Bibliography: pages 117-130. / Solventogenic clostridial strains were used extensively in the industrial acetone-butanolethanol (ABE) fermentation during the first half of this century. This fermentation was based predominantly on two substrates: corn mash (rich in starch) and molasses (rich in sucrose). Taxonomically distinct strains were used for ABE fermentations based on the two substrates. Although starch . utilization by amylolytic solventogenic clostridial strains had been investigated, sucrose utilization by saccharolytic solventogenic clostridial strains had not been comprehensively studied. This provided the impetus for the present study. The primary aim of the project was to provide a molecular and physiological characterization of the sucrose utilization system of Clostridium beijerinckii NCIMB 8052. The C. beijerinckii sucrose utilization operon, scrARBK, was cloned in three stages. In the first stage a C. beijerinckii genomic library was screened in Escherichia coli for clones able to utilize sucrose. Using this approach, a truncated scr operon was isolated. The remainder of the operon was isolated in the second and third stages by recovering a plasmid integrated into the scrB gene, and inverse-PCR respectively. The four genes of the scrARBK operon were prowsed to encode an Enzyme1IBC1ucrose protein of the PTS (ScrA); a transcriptional repressor of the GalR-Lacl family (ScrR), a sucrose-6-phosphate hydrolase of the glucosyl hydrolase family 32 (ScrB) and an ATP-dependent fructokinase of the ribokinase/pfkB sugar kinase family (ScrK).

A small, diverse group of angiosperms known as resurrection plants display vegetative desiccation tolerance and can survive loss of up to 95% of cellular water, a feat only seen in the seeds and pollen of other angiosperms. Xerophyta humilis is a resurrection plant native to Southern Africa that has been the target of previous transcriptomic and proteomic studies into the mechanisms of plant desiccation tolerance. The aim of this study was to investigate the hypothesis that vegetative desiccation tolerance is derived from the networks that control desiccation tolerance in seeds and germinating seedlings in angiosperms, particularly the epigenetically silenced seed maturation genes. Germinating seedlings of X. humilis and the related resurrection plant X. viscosa were found to be VDT from the earliest stages of germination, and exhibited the characteristic vegetative trait of poikilochlorophylly as seen in mature leaves. The X. humilis desiccation transcriptome comprising 76,768 distinct gene clusters was successfully assembled from sequencing samples at five relative water contents (100%, 80%, 60%, 40% and 5%) to identify the networks activated in response to water loss. Desiccation was associated with successive waves of transcription factor induction, as well as widespread down-regulation of histone modification enzymes. Many seed-specific genes, such as late embryogenesis abundant (LEA) proteins, seed storage proteins and oleosins, were induced in vegetative tissue. LEA transcripts in particular were highly up-regulated during desiccation, and the large number of distinct LEA transcripts (over 150) suggests possible LEA gene expansion in Xerophyta compared to desiccation-sensitive plants. Components of the PYL/SnRK2/ABF ABA-signalling pathway were also induced, although the ABF transcription factors activated in response to desiccation were most similar to those induced by drought in A. thaliana rather than seed maturation. Of the canonical seed master regulators (such as the LEC1/ABI3/FUS3/LEC2 network and ABI5) only three ABI3 transcripts were expressed, all of which encoded proteins lacking the seed motif-binding B3-domain. The results of this study suggest that vegetative desiccation tolerance in X. humilis is not associated with re-activation of seed master regulators in vegetative tissue, but may instead involve activation of seed genes by vegetative drought response regulators.

