Metal hyperaccumulating plants accumulate exceptionally high concentrations of metal ions in their above ground tissues and are defined as containing 1000 μg/g dry mass Co, Cu, Cr, Pb, Zn or Ni. This is remarkable because plants typically only require small amounts of these metals for survival, such as 0.004 μg/g Ni and 15-20 μg/g Zn. Scientific investigation has sought to understand the mechanisms underpinning hyperaccumulation in order to apply them in the phyto-technological processes of phytoremediation (removal of metal pollutants from the environment) and phytomining. However, little is known about the molecular mechanisms underlying Ni hyperaccumulation despite the fact that Ni hyperaccumulators account for almost three quarters of all known hyperaccumulating species. A comparative RNA-Seq experiment carried out on Ni accumulating and non-accumulating populations of the South African Ni hyperaccumulator Senecio coronatus (Asteraceae) identified a number of putative transport proteins that are constitutively upregulated in the hyperaccumulator plants. This MSc project focused on two of these, iron regulated 2 (ScIREG2) and iron regulated transporter 1 (ScIRT1), and aimed to validate the RNA-Seq derived nucleotide sequences, test for Ni transport activity and determine their sub-cellular localisation. Full-length ScIREG2 and ScIRT1 protein coding sequences were obtained using RT-PCR and conformed to the predicted sequences derived from the RNA-Seq data. Heterologous expression of ScIRT1 in yeast consistently conferred an increased Ni resistance phenotype to yeast across a variety of experimental conditions, suggesting that this protein is capable of transporting Ni, and may function as a Ni export protein in yeast. In contrast, the results obtained from heterologous expression of ScIREG2 were variable and thus inconclusive. An attempt was made to determine the subcellular localization of ScIRT1 using transient expression of an ScIRT1-YFP fusion protein in onion cells. While inconclusive, a YFP signal was detected in these cells, and appeared to localise to the plasma membrane. The work conducted serves as a pilot study to optimize the experimental systems necessary to identify Ni transporters from S. coronatus. These experimental systems can now be applied to characterise the remaining transport proteins identified in the RNA-Seq analysis.

The klipspringer, Oreotragus oreotragus, occurs from the Western Cape of South Africa through to north-east Africa. Anthropological factors such as hunting have eradicated the klipspringer in parts of its former range and efforts have begun to reintroduce klipspringer back into these areas. This has highlighted the need for the proper understanding of the genetic variation, phylogeographic and population structure of the species, as well as the genetic validity of the 11 subspecies designations. Mitochondrial control region and cytochrome b sequence data were used to investigate these topics using blood, faecal and museum specimens. Cytochrome b and control region fragments were amplified in 83 and 60 samples respectively, out of a total sample set of 89 individuals. The generation of authentic mitochondrial DNA control region fragments proved difficult, with both Numt and PCR recombinant sequences identified after amplification. High levels of genetic variation were observed in the klipspringer, with cytochrome b and control region haplotype diversities of 0.78 and 0.98 respectively. Phylogenetic and network analyses showed the distinct geographical clustering of individuals into two major groups, south/south-western (S/SW) and east/north-eastern (E/NE), with the S/SW further split into two divergent groups. This suggests that the two S/SW groups were separated and isolated as a result of shifting climatic conditions in the late Pl iocene/early Pleistocene, after which secondary contact occurred and klipspringer moved upwards from southern Africa to colonise the east. The results from this study have conservation implications with respect to translocation policies, suggesting that translocations should only occur within the geographic groups identified in order to avoid outbreeding depression.

