The broad-host-range Thiobacillus ferrooxidans plasmid pTF-FC2 is stably inherited over many generations despite a low copy number. The pas genes which lie between the repB primase and the repA helicase encode a proteic plasmid stabilisation system and are capable of stabilising the unstable heterologous R1 replicon present on p0U82. The deletion of the pas genes has been shown not to change the copy number of mutant plasmids. This suggested that the pas genes are not involved in replication and function as a stabilisation cassette. The pasA gene encodes an antidote, the pasB gene a toxin which exerts a bacteriocidal effect in an E. coli host and the pasC gene a protein which moderates the toxic effect of PasB. The PasC is unique in proteic plasmid stabilisation systems and reduces PasB toxicity only in the presence of PasA. PasA is able to repress the pas promoter and the addition of PasB increases this repression. PasC has been shown not to effect the pas promoter by itself. In the presence of PasA and PasB, PasC reduces the ability of PasA and PasB to repress the pas promoter. PasC is thought to stabilise the interaction of PasA and PasB and in doing so reduces their ability to function as repressors of the pas operon. The IncQ plasmid RSF1010 which has similarity to pTF-FC2 has two genes in a position analogous to the pasA, pasB and pasC genes. These genes have been found to be unable to function as a plasmid stabilisation system.

Rift Valley fever (RVF) is a haemorrhagic fever agent caused by an infection with an enveloped negative-stranded RNA Rift Valley fever virus (RVFV). It belongs to the genus Phlebovirus in the family Bunyaviridae. The virus is spread by infected mosquitoes and affects ruminants and humans, causing high numbers of neonatal fatalities in animals and occasional fatalities in humans. It is endemic to parts of Africa and the Arabian Peninsula, but is described as an emerging virus due to the wide range of mosquitoes that could spread the disease into non-endemic areas, posing serious health and agricultural problems. The disease can be prevented by vaccination, but there is currently no Food and Drug Administration-approved RVFV vaccine that can be used outside endemic areas, while there are two live attenuated vaccines available for use in endemic areas. These vaccines have the potential for reversion, and are therefore not recommended for use in countries where RVFV is not endemic. This indicates the need for more RVFV vaccine research and development. This work focused on the development of a RVFV vaccine candidate that would allow for differentiation between infected and vaccinated animals as well as humans.

Includes bibliographical references. / The acetone- butanol-ethanol (ABE) fermentation was one of the first fermentation processes to be industrialized on a large scale, and the dominant product, butanol is particularly significant due to its potential as a modern day fuel additive or fuel extender in the petrochemical industry. A collection of 19 solventogenic Clostridium beijerinckii and 11 Clostridium saccharobutylicum strains isolated from the National Chemical Products (NCP) ABE fermentation plant in Germiston, South Africa, were classed according to species by a quick species-specific colony PCR and by rifampicin screening methods respectively. The speciesspecific PCR aims to provide a rapid means of assessing any contamination of an ABE batch fermentation by differentiating between C. saccharobutylicum and C. beijerinckii species. Random Amplification of Polymorphic DNA (RAPD) analysis generated four C. beijerinckii and two C. saccharobutylicum strain groups respectively. Multilocus Sequence Typing (MLST) was developed for a smaller selection of strains and showed a further two strain groups within the NCP C. beijerinckii strains and three groups within the C. saccharobutylicum strains.