Cervical cancer caused by infection with Human papillomavirus (HPV) is the 4th most common cancer in women globally, and results in an estimated 266 000 deaths every year. Current vaccines are based on the immunodominant L1 major capsid protein, which assembles into virus-like particles (VLPs) that are highly effective in type-specific prevention of cervical infection. However, these vaccines are produced in expensive cell culture systems, are type-specific and do not induce the regression of established infections. The cervical cancer burden (~80%) is mainly in developing countries due to limited healthcare resources, therefore there is a need for more broadly protective and affordable vaccines. Plants provide an alternative platform to produce cheaper vaccines, given their scalability, rapid production and low risk of contamination. The L2 minor capsid protein has sequence regions that are highly conserved across several HPV types, and HPV-16 L2 peptides 108-120, 65-81, 56-81 and 17-36 have been shown to elicit cross-neutralising antibodies. To increase the immunogenicity of L2, second-generation L1:L2 chimaeric VLP (cVLP) vaccines have been investigated. In this study, the 4 L2 peptides above were used to generate plant-produced HPV-16-derived L1:L2 chimaeras. The L2 epitopes were substituted into the DE loop of HPV-16 L1 at position 131 (SAC) or between the helix 4 and β-J structural region at position 431 (SAE). All chimaeras were transiently expressed in Nicotiana benthamiana via Agrobacterium-mediated transfer. Optimisation of expression was conducted by comparing protein expression levels over several days using 4 plant expression vectors, with the highest yields obtained by targeting protein to the chloroplast or with the use of a self-replicating vector. The chloroplast targeted SAC chimaeras predominantly assembled into higher order structures (T=1 VLPs and T=7 VLPs), whereas SAE chimaeras assembled into capsomeres or formed aggregates, indicating that the length, sequence and substitution position of L2 epitopes affects VLP assembly. All SAC chimaeras in addition to SAE 65-81 (smaller epitope not previously tested in chimaeras) were used in vaccination studies in mice, and their immunogenic potential analysed in pseudovirion-based neutralisation assays (PBNAs). Of the 7 heterologous HPVs tested, cross-neutralisation was observed with HPV-11, -18 and -58. Only the anti-SAE 65-81 serum showed neutralisation of homologous HPV-16, suggesting that antibodies detected from all candidate vaccines were mostly non-neutralising, and that the position of the L2 epitope display is critical to maintaining L1-specific neutralising epitopes. Lastly, to address the lack of therapeutic efficacy of current vaccines, I aimed to develop a novel E7 DNA vaccine delivered by plant-made pseudovirions (PsVs). A geminivirus-derived self-replicating plasmid encoding a shuffled E7 (E7SH) sequence that has no transformation ability but contains natural cytotoxic T-lymphocyte epitopes, was constructed using Goldenbraid technology and co-expressed in plants with HPV-16 or HPV-35 L1- and L2-encoding expression vectors. The pseudogenome was successfully encapsidated into plant-made PsVs. These PsVs were capable of infecting mammalian cells and encapsidated replicons expressed E7SH showing the promise of this candidate vaccine as a future combination prophylactic and therapeutic vaccine.

Vertebrate limbs classically illustrate the morphological diversity of homologous structure. Bat limbs exemplify this, having strikingly divergent limbs: wings with asymmetrically elongated digit elements, supporting expansive membranes; and hindlimbs with short, symmetrical, free digits. An understanding of the genes, interactions and events that shape bat limbs, will inform conventional models of development. This dissertation characterised differential expression of Meis2, in the context of interdigital regression, and the 5'HoxD genes in the context of digit formation in developing bat autopods (CS15 - CS18). Meis2 is involved in limb proximodistal patterning, and has been shown to promote proliferation, and survival of cells in other developmental contexts. Meis2 had strong expression in the expanding bat forelimb interdigits, with lowered expression in mouse and bat hindlimb interdigits, and did not correspond with Hoxa13 expression, which was reduced in the forelimb. Autopod expression was independent of retinoic acid (RA) signalling, with genes involved in RA synthesis ( Rdh10 , Aldha2 ) , degradation ( Cyp26b1 ) and signalling (Rar β) expressed in bat limbs. Altered expression patterns of Aldha2 and Cyp26b1, indicate that this pathway may be modulated in the forelimb. Meis2 is suggested to play a role in interdigital tissue retention, enhancing cell proliferation and contributing to wing expansion. 5'HoxD genes (Hoxd10 - 13) are involved in limb patterning, digit formation and growth. Their modular autopod expression domains correspond to the bat skeletal element phenotype, with strong overexpression of Hoxd10 - 11 (and to a lesser extent Hoxd12) in the forelimb posterior elements (digits II - V), which are highly elongated, and a loss of expression of these genes in hindlimb digits. These genes were not expressed in a typical reverse collinear relationship, with absolute q PCR revealing highest expression of Hoxd10. While the coding protein sequence of these genes appeared highly conserved between bats and other mammals, several changes were found in the CsC region of the digit enhancer Prox, some of which were associated with alterations in transcription binding sites. These findings indicate that Hoxd10 - 12 expressions contribute to the altered skeletal element morphologies of bat forelimbs and hindlimbs. This study makes a valuable contribution to the growing body of work that explores bat limb development and the evolutionary adaptation s of these unique structures.

