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.

The targeting of liposomes to cells and the delivery of the liposomal contents into the cells have been investigated using either α-melanocyte stimulating hormone or Ricin-B-chain as ligands for promoting the binding of liposomes to cells. α-melanocyte stimulating hormone has been conjugated to liposomes and to Ricin-A-chain via the Lys₁₁ residue without significant loss of biological activity. The resulting conjugates were found to bind to Bl6 melanoma cells which express receptors for the hormone. Hormone targeted ricin was shown to be toxic to the cells, indicating receptor mediated internalisation of the conjugate. The hormone targeted liposomes however were unable to mediate the delivery of cytotoxic levels of methotrexate. Ricin-B-chain, a lectin which mediates membrane translocation of the toxic ricin-A-chain, was examined for its applicability for targeting of liposomes to cells. This lectin was shown to promote the binding of liposomes to cells and to mediate the delivery of cytotoxic concentrations of methotrexate. Further evidence of functional ricin-B-chain mediated intracellular delivery of the liposomal contents was shown by liposome mediated transformation of cells, and delivery of nuclease into the cell resultin in digestion of genomic DNA. The study demonstrates that α-melanocyte stimulating hormone is unsuitable as a ligand by which to achieve delivery of large quantities of material into cells, although cell-specific targeting can be achieved. Ricin-B-chain is however ideally suited for this task, though is less cell-specific. This finding may be of use in studies in which investigators wish to achieve intracellular delivery of compounds.

Haliotis midae is an economically important South African abalone species which has been cultured since the 1990s. H. midae farming constitutes approximately 93% of the country's total marine aquaculture industry. However, the slow growth rate of this animal and the potential for disease outbreaks remain a concern for farmers. Therefore, government and private institutions have joined efforts to investigate ways of enhancing abalone production, to increase abalone growth rates and improve disease resistance. Several studies have shown that probiotic microorganisms can significantly increase the growth rate and disease resistance of H. midae. To date, no comprehensive studies have been conducted to characterise these physiological improvements at the molecular level. Thus, the aim of this study was to evaluate the effect of a probiotic-supplemented diet on the haemocyte proteome in H. midae. Two probiotic-strains, Vibrio midae SY9 and Debaryomuces hansenii AY1, were introduced into H. midae via a kelp-based feed. Changes in the haemocyte proteome were analysed using isobaric tag for relative and absolute quantification (iTRAQ) coupled with LC-MS/MS. A total of 128 haemocyte proteins were identified. Proteins that were found to vary significantly in their expression levels in haemocytes sampled from abalone fed the probiotic-supplemented diet were identified as COP9 signalosome subunit 4, phosphorylase, T-complex protein 1 subunit gamma, V-type proton ATPase subunit B, Rab 1 and Ra-related protein Rab 1A. Differential expression of COP9 signalosome subunit 4 (up-regulated) and Ras-related protein Rab 1A (down-regulated) was confirmed by western blot analysis. Bioinformatics analysis revealed proteins with immune class GO terms that functioned in metabolism, apoptosis, cell adhesion, immune response, stress response, and response to endogenous and external stimulus. Hierarchical clustering analyses showed that proteins with similar expression patterns mostly belonged to the same immune classes. Analysis of protein interaction networks indicated that all the differentially expressed proteins may indirectly interact with each other. It was also found that the neurotrophic tyrosine kinase receptor was the central molecule within the interaction network, suggesting that this protein may play a crucial role within the protein interaction network that contains all the differentially expressed proteins. Biochemical pathway analysis indicated that phagosomal maturation was the most significant canonical pathway identified, in which V-type proton ATPase and Ras-related protein Rab have fundamental importance. Changes in Ras-related protein Rab 1A expression were further investigated in the cytosolic and membrane fractions of haemocyte cells using western blot analysis and cellular immunochemistry. The expression of this protein was found to be down-regulated both in cytosolic and membrane fractions from haemocytes sampled from H. midae fed a probioticsupplemented diet. Although an association between Ras-related protein Rab 1A and F-actin (cell cytoskeleton) was not detected, confocal microscopy confirmed Ras-related protein Rab 1A down-regulation. Thus, results from this study suggest that Ras-related protein Rab 1A may play a key role in H. midae immune response, when this species of abalone is fed with a probiotic-supplemented diet. This is the first time that a large-scale proteomics approach has been used to investigate proteome changes in haemocytes sampled from H. midae fed a probiotic-supplemented diet. The findings of this study, regarding the protein profile, interaction networks, molecular pathways and a putative molecular indicator of H. midae immune response, provide a foundation from which future studies can be conducted in order to increase our understanding of how probiotics affect the abalone immune system.

