Includes bibliographical references (leaves 129-146). / Human papillomavirus (HPV) infection, specifically with oncogenic types, has been implicated in effectively all cervical cancer cases. Cervical cancer is a global health burden, especially in the developing world. Up to 18 types of HPV are considered oncogenic, of which HPV -16 and -18 cause 70% of cervical cancer cases worldwide. Two vaccines are available on the market: Gardasil(R), targeted against HPV -16, -18; -6 and -11, and Cervarix(TM), against -16 and -18. Both vaccines are based on the L1 capsid proteins of the types they are targeted to and are efficient, pro- phylactic, typespecific vaccines. However, two problems remain: they do not protect against nonvaccine types, that may cause a significant proportion of cancers specifically in African and HIV- positive populations, and they cannot be used to treat existing infections. We designed eight different chimaeric vaccines.

Includes abstract. Includes bibliographical references (leaves 109-129). / Cervical cancer is the second most common cancer affecting women and in most developing countries it remains the leading cause of cancer deaths. In South Africa, more than 3 400 women succumb to the disease every year and 1 in 31 women develop cervical cancer. The causative agent for cervical cancer is the Human papillomavirus (HPV). High-risk (carcinogenic) HPV types have been linked with 99% of the incidences of cervical cancer. The most common types identified in almost 70% of cervical cancer cases worldwide are HPV 16 and 18. HPV infection is very common in young healthy women and most immunocompetent individuals can clear HPV infection. However, in immunosuppresed women, clearance by host immune system is impaired. In addition, multiple HPV infections are quite common in women with Human immunodeficiency virus (HIV) infections. The objectives of this study were to identify HPV types in South African women who also had HIV infection, and secondarily, to determine if recombination of HPV genomes occurs. Determining the HPV types circulating in this country is important to enable identification of most common HPV types, in order to guide the development of vaccines against HPV infection. HPV genotyping was performed by the commercial Roche Linear Array HPV Genotyping Test.

Includes bibliographical references. / Nitrilases catalyse the conversion of a nitrile to its corresponding acid and ammonia by the addition of two water molecules. Cyanide dihydratases, a subgroup of nitrilases, specifically hydrolyse cyanide to formic acid and ammonia. Nitrilases are found in a diverse collection organisms that includes plants, bacteria and fungi. They form one branch a superfamily of structurally related enzymes that are believed to have in common a unique cys-glu-Iys catalytic triad. Many nitrilases exiat as a large molecular weight oligomers of more than 300kDa. In the current study the structures of two cyanide dihydratases, from Pseudomonas stutzeri AK61 and Bacillus pumilus Cl, have solved at a resolution 2.9nm and 32nm respectively by single particle reconstruction from electron micrographs of enzyme particles stained in uranyl acetate. Each enzyme consists of a spiral structure of well-defined length. It is proposed that this arrangement of subunits occurs in many other nitrilases and that a number of unexplained observations in the literature can reconciled by this model.

Malaria is a leading cause of morbidity and mortality worldwide, and results in approximately 600,000 deaths annually. The life cycle of the parasite is complex, and has several distinct stages of development. The transitions between these stages are brought about through tightly controlled and highly synchronized changes in gene expression. Plasmodium falciparum causes the most lethal form of malaria in humans. The parasite is particularly virulent as it is able to evade immune detection by the infected host. This virulence is directly related to the expression of variable antigens on the surface of infected red blood cells. The control of gene expression is known to be largely regulated via RNA Polymerase II (RNAPII) transcription initiation, but in P. falciparum the underlying mechanisms have not been determined. This primarily because very little is known about both the key protein factors and DNA elements which guide the assembly of RNAPII components into the transcription initiation complex. Bioinformatics studies have shown that there is very little amino acid sequence conservation between human and Plasmodium RNAPII transcription initiation components. Together with the observation that the Plasmodium genome has an extremely high A+T content, this suggests that Plasmodium may have specific mechanisms to initiate transcription, which could be targeted by novel anti-malarials. The general transcription factor TFIIB and the TBP-like protein (TLP) are key proteins involved in the recognition of the core promoter, and the initiation of RNAPII transcription initiation complex assembly. TFIIB stabilises DNA binding of the primary promoter recognition factor, TATA-box binding protein (TBP), and is involved in promoter recognition through interactions with specific DNA sequences up- and downstream of the TBP DNA binding site. TBP-like protein is a member of the TBP protein family that has been implicated in life cycle stage specific gene transcription initiation in various eukaryotic model organisms. This research study reports the first successful expression and purification of recombinant epitope-tagged Plasmodium falciparum TFIIB and TLP proteins. Preliminary assays demonstrate DNA-binding activity for the recombinant Plasmodium TBP-like protein, and suggest DNA-binding activity in Plasmodium TFIIB protein, which has not been demonstrated before in eukaryotic TFIIB.