Bibliography: pages 108-128. / Cucumber mosaic virus (CMV) and tobacco necrosis virus (TN V) often occur in mixed virus infections in South Africa. Both viruses are of economic importance because of their world-wide distribution, extensive host range and their effects on yields of agriculturally important crop plants. The complete cDNA sequences of CMV-Wemmershoek (CMV-Wem) coat protein (CP) and TNV-F5P CP genes were cloned and subjected to sequence analysis. CMV-Wem is closely related to CMV-WL and CMV-Q, and therefore falls into CMV subgroup II. Similar analysis showed that TNV-F5P is closely related to TNV-A. By characterizing and sequencing these clones the authenticity of the CMV and TNV CP genes was also determined, prior to sub cloning into the appropriate vectors for expression in E. coli and tobacco. Constructs containing both the full-length CP genes of CMV-Wem and TNV-F5P were subcloned in frame with the malE gene, encoding the maltose binding protein (MBP), in the IPTG-inducible pMALTM vector system, and expressed in E. coli. Through immunological detection the authenticity of both CPs was confirmed. The CMV CP translation product expressed in E.coli was used as an antigen to raise antiserum free from contaminating plant host-specific antibodies. The CP genes of both viruses were individually cloned in both orientations (sense and antisense) in Agrobacterium tumefaciens Ti-plasmid-based binary and cointegrate vectors. The study was then extended to include engineering doubly transgenic plants. In order to determine whether the full-length CP is required to mediate virus resistance, a truncated form of the TNV CP was generated by deleting 83 amino acids from the C-terminus. Transgenic Nicotiana tabacum cv Petit Havana SRl plants containing one of a number of different forms of CMV and TNV CP nucleotide sequence were generated. In whole plant studies, mechanical inoculation of Ro lines with CMV-Wem resulted in more than 50% of the CMV CP-sense (CP+) and CP-antisense plants not developing visible systemic disease symptoms. In both the CMV CP+ and doubly transgenic plants CMV-Wem accumulation was delayed, but virus was found to accumulate in the inoculated leaves over time. The CMV CP+ lines showed excellent protection against CMV-Q, but showed only a delay in symptom production when inoculated with CMV -Y, from subgroup I.

The broad-host-range Thiobacillus ferrooxidans plasmid pTF-FC2 is stably inherited over many generations despite a low copy number. The pas genes which lie between the repB primase and the repA helicase encode a proteic plasmid stabilisation system and are capable of stabilising the unstable heterologous R1 replicon present on p0U82. The deletion of the pas genes has been shown not to change the copy number of mutant plasmids. This suggested that the pas genes are not involved in replication and function as a stabilisation cassette. The pasA gene encodes an antidote, the pasB gene a toxin which exerts a bacteriocidal effect in an E. coli host and the pasC gene a protein which moderates the toxic effect of PasB. The PasC is unique in proteic plasmid stabilisation systems and reduces PasB toxicity only in the presence of PasA. PasA is able to repress the pas promoter and the addition of PasB increases this repression. PasC has been shown not to effect the pas promoter by itself. In the presence of PasA and PasB, PasC reduces the ability of PasA and PasB to repress the pas promoter. PasC is thought to stabilise the interaction of PasA and PasB and in doing so reduces their ability to function as repressors of the pas operon. The IncQ plasmid RSF1010 which has similarity to pTF-FC2 has two genes in a position analogous to the pasA, pasB and pasC genes. These genes have been found to be unable to function as a plasmid stabilisation system.

