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.

Includes bibliographical references. / In angiosperms, desiccation tolerance, a genetic trait that enables tissues to survive loss of more than 95% of cellular water is widely observed in the seeds, but is only found in the vegetative tissues of a small group of species known as the resurrection plants. Xerophyta humilis is a small resurrection plant indigenous to Southern Africa. In this study, the hypothesis that vegetative desiccation tolerance is derived from an adaptation of seed desiccation tolerance was tested by characterizing changes in the transcriptome of X. humilis leaves during desiccation. The mRNA transcript abundance of a set of 1680 X. humilis genes was analyzed at 6 different stages of water loss in the leaves of X. humilis. Functional enrichment analysis showed that genes that were down-regulated during desiccation were over-represented with genes involved in photosynthesis, cellular developmental processes, as well as transcription regulator activity. Three distinct clusters of up-regulated genes were identified. The earliest set of up-regulated genes were enriched with genes associated with the turnover of proteins and the simultaneous synthesis of proteins required for protection. Enrichment also included genes associated with lipid body synthesis, as well as the transport of storage proteins to vacuoles. Two groups of late desiccation up-regulated genes were also identified, their expression only increased at later stages of desiccation and remained high in the desiccated leaves.

West Nile virus (WNV) is a widely disseminated flavivirus, with a geographical range that now includes Africa, America, Europe, the Middle East, West Asia and Australia. The virus is vectored by Culex mosquitoes and is maintained in a bird-mosquito transmission cycle with hundreds of bird species acting as reservoir hosts. In humans, infections can develop into febrile illness and severe meningoencephalitis and to date, there is no treatment or vaccine available. In horses, approximately 20% of infections are symptomatic, of which 90% of cases involve neurological disease, with 30-40% fatality rates. Several veterinary vaccines specific to the lineage 1 WNV strains are commercially produced in America and Europe, however, these vaccines are not easily obtainable for low and middle-income countries (LMIC) due to their high cost and that associated with importation as well as the need for annual vaccination. Due to continuous global disease outbreaks in birds, humans and horse populations with no preventative measures for humans, WNV poses a major public health threat, especially in naïve populations. The development of a vaccine that contributes to the ‘One Health' Initiative could be the answer to prevent the spread of the virus and control the disease. Current veterinary vaccines are produced in expensive cell culture systems that require sterile conditions, high-level biosafety facilities and trained personnel for their preparation. Transient plant-based expression systems have proven to be a very cost-effective means of making complex proteins. Plants can produce and modify proteins in a similar manner to mammalian cells and production does not require sterile conditions or specialised facilities. We propose that plants could be a viable means of making feasible, low-cost reagents for WNV, specifically virus-like particles (VLPs) for use as vaccines in South Africa and other LMIC. In this study, we set out to develop two particulate candidate vaccines based on a virulent South African WNV strain using Nicotiana benthamiana as the expression platform. We aimed to develop the first candidate vaccine by exploiting the virus's ability to form noninfectious VLPs by expressing only the WNV membrane (prM – precursor, M – matured) and envelope (E) proteins. Infiltration of these recombinant plasmids into plants yielded no protein expression unless co-expressed with the human chaperone protein calnexin (CNX), upon which expression of both M and E proteins were observed. We investigated the assembly of prM and E into VLPs by transmission electron microscopy (TEM), however, purification of these particles proved difficult with poor reproducibility and VLP yield. This led to the development of an alternative candidate vaccine making use of the antigendisplay technology based on the SpyTag (ST) and SpyCatcher (SC) peptides. The immunodominant epitope of the WNV E protein, domain III (EdIII), was selected for antigen display. Two constructs of the EdIII gene were generated, one with the SC peptide on the 5'- (SC-EdIII) and the other on the 3' end (EdIII-SC). Both SC-EdIII and EdIII-SC proteins were successfully expressed in the presence of the human chaperone protein calreticulin, and purified with yields of 9 mg/kg and 69 mg/kg fresh leaf weight (FLW), respectively. The VLP core selected for the display of the SC-linked EdIII proteins comprised the coat protein of the bacteriophage AP205 with the ST peptide linked to its N-terminus (ST-AP205). Spytagged-VLPs were purified by density gradient ultracentrifugation at a yield of approximately 50 mg/kg FLW. The purified SC-linked EdIII proteins and ST-AP205 VLPs were coupled in vitro, but successful complex formation of AP205:EdIII was only observed between ST-AP205 and EdIII-SC and not when the SC peptide was located on the N-terminus of EdIII. We further demonstrated the successful complex formation of AP205:EdIII in vivo by coinfiltration of the EdIII-SC and ST-AP205 constructs, as well as by extracting leaves of plants infiltrated individually with either of the constructs. Due to the ease of purification and the high yields of AP205:EdIII achieved, the co-extraction process was optimised to obtain the best coupling yield possible by evaluating different FLW extraction ratios and the formation of VLPs was confirmed by TEM. The optimal co-extraction process was established at a FLW ratio of 1:2 ST-AP205 to EdIII-SC yielding approximately 23 mg/kg AP205:EdIII/FLW processed. In this study, we describe the successful production of two particulate candidate vaccines. The first is based on the expression of the WNV prM and E genes in the presence of human CNX and the second is based on the ST/SC antigen-display technology. These outcomes exhibit the potential plants have of being used as biofactories for making significant pharmaceutical products for the ‘One Health' Initiative and could be used to address the need for their local production in LMIC.

