Satellite cells are quiescent skeletal muscle specific stem cells that are activated in response to injury to aid in muscle repair and regeneration. The interaction of hepatocyte growth factor (HGF) with these cells is crucial for their activation. However, to date, research on the effect of HGF on skeletal muscle satellite cells has yielded conflicting data. Clarity is therefore required as to its effect on downstream myogenic processes. Furthermore, mouse and rat cell lines and primary culture have been widely used for in vitro studies to investigate the effect of HGF on skeletal muscle physiology and disease; very few studies have been carried out in primary cultured human skeletal myoblasts. As a result, we aimed to investigate and compare the effect of HGF (2, 10 and 50 ng/ml) on mouse C2C12 myoblast versus primary culture human skeletal myoblast (HSkM) proliferation, migration and differentiation. Proliferation was assessed via both cell counts and crystal violet assay, while migration was investigated using the scratch assay. Differentiation was determined via analysis of expression patterns of transcription factors implicated in myogenic commitment (i.e. Pax7, MyoD) as well expression of the structural protein Myosin Heavy Chain (MyHC). We demonstrate a dose-dependent effect of HGF on myoblast proliferation whereby an increase in proliferation was detected in response to 2 ng/ml HGF, whilst 10 ng/ml HGF resulted in a reduction in proliferation capacity of both C2C12 and HSkM myoblasts. Interestingly, the reduction in proliferation in response to 10 ng/ml HGF was accompanied by a down-regulation in Pax7 expression during differentiation of both mouse and human myoblasts. HGF also affected myoblast migration and differentiation in a dose-dependent manner that was inversely proportional to proliferation. HGF (10 ng/ml) stimulated an increase in myogenic commitment and terminal differentiation of C2C12 and HSkM myoblasts as reflected by the increased percentage MyoD positive cells, improved fusion and greater MyHC expression. C2C12 myoblast migration was also stimulated at this HGF concentration, but reduced in response to the lower HGF (2 ng/ml) dose. The decrease in proliferation following incubation with 10 ng/ml HGF, allows cells to exit proliferation into either a mode of migration or differentiation. Our data confirms the importance of HGF during myogenesis and highlights the sensitivity of satellite cells to changing HGF concentration. This has implications in the regulation of skeletal muscle wound repair. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

Human malaria, caused by four species of the intracellular protozoan parasite Plasmodium, is a major health and economic burden in the tropics where the disease is endemic. The biotindependent enzyme acetyl-CoA carboxylase catalyses the commitment step in de novo fatty acid biosynthesis in several organisms. Acetyl-CoA carboxylase is a target for anti-parasitic drug development due to its relevance in membrane biogenesis. This study describes the detection of acetyl-CoA carboxylase and the partial characterisation of the biotinoyl domain of the enzyme of the mouse malaria parasite, Plasmodium yoelii. Acetyl-CoA carboxylase mRNA was detected by RT-PCR performed on total RNA isolated from P. yoelii 17XL-infected mouse erythrocytes using primers designed from PY01695 ORF of the Plasmodb-published MALPY00458 gene of P. yoelii 17XNL. The RT-PCR was confirmed by sequencing and comparative analysis of the sequenced RT-PCR cDNA products. Northern blot analysis performed on total RNA using probes designed from a 1 kb region of the gene showed that the transcript was greater than the predicted 8.7 kb ORF. An immunogenic peptide corresponding to the P. yoelii theoretical acetyl-CoA carboxylase sequence was selected using epitope prediction and multiple sequence alignment algorithms. The immunogenic peptide was coupled to rabbit albumin carrier for immunisation in chickens and the affinity purified antibody titre was approximately 25 mg. The anti-peptide antibodies detected a 330 kD protein in P. yoelii lysate blot, which corresponds to the predicted size of the enzyme. The enzyme was also detected in situ by immunofluorescence microscopy using the anti-peptide antibodies. A 1 kb region of the P. yoelii acetyl-CoA carboxylase gene containing the biotinoyl domain was cloned and expressed in E. coli as 66 kD GST-tag and 45 kD His-tag protein. Both recombinant biotinoyl proteins were shown to contain bound biotin using peroxidaseconjugated avidin-biotin detection system. This suggested in vivo biotinylation of the recombinant P. yoelii biotinoyl protein, possibly by the E. coli biotin protein