Young women in areas of high HIV risk are more vulnerable to new HIV infections compared to men. With the development of programmes using anti-retrovirals (ARVs) to prevent HIV infection, the concurrent use of hormonal contraception and ARVs is likely to increase in young women. Products delivering ARVs and hormonal contraceptives intravaginally, are in development, and aim to simultaneously prevent unintended pregnancies and prevent HIV infection. However, little is known about the combinatorial effects of ARVs and hormonal contraceptives in the female genital tract. The mucosal surface in the female genital tract is the initial site of HIV entry and viral replication. Understanding the effect of hormonal contraceptives and ARVs is relevant to the potential safety of these for long term use. The present study aims to investigate the effects of the ARVs dapivirine (DPV), tenofovir disproxil fumarate (TDF) and a panel of progestins, alone and in combination with each other, on the expression of immune function genes relevant to HIV acquisition, on steroid receptor activity, and on HIV replication. Analysis of expression of select pro- and anti- inflammatory genes revealed that, DPV exerts gene-specific pro-inflammatory and cytotoxic effects on cervical cells and tissue. Co-stimulation with the progestin medroxyprogesterone acetate (MPA) revealed that MPA can potentiate and inhibit the DPV-induced pro-inflammatory effects, in a gene-specific manner in cervical cells. DPV was shown to alter the efficacy and potency of an androgen receptor (AR) ligand, demonstrating its ability to influence AR activity. Furthermore, DPV inhibited viral replication but also increased HIV co-receptor expression in the absence and presence of MPA. In contrast to DPV, TDF had no effect on the expression of pro-inflammatory immune function genes or HIV co-receptors in the absence and presence of MPA. TDF had no effect on endogenous steroid receptor levels in cervical cells and showed no significant effects on steroid receptor transactivation. In summary, data from the present study show that TDF may be the better choice of ARV for combinatory usage with hormonal contraceptives. An association between genital inflammation and HIV acquisition has previously been established and thus the potentiated pro-inflammatory effects of DPV may potentially change the microenvironment of the female genital tract to favour increased risk of HIV or other genital tract infections, when used alone and in combination with hormonal contraceptives such as MPA. Furthermore, DPV's influence on steroid receptor activity points towards potential for increased side effects due to off-target AR effects. These results have serious implications for women using hormonal contraception who may consider the use of DPV as a microbicide. However, whether in vitro results will occur in vivo, remains to be established.

Includes bibliographical references (leaves 117-125). / Structural and molecular dynamics analysis of the glutamine synthetase from E. coli indicates that a possible mechanism by which the adenylylation/deadenylylation of the enzyme affects the enzyme specificity for either MgATP or Mn2ATP and NH4+ or NH3, is by switching between two putative serine protease-like catalytic triads. Site-directed mutagenesis of a number of residues identiï¬ ed as playing a role in these catalytic triads, led to the following observations. Both Ser52 and Ser53 were important for the catalytic activity of the enzyme. It was determined that the Ser52 residue appeared to have adenylylated functionality and the Ser53 residue, deadenylylated functionality. Evaluating these serine mutant enzymes in the presence of the serine protease inhibitors, AEBSF and PMSF, led to the conclusion that Ser52 and Ser53, did, indeed, appear to form part of a catalytic triad, as the activity of the enzymes were inhibited in the presence of the inhibitors. When both serine residues were removed in a single mutant, activity was not signiï¬ cantly inhibited by either inhibitor. His210 and His211 were found to be equally important to the functionality of the enzyme, and the results, in consultation with the enzyme model, led to the conclusion that the His210 residue had deadenylylated activity and the His211 residue had adenylylated activity. All the potential acid residues, when removed, had an effect on activity, but this was to be expected as all had previously been identified in the literature as important in the active site of the glutamine synthetase from E. coli. Again, consultation of the model led to the conclusion that the two acid residues that filled the function of the acid residue in each catalytic triad, were Glu129 for the adenylylated from of the enzyme, and Glu357 for the deadenylylated form of the enzyme. These catalytic triads are believed to be comprised of Ser52', His211 and Glu129 for the adenylylated form of the enzyme, and Ser53', His210 and Glu357 for the deadenylylated form of the enzyme. Possible model mechanisms for the both the adenylylated and deadenylylated forms of the enzyme are proposed.

