As a consequence of an immobile lifestyle, plants have had to evolve appropriate perception mechanisms and responses to diverse environmental stresses. Stress can be the result of both biotic and abiotic agents and the ORTHOLOG OF SUGAR BEET HS1 PRO-1 (HSPRO 1) and HSPRO2 genes were previously shown to be induced in response to several stresses including infection with Pseudomonas syringae and drought stress in Arabidopsis thaliana. The aim of this study was to characterise the biological role(s) played by these proteins in Arabidopsis. Several bioinformatics approaches provided evidence that supported function of both genes in response to both biotic and abiotic stresses and identified potential regulatory elements that may drive HSPRO gene expression during stress responses. Accordingly, analysis of null hspro mutants revealed antagonistic functions of the two proteins in PAMP-triggered immunity to P. syringae infections of shoot tissues and osmotic stress tolerance in plant roots. HSPRO proteins have been shown to interact with a central integrator of stress and energy signalling, SUCROSE NON-FERMENTING-1-RELATED KINASE1 (SnRK1) and microarray analysis of the null mutants suggested potential roles in carbohydrate signalling. An array of energy responsive genes including a subset of SnRK1 targets were misregulated in hspro mutants under standard growth conditions supporting involvement of HSPRO in energy signalling. Mutant phenotype and gene expression analysis revealed that HSPRO2 may be of importance in energy perception as hspro2 seeds were hypersensitive to exogenous glucose during germination, and that perception and/or signalling of low energy status may require HSPRO2. Although HSPRO2 expression may be driven via perception of environmental stress cues, promoter-luciferase assays revealed a diurnal expression pattern of the gene that was driven by the circadian clock. However, phenotypic analysis did not reveal a requirement of HSPRO2 for normal clock modulation. Since stress perception typically causes fluctuations in energy levels, it is proposed that HSPRO genes are important for the integration of energy and stress signalling in an effort to maintain a homeostatic balance between coping with environmental stress and normal growth and development.

The relationship between progestin-only injectable contraception and risk of HIV-1 acquisition is controversial. Most clinical data suggests that the injectable contraceptive medroxyprogesterone acetate (MPA), unlike norethisterone enanthate (NET-EN), increases susceptibility to infections such as HIV-1. The first part of this thesis investigated the differential effects, molecular mechanisms of action and steroid receptor involvement in gene expression by MPA as compared to NET and progesterone (P4) in the End1/E6E7 and HeLa cell line models for the endocervical epithelium, a key point of entry for pathogens in the lower female genital tract (FGT). Quantitative real-time PCR analysis showed that MPA, unlike NET-acetate (NET-A) and P4, increases mRNA expression of the anti-inflammatory GILZ and IκBα genes. Similarly, MPA unlike NET-A, decreases mRNA expression of the pro-inflammatory IL-6, IL-8 and RANTES genes, and IL-6 and IL-8 protein levels. The predominant steroid receptor expressed in the cervical cell lines and primary endocervical epithelial cells is the glucocorticoid receptor (GR), and GR siRNA experiments show that the anti-inflammatory effects of MPA are mediated by the GR. Chromatin-immunoprecipitation results suggest that MPA, unlike NET-A and P4, represses pro-inflammatory cytokine gene expression in cervical epithelial cells via a mechanism involving recruitment of the GR to cytokine gene promoters, like the GR agonist dexamethasone (DEX). This is at least in part consistent with direct effects on transcription, without a requirement for new protein vi synthesis. This is the first study to show direct proof for a GR-mediated mechanism of action in anti-inflammatory effects of MPA. Dose response analysis shows that MPA has a potency of ~24 nM for transactivation of the anti-inflammatory GILZ gene and ~4 - 20 nM for repression of the pro-inflammatory genes, suggesting that these effects are likely to be relevant at injectable contraceptive doses of MPA. These findings suggest that MPA effects on genital mucosal immune function and susceptibility to infections are likely to be very different to those of NET and P4, when mediated by the GR The second part of this thesis investigated the effects of the virion associated HIV-1 protein, Vpr, on GR-regulated inflammatory genes in the presence of the ligands.