It is commonly stated that the cell is the smallest functional unit of life. By analogy, then, the immune cell is the smallest functional unit of the immune system. Natural killer (NK) cells are effector cells of the innate immune system that are responsible for mediating cellular cytotoxicity against virally infected or neoplastically transformed cells. Many phenotypically distinct subpopulations of NK cells have been discovered, usually by dividing cells on the basis of cell-surface markers. These subpopulations are typically described as related to activation or developmental status of the cells. However, how these distinct phenotypes correlate with behavior in e.g. NK–target interactions is less widely understood. There is therefore a need to study NK cell behavior down at the single-cell level. The aim of this thesis is to approach methods that quantitatively describe these single-cell-level behavioral differences of NK cells. Using a newly developed single-cell imaging and screening assay, we trap small populations of NK and target cells inside microwells, where they can be imaged over extended periods of time. We have performed experiments on both resting and IL-2-activated NK cells and quantified their cytotoxic behavior. One major discovery was that a small population of NK cells mediate a majority of the cytotoxicity directed against target cells. A particularly cytotoxic group of cells, which we termed “serial killers”, displayed faster and more effective cytotoxicity. Also, we identified differences between resting and activated NK cells in regard to their migration and contact dynamics. Activated NK cells were found to more readily adhere to targets cells than did NK cells freshly isolated from peripheral blood. Apart form migration and contact dynamics, we have also quantified killing behavior, where NK cells can be seen to exhibit a behavior we term multiple lytic hits on the basis of analyzing target cell fluorescence profiles. We have quantified these heterogeneities and developed tools that can be used to further study and elucidate differences in the behavior of single immune cells. These methods, and automated single-cell analysis methods, will likely play a more important role in the study of immune responses in the future. / <p>QC 20140611</p>

Porcine circovirus type 2 (PCV-2) is considered the major cause of porcine circovirusassociated diseases and is one of the major pathogens in swine producing countries. PCV-2 is a non-enveloped virus with a single stranded circular DNA genome of about 1.8 kb. This encodes the single capsid protein (CP) which is highly immunogenic, as well as a replication-associated protein. Recombinantly expressed CP can selfassemble into virus-like particles (VLPs) that are structurally and immunogenically very similar to native virions. Current commercially available diagnostic kits are VLPbased and are effective at detecting PCV-2 antibodies in sera. However, these diagnostic assays are expensive, therefore limiting their use in developing countries. Plant-based transient expression systems have recently been investigated to express PCV-2 CP for a cheaper diagnostic reagent. The aim of this study was to develop an inexpensive lateral flow device to be able to test for PCV infection in pig herds. Production of PCV-2 CP in Nicotiana benthamiana via transient Agrobacterium-mediated expression was optimised by comparing two expression vectors, pEAQ-HT and pCBP2, and VLPs were also expressed in Escherichia coli. VLPs produced in plants and in E. coli were used to set up a lateral flow device. In addition, various purification methods of VLPs such as ion exchange chromatography (IEC) and sucrose gradient ultracentrifugation were explored to obtain pure VLPs free of bacterial contamination. The VLPs were successfully expressed in N. benthamiana with both pEAQ-HT and pCBP2, and VLPs were subsequently purified on discontinuous sucrose gradients by ultracentrifugation. The assembly of the CP was assessed by transmission electron microscopy, which showed the presence of assembled VLPs. To further purify the VLPs IEC was used, and fully assembled VLPs which were free of contamination were prepared. Purified VLPs expressed in plants and E. coli were successfully used as coating antigen in lateral flow devices, which were able to detect PCV-2 CP antibodies in CP-immunised rabbit sera. E. coli-made VLPs showed higher affinity to PCV-2 antibodies compared to plant-made VLPs. In conclusion, this study has successfully demonstrated the potential to use a plantbased transient expression system to produce affordable diagnostic reagent, especially for developing countries. This is the first study that expressed PCV-2 VLPs using a pCBP-2 expression vector and used PCV-2 VLPs as a coating reagent in the development of a lateral flow test as a proof of concept.