Includes bibliographical references (leaves 84-94). / In this study the effects of M. flabellifolia polyphenols on growth of S. cerevisiae yeast strains was investigated. This study showed that M flabellifolia polyphenols inhibited growth of both the wild type and the Δhsp 12 yeast strains largely by binding protein in the growth medium. A decreased specific growth rate, reduced maximum biomass, and prolonged lag phase were observed for both strains.

Includes abstract. Includes bibliographical references (leaves 122-132).

Includes bibliographical references (leaves 95-106). / For bifidobacteria to survive and achieve colonisation, they have to interact with inhibitory host-produced substances such as bile salts. Another aspect which should be studied is the safety of the probiotic bacterium and risks of acquisition of genes for resistance to antimicrobial agents. Although bifidobacteria exhibit resistance to a wide range of antibiotics, little is known about the molecular basis for this resistance. The aim of this project was, therefore, to investigate the molecular mechanisms responsible for the resistance to antibiotics and bile salts observed in bifidobacteria, and more specifically, to determine whether efflux systems are involved in this resistance. Five Bifidobacterium spp. were exposed to a range of antimicrobial agents. These included ethidium bromide, the bile salt sodium glycocholate, and a range of antibiotics.

Bibliography: leaves 75-89. / The identification of multiple forms of gonadotropin-releasing hormone (GnRH) in a single species is becoming a common occurrence. The highly conserved chicken GnRH II is present along with one or two other GnRHs, composing a combination unique to particular species. This multifunctional peptide is widely distributed through the central nervous system and peripheral tissues. Also, endogenous GnRHs demonstrate distinct patterns of spatial expression within the brain, suggesting they may have separate functions. In addition to being the primary regulator of gonadotropin secretion in vertebrates, GnRH is also involved in the release of GH and prolactin and may fulfil a possible neuromodulatory role. GnRHs exert their actions through the stimulation of distinct GnRH receptors on pituitary gonadotrophs. The presence of multiple GnRH receptor subtypes has been demonstrated in several species and is likely to be a common characteristic of most vertebrates. This thesis describes the cloning and characterisation of GnRH receptors in two species of teleost fish, Haplochromis burtoni (cichlid) and Dania rerio (zebrafish). A type I GnRH receptor has previously been shown to exist in the cichlid. In the present study degenerate primers designed to extracellular loop three of the mammalian GnRH receptors were used to identify a second putative receptor subtype from cichlid (Haplochramis burtoni) genomic DNA. Furthermore, a near full-length cDNA, encompassing transmembrane domain 1 through to transmembrane domain 7 of the GnRH receptor, was cloned from cichlid RNA by reverse transcriptase PCR. This region of the receptor shares approximately 80% amino acid homology with corresponding regions of type III GnRH receptors previously identified in species of perciform fish. Partial sequences of a type IA and a type lB GnRH receptor have previously been identified in the zebrafish. Two sets of degenerate primers were used to elucidate the possible existence of a third receptor in the zebrafish using both genomic DNA and RNA. However, this strategy failed to result in the amplification of novel receptor subtypes in the zebrafish. Controversy surrounds the developmental origins of GnRH neurons and their temporal expression in relation to GnRH receptors. The zebrafish is a model organism, widely used for the study of reporter gene expression during development. Hence an attempt was made to isolate the zebrafish GnRH receptor genes using a genomic DNA library and identify the promoter regions for use as reporter genes in the study of GnRH and GnRH receptor expression during development. Southern blot analysis revealed six genomic clones with sequences homologous to zebrafish GnRH receptor cDNA. Comparison with genomic and cDNA sequences of other GnRH receptors revealed that those regions of the genomic clones that were sequenced only encoded exons 2 and 3. The presence of large introns in the GnRH receptor gene made it difficult to identify genomic clones containing the entire gene and the promoter region. The cloning of part of the zebrafish GnRH receptor genes will make their complete characterisation somewhat less problematic since an idea of their basic intron/exon structure has been obtained. Exons 2 and 3 of the zebrafish type IA and type IB GnRH receptor genes show a high degree of conservation when compared to the same regions of the goldfish type IA and type IB GnRH receptor cDNAs, demonstrating approximately 90% homology in both cases. In this study sequence information was obtained for the regions between transmembrane domains 4 and 7, and 3 and 7 of the zebrafish type IA and type IB GnRH receptor genes, respectively, and was subsequently used clone zebrafish GnRH receptor full-length cDNAs. This study describes the discovery of a type III GnRH receptor in the cichlid but suggests its presence may be restricted to only certain orders of teleost since a type III receptor was not identified in the zebrafish on this occasion. The information acquired from this study may help to reveal patterns, which relate the presence of particular GnRHs and GnRH receptors in single species to specific reproductive requirements.