ligase. The Proscan™ and the NetPhos 2.0™ algorithms were used to predict protein kinase phosphorylation sites on the biotin carboxylase and the carboxyltransferase domains of the enzyme. The three-dimensional structure of the biotinoyl and the biotin carboxylase domains were predicted using the SWISS-MODEL™ homology modelling algorithm. Homology modelling revealed a similarity in the 3D conformation of the predicted P. yoelii biotinoyl domain and the E. coli biotinoyl protein with negligible root mean square deviation. The model also revealed the possibility of inhibiting P. yoelii and falciparum acetyl-CoA carboxylases with soraphen A based on the similarity in conformation with S. cerevisiae biotin carboxylase and the stereochemical properties of the residues predicted to interact with soraphen A. This study demonstrated that malaria parasite expresses acetyl-CoA carboxylase and, combined with data on other enzymes involved in fatty acid metabolism suggests that the parasite synthesizes fatty acids de novo. This enzyme could be a target for rational drug design. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes numerous human pathologies. Biochemical evidence suggests that soluble misfolded proteins are retrotranslocated out of the ER, via the endoplasmic reticulum associated degradation (ERAD) pathway, for proteosome-mediated cytoplasmic degradation. Excess, misfolded- or insoluble proteins, are suggested to cause induction of “overflow” degradation pathways. For soluble proteins, overflow to vacuole-mediated destruction is suggested to occur via two Golgi-to-vacuole (Gvt) routes, the alkaline phosphatase (ALP), direct route, or, a carboxypeptidase Y- (CPY-), prevacuolar compartmentvacuole, indirect route, though only the CPY route is thought to degrade soluble proteins. Insoluble aggregate-containing structures are suggested to be degraded by engulfment by membranes of unknown origin and trafficking to the vacuole for destruction, via an autophagic pathway. To confirm biochemical evidence, wild-type (BY4742), autophagosome- (W303/ATG14), CPY- and autophagy pathway- (W303/VPS30), and proteosome (WCG/2) mutants of S. cerevisiae yeasts were transformed with a high expression pYES plasmid and mutant (Z) human alpha-1- proteinase inhibitor (A1PiZ), giving rise to the derivatives cells BY4742/Z, W303/ATG14/Z, W303/VPS30/Z and WCG/2/Z, respectively. Electron microscopy using gold labeling for A1PiZ, markers for the ER, the ERAD ER channel protein, Sec61, or the chaperone, binding protein (BiP), ALP for the ALP pathway, and CPY for the CPY pathway, was used. Overexpression of A1PiZ seems to result in targeting to the vacuole via a prevacuolar, CPY-like compartment (PVC, 200-500 nm), though CPY and A1PiZ appears not to colocalise, unconvincingly confirming collaborative biochemical data. Large amounts of A1PiZ localise in the cytosol, possibly indicating a largely proteasome-mediated degradation. ER-resident A1PiZ targeting to the vacuole seems also to occur by the budding of the ER and peripheral plasma membrane or ER membrane only. This occurs in all cells, but especially in ATG14 gene (ΔATG14) mutants, possibly indicating autophagosome-mediated degradation independence, in the latter mutants. The ATG14 mutation gave rise to crescent-shaped, initiating membranelike (IM-like) structures of approximately Cvt vesicle-diameter, possibly indicating that ΔATG14 blocks autophagosome- (500-1000 nm) and Cvt vesicle (100-200 nm) enclosure, after core IM formation. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

During the erythrocytic stage, the malaria parasite digests host cell haemoglobin into amino acids. Toxic haeme is released and is incorporated into an insoluble non-toxic crystal called haemozoin. Haemozoin is released into the blood stream along with the merozoites when the erythrocyte bursts and is phagocytosed by circulating monocytes and macrophages resident in tissues. Phagocytosed haemozoin impairs many functions of the monocytes, including antigen presentation and adhesion to T cells, differentiation and maturation to dendritic cells, erythropoiesis and thrombopoiesis, but stimulates the release of proinflammatory cytokines and activation of metalloproteinase 9 expression. In response to interferon-g secretion by T-helper cells subtype 1, monocytes secrete neopterin, which is used as a marker of a cell mediated immune response. Neopterin is an oxidation product of 7,8-dihydroneopterin, produced by the dephosphorylation of 7,8- dihydroneopterin triphosphate which results from the conversion of guanosine triphosphate that is catalysed by GTP-cyclohydrolase 1. Elevated plasma and urine neopterin levels have been detected in malaria infections