Human papillomavirus (HPV) is known to be the cause of anogenital and oropharyngeal cancers as well as genital and common warts. There are currently three prophylactic virus-like particle (VLP) based vaccines. These vaccines, however, do not offer protection against all HPV strains and cannot act therapeutically and so further vaccine development is still needed. The burden of HPV is also highest in low-income countries for which the vaccine costs are still quite high, and therefore alternative methods of vaccine production and testing are needed. HPV pseudovirions (PsVs) are synthetic viral particles that are made up of the L1 major and L2 minor HPV capsid proteins and encapsidate up to 8Kb of pseudogenome DNA without the need of an encapsidation signal. HPV PsVs are used to test neutralising antibodies elicited by vaccines, for studying the virus life cycle, and potentially for delivery of therapeutic DNA vaccines. HPV PsVs are typically produced in mammalian cells; however, it has recently been shown that HPV PsVs can be produced in plants, a potentially safer, cheaper and more easily scalable means of production. While, a current problem with plant HPV PsV production is low yields, research has shown that using pseudogenome DNAs between 5-7Kb increases yields of papillomavirus PsVs in mammalian cells. Therefore, the objective of this study was to determine the optimal pseudogenome size for encapsidation by plant produced PsVs, in order to increase the amount of PsVs in a sample as opposed to VLPs. Pseudogenome constructs encoding Enhanced Green Fluorescent Protein (EGFP )and ranging in size from 4.8Kb – 7.8kb were cloned into a geminivirus-derived replicating vector, transformed into Agrobacterium tumefaciens and then infiltrated into Nicotiana benthamiana along with plant expression vectors encoding the HPV 35 L1 and L2 capsid proteins. Particles were purified by iodixanol density gradient ultracentrifugation and the 27% and 33% fractions of this gradient analysed. Transmission electron microscopy (TEM) was used to confirm particle assembly and L1 expression was quantified by ELISA. Particles were disrupted with proteinase K and quantitative PCR was used to quantify the encapsidated DNA. Ratios of encapsidated DNA to L1 capsid protein were calculated for each of the PsV samples with different sized pseudogenomes, to account for batch-to-batch variation and as an approximation of which size pseudogenome is better encapsidated. Infective ability of the particles was analysed by incubating the PsVs onto HEK293TT cells and then checking for DNA delivery and protein expression by measuring EGFP expression by Western blots. The results showed that PsVs are found predominantly in the 27% fraction of the iodixanol gradient whereas the 33% fraction of the gradient appears to only contain VLPs. The data also indicated that the smaller pseudogenomes, were packaged more efficiently into PsVs as higher concentrations of encapsidated DNA and higher levels of EGFP expression were obtained when the 4.8Kb pseudogenome was used, compared to when the larger 5.8 - 7.8Kb pseudogenomes were used. Thus, the results showed that smaller pseudogenomes, around 4.8Kb, should be used for the plant production of HPV 35 PsVs as they are better packaged than larger pseudogenomes and thereby produce higher yields of functional PsVs.

Includes bibliographical references. / [Fix supervisors field.] Psittacine beak and feather disease, caused by a circovirus known as beak and feather disease virus (BFDV), is a threat to both wild and captive psittacine species. There is currently no vaccine against BFDV and safe and affordable vaccine candidates are needed to alleviate the disease burden caused by this virus. Production of the BFDV's major antigenic determinant, the capsid protein (CP), in the inexpensive and highly scalable plant expression system, could satisfy these requirements as a potential subunit vaccine. In this work, truncated CP (ÄN40 CP) was first expressed in E. coli to successfully generate anti-CP polyclonal antibodies. ÄN40 CP and full-length CP transient expression in tobacco (Nicotiana benthamiana) was optimised as fusions to elastin-like polypeptide (ELP). Fusion of CP or ÄN40 CP to ELPs of different lengths was shown to increase yield relative to unfused CP/ÄN40 CP. Free ELP and a GFP-ELP fusion could be purified by inverse transition cycling (ITC), using centrifugation and membrane filtration methods. A ÄN40 CP-ELP fusion expressed in plants could be partially purified and represents low-cost vaccine candidate against BFDV. A candidate DNA vaccine expressing ÄN40 CP was also evaluated for expression of the antigen in vitro and may prove useful in a prime-boost regimen together with one of the plant-produced vaccine candidates.

