the presence of aflatoxin B₁. It seems likely from these results that chronic mycotoxicoses are a common occurrence, particularly in the Black rural population and are not the sporadic rare event that is found in the first world countries. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1999.

African trypanosomosis is a parasitic disease in man and animals caused by protozoan parasites of the genus Trypanosoma. T. congolense, T. vivax and T. brucei brucei cause nagana in cattle. The variable nature of the parasite surface coat has hindered the development of an effective vaccine. An option for developing vaccines and chemotherapeutic agents against trypanosomosis is to target pathogenic factors released by the parasite during infection, namely an “anti-disease” approach. Two pathogenic factors released during infection are oligopeptidase B (OPB) and TcoCATL (congopain). TcoCATL, a major lysosomal cysteine peptidase, is a member of the papain family C1 cysteine peptidases. RNA interference (RNAi) was used to down-regulate the expression of TcoCATL in T. congolense IL3000 TRUM183:29-13 parasites in vivo during mouse infections. TcoCATL RNAi was monitored in infected mouse blood by comparing the hydrolysis of Z-Phe-Arg-AMC and parasitaemia between mice in which RNAi was induced and control mice. Mice infected with parasites induced for TcoCATL RNAi had lower parasitaemia when compared to control mice. An attempt was also made at deleting the entire CATL gene array in both T. congolense IL3000 and T. brucei 427 Lister strains. The second pathogenic factor studied, OPB, is a cytosolic trypanosomal peptidase that hydrolyses peptides smaller than 30 amino acid residues, C-terminal to basic residues. In order to evaluate the role that OPB play during disease, RNAi was also applied to knock-down the expression levels of OPB in T. brucei T7T and T. congolense IL3000 TRUM183:29-13 strains (TbOPB and TcoOPB respectively). Oligopeptidase B null mutant strains (Δopb) were also generated in T. brucei brucei Lister 427. An attempt was also made to generate OPB null mutants in T. congolense IL3000 parasites. Western blot analysis of the knock-down experiments using chicken anti-TcoOPB peptide IgY showed that only TbOPB levels were reduced in T. brucei T7T parasites induced for RNAi when compared to TcoOPB RNAi induced cultures. Quantitative assessment of a fourteen day induction experiment for OPB RNAi in T. brucei showed an 87% reduction in TbOPB levels when compared to levels on day one. There was no growth effect observed in T. brucei parasites cultured in vitro and induced for TbOPB RNAi. It was concluded that TbOPB is not necessary for the in vitro survival of T. brucei parasites, thus making the generation of OPB null mutants possible. Δopb T. brucei parasites were successfully generated and grew normally in vitro and were as virulent as wild type strains during infection in mice. Immunohistopatholgy of infected mouse testes revealed Δopb parasites in extra vascular regions showing that T. brucei OPB (TbOPB) is not involved in assisting T. brucei parasites to cross microvascular endothelial cells. Gelatin gel analysis of Δopb null mutants and wild type strains showed an increase in cysteine peptidase activity. Enzymatic activity assays were carried out to identify how closely related oligopeptidases are affected by knocking out TbOPB, and a significant increase of T. brucei prolyl oligopeptidase (TbPOP) activity was observed. However, western blot analysis did not show any increase of TbPOP protein levels in Δopb parasites, suggesting that either TbOPB is responsible for generating an endogenous inhibitor for TbPOP or that another POP-like enzyme might compensate for a loss in OPB activity in Δopb null mutants. This study made a significant contribution to an understanding of the interplay between different trypanosomal peptidases that are important pathogenic factors in trypanosomosis. It highlights the need to simultaneously target several trypanosomal peptidases to develop an effective vaccine or chemotherapeutic agents for African animal trypanosomosis. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

