Nitrilases (EC 3.5.5.1) catalyse the enantioselective hydrolysis of a variety of organic nitriles to the corresponding amide and/ or carboxylic acid. These reactions are important in the manufacture of fine chemicals, pharmaceutical intermediates, plastics and paints. Industrial uses of nitrilases are limited however, as wild-type enzymes suffer from limitations such as: a lack of control of the acid:amide ratio of products, and limited availability of substrate specificities.

Includes bibliographical references (leaves 224-243). / This thesis describes the development of a novel cytochrome P450 array format, the P450 Biochip that allows quantitative and truly high-throughput measurement of cytochrome P450-mediated turnover reactions in sub-nanolitre volumes.

Includes bibliographical references / Burn injuries are among the most devastating of all injuries and a major global public health crisis, with fire related burns accounting for approximately 265 000 deaths annually. The African continent, most especially Sub-Saharan Africa, has the second highest mortality rates (15% of global mortality rates). In South Africa, 3.2 % of the total population sustains burn injuries, with 50 % of these cases as children under the age of20 years. Studies have also shown that most of these incidences are prevalent within the age groups of 0-5 years, and account for the 3rd most common cause of mortality in children under the age of 15 years. In depth knowledge and understanding of cellular facets of wound healing has allowed for a greater stance in the interventions aimed at circumventing problems associated with development of effective wound defects treatment regimen. Burn treatment options are largely dependent on the degree and extensiveness of burns. A wide body of literature exists with regards to traditional as well as current treatment options. These include, for instance the use of various forms of skin auto-grafts. Despite such great success with all kinds of innovative ideas surrounding the use of autologous skin grafting, lack of available donor sites for skin grafts still remains a problem, more so in cases where patients suffer burns spanning more than 70% TBSA. This therefore has inspired the design and use of bioengineered skin substitutes as well as cultured/non-cultured autologous epidermal cells. Unfortunately, to date, no tissue engineering technique has fully been able to recapitulate the anatomy and physiology of the skin, or has attained the biological stability as well as achieving the aesthetic outcome. Several hurdles are yet to be overcome to achieve this. Amongst many, inclusion of melanocytes, other skin appendages as well as potential progenitor cells is some of the attributes of an ideal 3D skin equivalent. Therefore pigmented 3D skin constructs are of great interest as they address not only the issues of complete wound healing, but also the aesthetic outcomes. In light of this, correct keratinocyte to melanocyte ratios are also of great importance in designing such pigmented 3D constructs. Therefore the major aim of this study was to isolate skin melanocytes and keratinocytes, and co-culture them at different ratios in order to attain optimal pigment production and/or consequent improved wound healing outcome. To determine the best keratinocyte to melanocyte ratio to use in developing pigmented3D skin constructs, the following co-culture ratios were used: 5:1, 10:1 and 20:1.Proliferation assays were employed to further elucidate the growth dynamics of both human skin melanocytes and keratinocytes in either mono- or co-culture system. Secondly, FACS was used to develop a reliable technique to be used to separate the two cell types from a co-culture system in order to perform downstream analyses. Thirdly, to establish the roles of the co-cultured cells in wound healing (with regards to proliferation and migration), scratch wound healing assays were employed. Lastly, FACS was used to infer the effect of such ratios on pigment production. Our results demonstrated that keratinocytes, compared to melanocytes mono-cultures have higher proliferation capacity. On the contrary melanocyte's proliferation is up-regulated by the presence of keratinocytes in a co-culture, whereas higher numbers of melanocytes in co-culture with keratinocytes resulted in less proliferative keratinocyte phenotype. The FACS separation technique worked excellently in identifying keratinocyte population from melanocytes, with an almost 100% accuracy. This is shown by melanocytes being sorted as 93% of MART-1 + cells in a mono-culture, followed by an approximately 5:1 separation of keratinocytes from melanocytes (77% Kc and 17% Mc). In vitro scratch assays demonstrated that none of the co-culture ratios was significantly superior with regards to wound healing capacities and pigment production, in the absence of fibroblast-conditioned medium. In conclusion, the 5:1 co-culture ratio seemed to yield a non-significant, yet best outcome with regards to wound healing capacity (only in the presence of fibroblast-derived factors), thus conferring it as a potential optimal ratio of keratinocytes to melanocytes, to be used in development of our pigmented 3D constructs.