Plasmodium falciparum is the causative agent of the most severe form of malaria and continues to pose challenges to international healthcare, with high mortality rates and emergence of drug-resistant strains. Plasmodium falciparum has multiple sexual and asexual lifecycle stages within its Anopheline mosquito and human hosts, accompanied by distinct morphological changes. The complex lifecycle, along with the ability to adjust rapidly to different environmental niches, is governed by highly regulated and tightly synchronised changes in stage-specific gene expression. In eukaryotes, regulation of RNA Polymerase II transcription initiation is one of the main mechanism of gene expression control. Past research has revealed the presence of crucial elements of RNA Polymerase II (RNAPII) transcription machinery in P. falciparum, however, the precise transcription initiation mechanisms in P. falciparum remain to date undescribed. Bioinformatics studies have found very little homology between human and P. falciparum transcription factors. Furthermore, because of the extreme bias toward A/T in the Plasmodium genome, TATA-box and other core promoter elements that direct transcription initiation, cannot be determined with confidence in bioinformatics studies. Functional characterisation of the key protein factors involved in transcription initiation is a first important step towards the identification of core promoter elements and could reveal currently unknown eukaryotic transcription initiation mechanisms or new anti-malarial targets. In eukaryotes, TATA-binding protein (TBP) and transcription factor IIB (TFIIB) are the key protein factors involved in the core promoter recognition and RNAPII preinitiation complex (PIC) assembly. TBP nucleates PIC assembly by binding the TATA box, thereafter the TBP-TATA complex is further stabilised by TFIIB. In addition, TFIIB has a crucial role in RNAPII recruitment and transcription start site selection and is therefore deemed indispensable in eukaryotic transcription. P. falciparum TBP is the only PIC component in P. falciparum that has been functionally characterised to date, albeit to a very limited extent. This research study reports the successful expression and purification, as well as initial characterisation of DNA-binding activity of P. falciparum TBP and TFIIB. We observe PfTBP binding at multiple locations on putative P. falciparum promoters. We further report PfTBP-independent binding activity of PfTFIIB, that has not been previously observed under the conditions and has implications for novel DNA-binding mechanisms of PfTFIIB. Furthermore, we conclusively demonstrate the formation of a PfTBP-PfTFIIB-promoter ternary complex.

Bibliography: pages 182-223. / B. fibrisolvens H17c is a Gram-positive obligate anaerobe which has been found in the rumen of most ruminants. Strains of B. fibrisol vens have been reported to exhibit activity toward cellulosic and hemicellulosic substrates. The aim of this thesis was to screen a genebank of B. fibrisolvens H17c DNA and to isolate genes expressing cellulase and xylanase activity. Two genes encoding β-1-4- glucosidase (BglA) and endo-β-1-4-xylanase (XynB) were cloned in E. coli.