Includes bibliographical references (p. 119-130). / Production in farming of ostriches (Struthio camelus) is limited by the high mortality rate of ostrich chicks. Chicks which lack a well established microbiota are more susceptible to potentially fatal pathogenic infections. Therefore, the mortality rate may be decreased by establishing the correct gut microbiota by the use of ostrich specific probiotic strains. Five selected strains were conclusively identified and their mucin adhesion abilities characterised: Strain P1.2 was identified as Enterococcus faecalis; the identity of strain 5934.3.1 was confirmed to be Lactobacillus oris; Strains Lactobacillus brevis 512.3.1 and Lactobacillus oris 5934.3.1. The five selected strains were included in an in vivo probiotic feeding trial, where ostriches were treated with an encapsulated mixture of the five strains and/or the antibiotic tylosin.

Includes bibliographical references.

Includes bibliographical references (leaves 105-120). / In an attempt to establish a plant-based Human immunodeficiency virus-1 (HIV-1) subtype C neutralizing antibody stimulating vaccine, a Tobacco mosaic virus (TMV) derived vector was used to express recognized HIV neutralizing antibody epitopes. These epitopes were expressed on the surface of the TMV coat protein, which served as an ideal means of antigen display. This model antigen display system was capable of assembling into multivalent, highly repetitive structures thereby displaying many copies of the attached epitope in the assembled virion. Three TMV-based vectors were acquired, which were essentially identical, differing only in the position at which they could accommodate a foreign protein fusion. These vectors allowed the display of a foreign peptide at the N-terminus, Cterminus or 60S-loop regions of the TMV coat protein. all of which protrude on the surface of the assembled virion. The HIV V3 loop is recognized as the principal neutralizing domain, and was the neutralizing epitopes displayed by the vectors. The epitope sequences used were derived from a cohort of infected individuals in Durban, South Africa, who displayed broad cross-neutralizing V3-specific activity towards heterologous viral strains. The recombinant viral vectors were shown to efficiently infect the host Nicotiana benthamiana plants and assemble into multivalent recombinant virion structures as observed by transmission electron microscopy. However, the level of coat protein expression was significantly dependent on the position of the coat protein fusion as either the levels of V3 epitope expressed or TMV coat protein was found to vary between the different vector types, confirmed by means of immunoblotting and enzyme-linked immunosorbent assays. Immunogenicity analysis using a guinea pig model was used to assess the ability of the recombinant vectors to firstly establish a V3-specific immune response, and secondly to stimulate a virus-specific neutralization antibody response. As a result of time constraints only the C-terminal coat protein fusions were assessed in the guinea pig model. Inoculated guinea pigs displayed distinct and gradually increasing V3-specific immune responses after 2 boosts. Serum samples that displayed the strongest V3 peptide responses were then analyzed for their ability to neutralize HIV infection in HIV pseudovirion neutralization assays. Results for selected serum samples showed no HIV neutralizing activity above what could be recognized as background activity. Thus the candidate vaccine, although establishing a path for the assembly of a multivalent vaccine, failed in its attempt to stimulate a neutralizing antibody response. This study has nevertheless paved a direct path to the development of variations of this type of vaccine possibly using different and perhaps more effective epitopes for candidate vaccine purposes.

Bibliography: leaves 213-233. / Camponotus than the cytochrome oxidase II gene, based on almost all measures of phylogenetic utility. The primary hypothesis proposed to account for this observation is that these two mitochondrial genes are evolving under different evolutionary constraints. Specifically, the cytochrome oxidase II gene displays greater rate heterogeneity than the cytochrome b gene, thereby decreasing its utility for phylogenetic analyses. Combining sequence data from both genes resulted in more robust phylogenetic hypotheses, with the combined topologies displaying greater congruence with the cytochrome b topologies than those based on cytochrome oxidase II sequence data. The morphological data produced a topology that was congruent with that obtained from molecular data, and provided increased support for certain nodes in the context of a combined molecular-morphological framework. The hypothesis that subgeneric classifications within Camponotus do not accurately reflect phylogenetic relationships was supported by the molecular phylogenies. An exception to this hypothesis was the monophyly of the subgenus Myrmosericus, based on cytochrome b data. The morphological and behavioural data provided support for a monophyletic group comprising the four species assigned to the subgenus Myrmopiromis. However, although these four species associated together in a group based on combined cytochrome oxidase II and cytochrome b sequences, this group was paraphyletic in the combined molecular topology, with two species in subgenus Myrmopsamma also falling within this group.