The proteinase, cathepsin L, has been strongly implicated in the processes of tumour invasion and metastasis. A new purification method, three-phase partitioning, characterised in terms of the parameters which affected its fractionation of proteins, was found to simplify the purification of cathepsin L from sheep liver. This method, together with a novel cation-exchange step on S-Sepharose and molecular exclusion chromatography, enabled the enzyme to be purified to homogeneity, in a single-chain form. A further enzyme fraction was isolated as a proteolytically active complex with the endogenous inhibitor of cysteine proteinases, cystatin. Studies on the proteolytically active complex revealed that approximately 60% of it was covalently bound and proteolytically active, while the other 40% was non-covalently bound and proteolytically inactive, in the manner normally found for the binding of cystatin to cysteine proteinases. A cystatin fraction from sheep liver containing variants of cystatin B, was shown to be able to form complexes with free cathepsin L in vitro in a pH-dependent, rapid process, which was mildly stimulated by a reducing agent. Cathepsin L was also isolated from human spleen, but only as a protcolytically inactive complex, presumably also with cystatin(s). The complexed and free cathepsin L from sheep liver were analysed for their pH-dependent characteristics, and it was found that both forms of the enzyme were more active and stable at, or near, neutral pH, than would have been expected from published values. Specific polyclonal antibodies to pure sheep cathepsin L were raised in rabbits and chickens. The chicken egg yolk antibodies were of a much higher titre and were immunoinhibitory towards the enzyme, which the rabbit antibodies were not. Anti-peptide antibodies, raised in rabbits against a peptide sequence selected from the active site of human cathepsin L, were highly specific for cathepsin L and immunoinhibitory towards the enzyme. Together with the polyclonal anti-cathepsin L antibodies, they show promise for immunoinhibitory and immunocytochemical studies on the enzyme, and as potential anti-tumour drugs. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1990.

Jaagsiekte is a contagious cancer affecting the lungs of sheep. Although the etiological agent is Jaagsiekte retrovirus (JSRV), two other retroviruses viz South African maedi visna virus ( SA - OMVV) and a novel Bovine retrovirus (BRV) have been associated with or implicated in the jaagsiekte disease complex. JSRV was sufficiently purified from lung rinse material using a Freon extraction, Percoll density gradient centrifugation and chranatography on a Sephacryl column, its polypeptide composition was studied by gel electrophoresis and its morphology observed electron microscopically. Monoclonal antibodies were made against purified preparations of the virus. Two hybridomas were isolated that produced MAbs which appear to be tumour cell specific. A third hybridoma, called 4A1O, produces antibodies considered to be viral specific. These MAbs have been used in the development of JS specific immunoassays. A cross reaction between JSRV and a polyclonal serum against Mason Pfizer monkey virus (MPMV) was confirmed and used in a Western blot technique to identify, monitor and differentiate JSRV from other viruses. During the study of JSRV it became apparent that another retrovirus was often present in JS infected lungs. This virus, referred to as SA - OM1V I, is a novel South African isolate of maedi visna virus (MVV). As SA - OM1V I has physicochemical characteristics similar to JSRV, it was often found in purified JSRV preparations. Being a retrovirus it is also detected by the reverse transcriptase assay which was the only method used to assay and monitor for JSRV during the early stages of our work. Using a Westen blot technique and sera against MVV and MPMV it was possible to simultaneously detect and differentiate JSRV from SA - OMVV I. A method was also developed whereby the two viruses could be separated from each other during purification. The information gained and techniques developed whilst studyiing JSRV were also used to isolate and characterize BRV. This novel virus originated from bovine cells that had been co-cultivated with white blood cells from an ox suffering from malignant catarrhal fever. Three out of four sheep inoculated with BRV developed JS. It therefore had to be· ascertained whether this virus was related to JSRV or not. The comparative study revealed that BRV was biochemically and morphologically quite different fran JSRV. Interestingly, it was shown that serum against MPMV cross reacted with a 32 kd protein of BRV indicating a serological relationship between JSRV, MPMV and BRV. The possible role of BRV in the etiology of jaagsiekte remains to be elucidated. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1987.

No abstract available. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1996.