A clinical and molecular analysis of Clostridium difficile isolated from symptomatic patients at Groote Schuur Hospital was conducted in order to gain insight into the identity, epidemiology and pathogenesis of the various strains. C. difficile was detected and isolated by selective culture from stool specimens from 34 of the 162 symptomatic patients (20%). Three toxigenic-types were distinguished by PCR: A+B+ (47%), A-B+ (47%) and A-B- (6%), none of which harboured the binary toxin genes. Compared to the direct culture method, enzyme immunoassay-based detection tests (Meridian ImmunoCard and bioMérieux MiniVidas) were found to be lacking clinical sensitivity, while nucleic acid amplification tests (Hain Lifescience CDiff and Cepheid GeneXpert) were far more sensitive in the local clinical setting. PCR ribotyping identified all the A-B+ strains as PCR ribotype 017, which was the prevalent strain type (47%). Genotyping based on the tcdC gene and MLVA both grouped the ribotype 017 strains in a single clade. The antimicrobial susceptibility of all the isolates to metronidazole (MET), vancomycin (VAN), moxifloxacin (MOX) and erythromycin (ERY) were determined by the Etest method. All were sensitive to MET and VAN; however, four ribotype 017 strains displayed reduced susceptibility to MET. With regard to MOX, reduced susceptibly and full resistance were observed in 32% and 12% of the isolates, respectively, with all of these belonging to ribotype 017. MOX-resistant strains had a Thr82->Ile amino acid substitution in the GyrA enzyme and strains displaying reduced susceptibility to MOX had an Asp426->Asn amino acid substitution in GyrB. High-level resistance to ERY was observed in 47% of the isolates, which were primarily ribotype 017. ERY-resistant strains all harboured the ermB gene, suggesting that this was the genetic basis of the observed phenotype. Auto-aggregation analysis revealed that the ribotype 017 strains were significantly stronger auto-aggregators than the other ribotypes examined. Results of semi-quantitative RT-PCR analysis suggest that the expression level of the cwpV gene, encoding the CwpV protein, may play a role in auto-aggregation. In conclusion, this pilot study revealed that the GeneXpert method was the most accurate and sensitive technique for diagnosing CDI in the clinical setting at Groote Schuur Hospital. Ribotype 017 was the most prevalent strain type, and the antimicrobial resistance profile and increased auto-aggregation capacity of this ribotype may contribute to its high prevalence.

Bibliography: leaves 24-29. / Low temperature is one of the environmental factors that cause substantial crop losses in the world. Recent advances in the study of plants native from temperate regions, have established that cold acclimation is regulated at the gene expression leveL To study the function ofaXerophyta viscosa cold responsive protein that accumulates in plant cells under various environmental stresses, we have cloned the Xerophyta viscosa cold-responsive cDNA gene designed Xvcor, into a less complex host, Saccharomyces cerevisiae with the purpose of carrying out in vivo functional analysis. Sequence analysis showed that Xvcor encodes a 264 amino acid residue protein (Garwe et al 2002). The Hydropathy plot indicated that the XVCOR protein is highly hydrophobic and contains 6 transmembrane domains (Garwe et al 2002). In order to achieve high-level expression of Xvcor, the gene was placed under the control of a strong promoter (Phosphoglycerate kinase). Reverse-transcription PCR amplification revealed that the Xvcor transcript accumulated in yeast cells. However, SDS-PAGE analysis could not detect the predicted 29.6 kDa recombinant protein, suggesting that the translation might be hampered. Tests designed to measure the enhancement of stress tolerance between the recombinant and the control showed similar growth performance, confirming the absence of recombinant protein. A computer search for codon usage showed that the codon usage bias in Xvcor was low compared with that of highly expressed genes of S. cerevisiae. Together, these results suggest that the codon usage in Xvcor could influence its expression in yeast.

