Includes abstract. / Includes bibliographical references (leaves 136-158). / HIV-1 Gag virus-like-particles (VLPs) produced in various expression systems are potent stimulators of both cellular and humoral immune responses in animal models. The encapsidation of large concentrations of random cellular RNA species is known to accompany the assembly of HIV virus particles. This RNA plays a crucial role by serving as a molecular scaffold for the assembly of Gag structural proteins into particles. Non-pseudotyped VLPs that do not present any HIV envelope glycoproteins are regarded as inert particles as they contain no replicative nucleic acid and are presumed to be unable to deliver encapsidated RNA for expression in inoculated individuals. Live virus cellular entry studies have shown that non-pseudotyped Gag particles are destined for degradation in acidified vesicles subsequent to receptor independent cellular entry. In addition to host cell RNA incorporation, Gag VLPs produced in insect cell-based, baculovirus expression systems have been observed to incorporate the baculovirus-derived Gp64 envelope glycoprotein. Gp64 has been shown to be efficient at enabling the delivery and expression of genes from recombinant baculoviruses and other Gp64 pseudotyped live viruses in mammalian cell lines both in vivo and in vitro. This study, therefore, set out to establish for the first time whether inert, baculovirus-derived (Gp64 pseudotyped) Gag VLPs could mediate delivery and expression of randomly encapsidated RNAs in mammalian cell lines.

Includes abstract. / Includes bibliographical references (leaves 138-161). / This dissertation describes three major sets of experiments, all of which involved the construction and use of various reciprocal chimaeric MSV constructs. First, chimaeric viruses were used in genetic complementation-type experiments to investigate the biological significance of interactions between the two virion-sense open reading frames (ORFs) of MSV, their products, and the rest of the genome. Six chimaeric MSV constructs were made by reciprocally exchanging the ORFs encoding movement protein (MP) and coat protein (CP) individually, and in pairs, between MSV-Kom and MSV-Set, which share just 78% overall nucleotide identity. Analysis of symptomatology and infection efficiency of chimaeras and wild-type parental viruses revealed evidence of functionally relevant specific interactions between MSV MP and CP.

Bibliography: leaves 72-77. / Jasmonic acid (JA) is a plant hormone with diverse functions, ranging from development to stress responses. Moreover, a role for JA in mediating defence against pathogen attack has been established, seemingly specific against necrotrophic pathogens such as Botrytis cinerea. Despite these known roles of JA, it is not known exactly how JA activated downstream responses, such as induced gene expression. To further our understanding of JA signalling, this work aimed to identify new components involved in JA signal transduction. A novel screening method based on lack of anthocyanin accumulation after exogenous application of the methyl ester of JA, methyl jasmonate (MeJA), was employed. A recessive, monogenic mutant, jasmonic acid modified2 (jam2), was isolated from T-DNA activation tagged lines and characterized genetically and phenotypically. jam2 was found not to be T-DNA tagged as the T-DNA segregates independently of the mutation. jam2 is unlikely to be an anthocyanin biosynthetic mutant but shows delayed anthocyanin accumulation after exogenous MeJA treatment. Resistance to MeJA root growth inhibition is a phenotype shared by all JA insensitive mutants. Contrary to this, jam2, like Col-0, exhibits stunted root growth on MeJA. The expression of the antifungal peptide, PDF1.2 can be induced by exogenous MeJA treatment. To assess how PDF1.2 expression was affected in jam2, plants were treated with external liquid and vaporous MeJA. Interestingly, the PDF1.2 expression pattern after MeJA application (liquid or gaseous) was biphasic for Col-0, jam2 and jar1. However, compared to Col-0 and jar1, jam2 appeared to be affected in the first induction peak upon liquid MeJA treatment, whilst in the second after gaseous treatment. PDF1.2 expression can also be seen as a marker for JA-mediated defence responses. Upon infection with B. cinerea, jam2 and jar1 showed intermediate resistance and faster PDF1.2 expression, compared to Col-0 and coil-1. These findings suggest that jam2 is possibly involved in temporal regulation of anthocyanin accumulation and PDF1.2 expression after MeJA application and B. cinerea infection. Therefore, jam2 may define a novel component within the JA signalling pathway and further genetic and phenotypic characterisation could confirm this.