N-glycosylation plays an important role in Envelope (Env) function and may be involved in the modulation of the immune response to HIV-1 infection. In this study, we hypothesized that Env N-glycosylation may affect viral pathogenesis by influencing Env structure and function. Furthermore, we also postulated that differences in Env glycosylation could affect interactions between Env and DC-SIGN of dendritic cells (DCs), activating alternative signalling pathways which stimulate the release of different immune modulators. We generated pseudovirus of eighteen Env clones (PSVs) with variable number and position of potential N-glycan sites (PNGs) and compared their ability to infect TZM-bl cells, bind to Raji+ DC-SIGN cells, trans-infect TZM-bl cells when captured by either Raji-DC-SIGN cells or monocyte-derived dendritic cells (MDDCs) and modulate MDDC signaling by investigating the release of Interleukin-10 (IL-10) and other immune modulatory cytokines and MAPK activation. Entry efficiency, DC-SIGN binding and trans-infection varied widely across all clones. The level of IL-10 secreted by MDDCs in response to PSV stimulation varied 32-fold. The induction of IL-10 secretion by purified gp140 confirmed that Env was the viral component that stimulated the secretion of IL-10 via interaction with DC-SIGN and potentially other undefined receptors. PSV and purified gp140 stimulated MDDC signaling via ERK and JNK phosphorylation, while p38 was not activated. The addition of recombinant DC-SIGN lowered the levels of secreted IL-10 and ERK /JNK phosphorylation, suggesting that DC-SIGN plays a role in these responses. As Env mannosylation correlated with DC-SIGN binding, five highly conserved Env PNGs (241, 262, 386, 392, and 448) previously identified to carry high mannose type N-glycans and hence thought to be involved in DC-SIGN binding were deleted in two Env clones by site-directed mutagenesis to confirm their importance in Env function. The potential role of these PNGs in Env entry efficiency, DC-SIGN binding, trans-infection, induction of MDDC IL-10 secretion and activation of MAPK phosphorylation was determined. Deletion of these sites significantly affected the entry efficiency, DC-SIGN binding, trans-infection and MDDC IL-10 secretion, with one Env clone proving to be more sensitive to mutation than the other. This suggests that PNGs influence Env function in a clone-specific manner. As deletion of highly conserved PNGs abrogated Env function we used sequence analysis to identify PNGs involved in binding DC-SIGN and inducing MDDC IL-10 secretion. We grouped PSVs based on the presence or absence of specific PNGs in Env sequences and compared entry efficiency, DC-SIGN binding, trans-infection, stimulation of MDDC IL-10 secretion and induction of MAPK phosphorylation. Three Env PNGs were significantly associated with entry efficiency (N356, N392, and N674), and three sites (N289, N356 and 674) were significantly associated with trans-infection while N674 also influenced DCSIGN binding. The majority of MDDC donors secreted higher levels of IL-10 when stimulated with PSVs that carried PNGS at N130 (p = 0.0016) and N332 (p = 0.0039) and lacked N674 (p = 0.033). When Envs were graded on whether they had 0, 1, 2 or 3 of the PNGs (e.g. -130, -332, +674; -130, +332 and +674, etc.) those that carried either one of the PNGs or the entire induction motif (N130+ N332+ N674-) significantly stimulated MDDCs to secrete higher levels of IL-10 than those that completely lacked the motif (p = 0.0335 and p = 0.0304, respectively). As the presence of N674 was linked to reduction in all functions of Env, it is likely that the presence of an N-glycan at this site affected Env structure and could skew the analysis. Excluding N674 indicated that the presence of PNGs at position 130 and 332 was sufficient to induce significantly higher IL-10 release than those that had either none or one of these sites (p = 0.0053). When we determined whether N130 and N332 were enriched in subtype C acute infection Envs, these sequences were not enriched with PNGs at either N130 or N332 compared to chronic infection viruses. However, when IL-10 levels were compared between MDDC donors stimulated with PSV of either acute or chronic infection clones, those from early infection significantly enhanced MDDC secretion of IL-10 (p = 0.0039). This suggests that even though PNGs at 130 and N332 could be involved in inducing MDDC IL-10 secretion, it is not the only requirement for enhanced stimulation. Although Env differentially activated ERK and JNK phosphorylation, ERK phosphorylation did not correlate with IL-10 secretion, suggesting that this MAPK signaling pathway was not solely responsible for triggering the release of MDDC IL-10 and other regulatory cytokines. PSVs also stimulated the release of TNFα, IL-1β, IL-6, IL-8, MIP-1a, and MIP-1b while having no effect on IL-12 levels. This suggests that HIV-1 binding to DCs in the genital tract could change the dynamics of DC immune responses, deregulating their cytokines secretion and destabilising the Th0 cell differentiation to facilitate viral survival and thus productive clinical infection. We therefore conclude that HIV-1 variants differentially stimulate MDDCs to release immunosuppressive IL-10 and that transmitted founders could be better at modulating immune responses in the genital tract compared to chronic infection variants.

Bibliography: pages 127-144. / Bacillus brevis Alk 36 was isolated from soil during a screening programme for the selection of extracellular enzyme producing strains. A gene coding for an endo(1,3- 1,4 )-.8-glucanase (or lichenase) was cloned from B. brevis Alk 36 and expressed in Escherichia coli. The nucleotide sequence of this gene was determined and found to encode a protein of 252 amino acid residues. The amino acid sequence of the B. brevis lichenase gene showed only a 50% similarity to previously published data for Bacillus endo-(1,3-1,4)-β-glucanases. The enzyme exhibited some unique properties. The optimum temperature and pH for enzyme activity were 65-70°C and 8-10, respectively. When held at 75°C for 1 h, 75% residual activity was measured. The molecular mass was estimated to be 29 kDa and the enzyme was found to be resistant to sodium dodecyl sulphate (SDS). B. brevis Alk 36 was evaluated as a potential host strain for the efficient production and secretion of foreign proteins and was found to grow optimally between pH 8.0 and pH 9.5 and between 42°C and 52°C. B. brevis was successfully transformed using vector DNA and was found to produce relatively low levels of protease. In addition, it was evaluated as a possible protein hyper-secreting strain. However, using PCR technology, the highly conserved cell wall protein genes could not be positively identified in B. brevis Alk 36.