Rift Valley fever (RVF) is a haemorrhagic fever agent caused by an infection with an enveloped negative-stranded RNA Rift Valley fever virus (RVFV). It belongs to the genus Phlebovirus in the family Bunyaviridae. The virus is spread by infected mosquitoes and affects ruminants and humans, causing high numbers of neonatal fatalities in animals and occasional fatalities in humans. It is endemic to parts of Africa and the Arabian Peninsula, but is described as an emerging virus due to the wide range of mosquitoes that could spread the disease into non-endemic areas, posing serious health and agricultural problems. The disease can be prevented by vaccination, but there is currently no Food and Drug Administration-approved RVFV vaccine that can be used outside endemic areas, while there are two live attenuated vaccines available for use in endemic areas. These vaccines have the potential for reversion, and are therefore not recommended for use in countries where RVFV is not endemic. This indicates the need for more RVFV vaccine research and development. This work focused on the development of a RVFV vaccine candidate that would allow for differentiation between infected and vaccinated animals as well as humans.

Includes bibliographical references. / The acetone- butanol-ethanol (ABE) fermentation was one of the first fermentation processes to be industrialized on a large scale, and the dominant product, butanol is particularly significant due to its potential as a modern day fuel additive or fuel extender in the petrochemical industry. A collection of 19 solventogenic Clostridium beijerinckii and 11 Clostridium saccharobutylicum strains isolated from the National Chemical Products (NCP) ABE fermentation plant in Germiston, South Africa, were classed according to species by a quick species-specific colony PCR and by rifampicin screening methods respectively. The speciesspecific PCR aims to provide a rapid means of assessing any contamination of an ABE batch fermentation by differentiating between C. saccharobutylicum and C. beijerinckii species. Random Amplification of Polymorphic DNA (RAPD) analysis generated four C. beijerinckii and two C. saccharobutylicum strain groups respectively. Multilocus Sequence Typing (MLST) was developed for a smaller selection of strains and showed a further two strain groups within the NCP C. beijerinckii strains and three groups within the C. saccharobutylicum strains.

