THIS THESIS IS IN THE EXAMINATION PROCESS Copyright / Thesis (DPhil)--University of Pretoria, 2012. / Philosophy / unrestricted

THIS THESIS IS IN THE EXAMINATION PROCESS Copyright / Thesis (PhD)--University of Pretoria, 2012. / Psychology / unrestricted

Foot-and-mouth disease virus (FMDV), which is a member of the Aphthovirus genus of Picornaviridae, is an economically important animal virus that infects cloven-hoofed animals. At least two families of cell-surface receptors have been identified that mediate binding of FMDV to susceptible cells, i.e. integrins and heparan sulfate proteoglycans (HSPG). Whereas several tissue culture-adapted FMD viruses use heparan sulfate proteoglycans for internalization, field isolates of FMDV use integrins as receptors. A conserved amino acid sequence Arg-Gly-Asp (RGD), located in the surface-exposed flexible ƒÒG-ƒÒH loop of the viral outer capsid protein 1D, participates in binding FMDV to integrin receptors on the surface of susceptible cells. Sequence analyses of the 1D-encoding genome region of a SAT1 type FMDV field isolate, NAM/307/98, indicated the presence of a second RGD sequence upstream of the conserved G-H loop RGD sequence. Since FMDV is capable of binding to RGD-binding integrin receptors, the aim of this study was to investigate whether the second RGD motif in capsid protein 1D of NAM/307/98 may function as a ligand for receptor-binding in baby hamster kidney (BHK) cells. Towards this end, a cDNA copy of the genomic region encoding the external capsid proteins (1B-1D) of NAM/307/98 was cloned into pSAT2, a genome-length cDNA clone derived from the SAT2 strain ZIM/7/83. Transfection of BHK-21 cells with in vitro-transcribed RNA derived from the chimeric pNAM/SAT2 clone resulted in the recovery of infectious chimeric virus particles. The availability of such an infectious chimeric cDNA clone greatly facilitated the introduction of specific, targeted mutations in the 1D capsid-encoding region of NAM/307/98 in order to investigate the functional role of the second RGD sequence. Using the chimeric SAT1/SAT2 cDNA clone as template, the RGD codons in the G-H loop of NAM/307/98 were replaced with codons specifying a KGE sequence by a polymerase chain reaction (PCR)-based method of site-directed mutagenesis. The mutated DNA fragment was introduced into the pSAT2 infectious cDNA clone, transcribed in vitro, and the resulting RNA transfected into BHK-21 cells. The transfected cells were analyzed for cytopathic effect (CPE). In contrast to cells transfected with non-mutated RNA transcripts, from which infectious virus could be recovered, cells transfected with the mutant RNA transcripts showed cell lysis, but no CPE could be observed upon subsequent passaging of the resultant viruses on BHK cells. Notably, subsequent replacement of the KGE sequence with an RGD sequence in the mutant clones led to recovery of infectious viruses. Furthermore, RNA replication could be demonstrated with the mutant and non-mutant chimeric viruses, suggesting that virus particles were indeed present in the tissue culture supernatants of all transfected cells. Based on the results obtained during the course of this investigation, it was therefore concluded that the second RGD motif, situated upstream of the conserved RGD motif in the G-H loop of capsid protein 1D of NAM/307/98, does not function as a ligand for receptor-binding in BHK-21 cells. / Dissertation (MSc (Microbiology))--University of Pretoria, 2007. / Microbiology and Plant Pathology / unrestricted