Includes bibliographical references / The synthetic progestogens, medroxyprogesterone acetate (MPA) and norethisterone enanthate (NET-EN),are widely used in developing countries as injectable contraceptives, where disease burden is high.Levonorgestrel (LNG) is a common progestogen used in oral contraceptives and intrauterine devices. Some studies suggest that MPA, unlike NET, increases HIV-1 acquisition in women, while few studies have reported on the effects of LNG on HIV-1 acquisition. Whether MPA, NET and LNG differentially affect HIV-1infection and the expression of key genes relevant to HIV-1 acquisition via differential molecular mechanisms is key to understanding choice of progestogen contraceptive in young women at high risk forHIV-1 acquisition. The central hypothesis of this study is that the differential effects on host gene expression and HIV-1replication by the different progestogens is due to their differential selectivity to towards different steroid receptors, in particular the glucocorticoid receptor (GR). In order to investigate this hypothesis, the regulation of selected genes was investigated in cervical tissue explants from premenopausal, HIV-1negative, and contraception negative women and peripheral blood mononuclear cells (PBMCs) from women, by real time quantitative PCR, western blotting and Luminex assays, in response to physiologically relevant doses of progestogens. Infection assays were performed in the absence and presence of HIV-1using HIV-1BAL-RENILLA or HIVpNL4.3 infectious molecular clones (IMCs). The GR antagonist RU486 or GR siRNA knockdown was used to determine the role of the GR in modulating ligand-specific effects. PBMCs and primary cervical explants were chosen as useful models to assess the direct effects of these progestogens in both the systemic and in the local mucosal immune environments. In PBMCs, MPA like dexamethasone (DEX, a GR specific agonist), showed anti-inflammatory effects, decreasing pro-inflammatory interleukin (IL) 6, IL8 and regulated upon activation, normal T cell expressed and secreted(RANTES) levels and increasing anti-inflammatory glucocorticoid interacting leucine zipper (GILZ) gene expression levels, while NET, progesterone (P4) and LNG did not, after 48 hours. In primary ectocervical tissue explants, DEX, cortisol, MPA and P4 significantly repressed IL6 while only DEX, cortisol and MPA significantly repressed IL8 and increased GILZ gene expression levels after 48 hours. Steroid receptor expression was characterised in both PBMCs and ectocervical explants. GR was the only detectable steroid receptor protein expressed in PBMCs, while ectocervical explants expressed all the steroid receptors. The progesterone and estrogen receptor levels were higher in ectocervical explants from donors that were in the follicular phase compared to ectocervical explants from donors in the luteal phase of the menstrual cycle. In PBMCs, results suggested that differential gene expression by MPA versus NET and P4is mediated via the GR after 48 hours. Furthermore, it was observed that MPA and DEX, unlike NET, LNG and P4 increases HIV-1 replication in viable PBMCs, in the majority of donors. The increase in HIV-1replication in the MPA treated PBMC samples correlated significantly with an increased in IL6 mRNA levels.

Bibliography: p. 217-233. / A cyanide-degrading, Gram-positive, aerobic, endospore-forming bacterium was isolated from a cyanide wastewater dam by an enrichment technique and was identified as a strain of Bacillus pumilus. The bacterium was routinely cultured in Oxoid nutrient broth and rapidly degraded 100 mg/1 of free cyanide in the absence of added inorganic and organic substances. The ability to degrade cyanide was linked to the growth phase and was not exhibited before late-exponential/ early-stationary phase. Cyanide-degrading activity could not be induced in early exponential phase by the addition of cyanide or acetonitrile to 20 mg/1. Production of the cyanide-degrading activity required at least 0.01 mg Mn2+ /1 in the growth medium (lower concentrations prevented the development of strong cyanide-degrading activity and also resulted in poor growth of the organism). No induction of cyanide-degrading activity occurred when Mn2+ ions were added to late-exponential-phase cells (or a cell-free extract from these cells) which had been grown with a low endogenous concentration of Mn2+. However, Mn2+ ions could be added to cultures growing in low-Mn2+ broth as late as the mid-exponential phase of growth with no apparent reduction of the cyanidedegrading activity exhibited in stationary phase. Culturing the organism in Difeo nutrient broth resulted in poor growth and very low levels of cyanide-degrading activity; addition of Mn2+ to this medium did not significantly increase the levels of activity. Production of the cyanide-degrading activity required de novo transcription and translation. Cyanide-degrading activity was located intracellularly and cell-free extracts rapidly degraded cyanide (0.27 ± 0.08 μmole cyanide/min/mg protein at 30 °C in pH 7.4 phosphate buffer). Eight strains of cyanide-utilizing fluorescent pseudomonads were also isolated from activated sewage sludge by an enrichment technique and tentatively identified as strains of P. fluorescens and P. putida. These isolates could not degrade cyanide rapidly.