Includes abstract. / Includes bibliographical references (p. 136-165). / This thesis exposes previously ignored evolutionary characteristics of the synonymous sites of virus nucleotide sequences and contributes new findings to the understanding of the evolution of HIV-1 in relation to the human immune response.

Includes bibliographical references (leaves 108-123). / Corynebacterium glutamicum is a Gram-positive facultative aerobe particularly known for its industrial application in the synthesis of amino acids, such as L-glutamate and Llysine. The central metabolic pathways of this organism has been an area of much research by many groups. Linked to glycolysis is the synthesis of glycogen, previously considered a storage molecule of excess glucose. No information concerning the role of glycogen or its metabolism in C. glutamicum was known, and the aim of this work was to elucidate glycogen metabolism in this industrially important organism.

Includes abstract. / Includes bibliographical references. / A healthy and stable gastrointestinal microbiota is a vital feature of the innate immune system. It affords the host numerous health benefits and acts as a barrier against opportunistic gut infections. Probiotic bacterial supplements are, therefore, widely used in industry to promote good health. There is, however, a need to understand not only the factors underlying the health promoting capabilities of these bacteria, but also the intrinsic antimicrobial resistance mechanisms which these bacteria are known to harbour. These antibiotic resistance traits confer a competitive advantage on these bacteria over other bacterial species where they reside in the gut. It also allows them to survive during antibiotic therapy and they are able to continue conferring health benefits on the host. To better understand the mechanisms these bacteria utilize in conferring antibiotic resistance, genes which confer multidrug resistance by the active hydrolysis of ATP were studied here. These genes belong to the ATP-binding cassette (ABC) family of efflux transporters.

Includes abstract. / Includes bibliographical references (p. 155-174). / Corynebacterium glutamicum is widely used for the commercial production of a variety of amino acids, including L-lysine, L-glutamate and L-threonine. With the exception of Larginine, the biosynthesis and regulation of most of these compounds in this bacterium are relatively well characterised in the literature. The research presented here focuses on improving our understanding of the regulation of L-arginine biosynthesis in C. glutamicum. This was performed with the ultimate goal of creating strains capable of producing L-arginine commercially. A novel gene replacement system was initially used for the directed mutation of the Larginine biosynthetic gene cluster in C. glutamicum ATCC 13032. This was met with limited success, however, and the pK19mobsacB vector was thus adopted for further mutagenesis of this region.

Includes bibliographical references (leaves 126-140). / In our search for new anti-tuberculosis lead molecules, five medicinal plant species, Olea capensis (L.l, Tulbaghia alliacea (L.), Inula graveolens (L.), Leyssera gnaphaloides (L.), and Buddleja saligna (L.) were collected in Cape Town and surrounding area and investigated for antimycobacterial activity following report of their therapeutic use in traditional medicine to treat infectious diseases such as tuberculosis. A bioassay guided fractionation of the acetone/water (4:1) crude extracts of O. capensis (leaves) and T. alliacea (rhizomes) showed no activity against Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 252923, and Mycobacterium aurum A+. In contrast, the orgamc fractions (hexane, dichloromethane) of the acetone/water (4: 1) crude extracts of 1. graveolens, L. gnaphaloides, and B. saligna exhibited significant activity against M. tuberculosis H37Rv, M. avium 25291, M. microti ATCC 19422, and M. scrofulaceum ATCC 19987. The isolation and structure determination of the bioactive led to the identification of pentacyclic triterpenoids, ursolic acid (UA) and oleanolic acid as major antitubercular constituents of B. saligna, L. gnaphaloides, and 1. graveolens. The in vitro cytotoxicity assays of the isolated bioactive constituents showed no cytotoxicity against Chinese Hamster Ovarian (CHO) cells line. Subsequently, given the pharmaceutical value of the above finding, a survey on structure activity of pentacyclic triterpenoids was conducted. It was was found, for instance that selective substitutions at C-3 and/or C-28 and the double bond at UA, OA and betulinic and (1) BA) were made in order to improve anti-tumour and anti-HIV activity. However, thought a great number of modified bioactive pentacyclic triterpenoids is reported, none was tested against Mtb. Therefore, this study also explored a new synthetic route (scheme 1) toward a generation of (5), which may allow improving antitubercular, anti-HIV or anti-tumour activity, and/or specificity.