Rhabdomyosarcoma (RMS) forms in skeletal muscle and is the most common soft tissue sarcoma in children and adolescents. Current treatment is associated with debilitating side effects and treatment outcomes for patients with metastatic disease are dismal. Other than a need for alternative and more effective therapies there is also a growing appreciation for the need to understand the molecular underpinnings of RMS with the aim of identifying, in part, novel targets to develop highly specific and effective treatments with negligible adverse effects. The aim of this study was to identify novel drugs for the treatment of the two major RMS subtypes viz alveolar (ARMS) and embryonal (ERMS) RMS and to do so it adopted a two-pronged approach. Firstly, a novel binuclear palladacycle, AJ-5, was investigated for its anti-cancer activity and its mechanism(s) of action in RMS cells. The second approach involved a target-based drug repurposing strategy where a library of FDA-approved drugs was screened to identify leads that were able to negatively regulate the oncogenic TBX2 and TBX3 transcription factors that are known drivers of RMS. The binuclear palladacycle, AJ-5, was recently shown to exert potent cytotoxicity in melanoma and breast cancer and to present with negligible adverse effects in mice. To investigate the anti-cancer activity of AJ-5 in RMS cells, MTT assays were firstly performed in ERMS and ARMS as well as 'normal' cells. IC50 values determined from these experiments showed values of ≤ 0.2μM for the RMS cells and a favourable selectivity index of > 2. Clonogenic and migration assays showed that AJ-5 inhibited the ability of RMS cells to survive and migrate respectively. Western blotting revealed that AJ-5 induced levels of key DNA damage response proteins (γH2AX, p-ATM and p-Chk2) and the p38/MAPK stress pathway. This correlated with an upregulation of p21 and a G1 cell cycle arrest. Annexin V-FITC/propidium iodide staining revealed that AJ-5 induced apoptotic and necrotic cell death. Apoptosis was confirmed by the detection of cleaved PARP and increased levels and activity of cleaved caspases-3, -7, -8 and -9. Increased levels of necroptotic markers p-RIP3 and p-MLKL and inhibition of necroptosis with necrostatin-1 with a corresponding significant increase in cell viability suggests that AJ-5 is also capable of triggering a form of programmed necrosis. Furthermore, AJ-5 reduced autophagic flux as shown by reduced LC3II accumulation in the presence of bafilomycin A1, and a significant reduction in autophagosome flux J. Pharmacokinetic studies in mice show that AJ-5 has a promising half-life and that its volume of distribution is high, its clearance low and its intraperitoneal absorption is good. With the intention of improving the drug-like properties of AJ-5, specifically its water solubility, a derivative of AJ-5, BTC2, was synthesised and identified to display comparable anti-cancer activity against ERMS and ARMS cells. Together these findings suggest that AJ-5 and BTC2 may be effective chemotherapeutics with a desirable and novel mechanism of action for treating drug resistant and advanced RMS. The highly homologous T-box transcription factors TBX2 and TBX3 have both been implicated as key drivers of RMS and they have been identified as novel therapeutic targets for the treatment of this sarcoma subtype. Indeed, TBX2 or TBX3 overexpression in normal myoblasts inhibits muscle differentiation and overexpression and knock-down cell culture and mouse models show that RMS cells are addicted to them for their cancer phenotype. However, targeting transcription factors is notoriously challenging because unlike enzymes they do not have catalytic activity and deep binding pockets to which small molecule inhibitors can be designed which is further exacerbated by the length of time and costs associated with de novo drug development. Therefore, this study adopted a novel strategy to circumvent these challenges by combining a drug repurposing with a targeted approach to TBX2/3. Briefly, a high throughput cell-based immunofluorescence screen was designed and conducted to identify FDA-approved drugs that could negatively regulate TBX2 and/or TBX3 protein levels or nuclear localisation. Cells were engineered to express induced exogenous FLAG-tagged TBX2 and TBX3 using a Tet-On system and they were screened with the Pharmakon 1600 drug library at a concentration of 10μM. 'Hits' were identified by z-scores and amongst these, niclosamide, piroctone olamine and pyrvinium pamoate were validated to be potent inhibitors of TBX2/3 and were shown to display anti-cancer activity in RMS. These drugs have the potential to be repurposed for the treatment of RMS and other TBX2/3 driven cancers either as single agents or in combination with currently used chemotherapeutics.

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