Pigment formation in melanocytes is the end-point of a series of biochemical reactions involving numerous melanocyte-specific proteins including, inter alia, the enzymes tyrosinase, tyrosinase-related protein-1 (TRP-1 ), tyrosinase-related protein-2 (TRP-2) and the melanosomal protein encoded by the P gene. The function of tyrosinase and TRP-2 have recently been clarified, but the roles of TRP-1 and the P protein remain unknown. The first aim of this study was to examine the expression of these proteins at a transcriptional and translational level in order to provide more insight into possible mechanisms which may lead to changes in melanoma cell differentiation. Three human melanoma cell lines (Mel 1, Mel-2 and Mel-3) with varying levels of pigmentation (highly melanised to amelanotic) were examined by enzyme assays and RNA quantification methods. The results showed gene expression of all four genes in the highly melanised Mel-1 and amelanotic Mel-3 cell lines. TRP-1 and TRP-2 were not expressed in the melanised Mel-2 cell line. These results suggest that there is no correlation between tyrosinase gene expression and level of pigmentation in these cell lines. In addition, they show that the level of pigmentation of human melanoma cell does not necessarily correlate to the level or pattern expression of the tyrosinase gene family. Furthermore the results of the present study show that the P gene is expressed at high levels in all the melanoma cell lines, irrespective of their level of pigmentation . The second broad aim of this study was to determine the effect of melanocytestimulating hormone (a melanogenic stimulator) on melanogenesis in Mel-1 cells. Mel-1 cells, which were exposed to 10⁻⁷ M MSH for 6 days, showed no change in tyrosinase mRNA levels, but the mRNA levels of TRP-1, TRP-2 and the P gene were reduced. This suggested the presence of a possible co-ordinated down-regulatory mechanism in the Mel-1 cells under the influence of MSH.

Wounding of skin initiates a complex series of overlapping cellular events that culminates in the formation of a scar. The negative consequence of most cutaneous scars is the lack of adequate repigmentation of the neoepidermis. In order to effect successful wound healing by restoration of effective skin barrier function, keratinocytes must proliferate and migrate to cover the gap created by the wound in an efficient and timeous manner. Melanocytes are not thought to actively participate in wound repair and closure, since they are primarily responsible for maintenance of normal skin pigmentation. Mechanisms of keratinocyte migration during the re-epithelialisation phase of cutaneous wound healing have been well documented. However, it is not clear how melanocytes contribute to neoepidermis formation and repigmentation. Provision of an optimal epidermal milieu would enable restoration of pigmentation of cutaneous scars for a satisfactory cosmetic outcome. This dissertation contributes to the understanding of melanocyte participation and proliferation during re-epithelialisation of skin using an in vitro skin organ culture model of wound healing. To determine whether the culture model used in this study was reliable, skin explants were cultured over a period of 12 days, fixed and processed for histological analysis. By measuring the lengths of the developing epidermal tongues, it was noted that epiboly was the initial event that occurred at the wound edges. After day two of culture, growth of the epithelial tongues was observed from the wound edges, which peaked at day five. The length of the epidermal tongues remained constant for the remaining culture period up to day twelve, at which stage complete wound closure was observed in some samples. Histological analysis of the tongues revealed that the cultured skin remained viable for the duration of culture period. To establish to what extent keratinocyte proliferation contributed to the growth of the tongues, immunohistochemical staining using the proliferation marker, Ki67, was used. Results show that after an initial lag phase, with no detectable cell proliferation at the wound edges, dividing keratinocytes were seen at day two post-wounding. The number of dividing keratinocytes peaked at day five, where after the numbers remained constant until day twelve of culture. This result supports the validity of the culture model. To uncover whether melanocytes re-enter the neoepidermis during wound during re-epithelialisation, the number of melanocytes per unit of basement membrane was determined using immunohistochemical staining. The melanocyte-specific marker, MART-1/MelanA, was used in conjunction with the proliferation marker, Ki-67, in an optimised dual immunolabelling protocol {Petersen, 2012 #397} to establish whether melanocytes proliferate during re-epithelialisation of wounds. Melanocytes along the basal layer of the epithelial tongues and in the normal epidermis were located and counted. Basal melanocytes (MelanA+) were seen from day two onwards in the normal epidermis and from day five onwards in the developing epidermal tongues. However, at days ten and twelve of the culture period. dividing melanocytes (Ki67+/MelanA+) were seen in the epidermis in locations: proximal to the wound area, at the wound edge and in the developing tongues. This result suggests that melanocyte entry into the wound is delayed until after keratinocyte proliferation has brought about the beginning of neoepidermis formation and growth. This result also demonstrates the proliferation potential of differentiated melanocytes and that de-differentiation of melanocytes can occur under favourable culture conditions as can be seen in this culture model.