Psittacine beak and feather disease (PBFD), the most prevalent viral disease affecting psittacines, is caused by beak and feather disease virus (BFDV). An outbreak of the disease has been reported in wild endangered Cape parrots (Poicephalus robustus), which is endemic to South Africa. No treatment or vaccine is commercially available. In this study, an investigation into the outbreak was undertaken. BFDV diversity was assessed and viral load and clinical signs correlated. A plant-produced BFDV subunit vaccine was produced in parallel with a corresponding challenge model. Cape parrots were assessed and 53 blood samples collected. Viral load was determined using quantitative real-time PCR (qPCR), and 22 BFDV full-length genome sequences acquired to infer phylogenetic relatedness. The capsid gene (cp) was optimised for transient Agrobacterium-mediated expression in whole-plant Nicotiana benthamiana (N. benthamiana). Virus-like particles (VLPs) were purified and analysed using transmission electron microscopy. Virions from a Palm cockatoo (Probosciger aterrimus) were purified and a BFDV dsDNA molecular clone was synthesised and replication assessed in 293TT mammalian cells and N. benthamiana using rolling circle replication and qPCR. Two distinct BFDV phylogenetic clusters were reported for Cape parrots, and a direct correlation was seen between viral load in the blood and clinical signs in PBFD-afflicted birds. The CP was successfully expressed in N. benthamiana, and increased through optimisation of Agrobacterium infiltration density and the inclusion of the NSs silencing suppressor. The CP formed VLPs, which were shown to be morphologically similar to infectious virions. The dsDNA molecular clone was shown to replicate autonomously in mammalian 293TT cells, and in plants with the assistance of the Bean yellow dwarf virus replication associated protein (Rep). BFDV genetic diversity in Cape parrots highlights the importance of ensuring new strains are not inadvertently introduced into the wild. This is the first systematic investigation of virus diversity in Cape parrots and assessment of BFDV viral load in a wild psittacine population. The CP was successfully produced in planta and presence of VLPs suggests the possibility of developing pseudovirions. This is the first reported replication of BFDV in tissue culture, and will greatly expand the scope of available research.

Includes bibliographical references / Reciprocal modulation between the glucocorticoid receptor (GR) and gonadotropin-releasing hormone (GnRH) signalling pathways is a potential mechanism for integrating cellular responses to stress with reproductive function. This study investigated if membrane rafts play a role in GR and GnRH receptor (GnRHR) crosstalk and explored the mechanism involved in the mouse pituitary gonadotrope LβT2 cell line by dexamethasone (Dex), GnRH and both together.

Includes bibliographical references. / In the past, commercial quantities of G. gracilis were harvested from Saldanha Bay until population collapses of the natural resource necessitated the need for research into alternative aquaculture programs for G. gracilis cultivation. One of the reasons for the G. gracilis population collapse was attributed to adverse conditions during summer which led to thermal stratification of the water column and subsequent nutrient limiting conditions. Inorganic nitrogen has been identified as the major nutrient factor limiting growth and production of G. gracilis populations in South Africa. Although the physiological mechanisms implemented by G. gracilis which allow adaption to low nitrogen environments have been investigated, not much is known about the molecular mechanisms which underlie these adaptions. Thus, it is necessary to elucidate the molecular basis of these adaptions in G. gracilis to complement the existing physiological data.

Includes bibliographical references / Haliotis midae is a commercially important abalone in South Africa, previously harvested from a stable, quota-managed fishery. However, the combined effects of overharvesting, increased illegal catches and negative environmental factors led to a collapse in wild populations in the mid-90s. Consequently, land-based aquaculture of H. midae has grown significantly in South Africa, to satisfy the global demand for abalone and alleviate pressure on wild stocks. Unfortunately, disease outbreaks have had a severe impact on the abalone aquaculture industry internationally and remain one of the single biggest factors contributing to economic loss. Understanding the effects of pathogen infection of abalone is therefore crucial to mitigating and controlling infection outbreaks on farms. Despite this, the molecular mechanisms underlying the immune response of H. midae remain obscure. High-throughput proteomics, a powerful tool to analyse global protein expression changes, can provide an integrated view of the immune system. Thus, this study aimed to elucidate the haemocyte proteome of H. midae and gain insight into regulatory molecular pathways underlying innate immunity. In this study, a comparative shotgun proteomics approach using isobaric tagging for relative and absolute quantification (iTRAQ) coupled with LC-MS/MS was employed to investigate H. midae proteome changes in response to Vibrio anguillarum challenge. A preliminary iTRAQ challenge trial was conducted which identified a putative early (1 and 2 hours post-injection) and late (48 hours post-injection) proteome response to bacterial-challenge. Using these time points, four independent challenge trials were conducted and analysed by iTRAQ and the results combined to produce a high-confidence dataset with good quantitative reproducibility for statistical analysis. A parallel set of experiments was conducted using mock-infected samples.