Clostridium acetobutylicum P262 is an endospore-forming Gram positive anaerobic bacterium, and for many years this organism has been used in the industrial fermentation for the production of acetone and butanol from carbohydrate substrates. The aims of this thesis included studies on small phosphorylated molecules involved in energy metabolism and cell differentiation, and an investigation into the genetics and molecular biology of C. acetobutylicum electron transport genes. To facilitate quantitation of nucleoside triphosphates in extracts of C. acetobutylicum, a chromatographic data acquisition and analysis system was constructed. Samples were prepared from C. acetobutylicum cultures by treatment with formic acid, and nucleotides contained in these extracts were separated by strong anion exchange HPLC. The developed manual integration system features the ability to collect and store chromatographic data, allowing for multiple integration using different calibration curves. Nucleoside triphosphate profiles were obtained from batch fermentations of the C. acetobutylicum P262 wild type, sporulation deficient (spo-1) and solvent deficient (ds-1) strains. The nucleoside triphosphate profiles of the wild type and spo-1 mutant were similar and were characterized by a trough in nucleotide levels which occurred just prior to the pH break point, the onset of the stationary growth phase, clostridial stage formation and the transition from the acidogenic to the solventogenic phase. The nucleoside triphosphate concentrations during the exponential growth phase were much lower than those found during the stationary phase. Exponential phase nucleotide levels in the cls-1 mutant were comparable to those observed in the wild type and spo-1 mutant. Unlike the wild type and spo-1 strains, the cls-1 mutant, which does not switch to solventogenesis, did not demonstrate an increase in nucleotide levels after the cessation of cell division. The involvement of nucleotide levels, particularly that of GTP, in the differentiation of C. acetobutylicum was indicated by the effect of inhibitors, which have been shown to decrease ribonucleotide levels in other organisms and cause an increase in sporulation. The antibacterial agent metronidazole, was used as a tool for the isolation of C. acetobutylicum electron transport genes. Since it was desired to clone these genes in Escherichia coli, and investigation into the activation of metronidazole by E. coli strains was necessary. E. coli strains with lesions in their DNA repair systems were more susceptible to metronidazole than wild type strains. However, it has been reported that DNA repair deficient strains of E. coli that also had a diminished ability to reduce chlorates and nitrates were no more susceptible to metronidazole than their wild type parents (Jackson et al., 1984; Yeung et al., 1984). To isolate a suitable E. coli cloning host for the selection of C. acetobutylicum electron transport genes which activated metronidazole, transposon mutagenesis of the recA E. coli strain CC118 with TnphoA, was used to construct the recA, metronidazole resistant E. coli strain Fl9. F19 was shown to have diminished nitroreductase activity, which was presumed to be responsible for the metronidazole resistant phenotype. However, the recA mutation renders E. coli F19 highly susceptible to the reduced toxic intermediates of metronidazole. The E. coli F19 recA, nitroreductase deficient mutant was used for the isolation of C. acetobutylicum genes on recombinant plasmids which activated metronidazole. Twenty-five E. coli F19 clones which contained different recombinant plasmids were isolated. The clones were tested for nitroreductase, pyruvate-Fd­oxidoreductase and hydrogenase activities. Nitroreductase and pyruvate-Fd­oxidoreductase activity was not demonstrated in any of the isolated clones, and only one clone tested positive for hydrogenase activity. DNA hybridization and restriction endonuclease mapping revealed that four of the C. acetobutylicum insert DNA fragments on recombinant plasmids were linked in an 11.1 kb chromosomal fragment. It was determined that this 11.1 kb fragment contained at least two regions responsible for activating metronidazole. The one gene responsible for making E. coli F19 extremely sensitive to metronidazole was localized to a 2 kb region. The nucleotide sequence of this 2 kb region was determined and two truncated open reading frames and one complete open reading were present. The complete open reading frame was shown to be responsible for activating metronidazole. The deduced amino acid sequence of this open reading frame was determined to be 160 amino acids in length, and database searches showed good similarity to flavodoxin proteins from many organisms. Based on alignments to the amino acid sequences of these flavodoxins, as well as the fact that Chen and Blanchard (1979) reported that reduced flavodoxin can transfer electrons to metronidazole, the sequence corresponding to this C. acetobutylicum metronidazole activating gene was identified as coding for a flavodoxin gene. The role of flavodoxin in C. acetobutylicum and other organisms is presented. Possible relationships between the cloned C. acetobutylicum flavodoxin gene and metronidazole sensitivity are discussed.

In a murine model of Leishmania major infection, susceptible BALB/c mice develop a detrimental Type 2 immune response characterized by the production of interleukin (IL)-4 and IL-13, which single through a common receptor, the IL-4 receptor alpha chain (IL-4Rα). Forkhead box P3 (Foxp3⁺) Regulatory T (Treg) cells are an unique subset of CD4⁺ T cells that play important immunomodulatory roles maintaining a balance between Type 1 and Type 2 immune responses. During L. major-induced cutaneous leishmaniasis, Treg cells accumulation at the site of infection has been implicated in suppressing a detrimental Type 2 immune response by modulating early interleukin (IL)-4 production, however it remains unclear if IL- 4Rα mediated signalling on Treg cells play a significant role in this process. To investigate this further, a novel BALB/c model was utilized in which the IL-4Rα chain was conditionally knocked out on Treg cells (Foxp3ᶜʳᵉIL-4Rα⁻^/ˡᵒˣ mice). We demonstrated that the differential IL- 4Rα deletion efficiency in male (approximately 102 %) and female (approximately 32%) was maintained during L. major infection. Foxp3ᶜʳᵉIL-4Rα⁻^/ˡᵒˣ male mice, which had a greater degree of IL-4Rα deletion on Foxp3⁺ Treg cells, developed significant footpad swellings and ear swellings, increased parasitic burdens at the site of infection and within draining lymph nodes. This hypersusceptible phenotype observed in Foxp3ᶜʳᵉIL-4Rα⁻^/ˡᵒˣ BALB/c male mice was accompanied with an increased Treg cell activity and amplified Type-2 immune response with an increase in IL-4, IL-10 from L. major-infected lymph node samples and IgE antibody secretion in L. major infected serum samples. Flow cytometry analysis revealed that a L. major-induced Indoleamine 2,3 dioxygenase (IDO)-mechanism could allow for increased Leishmania replication. Collectively, these data suggest a protective role for IL-4Rα signalling on Treg cells in suppressing a detrimental Type 2 during cutaneous leishmaniasis.