Includes bibliographical references. / Although the South African abalone, Haliotis midae, has been commercially harvested since 1949, successful cultivation of this species only began in the 1980s. Since then, the abalone mariculture industry has expanded dramatically and currently produces between 500 and 800 tons of abalone per year with a net farm gate value of approximately R125 million. However, disease has had a severe impact on the international aquaculture industry and is anticipated to become an increasingly important factor, together with the slow growth rate of H. midae, that will negatively impact on the further development and success of the local abalone mariculture industry. Thus, the future of H. midae mariculture in South Africa depends in part on the development of methods to enhance the growth rate and disease resistance of farmed H. midae. Erasmus et al. (1997) showed that abalone enteric bacteria enhanced digestive efficiency by secreting polysaccharolytic enzymes and it was suggested from these results that these bacterial enzymes could affect the growth rate of abalone. Furthermore, an overwhelming body of evidence has shown that probiotic microorganisms can significantly improve the growth rate and disease resistance of aquacultured animals. The aim of this study was to isolate enteric microorganisms from H. midae that are capable of hydrolyzing the various protein and starch substrates included in formulated abalone feeds. Upon identification, the selected microbes would be tested for their ability to colonize the digestive tract, improve digestion, growth and immunity of farmed H. midae.

Foot-and-mouth disease, also known as FMD, is caused by the aphthovirus Foot-and-mouth disease virus (FMDV), which is a highly contagious disease of cloven-hoofed animals. It is endemic in Africa, parts of South America and southern Asia. In South Africa, the disease is controlled essentially through prophylactic vaccination. Current vaccines on the market are chemically inactivated virus strains. However, these are not considered ideal due to the possibly insufficient inactivation which could fail to render the virus harmless. Research on recombinant vaccines which obviate the need for high biosafety requirements for vaccine preparation has shown that recombinant FMDV virus-like particles (VLPs), devoid of viral genetic material, are an ideal vaccine candidate as they are as immunogenic as the virions themselves when administered to animals. These VLPs are formed by the assembly of the FMDV capsid proteins VP0, VP1 and VP3, which are generated upon the proteolytic cleavage of the capsid precursor protein P1-2A, by the FMDV 3C-protease. The expression platforms used to co-express and produce the component capsid proteins and the protease are usually mammalian, insect or E. coli cells. The use of these expression systems requires extensive bioreactor infrastructure and sterile conditions for vaccine preparation which are costly. In addition, some studies have shown how the co-expression of the 3C-protease can prove to be deleterious when expressed at a high concentration in expression systems. In order to circumvent this, and encourage more efficient production of the capsid proteins and subsequent VLP assembly, researchers have shown that the levels of the 3C-protease can be down-regulated by introducing mutations in the 3C gene or a ribosomal frameshift in the gene sequence which subsequently reduce its deleterious effect. Our laboratory has previously shown that similar FMDV VLPs can be assembled by the expression of FMDV P1-2A (referred to as oP1-2A, in this study), in the absence of the 3C-protease in the plant Nicotiana benthamiana, albeit in low amounts. This platform does not require high biocontainment facilities for the production of recombinant proteins and VLPs and the process is easily scalable. This study centers mainly on optimisation of the FMDV capsid protein expression in N. benthamiana in order to increase VLP yields. I first used codon-optimisation as an approach to improve expression of the capsid proteins and compared expression in the presence and absence of the 3C-protease, using mP1-2A-3C (a new codon-optimised construct), mP1-2A and oP1-2A. Electron microscopy (EM) showed that VLPs resulting from the expression of both mP1-2A-3C and mP1-2A were very low in yield, and irregular in shape and size compared to those produced using oP1-2A. The stability of the plant-produced VLPs was assessed by counting numbers of VLPs, when it was seen that expression of mP1-2A-3C compared to oP1-2A produced an average of 1 VLP per field of view versus 3 VLPs per view, for the same magnification. Furthermore, maturation trials at room temperature was performed on the oP1-2A VLPs, whereby a time-point between 30 to 45 minutes was considered ideal to produce stable VLPs. It is also known that FMDV, unlike the other members of the Picornaviridae family, is acid and heat-labile. The second aim of this study was to promote the stability, and hence encourage the amount of the VLPs produced, by engineering acid and heat-resistant mutants, namely, VP1 N17D, VP2 H93C and VP1 N17D/VP4 S73N using site-directed mutagenesis. A fourth mutant, VP3 A118V which is acid sensitive was used as a control in downstream experiments. The mutants were subjected to a lower than normal pH and a higher than normal temperature. Expression of oP1-2A from the pH and heat assays was assessed to be better than its mutants. The optimum VLP count of 3 VLPs per field of view, was achieved from expression of oP1-2A, after treatment at pH 6.2, compared to 2 VLPs or 1 VLP per field of view for the other mutants tested under all the different conditions. The final aim of this study was to test the immunogenicity of the VLPs from expression of oP1-2A in Balb/C mice. Due to the low yields of VLPs obtained from purification through a continuous gradient, a partial purification method was adopted. Two experimental groups of animals were either vaccinated with P1-2A VLPs or with adjuvanted P1-2A VLPs. A control group was administered with partially-purified plant extract, previously infiltrated with pEAQ-HT. The two experimental groups elicited a marginal increase in humoral immune response at 41 days post vaccination (dpv), which increased significantly at 58 dpv. To my knowledge, this is the first study showing that VLPs produced from expression of FMDV P1-2A only, in tobacco plants, can withstand otherwise degradative acidic and heat conditions. This characteristic has potential for extending the shelf-life of such a candidate vaccine. I also implemented maturation steps to further promote the stability of such VLPs. Finally, the partially purified VLPs showed that they stimulate a significant FMDV P1-2A-specific immune response, particularly in combination with the adjuvant Montanide suggesting that it has potential as a candidate FMDV vaccine.