Current management options for cutaneous burn wounds, including split thickness skin grafts and cultured epithelial autografts, generate an epithelial barrier which lacks a dermal layer and skin adnexae including hair follicles and sebaceous glands. This results in a loss of pliability and contractures that cause functional and cosmetic impairment. Embryological hair follicle morphogenesis results from a complex series of mesenchymal-epithelial interactions and to date a method of generating de novo folliculogenesis from human cells has yet to be accomplished. Existing models rely on combining 'inductive’ dermal and 'receptive’ epithelial components and placing them within a suitable model. Epithelial cells are easily obtainable from skin biopsies therefore obtaining sufficient quantities of 'trichogenic’ dermal cells remains the most significant challenge of this approach. The main aim of this project is to contribute to the achievement of de novo folliculogenesis by generating dermal papillae (DP) like-spheroids using adipose derived mesenchymal stem cells (ASCs) that, when combined with responsive epithelial cells, would be capable of inducing hair follicle formation. ASCs were directed towards a hair follicle DP-like fate by culture using the hanging drop method and exposure to Wnt, mimicking signalling and mesenchymal condensation in embryological hair follicle induction. Gene expression analysis using RT-PCR showed that the DP-cell marker Versican is expressed at high levels in ASCs under routine culture conditions and the exposure of ASCs to Wnt results in a more than threefold increase in this expression. These results suggest that Wnt/β-catenin signalling may regulate DP cell aggregative growth through modifying versican expression possibly through binding of β-catenin to the TCF transcription factor complex. Culture of ASCs using the hanging drop method produces spheroids similar in size to human hair follicle DP. Histology of these spheroids demonstrates viable cells that flatten around the outside. The spheroids grow out when replated onto Matrigel in a 3D culture model and exhibit a morphology similar to that of primary hair follicle DP cells. Analysis of mRNA expression demonstrates that Versican expression is significantly upregulated in DP-like spheroids in the absence or presence of Wnt demonstrating that Versican may be responsible for both induction and maintenance of mesenchymal cell condensates. Alpha smooth muscle actin is expressed in low levels in ASC spheroids compared to ASCs in a monolayer and this may reflect a 'migratory’ myofibroblast like phenotype of ASCs in a monolayer similar to cells with the hair follicle dermal sheath. The addition of Wnt to ASC spheroids has no additional effect on Versican expression possibly reflecting a negative feedback loop resulting from high local concentrations of endogenous Wnt expression from ASCs. The results of this study show that spheroid cell culture and exposure to Wnt of ASCs results in cell clusters with similar morphology and gene expression to hair follicle DP cells. The novel method of DP-like cell generation described in this study makes use of cells that are readily obtainable from patients and require minimal time and manipulation in culture and therefore could potentially be rapidly translatable to clinical trials.

Includes bibliographical references (leaves 162-170). / Nanotechnology is defined as the technology that allows to fabricate nanoscale materials and manipulate them at the molecular or atomic level. Non-biotechnology is one of the sub-disciplines of nanotechnology. Materials used for nano-biotechnology are macromolecular materials found in nature with specific mechanical and biological functions. One of the most important materials is deoxyribonucleic acid (DNA). DNA has several unique properties that make it a good candidate for nanotechnology research. The primary aim of this research is to use DNA three-way junctions as building blocks and to assemble them into a huge DNA networks through self assembly followed by DNA ligation. The second step aims of assembling six DNA three-way junctions with different sticky end sequences to from an opto-mechanical hexagonal DNA nano-switch. The switching mechanism in the righ is based on the conformational change of B- to Z- DNA. This change is influenced by the environmental cation concentrations. The third approach presented here aims to create meta-stable DNA nanostructures based on a DNA hairpin loop that can mimic the enzyme-substrate binding mechanism. This mechanism is demonstrated using a "strand displacement strategy" (SDS). Lastly, we employed a novel way of organizing hairpin loop DNA structures based on the orientation of consecutive hairpin loop DNA structures "on top" of one another.