The findings presented in this study give evidence for an ABA-mediated stomatal closure in response to initial dehydration of X. humilis, however, from 60% RWC onwards, stomata were observed to open again. This supplies further support for the hypothesis that resurrection plants actively lose water once protection is accumulated in order to minimize ROS activity and put a stasis on unregulated metabolism.

Includes abstract. / Includes bibliographical references (leaves 66-77). / Genes within the Major Histocompatibility Complex (MHC) of vertebrates code for proteins that are involved in antigen recognition and activation of the adaptive immune response. The hallmark of the MHC is the extremely high levels of polymorphism found at loci. A diverse array of mechanisms have been proposed to explain the generation and maintenance of diversity at MHC loci, including the processes of gene conversion, genetic drift and selection; in the presence of many pathogens balancing selection is thought to be the dominant mechanism by which selection operates. Amino acid substitutions within the peptide-binding region (PBR) of MHC genes further supports the hypothesis that positive selection enhances amino acid diversity in the PBR, such that natural selection will favour PBR diversity in natural populations. This study investigated mechanisms that shape genetic diversity of MHC class II genes in a natural population of the Nile crocodile, Crocodylus niloticus. Using PCR-cloning-sequencing methodology, allelic diversity at MHC Class II genes was investigated and provides evidence for at least two Class II 13 gene families in the Nile crocodile. The Crni-OAB family is homologous to classical Class II vertebrate genes; high levels of both allelic and amino acid diversity characterise this gene family and a strong signal of balancing selection acts to maintain functional diversity. The second family, Crni-OBB, most likely represents a non-classical Class II locus in crocodiles and was characterized by reduced levels of diversity. Analysis suggests that Crni-OBB loci have evolved in a divergent manner to those of the Crni-OAB as balancing selection was not detected within the putative PBR. Results from this study suggest that duplication followed by a recombination event has most likely led to the formation of two distinct crocodilian Class II 13 gene families. Secondly, the relative contributions of balancing selection and random genetic drift in the evolution of extant MHC diversity are examined in a natural population of the Nile crocodile. Temporal variation in allele frequencies for MHC and microsatellite loci was assessed in four successive cohorts of crocodiles from the Okavango Delta, Botswana. Results from this study suggest that a combination of short-term neutral forces such as random genetic drift, together with longer-term selection influence variation at Class II loci in the Okavango Nile crocodile. Loci within the MHC of the Nile crocodile appear to be evolving within a dynamic framework of selection, random genetic drift and recombination. This study is the first of its kind to investigate the respective influence of demography and selection on allele frequencies in a natural population of crocodilians.

Includes abstract. / Includes bibliographical references (leaves 75-86). / The presence and expressIon patterns of orthologues of LEA group 1 genes has been characterised in the resurrection plant, Xerophyta humilis. The group I LEAs (Em I and Em6) were first identified as proteins that were abundantly and specifically expressed during the desiccation and germination phase of angiosperm seed development. The group I LEA genes are characterised by the presence of one or more tandemly repeated 20-amino acid motifs that are particularly rich in Gly residues.

No description available.

Includes abstract. / Includes bibliographical references. / On healing, partial-thickness burn wounds usually result in depigmentation of the skin. This is due to the loss of melanocytes. The lack of pigmentation in the healed wound is particularly prominent in dark-skinned individuals and could result in serious psychosocial consequences such as low self-esteem, stigmatisation and discrimination among sufferers. Methods aimed at investigating rapid and efficient repigmentation in wounded skin are therefore pertinent. The aims of this study were two-fold: i) To promote melanin synthesis in human skin cells using different ratios of human melanocytes (Mc) to keratinocytes (Kc) in an in vitro co-culture system, in order to ensure pigmentation of the skin and, ii) To understand cellular mechanisms that contribute basic scientific knowledge towards clinically improved wound healing.