[Not OCR'd]

Bibliography: pages 304-309. / The field of metal determination as a part of pollution studies, has been critically examined and metal pollution may be defined in one simple statement: The presence of metal binding proteins confirms toxic metal pollution. It has been shown that current methods of metal determination in biological systems are of little use. This has been illustrated by both a review of metal concentration in Southern African coastal water, sediments and biotopes, and by a comparative baseline study of organisms from Gough Island and Mar ion Island. These showed that extrapolation of results from one geographical area to another are invalid and that this interpretation is made difficult by factors such as age, sex, size life stage of the organisms. Furthermore, it was shown that many reports on metal pollution do not even mention fundamental information such as the size or the sex of the animals. Metal pollution could be linked to metal binding protein through an independent pollution er i ter ia, for example, the out of season moulting of crayfish. The new definition of metal pollution has then been tested by application to five different organisms (crayfish, Jasus lalandii; hermit crab, Diogenes brevirostris; shrimp, Palaemon pacificus; black mussel, Choromytilus meridionalis and limpet, Patella granularis) kept under identical conditions and it was shown that a much more meaningful interpretation of the results could be made. The new definition was al so tested with two naturally occurring metal accumulating organisms (whelk, Bullia digitalis and "kikuyu" grass) and it was shown that dramatic increases in metal may not necessarily be toxic. It was concluded that less effort and time should be spent on metal analysis in determination of metal pollution and attention should rather be directed to the presence or absence of metal-binding proteins.

Bluetongue is a disease of domestic and wild ruminants caused by Bluetongue virus (BTV). It has caused several serious outbreaks, the most recent occurring in Northern Europe in 2006 during which high mortality rates of livestock were reported. The only vaccines currently approved and commercially available for use are live-attenuated or inactivated virus strains and although these are effective, there is the risk of reversion in the case of live-attenuated strains to more virulent forms by recombination. Another drawback associated with the use of live-attenuated virus vaccines is that they are not DIVA (differentiate infected from vaccinated animals) compliant, this means that naturally infected animals cannot be distinguished from vaccinated animals. Recombinantly produced vaccines would be preferable to minimize the risks associated with live-attenuated virus vaccines and also enable the development of candidate vaccines that are DIVA-compliant. A number of recombinant vaccine candidates have been developed against BTV, with the most promising vaccine consisting of BTV virus-like particles (VLPs). BTV VLPs were successfully produced in insect cells by the co-expression of the four BTV capsid proteins (VP2, VP3, VP5 and VP7). Sheep vaccinated with insect cell-produced BTV VLPs were shown to be protected against challenge with wild type virus. However, the high costs associated with the production and scale-up of BTV VLPs in insect cells has possibly limited their widespread application. Plants – such as N. benthamiana – provides a safe, efficient and cost effective system for the production of recombinant proteins. In this study the best plant expression vector with which to co-express the four BTV serotype 8 (BTV-8) VPs – which direct formation of BTV-8 VLPs – was identified. Expression and purification of the BTV-8 VLPs was optimised with the aim of producing a VLP-based vaccine for BTV-8. It was further undertaken to develop two novel second generation plant-produced protein body (PB) vaccines that are DIVA compliant. Mice were immunised with the plantproduced VLP and PB vaccines in order to analyse their ability to elicit humoral immune responses.