Includes bibliographical references (leaves 61-84). / The circadian system has a regulatory role in almost all aspects of a plant's life. In Arabidopsis thaliana, almost 36% of the genome has been shown to be circadianly regulated and many genes that are circadianly regulated have been shown to be light responsive or involved in light responses. Rhythmic histone acetylation has been demonstrated in the promoter of TIMING OF CAB EXPRESSION1 (TOC1). Here, I used semi-quantitative Reverse Transcriptase Polymerase Chain Reaction (semi-quantitative RT -PCR) to investigate which enzymes are involved in the rhythmic expression of TOC1. I also determined whether loss-of-function histone acetylation and methylation mutants could affect the overall functioning of the circadian oscillator by measuring their circadian leaf movement and delayed fluorescence (DF) rhythms. GCN5/ HAG1 mutant plants (gcn5) exhibited erratic TOC1 expression in both constant dark (DD) and constant light (LL) conditions. Although TOC1 expression appeared to be rhythmic in both DD and LL conditions, the waveform of the rhythm was altered in TATA-binding protein associated factor 1 (taf1) mutants. This suggested that TAF1 and GCN5 might play different roles in the rhythmic histone acetylation affecting TOC1 expression. DF data and leaf movement data indicated that both TAF1 and GCN5 might play a role in the overall functioning of the A. thaliana circadian clock. Arrhythmic TOC1 expression and DF was observed in histone deacetylase 1 (hd1) mutants, suggesting that HD1 is not only involved in the rhythmic histone deacetylation affecting TOC1 expression but in the overall functioning of the circadian clock. Semi-quantitative RTPCR, DF and leaf movement studies demonstrated that CURLY LEAF (CLF), a histone methylase is involved in both the histone methylation affecting TOC1 expression and in the overall functioning of the A. thaliana circadian clock.

Concurrent use of anti-retroviral drugs (ARVs) and progestin-based hormonal contraceptives is widespread. During times of stress and during glucocorticoid (GC) therapy, intracellular ARVs are in the presence of high concentrations of GCs, which regulate all aspects of immunity and inflammation via the glucocorticoid receptor (GR). However, the reciprocal modulation of ARV and steroid intracellular functions is relatively unexplored. In this study, the effects of the ARVs tenofovir disoproxil fumarate (TDF), dapivirine (DPV), and maraviroc (MVC) on activation of the GR and GR-regulated mRNA expression were investigated, in the absence and presence of select GR ligands. The effects of TDF and DPV on GR protein levels and phosphorylation were also determined. The inhibitory activity of these ARVs on HIV-1 infection in the presence of the progestins medroxyprogesterone acetate (MPA) and levonorgestrel (LNG), and a GR agonist, dexamethasone (DEX) was also assessed. This study shows that (0.01 nM-10 µM) TDF, DPV and MVC do not transactivate reporter gene expression via the unliganded GR exogenously expressed in the steroid receptor-deficient U2OS human osteosarcoma cell line, or alter the reporter gene transcriptional activity of (100 nM) MPA or LNG via the GR in these cells. However, (1 µM) TDF and DPV modulate the reporter gene transcriptional efficacy of (0.01 nM-10 µM) DEX via the GR. In the U2OS cell line model, (1 µM) TDF, but not DPV significantly decreased (1µM and 10µM) DEX-induced mRNA expression of the anti-inflammatory glucocorticoid-induced leucine zipper (GILZ) gene. TDF also appeared to decrease (1 µM) cortisol (CORT)- and MPA-induced GILZ mRNA expression. This may be mediated by the apparent increase in (100 nM and 1µM) DEXinduced phosphorylation at Serine 226 on the GR, observed in the presence of (1µM) TDF in this study. DPV and TDF (at 1µM) did not significantly alter GR protein levels, or cell-viability in the absence and presence of (100 nM) DEX, CORT or MPA in U2OS cells. However, (1 µM) DPV and TDF alone, significantly altered cell viability in peripheral blood mononuclear cells (PBMCs). In PBMCs, (1 µM) TDF, MVC and DPV alone altered basal GILZ mRNA expression and had variable, donor-specific effects on interleukin (IL)-6, IL-8, and interferon (IFN)-γ gene expression. In PBMCs from some of the nine donors tested, these ARVs had proinflammatory effects which may undermine their efficacy at preventing HIV-1 acquisition in pre-exposure prophylaxis products. Moreover, the ARVs proinflammatory effects may negatively impact HIV-1 disease progression and increase the risk of non-AIDS mortality in individuals using the ARVs therapeutically. Neither (1 µM) DPV, TDF nor MVC significantly altered the effects of (100 nM) DEX on the immunomodulatory genes assessed in PBMCs. DEX, MPA and LNG (at 100 nM) did not affect the anti-HIV-1 activity of the ARVs (at 1 µM) in PBMCs from the majority of the three donors tested in this study. Taken together, the results show that ARVs can modulate GR activity in an ARV-, steroid-, gene- and cell-specific manner, while the steroids investigated did not modulate ARV anti-HIV-1 activity.