Includes abstract. / Includes bibliographical references. / Myasthenia gravis (MG) is an autoimmune disease in which pathogenic antibodies block, target or destroy the acetylcholine receptors of the muscle endplate resulting in failure of neuromuscular transmission and fatigable weakness. We have previously shown that a subpopulation of South African MG patients of African genetic ancestry develop a severe extraocular muscle (EOM) phenotype whilst receiving standard immunosuppressive drug therapies. This phenotype associates with a functional c.-198C>G SNP (C>G SNP) in the regulatory region of decay accelerating factor (DAF), a complement regulatory protein, which is essential for protection against complementmediated damage. MG patients are treated with prednisone as the first-line immunosuppressant and frequently, an additional steroid-sparing agent, such as azathioprine, cyclosporin A or methotrexate. We hypothesised that MG patients with the C>G SNP when treated with immunosuppressant drugs, may have lower DAF protein levels contributing to increased susceptibility to autologous complement-mediated damage at their EOMs. This study tests this by comparing the effect of prednisone, azathioprine, cyclosporine and methotrexate individually and prednisone in combination with each of these steroid-sparing agents on wild-type and C>G DAF protein (western blotting) and mRNA (quantitative real time PCR) levels and promoter activity (luciferase reporter assays). These experiments were performed in EBV transformed lymphoblastoid cell lines from control individuals with wild-type DAF and MG patients carrying the C>G SNP. As a more representative model for this study the experiments were repeated in mouse skeletal muscle cells because there was no available EOM cell line. In support of the hypothesis of this study, prednisone, cyclosporin A and azathioprine individually was shown to repress C>G DAF protein levels while having either no effect on wild-type DAF or slightly activating it. Importantly, methotrexate was the only drug that was able to increase C>G DAF lymphoblast protein expression and therefore holds promise as a potentially effective treatment for MG patients with the C>G SNP. Moreover, using a calcein assay, clinically relevant doses of prednisone in combination with MG patient sera was shown to significantly increase the susceptibility of C>G DAF lymphoblasts to cell lysis (82% C>G DAF lymphoblasts vs. 9% wild-type DAF lymphoblasts). These results suggest that MG patient sera contain factor(s) that together with prednisone may also be responsible for the susceptibility of the EOMs in these patients to injury. The results show that the levels of DAF protein and mRNA did not always match which suggests that the drugs tested may regulate the DAF protein at a posttranscriptional level. In a mouse skeletal muscle model, Daf (equivalent to human wild-type DAF) protein expression was consistently repressed by prednisone treatment but activated by cyclosporine A, azathioprine and methotrexate. Furthermore, co-treatment of prednisone with either of the steroid-sparing drugs showed that azathioprine, and low doses of cyclosporin A and methotrexate were able to overcome the repressive effect of prednisone-only treatment on Daf expression. These observations indicate that the regulation of Daf may be species and/or cell-type specific. Together the results from this thesis suggest that in EOMs, where DAF is already downregulated, compared to other muscles, our MG patients with the C>G SNP may have an increased susceptibility to complement-mediated damage when treated with prednisone, which further represses C>G DAF expression.

Bibliography: leaves 126-145. / Expression of the nefgene from HIV-1 subtype C in Nicotiana benthamiana was carried out using a TMV -based vector with the aim of developing a plant-based candidate vaccine for HIV-1. The nef gene of the DU151 isolate of mV-l subtype C taken from a recently seroconverted individual was amplified by PCR with a deletion of 59 amino acids from the cytotoxic N-terminal. The amplified gene was inserted into a bacterial expression vector pProEXHTb for rapid expression of Nef protein, which was used as a diagnostic tool in the development of an indirect ELISA assay for detection of Nef in Nicotiana benthamiana. An indirect ELISA assay was developed using a commercially available polyclonal anti Nef antiserum raised in sheep. The role of codon optimization in expression of Nef in benthamiana was investigated. A synthetic nef gene was constructed based on the codon usage of benthamiana. The plant codon optimized gene and the wild type nef genes were inserted into the TMV -based vector pBSG1057. RNA transcripts from both constructs were used to infect young benthamiana plants. Expression of nefmRNA was confirmed by RT -PCR analysis of total RNA extracted from plants inoculated with respective constructs. The Nef protein was expressed at low levels which were detectable by ELISA. Nef was detectable by Western blot after concentration of plant extract using a membrane filter device. Quantitative analysis of Nef expression in plants was done by western blot on concentrated plant extract from three separate infections. Codon optimization of the nef gene improved the expression of Nef by a factor of about two.