Banana is an important staple food crop however, production of the world’s most widely planted variety, the Cavendish banana, is threatened by a devastating fungal disease, called Fusarium wilt. Fusarium wilt of Cavendish bananas in the tropics is caused by Fusarium oxysporum f. sp. cubense (Foc) ‘tropical’ race 4 (TR4), while a variant of the fungus causing the disease in the subtropics is called Foc ‘subtropical’ race 4 (STR4). The incidence of Fusarium wilt in the subtropics is usually aggravated after winter, which suggests that the plant is predisposed to Foc STR4 during cold temperatures. The objective of this study was to investigate the molecular processes, in both Foc and banana, which contribute to Fusarium wilt development under cold stress. cDNA-AFLP expression profiling was used to elucidate the transcriptome of Foc STR4, Foc TR4 and non-pathogenic F. oxysporum isolates on minimal medium (without carbon source). This resulted in the identification of 229 unique gene fragments which included transcript derived fragments (TDFs) encoding for chitinase class V (chsV), GTPase activating protein, Major Facilitator Superfamily (MFS) multidrug transporter and serine/threonine protein kinase (ste12) genes. We speculate that those genes play a role in escaping host defence responses, and result in cell wall degradation. Pathogenicity-related genes identified in other formae speciales of F. oxysporum, such as the sucrose non-fermenting, F-box protein required for pathogenicity genes (frp1) and cyp55, were significantly up-regulated in Foc STR4 and Foc TR4, but not in F. oxysporum isolates non-pathogenic to banana. We suggest that these genes are important for the Fusarium wilt pathogen to enter the host xylem tissue, as they regulate the abundance of cell wall degrading enzymes. The increase in expression of cyp55 in pathogenic F. oxysporum may give the pathogen the ability to regulate the nitrogen response pathway, which is essential for pathogenicity. This study provided the first identification of genes in Foc that potentially contribute to pathogenicity in banana. Cavendish banana plants subjected to cold temperatures and inoculated with Foc resulted in a significant increase in disease severity. Visual symptoms, however, only appeared in inoculated plants after they were transferred to 28oC. Transcriptome analysis showed that several general defence mechanisms are activated in Cavendish bananas infected with Foc. An important finding was that expression of defencerelated genes was delayed in cold-treated plants, which enhance disease severity. More specifically, the induction of PR genes (PR-1, PR-4, PR-6 and PR-10), C4H, involved in phenylpropanoid pathway and thus important for phytoalexin and lignin production, and Hin1, involved in the hypersensitive response, was significantly suppressed at an early stage during cold stress. This potentially provides an opportunity to Foc STR4 to invade the xylem and progress within the vascular bundles before plant defences are activated. Disease development mainly occurs at 28oC, as the pathogen prefers higher temperatures for optimal growth and sporulation. At this temperature, movement of water through the vascular vessels of the roots and pseudostem is increased, resulting in the more rapid wilting of affected plants. Thus, cold stress may enhance infection of Foc STR4. / Thesis (PhD)--University of Pretoria, 2013. / gm2013 / Microbiology and Plant Pathology / Unrestricted

Dissertation (MSc)--University of Pretoria, 2010. / Chemistry / unrestricted

Thesis (Ph. D.)--University of Wisconsin--Madison, 1972. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

A network is a system that involves movement or flow of some commodities such as goods and services. In fact any structure that is in the form of a system of components some of which interact can be considered as a network. In network design the problem is often to construct economical and reliable networks which satisfy certain requirements and which are optimal according to some criterion such as cost, output or performance. Graph theory is useful when the requirements of the network can be expressed in terms of graph parameters, usually as bounds. Some of the graph parameters that have been considered include: degree; distance; diameter; and connectivity. Problems with these parameter restrictions are usually from a class of NP-complete problems with instances that require exponential computer time to solve by available algorithms.The major focus of this thesis is to develop fast and efficient heuristics for some of these NP-complete problems. The two main topics analysed are Restricted Spanning Trees and Graph Partitioning. The aim of the Restricted Spanning Trees section is to construct the most efficient spanning tree (connected network) subject to various degree constraints. These degree constraints imposed are usually in the form of an upper bound. The upper bound represents the maximum number of connections allowed on a particular vertex. The Graph Partitioning section considers the problem of clustering vertices of the graph into sets such that the overall cost of the edges in the different sets is minimised.Chapter 1 provides the notation and terminology used throughout the thesis and a review and summary of the thesis.A literature review of related work that has been carried out to date is presented in Chapter 2. Some of the more promising results are discussed. The first part of the chapter surveys work related to the Restricted Spanning Tree problem. ++ / Analysis of both exact and heuristic methods is given. The second part of Chapter 2 provides a survey of the Graph Partitioning problem. We discuss the many different approaches that have been proposed to solve this problem. The quality of computational results achieved is discussed.Chapter 3 considers the Degree Constraint Minimum Weight Spanning Tree problem. This problem arises in networks where a given terminal is only allowed connections to a maximum number of specified terminals. We consider a number of cases including: same degree constraint on each vertex; different degree constraint on some vertices; and when the degree constraint is only on one or two vertices. A number of heuristics are developed and implemented and compared against an exact Branch and Cut algorithm. Our computational results demonstrated the value of our better performing heuristics.Chapter 4 considers the complexity of the (1,k)-tree problem. This problem is defined m given a graph G with maximum degree k find a spanning tree T with all vertices having degree 1 or k. Analysis is done on graphs with maximum degree 3, 4 and 5. Results establishing that the (1,3)-tree and (1, 4)-tree problems are NP-complete are presented. Further consideration is also given to the complexity of spanning trees with degree from the set { 1, 3, 5}. Analysis is also carried out on the number of degree one vertices in the (1, k)-tree. Presentation of heuristic procedures to solve this NP-complete problem concludes the chapter.Chapter 5 is devoted to the Graph Partitioning problem. A number of heuristics are presented and extensive computational work carried out. Computational findings support the usefulness of the heuristic methods both in terms of quality and time.We conclude this thesis by detailing some future work that can be carried out.

