Includes bibliographical references (leaves 134-151). / The glucocorticoid receptor (GR) is a ligand-activated transcription factor which, due to its central role in anti-inflammatory responses, is a target of many therapeutically prescribed drugs. The GR undergoes multiple post-translational modifications, including phosphorylation and acetylation; however the role of GR acetylation in transactivation is unclear.

Includes abstract. / Includes bibliographical references (leaves 170-209). / The Xerophyta viscosa aldose reductase cDNA (XvAld1) was isolated from a dehydration library. Gene transcripts that are upregulated during stress are normally involved in protection and/ or adaptation, leading to stress tolerance. The genomic organisation of XvAld1 was characterised using Southern blot analysis and DNA sequencing. The results revealed more than one copy of the gene with a complex banding pattern that was partially resolved by sequencing. The sequencing of PCRamplified genomic clones showed that the gene is organised into nine exons and eight introns spanning ~2.9 kb. The observed nucleotide differences between the sequenced clones could reflect polymorphisms between different copies of the gene. An 870-bp clone of the 5′ untranslated region, matching the 5′ leader sequence on the XvAld1 cDNA was analysed for cis-acting response elements. Many of the sequence motifs matched those for hormonal regulation, organ specific expression, dehydration, high or low temperature responses, light and phytochrome responsiveness, wounding, as well as G-box, CAAT and TATA-boxes.

Includes bibliographical references. / Gracilaria gracilis is a commercially important alga, previously harvested from the wild South African population in Saldanha Bay as a feed for marine organisms and as a source of commercially important agar. Since 1974 however, a number of sporadic population collapses has lead to the destruction of this once flourishing resource. After numerous failed attempts at re-establishing this industry, the need to develop an alternative farming strategy became evident. In order to devise such a solution, a better understanding of the tolerances and responses of this alga to the environmental parameters responsible for the downfall of the population is required. Although the exact reasons remain unclear, Jaffray et al., 1997 have reported that increased water temperature in Saldanha Bay may be a contributing factor as the population collapses have repeatedly occurred during summer months. Thus the effect of heat stress on G. gracilis has been selected for this study.

Includes bibliographical references (leaves [123]-141). / Bacteroides species are Gram-negative obligate anacrobes that live in the gastrointestinal tract of mammals and are thought to account for approximately 30% of the colonic microbiota. Certain Bacteroides species, such as B. fragilis and to a lesser extent B. thetaiotaomicron, can become opportunistic pathogens and cause severe infection. The antibiotic of choice for treating such infections is metronidazole, a DNA damaging agent. Metronidazole enters the bacterial cell as an inert prodrug, and is activated by cellular reduction into a cytotoxic compound which is thought to cause DNA strand breaks. Certain metronidazole resistant B. fragilis strains have been described, where the drug was not reduced inside the cell due to decreased activity of the metabolic enzymes which are involved in this process. Little is known about the mechanisms involved in repair of metronidazole damage and the potential for resistance. In this study, two difIerent approaches were used to isolate and analyse Bacteroides genes involved in metronidazole resistance, with emphasis on DNA repair genes. These methods were transposon mutagenesis of Bacteroides, and functional complementation of E. coli metronidazole sensitive mutants with genes from B. fragilis.

Includes bibliographical references. / Proteas have been extensively cultivated and are grown as floricultural crop plants in many parts of the world, including South Africa. However, the factors that influence the initiation of flowering in Protea have not been identified. From data gathered by the Protea Atlas Project it is evident that Protea spp. have greatly varying flowering times. Furthermore, flowering times between Protea spp. and their hybrid cultivars are also very different. Towards a better understanding of the factors involved in floral initiation in this cultivated crop, three aspects of flowering were investigated in this study. The carbon input into Protea inflorescence development was determined by measuring respiration rates and weights of developing structures. By manipulating source-sink ratios in plants, the carbon assimilatory capacities to support inflorescences were investigated in three cultivars and one wild-grown species of Protea which develop different sized flowers. As some Proteas flower in response to seasonal change, an orthologue of the floral inducer FLOWERING LOCUS T (FT), ProteaFT (ProFT), was isolated from ‘Carnival’ (P. compacta x P. neriifolia) and its expression pattern followed diurnally and seasonally. Finally, the functions of paralogous genes of Protea LEAFY (ProLFY) from ‘Carnival’ displaying sequence similarity to the meristem identity gene LEAFY from Arabidopsis thaliana, were investigated through heterologous expression studies in A. thaliana.

Includes abstract. / Includes bibliographical references (leaves 131-169). / The ability of tissues to survive almost complete loss of cellular water is a trait found throughout the plant kingdom. While this desiccation tolerance is common in seeds of most angiosperms it is rare in their vegetative tissues. Xerophyta humilis (Bak.) Dur and Schintz belongs to a small group of resurrection angiosperms and it possesses the ability to withstand extreme desiccation of greater than 90% in both its seeds and vegetative tissues and return to active metabolism upon rehydration. We have tested the hypothesis that vegetative desiccation tolerance in angiosperms has evolved as an adaptation of seed desiccation tolerance.