Includes bibliographical references. / Xerophyta viscosa (Baker) is a resurrection plant with the ability to survive desiccation and rehydrate upon watering with minimal tissue damage. XVEF was isolated by differential screening of aX viscosa dehydration stress cDNA library. Reconstruction of the full length XVEF cDNA was conducted utilizing overlap extension PCR. XVEF codes for a putative calcium-binding protein and sequence analysis indicated that it has a 708 bp ORF corresponding to a protein with a molecular mass of26.95 kDa, has a conserved calcium-binding EF-hand motif, potential phosphorylation sites, a pI of 6.49 and a putative conserved transmembrane domain spanning residues 90-107. Northern blot analyses of total RNA indicated that XVEF transcript increased 48 hours after 100/-lM ABA application and was present between 12 and 48 hours in response to a low temperature stress (4°C). A second gene, XvCaM, was isolated from a low temperature (4°C) cDNA stress library. Sequence analysis indicates that it has a 450 bp ORF corresponding to a 16.39 kDa protein, a pI of 3.90 and potential phosphorylation sites. It apparently encodes a calcium-binding protein with putatively three EF-hands which showed the highest similarity to plant calmodulins. XVCAM was heterologously expressed in E. coli as a fusion protein with a 6X His-tag and it was shown to be a functional protein that binds Ca2+ by utilizing a 45CaCh overlay assay. Northern blot analyses of XvCaM using total RNA under low (4°C) and high temperature treatment (42°C) showed constitutive expression levels of the transcript. Under the dehydration/rehydration treatment transcript levels decreased between 40% R WC dehydration and 26% RWC rehydration. The northern blot conducted with polysomal RNA isolated from 150 mM NaCI treatment also showed constitutive expression. Western blot analyses using anti-XVCAM polyclonal antibodies showed that the protein accumulated at 24 hours during the NaCI stress and at 15% RWC (dry) to 40% RWC (rehydration) during the dehydration/rehydration stress. The northern and western analyses results suggest that XVCAM undergoes post-translational modifications and XvCaM mRNA is possibly stored for rapid recovery processes upon rehydration. These results indicate that XVEF and XvCaM are possibly calcium-binding proteins most likely involved in modulating stress-responsive calcium-signaling cascades.