A total of 56 presumptive actinobacterial strains was isolated from three different samples taken from the Silvermine Nature Reserve (Table Mountain National Park, Cape Town), namely, cave soil, the wall of the cave and sediment from the shallow waters of a reservoir. Twenty nine (29) isolates were successfully identified to the genus level by 16S-rRNA gene analysis: one Micrococcus strain, one Streptacidiphilus strain, one Micromonospora strain and 26 Streptomyces strains. The phylogenetic position of each identified strain within its genus was investigated by generating a phylogenetic tree based on its 16S-rRNA gene sequence. Further analysis of the Streptacidiphilus strain was conducted based on the gyrB gene. Metagenomic analysis was used to further analyse the actinobacterial diversity of the freshwater reservoir sediment from the Silvermine Nature Reserve. A total of 97 16S-rRNA gene clones was obtained from the reservoir sediment sample, RS1, using actinobacteriumspecific 16S-rRNA gene primers S-C-Act-0235-a-S-20-F and S-C-Act-0878-a-A-19-R and each clone was identified using the EzBioCloud database. Analysis based on unique phylotypes in the clone library revealed that 80% of the clone library was composed of actinobacterial strains belonging to the orders Acidimicrobiales, Streptomycetales, Streptosporangiales, Corynebacteriales, Sporichthyales and the family Jatrophihabitandaceae (the remaining 20% was identified as non-actinobacterial strains). The percentage composition of the actinobacterial clonal diversity for each order was as follows: Acidimicrobiales, 56%; Streptomycetales, 29%; Streptosporangiales, 9%; Corynebacteriales, 4%; Sporichthyales, 1% and family Jatrophihabitandaceae, 1%. Rarefaction analysis revealed that the total actinobacterial diversity of the sample was not represented in the clone library. Therefore, further sampling and analysis of the sample site would uncover greater actinobacterial diversity. Thirty seven (37) putative actinobacterial isolates of the 56 that were isolated from the Silvermine Nature Reserve were screened for antimycobacterial activity against the non-pathogenic Mycobacterium aurum strain A+ using a standard over-lay method. A total of five identified 2 actinobacterial isolates (Streptomyces strains RS6, RS7, RS9, RS13 and RS15) and an unidentified actinobacterium, strain RS4, demonstrated very strong antimycobacterial activity (zone of growth inhibition of over 3000 mm2 ). In addition, 15 of the 37 strains were active against Staphylococcus aureus ATCC 25923 and three were active against Escherichia coli ATCC 25922. Streptomyces strains CS1, CS3, CS12, CS18, CS19, CW5, RS3, RS6, RS9, RS13 and RS15, displaying varying strengths of antimycobacterial antimicrobial activity, were selected for antibiotic extraction from culture broths. The resulting crude extracts were subjected to spot bioautography to test for antibacterial activity. The organic compounds extracted from the cell mass of Streptomyces strain CS3 and the broth fraction of Streptomyces strain RS3 demonstrated strong activity against M. aurum strain A+. Furthermore, the crude extracts of 15 actinobacterial isolates (Micromonospora strain RS10 and Streptomyces strains CS1, CS3, CS12, CS18, CW2, CW5, RS3, RS6, RS7, RS9, RS13, RS15, RS18 and RS19) were additionally tested for antiplasmodial activity against Plasmodium falciparum strain NF54. Seven of these strains showed activity against Plasmodium namely, Streptomyces strains CW2, CW5, RS3, RS7, RS13, RS15 and RS19. Streptomyces strains CW2, CW5 and RS7 displayed the strongest activity against P. falciparum strain NF54 with IC50 values below the guideline threshold of 1000 ng/mL (strain CW2 culture broth crude extract: IC50 40 ng/mL, strain CW5 culture broth crude extract: IC50 128 ng/mL and strain RS7 culture broth crude extract: IC50 70 ng/mL).