Bibliography: pages 119-137. / Butyrivibrio fibrisolvens HI 7C is an important anaerobic bacterium which occurs in the rumen of most ruminants. A key factor affecting the growth of this bacterium is the availability of nitrogen sources, particularly in the form of ammonia. The aims of this study were to attempt the isolation of a gene/genes involved in the regulation of the B.fibrisolvens type III glutamine synthetase (GS), which is a key enzyme involved in ammonia assimilation in rumen bacteria. An existing B. fibrisolvens gene bank, as well as the B. fibrisolvens glnA gene cloned onto a low 11 copy number plasmid, were used to generate a heterologous, Escherichia coli- based, two plasmid, in trans system. This system was used to isolate an E. coli clone which showed increased GS activity levels (2.5-fold) and a retarded growth rate phenotype in complete media.

Bibliography: pages 105-115. / A cloning system, using metronidazole as a screening tool and E. coli FI9 as a selection host, was previously established to clone C. acetobutylicum P262 electron transport genes which may play a role in solvent metabolism in this bacterium. In theory, metronidazole would be reduced under anaerobic conditions to a cytotoxic intermediate by C. acetobutylicum P262 electron transport genes or reductive enzymes cloned into a recombinant plasmid. This intermediate would kill the host E. coli FI9. One C. acetobutylicum P262 clone, pMETIOB, was found to render the E. coli strain FI9 sensitive to metronidazole, under anaerobic conditions. A number of subclones of the 2.56kb C. acetobutylicum P262 insert DNA were generated in Bluescript pKS and pSK. A range of exonuclease III deletions of this C. acetobutylicum P262 insert DNA were also generated which were shown to lose the metronidazole sensitivity phenotype on progressive deletion of the insert DNA. In vitro and in vivo protein transcription/translation experiments failed to reveal a protein product that was related to the metronidazole sensitivity phenotype. DNA hybridization confrrmed that the insert DNA of pMETIOB hybridized to C. acetobutylicum P262 chromosomal DNA, but not to E. coli chromosomal DNA. The nucleotide sequence of a 933-bp fragment of the C. acetobutylicum P262 insert DNA was determined.

