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.

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.

Drought-induced senescence is a degenerative process that involves the degradation of cellular metabolites and photosynthetic pigments and uncontrolled dismantling of cellular membranes and organelles. Angiosperm resurrection plants display vegetative desiccation tolerance and avoid drought-induced senescence in most of their tissues. Developmentally older tissues, however, fail to recover during rehydration and ultimately senesce. Comparison of the desiccation-associated responses of older senescent tissues (ST) with non-senescent tissues (NST) will allow for understanding of mechanisms promoting senescence in the former and prevention of senescence in the latter. In the monocotyledonous resurrection plant Xerophyta schlechteri (Baker) N.L.Menezes, leaf tips senesce following desiccation, whereas the rest of the leaf blade survives. This study characterised structural, metabolic and transcriptional changes in ST and NST at varying water contents during desiccation and rehydration. Light and transmission electron microscopy was used to follow anatomical and subcellular responses, and metabolic differences were studied using gas chromatography-mass spectrometry and colorimetric metabolite assays. These results show that drying below 35% relative water content (0.7 gH2O/g dry mass) in ST resulted in the initiation of age-related senescence hallmarks and that these tissues continue this process after rehydration. Analysis of the transcriptome was done using RNA-Seq, which was subject to differential expression analysis and network analysis to elucidate the potential mechanisms for senescence regulation in this species. Significantly increased transcription of senescence associated genes was observed in the air dry sampling point, indicating that initiation of cellular death occurred below 20% RWC. Network analysis based on Pearson correlation revealed a high degree of clustering of these genes, suggesting co-regulation. The majority of these genes had two enriched motifs in their upstream regions, identified as binding sites for WRKY and other transcription factors. A model integrating these observations is presented, with insights into how senescence is initiated in ST and repressed in NST.

Histone F3 (III) from chicken erythrocytes was isolated by selective extraction from nucleoprotein with ethanolic-HCl and purified by a single gel filtration step. This protein was found to be homogeneous by the following criteria: gel filtration, electrophoretic mobility, N- and C-terminal amino acid residues and amino acid analysis. The primary structure of this histone was established without resorting to the use of overlapping sequences. This has been achieved with specific chemical cleavages rather than enzymatic degradations chosen and applied, first to the original protein chain, and subsequently to the generated polypeptides, to yield sets of not more than 3 peptides in any single cleavage. Their relative position in the protein or polypeptides became evident after comparison of the N- and C-terminal amino acids in the cleavage products and the uncleaved starting material. The simplicity of the peptide mixture after each cleavage, resulting in easy separation of the peptides, together with the highly efficient Edman degradation of automatic sequencing, allowed a rapid and relatively nonlaborious primary structure determination. Finally, the amino acid sequence is compared with those of protamines and other histones. The evolution and the structure of this protein in relation to DNA is briefly considered.

Background: Circadian rhythms are internally driven biological variations that fluctuate with a period of approximately 24 hours, even in the absence of external environmental time cues. These rhythms enable organisms to synchronise their internal clock time with external environmental time. This ensures appropriate timing of biological and metabolic processes, and allows anticipation of daily changes in the environment. Circadian rhythms also play an important role in sports in terms of optimising performance time-of-day and aiding adjustment to global time zone changes. Thus, performance, which is under the control of the athlete, may be impacted by event time-of-day scheduling in the new time zone. It has previously been shown that individual sport athletes in South Africa tend to be morning-types and carry the PERIOD3 (PER3) Variable Number Tandem Repeat (VNTR) 5-repeat allele, which has been associated with a preference for mornings. The distribution of the PER3 VNTR polymorphism in combination with an individual's preference for mornings or evenings has not yet been described in team sports. Differences in the PER3 VNTR genotype between team and individual sport athletes are expected, given that individual sport athletes may be free to choose the time-of-day at which they train. In contrast, team sport athletes usually train in groups, thus these individuals may not have the flexibility to choose their preferred training times. There are notable inter-individual differences in adjustment to jet-lag after time zone changes. A possible genetic candidate that may be responsible for some of this variation is the PER3 VNTR gene. This gene consist of two alleles corresponding in size to 4-repeats (PER34) or 5-repeats (PER35). Individuals are either homozygous for the 4-repeat allele (PER⁴⁄⁴) or the 5-repeat allele (PER3⁵⁄⁵), while others are heterozygous for the PER3 gene (PER34/5). The PER3 VNTR genotype might explain individual sensitivity to bright light and has been reported to be associated with sleep pressure- an increase in the brain's pressure and need for sleep, following an extended period of awakening. Individuals homozygous for the longer variant of the gene (i.e. PER3⁵⁄⁵) experience a higher sleep pressure during extended wakefulness. The PER3⁵⁄⁵ genotype has been reported to be more sensitive to the alerting and melatonin suppression effects of blue enriched light than the PER⁴⁄⁴ genotype. Aims: Therefore, the aim of Study 1 was to compare the chronotype and PER3 VNTR genotype distribution of South African Super Rugby players to individuals of low physical activity (i.e. those who are physically active ≤2 times per week). The aim of Study 2 was to determine whether PER3 VNTR genotype might contribute to inter-individual variation in the extent to which game involvement and quality of play are affected following trans-meridian travel. Further, the aim of Study 3 was to compare the impact of time zone travel during the 2012 Super Rugby competition in South African players genotyped as PER⁴⁄⁴, PER34/5 and PER3⁵⁄⁵ on the incidence of illnesses and injuries. Lastly, the aim of Study 4 was to compare the extent to which individuals genotyped as PER⁴⁄⁴ or PER3⁵⁄⁵ respond to appropriately-timed blue light exposure in order to resynchronise their circadian rhythm, following simulated eastward travel, based on changes in dim-light melatonin onset and cortisol circadian phases.

Includes bibliographical references. / The abalone Haliotis midae has been fished to near-extinction on the South African coastline, primarily to satisfy a growing international market. In order to meet demands, H. midae has been produced in South Africa by aquaculture for several decades, and the South African abalone aquaculture industry continues to expand. Internationally, abalone aquaculture has been actively affected by the outbreak of bacterial and viral diseases, which spread rapidly and lead to high abalone mortality. There is evidence that environmental stresses on abalone farms may lead to immunosuppression, and thereby increase the severity of disease outbreaks. The water temperature on abalone farms fluctuates seasonally, and increased abalone mortality has been associated with warmer water during the summer months. However, the molecular mechanisms affecting the abalone during exposure to stress remain unclear. With advances in proteomics technology, it is possible to identify and quantify the expression of several hundred proteins simultaneously. This study therefore aimed to gain insight into the H. midae stress response by using proteomic tools to identify proteins that are differentially regulated in haemocytes during exposure to acute heat stress. Identifying which biochemical pathways are involved in the abalone stress response will give some insight into the molecular mechanism by which H. midae responds to heat stress.