and are associated with severe anaemia, respiratory distress, peak temperatures as well as fever- and parasite-clearance times. It has also been reported that monocytic U937 cells treated with P. falciparum-infected red blood cell lysate secrete elevated levels of neopterin. Antimalarial drugs are known to modulate the functions of monocytes, including inhibition of cytokine release, changes in phospholipid metabolism, decrease in expression of cytoadherance receptors as well as TNF receptors and MHC Class I and II molecules, changes in the production of reactive oxygen and nitrogen intermediates, and decreased phagocytosis. However, the effects of antimalarial drugs on haemozoin phagocytosis and GTP-cyclohydrolase 1 mRNA expression by monocytes are unknown. This study aimed to determine the effects of seven antimalarial drugs, amodiaquine, artemisinin, chloroquine, doxycycline, primaquine, pyrimethamine and quinine, on the phagocytosis of latex beads and b-haematin, a synthetic equivalent of haemozoin. Phagocytosis of b-haematin and latex beads by two monocytic cell lines, J774A.1 and U937, as well as peripheral blood mononuclear cells were monitored by enumeration and a novel spectrophotometric method. Patterns of inhibition and activation differed with each cell type investigated, due to the differing stages of cell differentiation. In general, artemisinin, primaquine, pyrimethamine and quinine activated the phagocytosis of b-haematin, whereas amodiaquine and chloroquine inhibited b-haematin phagocytosis. Doxycycline had different effects on each cell type investigated. Artemisinin, chloroquine, primaquine and quinine inhibited latex bead phagocytosis. The remaining drugs had minimal effects on latex bead phagocytosis. Thus, the effects of antimalarial drugs on monocyte phagocytosis appear to be dependent on the substance being phagocytosed. The effects of antimalarial drugs, b-haematin, latex beads, non-infected- and P. falciparuminfected cell lysates on interferon-g-induced neopterin secretion by U937 cells was monitored by GTP-cyclohydrolase 1 mRNA expression using quantitative PCR. Artemisinin, primaquine and quinine down-regulated the interferon-g-induced expression of GTPcyclohydrolase 1 mRNA, but by no greater than 1.7-fold. b-haematin up-regulated mRNA expression by 1.2-fold whereas P. falciparum-infected red blood cell lysate down-regulated the mRNA expression of GTP-cyclohydrolase 1 by 1.6-fold. Quinine and artemisinin, currently used to treat malaria, increased b-haematin phagocytosis suggesting that quinine and artemisinin might promote increased phagocytosis of infected red blood cells and enhance clearance of the parasite from circulation. Increased b- haematin phagocytosis also reduces ICAM-1 expression on the monocyte surface, thereby leading to reduced cytoadherance and sequestration, thus increasing the number of circulating monocytes to phagocytose infected red blood cells. Down regulation of GTPcyclohydrolase 1 mRNA expression by quinine and artemisinin suggested that the drugs reduce the responsiveness of the monocyte to interferon-g. Thus, quinine and artemisinin might also decrease the production of interferon-g-induced proinflammatory cytokines by monocytes, and potentially play a role in maintaining the balance between the pro- and antiinflammatory cytokines that determines the progression from acute to severe malaria. Therefore, in addition to the drug’s ability to kill the malaria parasite, the immunomodulatory effects of the antimalarial drugs may play a role in controlling the pathophysiology associated with the malaria infection. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

Post-harvest cane deterioration in the South African sugar industry results in significant revenue loss that is estimated to be in the region of ZAR 60 million per annum. Despite these large losses, precise biological data relating to the process of cane deterioration under South African conditions is limited. Severity of deterioration is influenced by a number of factors, including the length of the harvest-to-crush delay (HTCD), ambient temperature and harvesting practices. For example, burning of cane prior to harvest may result in rind splitting, which provides entry for microbes, particularly Leuconostoc mesenteroides that may exacerbate deterioration. The effect of these factors on deterioration was examined by quantifying the biochemical and microbiological changes that occur in sugarcane stalks after harvest, with the influence of length of HTCD, degree of L. mesenteroides infection and ambient temperature receiving attention. The primary novelty of the work resides in the analysis of deterioration under tightly regulated temperatures, which