Includes bibliographical references (p. 78-90). / Bactereroides fragilis is a gram-negative, non spore-forming, obligate anaerobe of the human intestinal microbiota. It is, however, an opportunistic pathogen and has been ranked as the most prevalent isolate in cases of anaerobic septicaemia. Similar to most bacteria, ammonium is assimilated in B. fragilis through the action of glutamine synthetase (GS). Glutamine is vital to nitrogen metabolism as it serves as a precursor to many secondary metabolites. GS enzymes are, therefore, vital to the growth of the organism and many prokaryotes are known to possess two or more isoforms of the enzyme. In addition, GS expression and regulation is usually tightly regulated in concert with the availability of nitrogen. Previous studies have identified a single GSIII encoding gene (glnN) in B. fragilis. In this dissertation, an additional ORF coding for a putative GSI enzyme in B. fragilis was identified, isolated and functionally characterized. A putative regulatory protein was also identified and its functional contribution to nitrogen metabolism was determined, in order to extend our understanding of nitrogen assimilation in B. fragilis.

Maize is a socially and economically important crop in Africa (and worldwide) that is severely affected by many fungal pathogens. The pathogen Fusarium verticillioides causes Fusarium ear rot in maize, a disease that greatly reduces quantity and quality of annual maize yields. The pathogen produces mycotoxins called fumonisins, which have been linked to adverse health effects in both humans and animals. Maize produces terpenoid phytoalexins, which are antimicrobial compounds that directly reduce the growth of many fungal pathogens including F. verticillioides. Two families of maize phytoalexins, termed kauralexins and zealexins, have been characterized. Key genes putatively involved in the biosynthetic pathway of these phytoalexins have been identified from the rice model and subsequent studies on maize. This research aimed to evaluate the correlation between phytoalexin accumulation and fungal growth in diverse southern African maize lines in response to F. verticillioides inoculation. Maize lines were inoculated with F. verticillioides using a seed soak inoculation method and grown in vitro for up to two weeks. The harvested tissue was analysed for fungal growth using quantitative PCR, putative phytoalexin biosynthetic gene expression using RT-qPCR and phytoalexin accumulation using gas-chromatography mass spectrometry. Furthermore, an endophyte growing in one of the maize lines was isolated and identified as Trichoderma asperellum. Trichoderma spp. are used as biocontrol agents against many fungal pathogens, although research on the specific antagonistic effect of T. asperellum on F. verticillioides is limited. Phytoalexin accumulation in maize containing endophytic T. asperellum was compared to maize inoculated with F. verticillioides. In vitro competition assays were performed to analyse the antagonistic effect of T. asprellum on F. verticillioides. Results from this study show that inoculation of maize lines with F. verticillioides induces the accumulation of total phytoalexins, and more specifically the accumulation of total kauralexins. Putative phytoalexin biosynthetic genes are also up-regulated in response to inoculation. Maize growing with a T. asperellum endophyte accumulated phytoalexins to the same levels as F. verticillioides, suggesting that T. asperellum induces a defence response that 'primes' the plant for further infection. In vitro competition assays between F. verticillioides and T. asperellum showed that T. asperellum significantly inhibits F. verticillioides growth. These results will aid in the identification of maize lines that can be bred with increased resistance to F. verticillioides with the goal to reduce F. verticillioides incidence in southern Africa. Furthermore, analysis of the efficacy of T. asperellum as an antagonist against F. verticillioides may provide another method for disease reduction in the field.