Bibliography: leaves 60-73. / Eragrostis nindensis and Eragrostis tef are wild and domestic grasses respectively that belong to the subfamily Eragrostideae. E. nindensis is desiccation tolerant while E. tef is desiccation sensitive. The responses of these plants to water-deficit stress were studied using molecular and physiological approaches. A cDNA library of E. nindensis was screened to identify differentially expressed genes during dehydration. Physiological studies included monitoring changes in photosynthesis, respiration, ultrastructure and membrane integrity of plants during dehydration and rehydration. The differential screening of the cDNA library, using a radio-labelled cDNA from hydrated and dehydrated leaves respectively, revealed two genes, referred as Nin-19 and Nin-44, that were differentially expressed in dehydrated leaves of E. nindensis. These genes were sequenced and partially characterized. Nin-19 did not show considerable identity with any known genes and was not studied any further. Nin-44 was identified as a dehydrin-like gene with approximately 99 % identity to seven water-deficit stress responsive genes on a section of about 60 bp near the 3' end. As its sequence was found to represent a partial insert size, two forward and reverse primers were designed to find the full length through RT-PCR. Despite repeated attempts, no products that could be used in subsequent procedures were achieved using this technique. Hence, further characterization of this gene also could not be performed and different approaches were suggested. The physiological studies showed that E. nindensis is desiccation tolerant but E. tefis not, the latter dying below RWC of about 33 %. Difference among plants in physiological responses became evident after 6 days of dehydration treatment, which resulted in a decline of RWC to 65%, 39% and 33% in E. nindensis, E.tif(R) (red-seeded) and E. tef (W) (white-seeded) plants respectively. A significant decrease in photosynthesis, transpiration, stomatal conductance, and an increase in electrolyte leakage occurred in all species after 6 days of dehydration, but leaves of E. tef (W) did not recover from this level of dehydration when watered. Instead, new leaves were observed to re-grow from the stem nodes. The leaves of red-seeded variety of E. tef did recover fully from RWC of 39 %. After a further 3 days dehydration both varieties of E. tef died. On the other hand, E. nindensis was found to survive extreme water-deficit (-10 % RWC tested here) and recovered full physiological activity when watered. The electrolyte leakage study on these plants indicated major injury on E. tef(W), being intermediate in E. tef(R) and very low in E. nindensis, which coincided with the trend of declining in RWC and other metabolic activities measured. The ultrastructural study on E. tef varieties also showed evidence of the damage caused by dehydration, but the difference among these species was not significant enough to indicate the level of susceptibility of the plants to dehydration damage. The study demonstrated that E. tef varieties are not drought tolerant and showed a considerable difference in their responses to water-deficit stress with each other and with respect to E. nindensis. However, E. tef (R) seems to have a better control over transpiration and some form of repair mechanism operational at least until dehydration to 39 % RWC. This is proposed to be a better performing cereal in conditions of water stress. On the other hand E. nindnsis did not suffer major injury from the dehydration treatment and confirmed to be desiccation tolerant.

Bibliography: leaves 94-102. / Resistance to antibiotics poses a serious threat to healthcare and new drugs are needed. This is especially true for tuberculosis (TB), which is at epidemic levels in South Africa. Multidrug-resistance in Mycobacterium tuberculosis makes TB more difficult and expensive to treat and increases mortality rates. The surfaces of 12 seaweed species found in South African coastal waters were screened for the presence of antibiotic-producing actinomycetes. Of the 67 strains isolated, 26 exhibited antibacterial activity against Mycobacterium aurum A+ and for Enterococcus faecium VanA. These actinomycete strains were physiologically characterised. Three strains showing very strong antibacterial activity were further characterised by the use of chemical taxonomy, DNA sequencing and scanning electron microscopy and were shown to belong to the genus Streptomyces. A strain not showing activity was shown by the same methods to belong to the genus Micromonospora. Partial purification of the active compounds was carried out on the three strains exhibiting strong antibacterial activity. All were shown to produce moderately to highly polar compounds.