were designed to reflect diurnal variations typically experienced during summer and winter in the South African sugar belt. In addition, inoculation of mature internodes with a consistent titre of L. mesenteroides was used as a means to mimic a consistent level of infection of harvested stalks by the bacterium. Metabolites selected for analysis were those both native to the stalk and produced as by-products of microbial metabolism, viz. sucrose, glucose, fructose, ethanol, lactic acid, dextran and mannitol. Simulated HTCDs under summer temperatures resulted in increasing glucose and fructose levels with time, which contrasted to the approximately constant levels of these hexose sugars under winter conditions. Commonly referred to as ‘purity’ in an industrial context, precise determination of the concentration of these hexoses in cane consignments could potentially indicate the extent of deterioration. Despite the detection of a basal concentration of lactic acid in unspoiled cane, the observed increase in concentration of this organic acid over the simulated summer HTCD suggests that this metabolite could also potentially serve as an indicator for postharvest deterioration. In contrast, the investigation indicated that ethanol was an unsuitable biochemical marker for deterioration of L. mesenteroides infected cane. An inability to detect dextran and mannitol in the samples, combined with consistent sucrose levels and variable mill room data, suggest that extreme proliferation of L. mesenteroides is facilitated primarily by in-field practices, particularly the manner in which cane is prepared prior to harvest and transport to the mill. Bacterial proliferation and infection by L. mesenteroides of inoculated stalks were monitored by standard selective culturing techniques. Despite the limited detection of L. mesenteroides-associated metabolites, culture-based analyses revealed that the bacterium was the dominant bacterial species within the samples. A number of other bacterial species were isolated and identified, however the extent to which the total number of microorganisms proliferated was limited to a maximum of 1 x 105 colony forming units per gram of fresh tissue. In conjunction with these analyses, a molecular approach known as Polymerase Chain Reaction-Mediated Denaturing Gradient Gel Electrophoresis (PCR-DGGE) was undertaken to investigate the bacterial diversity patterns associated with deteriorating sugarcane stalks throughout the delay period. In contrast to the results obtained by means of the culture-based assays, PCR-DGGE revealed that L. mesenteroides was not the dominant bacterial population, and showed that the level of bacterial diversity was relatively consistent across the differing treatments and with time. The use of complimentary culture-dependent and cultureindependent analyses thus permitted the detection of this discrepancy and indicated the utility of PCR-DGGE in the determination of bacterial community structure of postharvest sugarcane tissue. The biology of post-harvest deterioration of green sugarcane stalks is highly complex, even under rigorously controlled temperature and infection regimens. The results of this study emphasize the important effects that harvest method and environmental conditions have on post-harvest sugarcane deterioration. Towards the formulation of industry-relevant recommendations for combating post-harvest deterioration, future work will strive to mimic the effects that harsh harvesting and transport practices have on the severity of the problem. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

The protozoan parasite Trypanosoma congolense is one of the aetiological agents of African animal trypanosomiasis that is transmitted by the tsetse fly. The parasite causes nagana in animals and affects livestock throughout sub-Saharan Africa. The toxicity of available drugs and the emergence of drug resistant parasites have affected the treatment of trypanosomiasis. Control of the disease has also been difficult due to ineffective vector control and the potential of trypanosomes to express hundreds of antigenetically distinct proteins on their surface. Vaccination against trypanosomiasis has been thought to be a possible control method. Since a vaccine based on variable surface proteins of the parasite is unlikely, research has been directed towards the identification of invariant pathogenic factors of the parasite as potential targets for therapy. Congopain, the major cysteine protease of T. congolense has been implicated in the pathology of the disease. Antibodies against congopain are known to contribute to the mechanisms of natural resistance to trypanosomiasis known as trypanotolerance