Malaria, caused by Plasmodium parasites, remains a leading cause for morbidity and mortality worldwide, resulting in more than 430 000 deaths annually. P. falciparum is responsible for the vast majority of severe malaria cases, accounting for more than 90% of malaria-related fatalities, predominantly in subsaharan Africa. The parasite has a complex life cycle, which involves transitioning between multiple distinct morphologies. The severity of the disease is brought about by the variable expression of parasite proteins on the surface of infected red blood cells. The substantial morphological changes, together with the variable expression of cell surface proteins, are governed by tightly controlled stage-specific changes in gene expression patterns. Understanding the regulatory mechanisms that govern these changes is crucial to fully understanding the parasites biology and pathology at the molecular level, a key step toward identifying targets for the development of much needed novel antimalarial drugs. Ultimately, all gene regulatory mechanisms converge to regulate the assembly and function of the RNA polymerase II (RNAP-II) transcription initiation complex composed of RNAP-II and the general transcription factors (GTFs). Bioinformatics analyses show that the RNAP-II GTFs in P. falciparum have greatly diverged from those studied in other eukaryotes, suggesting the existence of parasite-specific gene regulatory mechanisms, which have so far not been studied. This research project concerns the structure and function of P. falciparum TBP, TLP and TFIIA, key proteins involved in core promoter recognition, the first step in RNAP-II transcription initiation complex assembly. The work provides strong evidence for the existence of two different PfTFIIA complexes containing different PfTFIIA-γ subunits. The data further demonstrate that PfTBP and PfTLP DNA-binding activities differ distinctly from the classical TBP-DNA interactions seen in other eukaryotes and demonstrate interaction with and stimulation of PfTBP and PfTLP DNA-binding activity by one of the two PfTFIIA complexes. The work represents a first step towards understanding the regulation of transcription initiation in P. falciparum, gives first insights into Plasmodium-specific features, and provides a solid foundation for further investigations into this crucial aspect of malaria biology.

Includes bibliographical references / Eragrostis tef, commonly known as tef, is an important staple food and forage crop indigenous to Ethiopia. Tef plants are highly adaptable to abiotic stress conditions and are able to grow and produce grain yields under a wide range of environmental conditions, particularly under drought stress. In this study, tef plants were subjected to controlled dehydration stress treatment and physiologically characterised using relative water content (RWC), electrolyte leakage and chlorophyll fluorescence measurements, to establish critical water content stages for investigation of changes to the tef proteome in response to dehydration stress. Physiological testing showed tef viability to be retained to 30% RWC, however, further water loss to below 30% RWC, resulted in total loss of viability. Physiological characterisation with dehydration treatment showed a maximum leakage rate of 780 μS.min-¹.gdw-¹ and complete photosynthetic disruption with Fv/Fm and ɸPSII values decreasing to 0.2, below 30% RWC. Additionally, ultra-structural analysis using transmission electron microscopy showed extensive damage to the subcellular organisation of tef plant cells at water contents below 30% RWC. Based on these physiological data, it was decided to investigate the proteome of tef leaf dehydrated tissues at 50% RWC, as a non-lethal dehydration stress, as compared to hydrated tissues at 80% RWC. Proteomic analyses using iTRAQ mass spectrometry coupled to peptide OFFGEL fractionation and appropriate database searching with the Tef Extended and Liliopsida databases enabled the generation of three dataset results. These datasets, each contained a substantial amount of database matched proteins, where 5727 proteins for the Tef Extended (TE), 2656 proteins for the Tef Extended unique (TEU) and 4328 proteins for the Monocot unique (MU) datasets, were identified. Statistical analyses on peptide relative quantification values showed differential regulation of 211 proteins for the TE dataset, 111 proteins for the TEU dataset and 174 proteins for the MU dataset, in response to dehydration stress. A reciprocal BLAST search through the use of OrthoMCL with all three differentially regulated datasets (foregrounds) showed the TE foreground to provide the most comprehensive total protein coverage for further bioinformatics inference. Bioinformatics analysis using the programs Mercator, MapMan and Blast 2GO showed a diverse range of biological processes, where functional enrichment of GO-terms involved in biotic and abiotic stress response, signalling, transport, cellular homeostasis and pentose metabolic processes were enriched in tef high-abundance proteins. GO-terms linked to ROS producing processes such as photosynthetic reactions, cell wall catabolism, manganese transport and homeostasis, the synthesis of sugars and cell wall modification were enriched in tef low-abundance proteins. Additionally, KEGG analysis was used to observe tef proteins mapped to various biological pathways, of which the stress-responsive pathways, glutathione metabolism and ascorbate and aldarate metabolism were analysed in depth. Furthermore, biological validation of a few high-abundance proteins generated from iTRAQ analysis in the form of western blotting and relevant enzyme assays were conducted. The results showed the proteins fructose-bisphosphate aldolase (FBA), glutamine synthetase (GLN), functioning in plant maintenance 2 processes as well as the stress-protective antioxidant proteins, monodehydroascorbate reductase (MDHAR), peroxidase (POX) and superoxide dismutase (SOD) to be accumulated and further support iTRAQ findings. To date, this is the first study that has investigated the proteome profile of tef in response to dehydration.