Includes bibliographical references. / Knowledge of the biochemical and molecular mechanisms by which plants tolerate environmental stresses is necessary for genetic engineering approaches to improve crop performance. A unique feature of resurrection plants, such as Xerophyta viscosa, is their ability to cope with severe water loss of greater than 90%. A full-length cDNA library was synthesised from a cold stressed X viscosa plant. Sequencing and BLAST analysis revealed the identity of sixty genes. A type 2 peroxiredoxin (XvPrx2) was selected for further analyses as it was observed, by northern analyses, to be stress-inducible. The XvPrx2 protein was confirmed to be involved in the stress response by Western analyses. The XvPrx2 gene, which displays highest identity to a rice orthologue, has an open reading frame of 162 amino acids, and codes for a hydrophilic polypeptide of 162 residues with a predicted molecular weight of 17.5 kDa. The XvPrx2 polypeptide displays significant identity with other plant type II Prxs, with an absolutely conserved amino acid sequence proposed to constitute the active site of the enzyme (PGAFTPTCS). The XvPrx2 protein has a single cataly1ic cysteine residue at position 51 similar to Prxs from Oryza sativa and Candida boidinii. A mutated protein (XvV76C) was generated by converting the valine at position 76 to a cysteine resulting in a conformational change as determined by limited proteolysis. An in vitro DNA protection assay showed that, in the presence of either XvPrx2 or XvV76C, DNA protection occurred. In addition, an in vivo assay showed that increased protection was conferred on cell lines over-expressing either XvPrx2 or XvV76C. Several upstream promoter regions were identified for the XvPrx2 gene using the splinkerette method. Southern and two dimensional gel analyses revealed that multiple XvPrx2 homologues exist within the X viscosa genome. These homologues have similar pI values to Arabidopsis orthologues. Immuno-cytochemical data revealed that XvPrx2 is localised to the chloroplast, however, this could be attributed to cross reactivity with a chloroplastic homologue. Using YFP technology, the protein was observed to be expressed in the cytosol, and this location is supported by the absence of an upstream targeting signal in the XvPrx2 sequence. The XvPrx2 activity was maximal with DTT as electron donor and HzOz as substrate with t-BOOH being the next preferred. Using Trx£. coli a 2-15 fold lower enzyme activity was observed. The XvPrx2 activity with GSH was significantly lower and Grx had no measurable effect on this reaction. The XvV76C protein displayed significantly lower activity compared to XvPrx2 for all substrates assessed. Enzymatic kinetic parameter values determined for XvPrx2 using DTT as electron donor and HzOz as substrate were: Km = 45 IlM, V max = 278 Ilmol min-I.mg-I protein, kcat 6.173 x 103 s-1 and kcaJKm = 0.136 X 103 IlM-1.s-l. Based on knowledge-based models of XvPrx2 and XvV76C no structural differences were observed between the two molecules.

Word processed copy. / Includes bibliographical references (leaves 209-232). / This study attempted to further characterise OXI1 protein kinase. Confocal microscopy and subcellular fractionation studies revealed a cytosolic localisation pattern for OXI1. Employment of a bioinformatics approach confirmed the induction of OXI1 gene expression in response to a range of AOS generating stimuli. However, the transcriptional increase of OXI1 in response to salinity and heat appears to be of no biological significance since the oxi1 mutant did not display altered tolerance to these two stresses in comparison to wild type.

Summary in English. / Includes bibliographical references. / The substrate basis for the industrial acetone-butanol-ethanol (ABE) fermentations, has been agricultural products rich in starch or sucrose, and employed taxonomically distinct amylolytic and saccharolytic solventogenic clostridial strains respectively. There is evidence to suggest that the utilization of these substrates is subject to carbon catabolite repression. In Gram-positive bacteria, carbon catabolite repression is controlled by a global regulatory mechanism, central to which is an imperfect palindromic sequence, the cre element, which is recognized by a protein of the GalR-LacI family, the CcpA protein. A ccpA homologue, regA, has been previously identified in C. acetobutylicum NCP262 and successfully complemented a B. subtilis ccpA mutant strain. The sucrose operon from C. beijerinckii NCIMB 8052, scrARBK, has been characterised at the physiological and genetic levels with the ScrR repressor found to negatively auto-regulate the operon.

Includes abstract. / Includes bibliographical references. / Plants are emerging as cost friendly alternative production systems for a variety of pharmaceuticals. Numerous therapeutic proteins have been produced in plant systems (Giddings et al., 2000; Ma et al., 2003). Protein based microbicides,-namely, neutralising antibodies and peptide lectins- lend themselves to production in plants (De Muynck et al., 2010; Matoba et al., 2010; Sexton et al. 2006; O’Keefe et al., 2009). One of these lectins, namely Griffithsin (GRFT) was isolated from the blue green algae Griffithsia and is being developed as a leading anti-HIV microbicide peptide (Mori et al., 2005). As literature indicates, the optimal production of any protein is an empirical experimentation with different host systems, vector systems, codon optimisations and subcellular targeting. (Maclean et al., 2007; Yang et al., 2005). The latter sometimes results in unexpected locations which might reflect on an inherent property of the protein itself or specifically be associated with the plant organ involved (Chikwamba et al., 2003). This again can influence protein yield- and activity, and impact downstream purification. In this study we aimed to compare expression levels using both Zea mays (maize) and Nicotiana benthamiana (tobacco) with relevant vector technologies. We expressed GRFT in maize using an endosperm specific maize expression vector with and without a signal peptide. In tobacco we utilised both the pTRA binary vector and magnICON deconstructed viral vector system to express GRFT with different subcellular targeting signals.