by neutralising the pathogenic effects of the enzyme. Oligopeptidase B (OpdB), a trypanosomal serine protease has also been associated as a pathogenic factor of the disease. It is released into the host’s circulation by dead or dying parasites and retains its catalytic activity since it is insensitive to host serum inhibitors. In the present study, the catalytic domain of congopain (C2) and the use of alpha-2-macroglobulin (α2M) as an adjuvant were investigated for their potential use in an anti-disease vaccine. α2-Macroglobulin has been used to varying degrees to target different antigens to cells of the immune system and enhance their immunogenicity. A previous study showed that antibodies raised in rabbits against C2 complexed to α2M gave a higher percentage inhibition than antibodies made using C2 mixed with Freund’s adjuvant. In the present study, goats were immunised with C2 complexed with α2M to confirm the enhanced immunogenicity of C2 and the production of anti-C2 antibodies with superior inhibitory properties. Following immunisation, goats were challenged with T. congolense (strain IL 1180) and showed sustained antibody production during the two month infection period. Goat antibodies made using C2 in complex with α2M inhibited the hydrolysis of hide powder azure by C2 by 96%. Maximum inhibition of the hydrolysis of azocasein was observed to be 63% and hydrolysis of Z-Phe-Arg-AMC by C2 was inhibited by 73%. In order to determine the vaccine potential of OpdB, protein was recombinantly expressed as a glutathione-S-transferase fusion protein in the pGEX expression system and purified by glutathione agarose affinity chromatography and molecular exclusion chromatography. Since a small yield of protein necessitated several rounds of expression and extensive purification, OpdB was subsequently expressed as a His-tagged fusion protein in the pET bacterial expression system. Recombinant protein was easily purified using nickel chelate affinity chromatography. Purified OpdB was used with alum for the immmunisation of mice to produce antibodies capable of inhibiting enzyme activity. Following immunisation, mice were challenged with T. congolense (strain IL 1180) and also showed sustained antibody production following two months infection. Since all mice died, the administration of OpdB conferred no protection; however, anti-OpdB mouse antibodies inhibited 86% of OpdB activity against the substrate Z-Arg-Arg-AMC. In addition immunised mice were observed to survive 40% longer than control mice as they had previously been immunised with OpdB and were able to mount a rapid immune response against this pathogenic factor during infection. In general it could be concluded that immunisation of goats with C2 in complex with α2M produced antibodies with superior inhibitory properties. The immunisation of mice with OpdB and alum also produced inhibitory antibodies and previous administration of OpdB enabled mice to mount a rapid immune response against OpdB during infection. Antibody mediated enzyme inhibition demonstrates the potential use of C2 and OpdB as vaccines that may contribute to the development of an effective anti-disease vaccine. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

Tuberculosis (TB) is the fifth largest cause of death in South Africa, with one in ten cases being resistant to treatment due to the development of multidrug-resistance and extensively drug-resistance in the agent responsible for this disease, Mycobacterium tuberculosis. This pathogen has developed mechanisms to evade killing by immune cells such as macrophages. Mycobacterium smegmatis, a non-pathogen, that does not evade killing by the macrophage, is often used to gain a better insight into the bacteriocidal pathways used to kill mycobacteria, and those potentially blocked by M.tuberculosis. In such studies nitric oxide and “lysosomal” proteases have emerged as major bacteriocidal pathways. Studies on the role of aspartic protease, cathepsin D, in killing green fluorescent protein- (GFP-) tagged-M.smegmatis in J774 macrophages required antibodies that would not cross-react with mycobacterial antigens. These were raised in chickens, using alum and saponin as adjuvants, and porcine and human cathepsin D. Using such antibodies, quantitative colocalization analysis using ImageJ and the JACoP colocalization plugins showed a greater degree of colocalization between cathepsin D and LysoTracker Red DND-99 in M.smegmatis-infected J774 macrophages than in uninfected cells. This indicates the possible presence of active, bacteriocidal cathepsin D in acidic, and hence matured phagosomes. A higher colocalization between cathepsin D and LAMP-1 and cathepsin D and LAMP-2 in uninfected