During development of the eye, invagination of the optic cup gives rise to a double layered neuroepithelium, part of which differentiates into the retinal pigment epithelium (RPE). The molecular mechanisms which control differentiation of the RPE are not known. The present study was undertaken to determine 1) when induction of the RPE has occurred in chicken embryos and 2) to investigate whether contact with the presumptive neural retina (NR) is required for RPE differentiation. In order to investigate when RPE induction has occurred, early expression of two genes involved in pigmentation were investigated. Digoxigenin-labeled tyrosinase and tyrosinaserelated protein-2 (TRP-2) riboprobes were synthesised and used in ISH reactions on embryonic eye tissue. Tyrosinase transcripts were first detected at stage 19.5 (70-71 hours) and TRP-2 transcripts were detected a few hours earlier at stage 18.5 (67-69 hours) of embryonic development. These results indicate that induction has occurred by stage 18.5, approximately ten hours before distinct granules are visible in the RPE. The tyrosinase and TRP-2 transcripts were always localised first in the optical axis of the eye in the region where pigment granules are first present. This indicates that differentiation of the RPE proceeds from the optical axis of the eye cup outwards towards the periphery and that induction of the RPE may also proceed in this direction. To determine whether the presumptive NR is required for RPE induction, synthetic barriers were inserted into the uninvaginated optic vesicle of chicken embryos at stage 11 (40-45 hours) of development. The embryos were cultured in vitro until the optic vesicle had invaginated and sectioned to locate the barrier. Results suggest that contact with the presumptive NR may not be necessary for RPE induction.

Vitiligo is a common depigmenting skin disorder with devastating psychological implications. Although evidence strongly suggests that melanocytes die in various forms of vitiligo, it is also evident, at least in certain cases, that melanocytes/melanoblasts survive in an undifferentiated stem cell form and it is from these that re-pigmentation is assumed to occur. Treatment of this distressing condition remains 'hit-or-miss' and patients' responses are unpredictable. This study aims to integrate prognostic variables such as the molecular profile of vitiliginous lesions and the patient's epidemiological profile, to develop a reliable and sensitive prognostic test enabling clinicians to plan an individualized, rational therapeutic approach for their vitiligo patients. The analysis of tyrosinase and TRP-2 mRNA expression in vitiliginous skin using quantitative RT-qPCR revealed the presence of melanoblasts/melanocytes in 60% of patients (n=21). When this survival was calculated as a percentage of the mRNA expression in the pigmented positive control specimen, it was found to range from 4% to 46% survival. Patients were subsequently treated with either potent topical corticosteroid ointment or 5% khellin cream in combination with daily sunlight exposure for 3 months and their clinical response was then assessed and correlated to the lesional melanocyte status. A good response (>50% repigmentation) was found in 3/5 patients demonstrating both tyrosinase and TRP-2 gene expression, in only 1/4 patients demonstrating TRP-2 gene expression alone and in 0/3 patients demonstrating only tyrosinase gene expression. The presence of both tyrosinase and TRP-2 mRNA expression is therefore a significant positive prognostic indicator. A significant correlation (R2 =0.8919) was found between melanocyte survival and response to khellin therapy suggesting that therapeutic modalities stimulating melanocyte proliferation and migration should be employed in patients demonstrating a melanocyte/ melanoblast reservoir. Conversely, an absence of melanocyte/melanoblast markers is predictive of a poor response to treatment with 7/9 patients lacking lesional tyrosinase or TRP-2 mRNA expression demonstrating <25% repigmentation at the conclusion of the 3 month treatment period. Therapeutic options such as surgery or cosmetic camouflage techniques should be considered for these patients. The epidemiological profile was not found to be a significant prognostic factor in predicting treatment response.

Includes bibliographical references. / The red agarophytic alga, Gracilaria gracilis, occurs naturally within Saldahna Bay, South Africa. Gracilaria species are commercially exploited for their hydrocolloid agar, valued at US$132 million per annum (FAO, 2002). G. gracilis is thus a valuable resource for South Africa in terms of generating foreign currency through export. Nitrogen limitation occurring within Saldahna Bay during the summer to autumn months, however, is considered to be the major factor preventing commercial cultivation of G. gracilis. Algae possess various survival mechanisms that generally involve the activation or repression of various gene regulatory systems. Such regulatory systems aid in preserving energetic homeostasis at a cellular level, thereby allowing maximal algal survival during adverse environmental conditions. Microarray technology is a highthroughput global gene analysis tool, allowing for the simultaneous identification of thousands of differentially expressed genes in a single assay. Combined with genomic technologies such as genetic engineering, microarray technology has the potential to identify genes that confer enhanced in vivo tolerance to a specific stress. In the current investigation, microarray technology was utilized to identify genes differentially expressed in G. gracilis in response to nitrogen limitation. A total of 39 differentially expressed genes were identified. Ofthe 35 genes that were sequenced, 13 were assigned a putative function based on sequence similarity to genes deposited in various databases. The remaining sequences corresponded to either unknown hypothetical proteins or else had no significant homology to any of the sequences in the databases. Overall, the current investigation has provided a foundation for future research into the biological role(s) of each of the identified differentially expressed genes within their true isogenic background. Future studies may thus allow for improved crop tolerance and stability, enhancing aquaculture of this valuable resource in Southern Africa.