Plasmodium falciparum, the deadliest strain of human malaria, affected 200 million people and resulted in several hundred thousand deaths in 2017 (World Health Organization, 2018). A better understanding of the mechanisms of P. falciparum gene regulation can open novel avenues for the development of much needed new drugs. A key step in eukaryotic gene regulation is the process of transcription, which is largely uncharacterized in Plasmodium. Bioinformatic analysis identified putatuve P. falciparum orthologues of RNA polymerase II general transcription factors (Bing, 2014; Milton, 2017), including a TATA box-binding-like protein, PfTLP. Bioinformatic analysis suggested that PfTLP is a TRF2-type TBP-like protein. However, PfTLP differs in several aspects from previously characterized TRF2-type proteins. These differences are thought to be Plasmodium specific adaptations to the parasite’s intricate life cycle and AT-rich genome. This study investigates two Plasmodium-specific features of PfTLP. Firstly, DNA binding by eukaryotic TATA-box binding protein (TBP) is mediated by four evolutionary conserved phenylalanine residues, two of which intercalate into the DNA. These residues are absent in previously characterized TRF2-type TLPs, and consistent with this, these proteins lack detectable DNA binding activity (Duttke et al., 2014). In contrast, PfTLP, a TRF2-type TLP, has DNA binding activity, and all four of the DNA binding phenylalanine residues are conserved (Bing, 2014; Milton, 2017). The importance of evolutionary conserved intercalating phenylalanine residues F60 and F283 was investigated by generating mutant PfTLP proteins, carrying alanine substitutions, and analysing their DNA-binding properties. The results suggest that while both phenylalanine residues are important for PfTLP DNA-binding, only F60 is critical for stabilization of PfTLP/DNA complexes. Secondly, PfTLP possesses two low-complexity or intrinsically disordered regions (LCR1 and 2), which are absent in TLPs from model eukaryotes. These regions are located at the same positions within the two quasi-symmetrical repeats of the TLP core structure and show a non-random compositional bias towards a limited set of amino acids. A growing body of evidence supports the idea that low complexity or intrinsically disordered proteins mediate liquid-liquid phase separation (LLPS) (Alberti et al., 2019; Brangwynne et al., 2009; Elbaum-Garfinkle et al., 2015; Nott et al., 2015). Bioinformatic analysis revealed that PfTLP LCRs are enriched in asparagine and lysine, and that these regions are well conserved throughout Plasmodium TLPs. PfTLP LCRs were fused to fluorescent proteins and the fusion proteins were functionally characterized in liquid-liquid phase separating assays. The results demonstrate that PfTLP LCR1 is capable of mediating LLPS, at least under certain conditions in vitro.

The postulated model for the insulin - stimulated induction of mRNA efflux (Purrello et al., 1983) is based on the demonstrated binding of insulin to intracellular membrane structures (see chapter 2, section 2.2.1), and the in vitro effect of insulin on nuclear envelope phosphorylation, NTPase activity, and mRNA efflux (see chapter 5, section 5.1). These independent observations have led to the development of a model for the direct induction by insulin, at the level of the nucleus, of mRNA efflux (figure 1.1). However, the specific intracellular insulin binding has been inf erred from kinetic or morphological studies which have not identified a discrete membrane - bound polypeptide(s) as an insulin docking molecule in situ (Goldfine, 1981). Also, the stimulation of NTPase activity has only been established by monitoring the level of general ATP hydrolysis of nuclear envelope fractions in the presence and absence of insulin (Purrello et al., 1983). The scope of this thesis has been to further the understanding of this mechanism by attempting to a) unequivocally identify a specific nuclear envelope - associated insulin docking polypeptide in situ and b) to demonstrate that insulin directly affects the ATP - binding of nuclear envelope ATP - binding proteins. The latter would demonstrate a primary effect of insulin i.e. the modulation of the ATP - binding capacity of identified NTPases / protein kinases (or their release from some inactive storage form), and not a general phenomenon such as elevated ATP.