cells possibly indicates the recycling of these two markers from vesicles not containing killed bacteria. Propidium iodide (PI) labelling and loss of GFP fluorescence appeared reliable indicators of M.smegmatis death or viability, respectively, as myobacteria that took up PI also lost green fluorescence, while M.smegmatis that exhibited green fluorescence (viable) were not observed to take up propidium iodide (dead). Faint colocalization between cathepsin D, LAMP-1 and -2 with dead, and to a lesser extent with live M.smegmatis occurred. Besides intensity correlation values other colocalization programs indicate the absence of colocalization between these markers and dead M.smegmatis, but, together with in vitro killing experiments (cathepsin D, 0.0098 units/ml resulting in 59% killing in 4 h) these appear to indicate a possible role of cathepsin D in killing of M.smegmatis. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

Thesis (MSc)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Potato Virus Y (PVY) and Potato Leafroll Virus (PLRV) are two of the most destructive potato viruses capable of drastically diminishing crop yields by up to 80%. The presence of these viruses in planting material namely seed potato stocks are routinely diagnosed by enzyme-linked immunosorbent assay (ELISA) kits. The kits currently used by Potatoes South Africa are obtained from Europe. These kits have produced false positive and false negative results in the past. Potatoes South Africa required an ELISA that was reliable, cheap and specific for the detection of South African strains of the two respective viruses. In this study the viral coat protein genes were amplified by RT-PCR from a South African source of infected plant material. The PVY and PLRV coat protein genes were subsequently cloned into pGEM-T Easy vector and sequenced. The sequences of the two viruses were aligned and compared to corresponding viral coat protein gene sequences obtained from Genbank. Subsequently the two amplified and cloned coat protein genes of PVY and PLRV were sub-cloned into an expression system (pET-14b) to induce and express the respective recombinant viral coat proteins. The induction of the cloned coat protein genes yielded successful production of the recombinant PVY coat protein but the induction and expression of the recombinant PLRV coat protein was unsuccessful. The isolated recombinant PVY CP was then used to immunize a rabbit to produce highly specific anti-PVY CP immunoglobulins. The antiserum obtained from the rabbit was used to develop an ELISA to detect the presence of PVY in seed potato stocks in South Africa. The ELISA kit was subsequently used in preliminary trials to determine if the kit could detect PVY infected plant material. The initial results of the ELISA trials using PVY infected material obtained from Potatoes South Africa yielded positive results. / AFRIKAANSE OPSOMMING: Aartappel Virus Y (PVY) en Aartappel Rolblad Virus (PLRV) is twee van die mees vernietigende aartappel virusse wat ‘n oes tot 80% kan verlaag. Virus infeksie van plant materiaal tewete aartappelmoere word deur “enzyme-linked immunosorbent assay” (ELISA) toetsstelle bevestig. Die toetsstelle wat op die oomblik gebruik word deur Aartappels Suid- Afrika word in Europa vervaardig. Hierdie toetsstelle het vals positiewe en vals negatiewe resultate in die verlede gegee. Aartappels Suid-Afrika benodig toetsstelle wat betroubaar, goedkoop en spesifiek vir Suid-Afrikaanse virus stamme is. In hierdie studie is besmette plantmateriaal vanuit Suid-Afrika gebruik vir die amplifisering van virale mantel proteïen gene met behulp van RT-PCR. Die PVY en PLRV mantel proteïen gene was daarna in die pGEM-T Easy vektor gekloneer en nukleotied volgordes is bepaal. Die nukleotied volgordes is met ander PVY en PLRV gene vanaf Genbank vergelyk. Die twee ge-amplifiseerde en gekloneerde mantel proteïen gene van PVY en PLRV is uitgesny en gekloneer in ‘n ekspressie sisteem (pET-14b) om die mantel proteïen te produseer. Induksie van die gekloneerde mantel proteïen gene het gelei tot die suksesvolle produksie van ‘n PVY mantel proteïen, maar produksie van die PLRV mantel proteïen was onsuksesvol. Die geïsoleerde PVY mantel proteïen is vervolgens gebruik vir die immunisering van ‘n konyn vir die produksie van konyn anti-PVY antiliggame. Die antiserum verkry vanaf die konyn is gebruik vir die ontwikkeling van ‘n ELISA vir die identifisering van PVY infeksies in aartappelmoere. Voorlopige proewe is deurgevoer om te bepaal of hierdie ELISA PVY infeksies in plantmateriaal sou kon opspoor. Aanvanklike resultate toon dat die ELISA suksesvol PVY infeksies in plantmateriaal verkry vanaf Aartappels Suid-Afrika kan opspoor.