Fusarium verticillioides and Cercospora zeina are two economically important fungal pathogens of maize in Southern Africa. Phytoalexins are low molecular weight anti-microbial compounds produced in plants in response to pathogen infection. In maize, two classes of non-volatile terpenoid phytoalexins, viz. kauralexins and zealexins, play a role in fungal resistance. It has previously been shown that maize lines inoculated with either F. verticillioides or C. zeina induces kauralexin and zealexin accumulation. In addition, kauralexin metabolite accumulation and candidate kauralexin biosynthetic gene expression were highly correlated. In this study a mutant line with a Dissociation transposon element inserted into An2 was identified with the goal of stopping An2 from being expressed. The mutants were maintained in an inbred W22 maize line. Gene expression was compared between transposon-insertion mutants and wild type W22 at the seedling stage. A F. verticillioides and C. zeina inoculation assay was carried out on a segregating knock-down line. Phytoalexin accumulation, gene expression and disease susceptibility were subsequently examined in the mutants and wild type. F. verticillioides-inoculated mutants displayed significantly decreased kauralexin and zealexins accumulation and An2 gene expression. Fungal load and symptoms was greater in mutants than wild type controls. Kauralexin accumulation and An2 expression were negatively correlated with the quantified fungal load. C. zeina-inoculated mutants did not display significantly reduced kauralexin accumulation and An2 expression as An2 did not appear to be upregulated in the W22 maize line in response to C. zeina. This is likely due to a genetically-controlled leaf flecking phenotype in W22 leading to broad-spectrum resistance, as well as potentially impacting the jasmonic acid pathway. Lastly, an attempt was made to clone An2 towards A. thaliana transformation for overexpression analysis. Only a truncated section of An2 was able to be cloned into the expression vector.

Mammals have evolved a vast myriad of limb morphologies adapted for a wide range of activities. One of the most remarkable evolutionary adaptations of a mammalian limb is that of the forelimb wing of a bat used for powered flight. This capability evolved ~ 51 Mya from its arboreal ancestor without any fossil record of intermediate forms. To reconstruct how this transition occurred, an Evolutionary Developmental approach can be applied to investigate altered mechanisms present in bat limb development. Similar genes and signalling centres are present in both mice and bats, making mice a good model organism for comparison. This study used a pre-existing set of RNA-seq transcriptomes from three pivotal developmental stages (CS 15, CS 16 and CS 17) of bat development, to compare FL and HL gene expression. Of the list of differentially expressed genes, a subset was selected to characterise spatial expression patterns within the developing bat limb compared to mouse limbs by whole-mount in situ hybridisation. Five transcription factors: Lef1, Lhx8, HoxA10, Mllt3 and Tbx5, as well as two Long non-coding RNAs: Hottip and Tbx5-as1 were selected. Novel expression of Mllt3 was detected in FL autopods at CS15, in a region slated to expand with digit elongation. Lef1 in situ signal was more robust in HL autopods of CS 15 embryos compared to FLs and equivalently staged mice. Lhx8 displayed a strong signal in CS 16 and CS17 wrist tissue, as well as a faint signal in interdigital tissue in the FL autopods. The LncRNA Hottip displayed vastly different expression pattern between FL and HL, with staining being reduced in the digit and interdigital regions of the FL at CS 16L, whereas expression in the HL was robust in the digit, and even more so in the interdigital regions. The LncRNA Tbx5-as1, displayed a similar expression pattern to the known FL initiation transcription factor Tbx5 at late stages (CS 16L -CS 17L). Isoform characterization to validate the two LncRNAs, was performed on cDNA a CS 18L embryo. The cloned transcripts identified a new set of alternatively spliced isoforms for both LncRNAs. Unusual RNA-seq tracks in the HoxA10 locus were investigated using qPCR. It was discovered this region is variable amongst biological samples; however there is a large reduction in expression in this region from CS 15 to CS 16.