Cathepsin L, which has been implicated in many tissue degradative pathologies by virtue of its ability to degrade extracellular matrix components, was isolated by a novel, scaled-up protein purification method and purified to homogeneity in the single-chain form. In addition, the high molecular weight variant of cathepsin L covalently complexed with stefin B was isolated. Both cathepsin L and the complex were stable, in respect of their proteolytic activity, to the chaotropic agent urea, both showing enhanced activity in the presence of urea. Urea did not dissociate the complex. The suitability of cathepsin L for a purported extracellular role was addressed by investigating its pH optimum and pH stability. Cathepsins L and B are affected by ionic strength and so buffers of constant ionic strength (rather than constant molarity, and therefore varying ionic strength) were used in determining their pH optima and stability. Cathepsins L and B had apparent pH optima of pH 6.5 and 7.5, respectively, (measured with synthetic substrates) and, contrary to the previous belief, were substantially stable at physiological pH. In Hanks' balanced salt solution, a model of the extracellular fluid, they were shown to be active and stable, cathepsin L having a half-life of 179 s at pH 7.2 and 657 s at pH 6.8 (the peritumour pH). It was also shown that prior reductive activation of these enzymes increased their stability to extracellular conditions, supporting the hypothesis that the active site thiolate-imidazolium ion pair contributes to their stability. The nature of the bond between cathepsin L and stefin B in the covalent complex was examined, using CNBr cleavage, HPLC and amino acid sequencing. Stefin B was shown to be associated with residues 1-137 of cathepsin L via a reduction sensitive linkage which was deduced to be a thioester bond betwen Asp-71 of cathepsin L and Cys-3 of stefin B. Polyclonal antibodies to cathepsin L and stefin B-complexed cathepsin L were raised in rabbits and chickens, and characterised with respect to their suitability for immunocytochemical localisation of these forms of cathepsin L. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1998.

Bloodstream forms of African trypanosomes, which endocytose macromolecules exclusively through their flagellar pockets, contain an acid phosphatase (AcP) activity in this organelle. In the present thesis, AcP activity was demonstrated cytochemically in some intracellular vesicles and on the surface of Trypanosoma congolense as well as in the flagellar pocket. Unlike other trypanosomatids such as Leishmania spp. and Trichomonas spp., these trypanosomes, while viable, did not release this enzyme into the surrounding medium. In contrast to mammalian cells, the AcP in T. congolense was shown by cell fractionation to be a non-lysosomal enzyme. The enzyme was mostly recovered in the microsomal and cytosolic fractions which had 52.7% and 44.4% of the total activity, respectively. Further separation of the microsomal fraction showed an association of AcP activity with vesicles derived from the plasma membrane, Golgi apparatus and endoplasmic reticulum. After ammonium sulfate precipitation and chromatography on a succession of columns containing Sephacryl S-300, DEAE-cellulose and Sephadex G-75, two acid phosphatases (AcPi and ACP2) were produced from the cytosolic fraction. A membrane-bound acid phosphatase (ACP3) was isolated from the microsomal pellets extracted with Triton X-l 14 and subjected to the above chromatographic procedures. The molecular mass of AcP 1 was higher than 700 kDa. It had an isoelectric point of 4.7. AcP2 (pi 5.3) and AcP3 (pi 6.5) had molecular masses of 33 and 320 kDa, respectively. AcPi and ACP3 were strongly inhibited by vanadate while ACP2 was strongly inhibited by p-chloromercuribenzoate. None of the enzymes was inhibited by tartrate but all were inhibited by NaF. The Km values for each of the various substrates differed widely between the three AcPs indicating that the binding site of each enzyme was distinct. The best of all the substrates tested was para-nitrophenyl phosphate. On non-denaturing gels the enzymes exhibited very high molecular masses but on denaturing SDS-PAGE, two similar bands of activity, localised at 62 and 65 kDa, were observed in all three AcP preparations. Thus the three isolated enzymes may be derived from the same base 62 and 65 kDa units. Differences between enzymes may be derived from differential processing of the isoenzymes for different functions at different locations. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1997.