Bibliography: pages 170-194. / Maize streak virus (MSV) is the type member of the Mastrevirus genus of the Geminiviridae. As the causal agent of maize streak disease (MSD), MSV is the most significant pathogen of maize in Africa, resulting in crop yield losses of up to 100%. Transmitted by leafhoppers (Cicadulina spp.), MSV is indigenous to Africa and neighbouring Indian Ocean Islands. Despite maize being a crucial staple food crop in Africa, the average maize yield per hectare in Africa is the lowest in the world, leading to food shortages and famine. A major contributing factor to these low yields is MSD. To genetically engineer MSV-resistant maize using the pathogen-derived resistance (PDR) strategy, the viral replication-associated (Rep) protein gene was targeted, whose multifunctional products Rep and RepA are the only viral proteins essential for replication. Rep constructs had previously been made containing deleterious mutations in several conserved amino acid motifs. In this study, these mutants and the wild type Rep gene were truncated to remove key motifs involved in viral replication. A quantitative PCR assay was developed to determine the effects of the mutant and truncated Reps on viral replication in black Mexican sweetcorn (BMS) suspension cells. The MSVsensitive grass Digitaria sanguinalis was then transformed with Rep constructs that inhibited MSV replication in BMS, and transgenic lines were tested for virus resistance. Several plants of a D. sanguinalis line transgenic for a mutated full-length Rep gene showed excellent resistance (immunity) to MSV, but the transgene had negative effects on aspects of plant growth and development. Transformation with a mutated/truncated Rep gene resulted in healthy fertile transgenic D. sanguinalis plants, many of which showed good MSV resistance. Fertile maize (Hi-II) T 1 transgenic plants expressing the truncated/mutated Rep gene have been obtained, the offspring of which will be tested for resistance to MSV. Considering the success in achieving MSV-resistant D. sanguinalis, there is good reason to believe that the transgenic maize will too be resistant to MSV.

Bibliography: pages 187-194. / During the course of this study, the histones of the algae Olisthodiscus luteus were isolated, purified and fractionated. Identification of the histones was achieved by partial primary structure analysis. The histones Hl, H2A, H2B, H3 and H4 were found to be present in the O. luteus nucleus. The complete structure of H2A and H4 was determined. There is no evidence of the existence of the unique histone HO1 (Rizzo et al., 1985). Construction of phylogenetic trees suggests that the alga Olisthodiscus luteus diverged from the animal line. By sequence comparison, the most closely related histone sequence to the algae was found to be that of the echinodermata. An endosymbiotic event between an echinodermata ancestor and a primitive unicellular alga is hypothesised in an attempt to explain the smilarity between the histones.

Food security is one of the most important global challenges facing the world today, especially in the context of climate change. Research has been conducted into a unique group of plants, called “resurrection plants”, that can withstand up to 95% tissue water-loss without compromising viability by, inter alia, undergoing extensive metabolic reprogramming and suppressing senescence. In this thesis the African desiccation tolerant grass Eragrostis nindensis (Fical & Hiern) was used as a model plant to identify which biological processes are unique to senescence and critical for desiccation tolerance. When desiccated, the older leaves of E. nindensis senesce, whereas, the younger leaves recover fully upon rehydration, thereby displaying two phenotypes in a single species. Comparing these two tissue types can show how senescence upon abiotic stress is regulated. Differences in transcript abundances between the two tissue types during drying and rehydration was analysed through RNA-seq analysis, coupled with physiological quantitative traits, mass spectrometry analyses and immunoblotting. The transcriptome reflected a transcriptomic reprogramming towards desiccation tolerance by maintaining transcription of genes that control desiccation tolerance traits in both tissue types, however, only the desiccation tolerant (non-senescent) tissue appeared to suppress senescence and maintained translational control. It was hypothesised that the non-senescent tissues regulate and stabilise RNA. The older tissues were unable to suppress senescence, which resulted in cell death. Lipids accumulated in the non-senescent tissue, particularly unsaturated triacylglycerols. It was proposed that lipid droplets that accumulated during drying were stabilised through, in part, the protein expression of oleosin. These lipid droplets appeared to provide a mechanical stabilisation and energy-providing role in the non-senescent tissue. The transcription of genes that control desiccation tolerance traits was generally maintained in both tissue types, however, translation was prevented in the senescent tissue. The non-senescent tissue therefore appeared to engage in a regulation of senescence at the translational level, rather than a fine-tuned transcriptional regulation. The aim of this work was to provide a critical baseline for future studies working on E. nindensis, and desiccation tolerance and senescence in resurrection plants in general. Ultimately, understanding water-deficit stress in the context of senescence can help to improve drought resistance in crops to ensure food security, particularly in Africa.