Chikungunya fever is a non-fatal but highly debilitating disease that affects primates, birds and humans. The causative agent is the chikungunya virus (CHIKV), an arbovirus of the Alphavirus genus. CHIKV is responsible for the largest epidemic recorded for an Alphavirus, infecting an estimated 1.4 to 6 million patients worldwide. Furthermore, it has been recognised by the United States army as a potential biological weapon used for bioterrorism owing to the potential for infection via aerosol. CHIKV is primarily transmitted by infected Aedes aegypti mosquitos and is currently distributed in Africa, parts of Asia and South, Central and North America. As a result of the virus genetically adapting to infect the Aedes albopictus mosquito, its recent and rapid spread to non-endemic regions has occasioned increasing anxiety as well. Infection in humans presents as a sudden onset of fever, rash and severe arthralgia that persists for years. At present, there is no fast and effective diagnostic test to distinguish CHIKV from other similar viruses. This is a problem because viral infection displays the same symptoms as that of dengue, Zika, Ebola and yellow fevers while prognosis, patient care, and persistent symptoms of these viruses are very different. Usually, during the development of a diagnostic reagent, Biosafety Level 3 (BSL3) containment is required for purifying antigens from live viruses. These lab diagnostic tests are expensive to perform and, in regions facing a CHIKV epidemic, are inefficient due to their long waiting periods. This results in patients going undiagnosed or misdiagnosed and/or falling outside the window of prophylactic treatment. As such, a cheap and rapid diagnostic reagent to detect the presence of CHIKV antibodies would be most advantageous. In this study, two recombinant variants of the CHIKV E2 glycoprotein were expressed in Nicotiana benthamiana plants to assess their viability for use in a diagnostic reagent for CHIKV infection. Two versions of a tobacco sp. codon-optimised, 6xhis-tagged CHIKV E2 envelope glycoprotein gene were synthesised and cloned into the plant expression vector, pTRAkc-ERH. The E2 glycoprotein is a desirable protein candidate used for a diagnostic reagent as it is a major target for neutralizing antibody production against CHIKV during early infection. One variant contained a ~52 kDa full length E2 glycoprotein (CHIKV E2-HIS) while the other contained a ~49 kDa truncated E2 glycoprotein lacking its transmembrane domain (CHIKV E2ΔTM-HIS). Following this, an expression time trial was performed whereby the recombinant proteins were expressed in N. benthamiana plants via Agrobacterium-mediated small-scale 6 syringe-infiltration at different optical densities, OD600 = 1.0 and 0.5. To improve expression, both genes were co-infiltrated and co-expressed with a human chaperone proteins calreticulin (CRT) or calnexin (CNX), or a plant silencing-suppressor protein NSs. Expression of the recombinant protein variants alone showed low to undetectable levels of expression in plant leaves across 7 days post infiltration (dpi) for both ODs tested. CHIKV E2ΔTM-HIS yielded the highest levels of all combinations tested at an OD600 = 1.0 when co-expressed with CRT and harvested at 3 dpi. These parameters were used for subsequent scaling up and production of E2 using vacuum infiltration. Attempts at purifying CHIKV E2ΔTM-HIS proteins using Ni-NTA affinity chromatography and further investigation into the exposure of the 6xHis-tag on the native conformation of CHIKV E2ΔTM-HIS indicated that the 6xHis-tag was insufficiently exposed on E2 and thus inaccessible to facilitate purification by Ni-NTA affinity chromatography. Further attempts at purifying recombinant CHIKV E2ΔTM-HIS proteins by pH purification were also unsuccessful as large amounts of plant-protein contaminants present in all samples prevented adequate separation from CHIKV E2ΔTM-HIS. A different approach utilising ammonium sulphate precipitation facilitated separation of recombinant CHIKV E2ΔTM-HIS from some of the contaminating plant proteins in the 30 - 60% ammonium sulphate fraction; however, large amounts of recombinant CRT were co-purified with E2 in this fraction. Although expression of a candidate diagnostic reagent in plants for detecting CHIKV antibodies in the form of E2 glycoprotein was achieved, further research needs to be done to optimise a purification strategy for CHIKV E2ΔTM-HIS proteins.