N-glycosylation plays an important role in Envelope (Env) function and may be involved in the modulation of the immune response to HIV-1 infection. In this study, we hypothesized that Env N-glycosylation may affect viral pathogenesis by influencing Env structure and function. Furthermore, we also postulated that differences in Env glycosylation could affect interactions between Env and DC-SIGN of dendritic cells (DCs), activating alternative signalling pathways which stimulate the release of different immune modulators. We generated pseudovirus of eighteen Env clones (PSVs) with variable number and position of potential N-glycan sites (PNGs) and compared their ability to infect TZM-bl cells, bind to Raji+ DC-SIGN cells, trans-infect TZM-bl cells when captured by either Raji-DC-SIGN cells or monocyte-derived dendritic cells (MDDCs) and modulate MDDC signaling by investigating the release of Interleukin-10 (IL-10) and other immune modulatory cytokines and MAPK activation. Entry efficiency, DC-SIGN binding and trans-infection varied widely across all clones. The level of IL-10 secreted by MDDCs in response to PSV stimulation varied 32-fold. The induction of IL-10 secretion by purified gp140 confirmed that Env was the viral component that stimulated the secretion of IL-10 via interaction with DC-SIGN and potentially other undefined receptors. PSV and purified gp140 stimulated MDDC signaling via ERK and JNK phosphorylation, while p38 was not activated. The addition of recombinant DC-SIGN lowered the levels of secreted IL-10 and ERK /JNK phosphorylation, suggesting that DC-SIGN plays a role in these responses. As Env mannosylation correlated with DC-SIGN binding, five highly conserved Env PNGs (241, 262, 386, 392, and 448) previously identified to carry high mannose type N-glycans and hence thought to be involved in DC-SIGN binding were deleted in two Env clones by site-directed mutagenesis to confirm their importance in Env function. The potential role of these PNGs in Env entry efficiency, DC-SIGN binding, trans-infection, induction of MDDC IL-10 secretion and activation of MAPK phosphorylation was determined. Deletion of these sites significantly affected the entry efficiency, DC-SIGN binding, trans-infection and MDDC IL-10 secretion, with one Env clone proving to be more sensitive to mutation than the other. This suggests that PNGs influence Env function in a clone-specific manner. As deletion of highly conserved PNGs abrogated Env function we used sequence analysis to identify PNGs involved in binding DC-SIGN and inducing MDDC IL-10 secretion. We grouped PSVs based on the presence or absence of specific PNGs in Env sequences and compared entry efficiency, DC-SIGN binding, trans-infection, stimulation of MDDC IL-10 secretion and induction of MAPK phosphorylation. Three Env PNGs were significantly associated with entry efficiency (N356, N392, and N674), and three sites (N289, N356 and 674) were significantly associated with trans-infection while N674 also influenced DCSIGN binding. The majority of MDDC donors secreted higher levels of IL-10 when stimulated with PSVs that carried PNGS at N130 (p = 0.0016) and N332 (p = 0.0039) and lacked N674 (p = 0.033). When Envs were graded on whether they had 0, 1, 2 or 3 of the PNGs (e.g. -130, -332, +674; -130, +332 and +674, etc.) those that carried either one of the PNGs or the entire induction motif (N130+ N332+ N674-) significantly stimulated MDDCs to secrete higher levels of IL-10 than those that completely lacked the motif (p = 0.0335 and p = 0.0304, respectively). As the presence of N674 was linked to reduction in all functions of Env, it is likely that the presence of an N-glycan at this site affected Env structure and could skew the analysis. Excluding N674 indicated that the presence of PNGs at position 130 and 332 was sufficient to induce significantly higher IL-10 release than those that had either none or one of these sites (p = 0.0053). When we determined whether N130 and N332 were enriched in subtype C acute infection Envs, these sequences were not enriched with PNGs at either N130 or N332 compared to chronic infection viruses. However, when IL-10 levels were compared between MDDC donors stimulated with PSV of either acute or chronic infection clones, those from early infection significantly enhanced MDDC secretion of IL-10 (p = 0.0039). This suggests that even though PNGs at 130 and N332 could be involved in inducing MDDC IL-10 secretion, it is not the only requirement for enhanced stimulation. Although Env differentially activated ERK and JNK phosphorylation, ERK phosphorylation did not correlate with IL-10 secretion, suggesting that this MAPK signaling pathway was not solely responsible for triggering the release of MDDC IL-10 and other regulatory cytokines. PSVs also stimulated the release of TNFα, IL-1β, IL-6, IL-8, MIP-1a, and MIP-1b while having no effect on IL-12 levels. This suggests that HIV-1 binding to DCs in the genital tract could change the dynamics of DC immune responses, deregulating their cytokines secretion and destabilising the Th0 cell differentiation to facilitate viral survival and thus productive clinical infection. We therefore conclude that HIV-1 variants differentially stimulate MDDCs to release immunosuppressive IL-10 and that transmitted founders could be better at modulating immune responses in the genital tract compared to chronic infection variants.

Bibliography: pages 127-144. / Bacillus brevis Alk 36 was isolated from soil during a screening programme for the selection of extracellular enzyme producing strains. A gene coding for an endo(1,3- 1,4 )-.8-glucanase (or lichenase) was cloned from B. brevis Alk 36 and expressed in Escherichia coli. The nucleotide sequence of this gene was determined and found to encode a protein of 252 amino acid residues. The amino acid sequence of the B. brevis lichenase gene showed only a 50% similarity to previously published data for Bacillus endo-(1,3-1,4)-β-glucanases. The enzyme exhibited some unique properties. The optimum temperature and pH for enzyme activity were 65-70°C and 8-10, respectively. When held at 75°C for 1 h, 75% residual activity was measured. The molecular mass was estimated to be 29 kDa and the enzyme was found to be resistant to sodium dodecyl sulphate (SDS). B. brevis Alk 36 was evaluated as a potential host strain for the efficient production and secretion of foreign proteins and was found to grow optimally between pH 8.0 and pH 9.5 and between 42°C and 52°C. B. brevis was successfully transformed using vector DNA and was found to produce relatively low levels of protease. In addition, it was evaluated as a possible protein hyper-secreting strain. However, using PCR technology, the highly conserved cell wall protein genes could not be positively identified in B. brevis Alk 36.