In this work, the main emphasis of the research concerns the development of isolation and purification methods of biomolecules from biological fluids. Several separation techniques were incorporated in chromatographic gels to obtain multifunctional hybrid chromatographic separation media for proteins, peptides and amino acid isolation and purification.In the first part of the research, several chelating agents were synthesized and their effectiveness to purify immunoglobulins using Immobilized Metal Affinity Chromatography (IMAC) was investigated. Ethylenediamine triacetic acid (TED) with immobilized copper resulted in the most effective in terms of purification and protein capacities.The next part of the work involved the development of hybrid chromatographic media that combines protein specific adsorption with sharp controlled size access permeation. This was accomplished by incorporating two types of ligand derivatives, one that permits the permeation of only certain molecular size range compounds, and a second one that specifically binds target biomolecules among the compounds of that specific molecular size range. Hybrid systems included binding ligands for Immobilized Metal Affinity Chromatography (IMAC), Ion Exchange Chromatography (IEX) and Hydrophobic Interaction Chromatography (HIC) combined with a controlled access polymer at different densities such as polyethylene glycol (PEG) and dextran derivatives. In general, low grafting density of high molecular weight PEG was found to be as effective as high grafting density of low molecular weight PEG in the rejecting properties of the semi-permeable synthesized media.Theoretical and experimental batch adsorption studies were also performed with the hybrid media and a mathematical model was developed to study the uptake of proteins under specific conditions of controlled permeation.In the last stage of this work, chelating surfactants were synthesized and used as reversible affinity ligands on reversed phase adsorbents for protein separations.One of the main accomplishments of this research was the development of separation media for small molecular size compounds from larger molecules and from complex biological systems. Applications of special interest will include the isolation and purification of solutes, such as metal ions, toxins, drugs, biomolecules, including proteins, biotoxins, nucleic acids, peptides, hormones, and biomarkers from biological fluids (such as human serum, urine, etc.) and from aqueous solutions.

No description available.

Nigeria recently became the leading economy in Sub-Saharan Africa with a total GDP of 522.64 billion of US dollars (Tradingeconomics.com). As GDP increases, population rises and food demand intensifies. Within this context it is of critical importance to achieve food security. However, Nigeria heavily relies in exportations in order to meet the growing food demand, especially of meat products, a situation which is not desirable. The livestock industry, although one of the largest in Sub-Saharan Africa, still constrained by several endemic livestock diseases which result in annual economic loses for value of 140 million of US dollars (Fadiga et al., 2013). Within this group, bovine and porcine trypasosomiasis alone has been estimated to be responsible for 50 million of US dollars in economic loses in Nigeria (Fadiga et al., 2013). However, the real epidemiological situation, and hence the possibility of developing a rational control programme, remains largely unknown across the country due to the absence of large epidemiological studies. Majority of the trypanosomiasis research studies in Nigeria employ the Haematocrit technique or the Buffy coat technique and Giemsa stain as a diagnostic method. These techniques possess a high specificity but a much lower sensitivity than the molecular method employed in this research study. In fact, better epidemiological studies employing molecular techniques have been conducted in recent times such (Majekodumni et al., 2013a; Takeet et al., 2013) and results displayed much higher trypanosomiasis prevalence than previously detected by microscopy. In many sub-Saharan countries the majority of national livestock herds are owned by mobile communities; however, the trypanosome status of cattle owned by mobile pastoralist communities have been less thoroughly studied when compared to those of sedentary livestock keepers. In this doctoral work, the epidemiology of trypanosomiasis was studied, in transhumant herds located in two different Nigerian enclaves: the Kachia grazing reserve and the Jos Plateau, both located in North-central Nigeria. Within Kachia, the ecology appears to determine the presence of infection with a spatially differentiated distribution of the detected trypanosome species being observed across the reserve that appears not to be related to the migration of livestock. While upon the Jos Plateau, the current reduction in trypanosome prevalence suggests an abrupt change in the trypanosome infection rates in this part of the country. The hypothesis established in this doctoral work is that these epidemiologically different scenarios are the result of land pressures that have ultimately resulted in the habitat destruction of the vector. Longitudinal data was also collected in order to assess the effectivity of different formulations of synthetic pyrethroids for the combined control of trypanosomiasis and tick-borne diseases. Insecticide treated cattle represents at the moment the best long-term and cost-effective method for the control of the vector responsible for the transmission of trypanosomiasis, the tsetse fly. Since no data exist about the efficacy of the insecticide or the compliance of the pastoralist population with its application under migratory conditions, its performance was assessed in this doctoral work. In addition, animal health outcomes were monitored to stablish the possible relationship between clinical symptoms and disease outcome and socio-economic data relevant for the dynamics of disease such as migration trends, husbandry practices, awareness and administered treatment has been also analysed. The compiled information of this data will establish the risk associated with contracting the disease and provide further indications for the control of African bovine trypanosomiasis in the specific context of transhumant pastoral systems of sub-humid sub-Saharan African.