Thesis (MSc (Biochemistry))--University of Stellenbosch, 2009. / A Python package called PySUNDIALS has been developed which provides an interface to the suite of nonlinear di erential/algebraic equation solvers (SUNDIALS) using ctypes as a foreign function interface (FFI). SUNDIALS is a C implementation of a set of modern algorithms for integrating and solving various forms of the initial value problem (IVP). Additionally, arbitrary root nding capabilities, time dependent sensitivity analysis, and the solution of di erential and algebraic systems are available in the various modules provided by SUNDIALS. A signi cant focus of the project was to ensure the python package conforms to Python language standards and syntactic expectations. Multiple examples of the SUNDIALS modules (CVODE, CVODES, IDA and KINSOL) are presented comparing PySUNDIALS to C SUNDIALS (for veri cation of correctness), and comparing PySUNDIALS to various other comparable software packages. The examples presented also provide benchmark comparisons for speed, and code length. Speci c uses of the features of the SUNDIALS package are illustrated, including the modelling of discontinuous events using root nding, time dependent sensitivity analysis of oscillatory systems, and the modelling of equilibrium blocks using a complete set of implicit di erential and algebraic equations. PySUNDIALS is available as open source software for download. It is being integrated into the systems biology software PySCeS as an optional solver set, on an ongoing basis. A brief discussion of potential methods of optimization and the continuation of the project to wrap the parallel processing modules of SUNDIALS is presented.

Thesis (MSc)--University of Stellenbosch, 2008. / ENGLISH ABSTRACT: Potato virus Y (PVY) is responsible for considerable yield losses in the South African potato industry. The incidence of this virus has greatly increased over the past few years. Even more worrying is the variation of symptoms observed during PVY infection and the recent appearance of the more virulent PVYNTN strain in local fields. This project aimed to investigate the possible genetic variation within the viral genome and to establish the origin of strains. The project also aimed to establish a dependable, area specific enzyme-linked immunosorbent assay (ELISA) to replace the currently used ELISAs. Currently seed potato certification is done using ELISA kits imported from Europe. These kits were developed for the detection of overseas variants of PVY and the use thereof in South Africa has in the past lead to false negatives. Finally, this project set out to develop, optimize and establish a sensitive and reliable real-time reverse transcriptase polymerase chain reaction (qRT-PCR) detection protocol for PVY. In the first part of the study the coat protein (CP) gene of PVY isolates from plant material obtained from various parts of South Africa was amplified using RT-PCR. The resulting cDNA was then sequenced directly or cloned into a vector and then sequenced. The resulting sequences were aligned in a data matrix with international reference sequences, analyzed and grouped according to strain. Examination of the CP gene within this matrix as well as phylogenetic analysis revealed six main groups of PVY. These six groups included the traditional PVYN and PVYO groups and a recombinant group. Furthermore it also revealed variants of PVYN and PVYO. These mutants and recombinants pose a threat as they may lead to South African strains of PVY expressing coat proteins which vary from those found overseas. This may render the currently used European ELISA method of detection less effective and subsequently result in an increase in viral prevalence. This reinforced the need for a detection method based on local viral strains. Phylogenetic and Simplot analysis also confirmed that a recombinant strain between PVYN and PVYO had evolved and that PVYNTN was such a recombinant. The second part of the study aimed to develop and establish detection methods based on local variants of PVY. This included the development of ELISA and qRT-PCR detection methods of PVY. Previously amplified cDNA of the PVY CP gene was cloned into an expression vector and successfully expressed. Antibodies produced against the recombinant protein, when used in ELISA, however, failed to achieve