Oculocutaneous albinism is a congenital heritable disorder characterised by hypopigmentation of the eyes, hair and skin, together with visual acuity. Ten albinism have been photophobia, nystagmus and decreased different forms of oculocutaneous described. Of these, the tyrosinasepositive and rufous forms are particularly prevalent in Southern Africa. The tyrosinase-positive form manifests as two distinctly different phenotypes with some individuals developing pigmented freckles (ephelides) on their sunexposed regions and others never developing these freckles. To date, studies on the pathophysiology of tyrosinasepositive albinism have been restricted to examination of hairbulbs rather than skin biopsies. The present study utilises light and electron microscopical investigations of both hairbulb and skin melanocytes from the tyrosinasepositive and rufous forms of oculocutaneous albinism to elucidate the cellular aetiology of these disorders. In addition, melanocyte numbers were quantitated in the tyrosinase-positive skin to establish whether the observed hypopigmentation results from a decrease in the size of the melanocyte population. The melanocyte numbers in regions with ephelides were similarly quantiatated in order to see whether these freckles were a consequence of increased melanocyte numbers. The results show, for the first time, that the hypopigmentation observed in tyrosinase-positive albinos is not a consequence of melanocyte paucity and that the regions of ephelides do not contain more melanocytes. Ultrastructural studies show that regions of ephelides and non-ephelides are distinctly different. In regions of ephelides, numerous fully melanised stage IV eumelanosomes, in addition to unmelanised stage I melanosomes, were seen in the melanocyte cytoplasm. Both stage IV and unmelanised stage I melanosomes are transferred to keratinocytes. In ephelis-free regions the melanocyte cytoplasm was filled with numerous unmelanised stage I melanosomes with no evidence of stage IV melanosomes. This suggests that the defect underlying tyrosinase-positive albinism relates to the melanisation process rather than the process of melanosome assembly. In regions of ephelides, the melanocytes are able to produce numerous stage IV melanosomes, and, because these ephelides occur only on sunexposed regions, it is postulated that U.V. exposure induces a "back mutation" resulting in the restoration of the process of melanosome melanisation in these regions of skin. Numerous aberrant melanocyte organelles were also observed in tyrosinase-positive skin. These included dilated RER, distorted mitochondria and bloated Golgi. These ultrastructural observations support the recent report of a candidate gene for tyrosinase-positive albinism, which, it is speculated encodes an integral melanosomal membrane tyrosine transport protein. A defect in tyrosine transport into melanosomes would explain the observed incomplete melanisation of melanosomes in tyrosinase- positive albino skin. In rufous albinism, several aberrant melanosomal shapes were also seen, including "racquet", "crescent"and "comma"shaped melanosomes, all of which were fairly densely melanised. These melanosomes were also about 30% smaller than normal Negroid melanosomes. Upon transfer to keratinocytes, the melanosomes formed membrane-bound "rosette-like" clusters. These findings that the defect in rufous albinism seem to relates suggest to the melanosomal assembly process rather than the melanisation process.

Includes bibliographical references. / Bacteria that are capable of degrading alkane fractions, C12-C13 and C14-C17, were isolated from oil-contaminated soil collected from the petrochemical company, CALTEX Refineries, South Africa. A total of twenty-three environmental strains were isolated. A preliminary procedure, Nile Blue A straining suggested that twelve of the twenty-three environmental strains might accumulate polyhydroxyalkanoates (PHAs) in their cytoplasm, which is a good candidate for biodegradable plastics. The gene that catalyzes PHA polymerization, phaC, was detected using PCR in some of the environmental strains. The strains of interest were identified and characterized biochemically using various techniques and later sequenced by 16S rONA PCR. The environmental isolate 2 showed a 99 % identity to Pseudomonas aeruginosa BHP7 -6 and was for that reason given a name, Pseudomonas aeruginosa MB2SA. P. aeruginosa MB2SA was shown to possess a 0.5 kb internal fragment corresponding to the phaC gene and capable of degrading the alkane fractions, Cn-CI3 and CI4-CI7, effectively. P. aeruginosa MB2SA was shown to grow optimally in the long alkane fraction, Cw C17, and was further grown in pure alkanes, n-dodecane, n-tetradecane, and hexadecane for comparison. In addition, the strain, P. aeruginosa MB2SA, was grown in an appropriate medium for PHA synthesis and high yields of PHA were observed when both the long alkane fraction, Cw CI7, and pure alkane, hexadecane, were employed as sole carbon sources respectively.