Abstract Maize (Zea mays L.) is an important staple food crop in sub-Saharan Africa providing food security to millions of people. Fusarium verticillioides is an important fungal pathogen that infects maize and causes 'Fusarium Ear Rot' which decreases maize kernel yield and quality. In addition, the fungus produces mycotoxins which contaminate the kernel and upon ingestion have negative health consequences for both people and livestock. To this date, there is still no African maize line completely resistant to infection by F. verticillioides. In this study, an African maize line, Zea mays CML144, was infected with F. verticillioides using a soak-seed inoculation method and grown for two weeks under controlled conditions. Analysis of the morphological characteristics showed that compared to the control (mock-infected) maize, infected maize seedlings displayed signs of stunting with leaves shorter & thinner while roots were shorter and displayed visible signs of rotting. Control and infected maize plants were also characterised physiologically and biochemically. Electrolyte leakage experiments were conducted on the meristem regions of the plants after week one and two of infection and showed that leakage increased over time in both control and infected samples with no significant difference observed between the two groups. Biochemical characterisation by analysing superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) antioxidant enzymes showed an increase after the two weeks of infection, indicating a defense response by the plants in response to infection by the fungal pathogen. RNA-sequencing, the main aim of this study was conducted on control and infected plants after two weeks of infection to identify differentially expressed genes (DEGs) involved in F. verticillioides infection. The Illumina NextSeq 500 platform was used to sequence the transcriptome and quantify changes in gene expression. Analysis of the RNA-seq data using the Tuxedo suite of protocols revealed significant DEGs that were both up- and down-regulated in the infected samples compared to the control. Data analysis was conducted using the DNA subway online bioinformatics tool and these results were compared to those obtained using a separate analysis which also incorporated the Tuxedo suite of protocols. Bioinformatic analysis on the RNA-seq DEGs were performed using the agriGO analysis tool which revealed three significant Gene Ontology (GO) terms for both the up- and down-regulated genes, respectively, with the 'response to stimulus' GO-term (within the down-regulated genes) being of specific interest. Other GO-terms included response to chemical stimulus, carbohydrate metabolic process and ion bonding, which also played a role in the defense response when plants were infected by the fungal pathogen. Quantitative Real-Time PCR was performed on five DEGs that were either up- or down-regulated in response to F. verticillioides infection to validate RNA-seq data as well as the GO-analysis results. Quantitative Real-Time PCR was also used as a pre-validation (before RNA-seq) on shrunken-1, a down-regulated gene found in a previously conducted study. We observed that in response to infection by F. verticillioides, expression of shrunken-1 was down-regulated, however, this was not shown to be significant (p>0.05). The results in the current study and the identification of the genes in Zea mays CML144 responding to fungal infection will aid in the goal to develop a maize line completely resistant to F. verticillioides in Africa and in particular South Africa. This would provide improved food security and minimise health risks to the population in the long term. To our knowledge, this is the first study investigating F. verticillioides infection in the African maize line Zea mays CML144 using the soak-seed inoculation method.

Bibliography: pages 132-155. / In order for bacteria to survive in their environment they must continuely sense signals such as, presence of host organisms, chemical concentrations, or variationsin other physiological parameters. Many bacteria sense their environment through the use of a two component regulatory systems. These systems usually employ the use of two different proteins, a sensor protein and its cognate response regulator. Some bacteria can survive fluctuations in medium osmolarity through the use of a two component signal transduction system. In Escherichia coli and Salmonella typhimurium this two component system includes the EnvZ sensor protein and its cognate response regulator, OmpR. The two genes that code for these proteins are envZ and ompR genes respectively. The two genes together form the ompB operonrespectively. This operon regulates the expression of two outer membrane proteins, OmpF and OmpC in response to medium osmolarity in E. coli.Erwinia chrysanthemi has been found to be sensitive to desication. Proliferation of soft rot, caused by this organism, has also been associated with irrigation. E.chrysanthemi has also been observed to respond to changes in medium osmolarity. Evidence of an ompB operon was thus sought. Outer membrane proteins were isolated using sodium lauroylsarcosine. Three major outer membrane proteins were isolated, namely Ompl (37.5 kd), Omp2 (35.5 kd) and Omp3 (34.5 kd). Increase in medium osmolarity resulted in an increase in expression of Omp3, while Ompl was suppressed. This lends support to the presence of an ompB like signal transduction system in E. chrysanthemi. Growth temperature was shown to have no effect on the expression of the major OMP. Similarly, culture growth phase had no effect on major OMP expression. However, two induced OMP were present from mid log phase onwards.