the required levels of sensitivity. This prompted the development of qRT-PCR detection methods for PVY. Primer combinations for PVY were designed using the previously established CP gene data matrix. A reliable and sensitive SYBR® Green I based qRT-PCR assay was developed for the detection of PVY. The assay effectively detected all known South African variants of PVY. Furthermore, a Taqman® assay was developed for the detection of all variants of PVY. The Taqman® assay was 10 fold less sensitive and does not allow for amplicon verification through melting curve analysis, but it does add more specificity due to the addition of the probe. Although these qRT-PCR detection methods are still too expensive to replace the routine diagnostics done with ELISA, they do offer the opportunity to screen valuable mother material and confirm borderline cases in seed certification. / AFRIKAANSE OPSOMMING: Aartappel virus Y (PVY) is verantwoordelik vir aansienlike opbrengsverliese in die Suid-Afrikaanse aartappelindustrie. Die insidensie van infeksie deur die virus het drasties toegeneem oor die afgelope jare. Wat egter meer kommerwekkend is, is die groter variasie in simptome van PVY infeksie en die onlangse voorkoms ‘n meer virulente ras, PVYNTN. Hierdie projek poog om moontlike genetiese variasie van PVY te ondersoek en om die oorsprong van rasse op te spoor. Die projek het ook gepoog ook om ‘n bruikbare, betroubare en area spesifieke “enzyme-linked immunosorbent assay” (ELISA) toets te ontwikkel om die huidige ingevoerde ELISA te vervang. Hierdie toetse is ontwikkel om oorsese variante van PVY op te spoor en die gebruik daarvan het in die verlede gelei tot vals negatiewes. Verder is daar ook ondersoek ingestel na die ontwikkeling van ‘n sensitiewe en betroubare “real-time reverse transcriptase polymerase chain reaction” (qRT-PCR) protokol vir die opsporing van PVY. In die eerste deel van die studie is die mantelproteïen geen van PVY isolate vanuit plant materiaal geamplifiseer deur die gebruik van RT-PCR. Hierdie materiaal is vanaf verskeie streke in Suid-Afrika ontvang. ‘n Volgordebepalingsreaksie is uitgevoer op gekloneerde of ongekloneerde cDNA verkry uit die RT-PCR. DNA volgordes is in ‘n data matriks geplaas en vergelyk met internationale volgordes om die plaaslike isolate te analiseer en te groepeer. Deur vergelyking en filogenetiese ontleding kon ses hoofgroepe van PVY geïdentifiseer word, wat tradisionele PVYN en PVYO, sowel as ‘n rekombinante ras en variante binne die tradisionele PVYN en PVYO groepe ingesluit het. Rekombinante en mutante kan veroorsaak dat Suid-Afrikanse rasse van PVY mantelproteïene uitdruk wat afwyk van die oorsese rasse wat tot gevolg mag hê dat die ELISAs van oorsee minder effektief kan wees en kan lei tot verhoogde virus voorkoms. Die realiteit en gevaar versterk die gedagte dat ‘n deteksie metode gebaseer op plaaslike virusse absoluut krities is. Filogenetiese sowel as Simplot analise het bevestig dat ’n mutante ras tussen PVYN en PVYO ontstaan het en dat PVYNTN ’n rekombinante ras is. Die tweede deel van die studie was daarop gemik om deteksie metodes te ontwikkel wat gebaseer was op plaaslike variante van PVY. Dit sluit die ontwikkeling van ELISA sowel as qRT-PCR deteksie van PVY in. Voorheen geamplifiseerde cDNA is in ‘n ekspressievektor gekloneer en suksesvol uitgedruk. Teenliggaampies teen die rekombinante proteïen, indien in ELISA aangewend, kon egter nie die nodige sensitiwiteit oplewer nie. Dit het aanleiding gegee tot ontwikkeling van qRT-PCR deteksie metodes. Inleier kombinasies vir PVY was ontwikkel deur die gebruik van die bestaande mantelproteïen geen data matrikse. ‘n Betroubare en sensitiewe SYBR® Green I qRT-PCR deteksie protokol was ontwikkel vir die effektiewe deteksie van alle bekende Suid-Afrikanse rasse van PVY. Verder is ‘n sogenaamde “Taqman®” protokol ook ontwikkel vir deteksie van alle rasse. Die “Taqman®” protokol was 10 voudiglik minder gevoelig and laat nie bevestiging deur smeltkurwe analise toe nie, maar verleen meer spesifisiteit deur die toevoeging van die “Taqman® probe”. Hierdie qRT-PCR deteksie metodes is tans te duur om as roetine diagnostiese toetse te gebruik en kan dus nie ELISA vervang nie, maar hulle bied wel die geleentheid om waardevolle moeder materiaal te toets en grensgevalle in aartappelsaad sertifisering te bevestig.