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Abstract Background: Streptococcus pneumoniae (pneumococcus) is one of the leading causes of morbidity and mortality. It accounts for over 500 000 case fatalities in children annually. Its invasive nature is not well understood; however, progress has been made with the discovery of the major virulence factor: the polysaccharide capsule. The biochemical properties of the capsule have allowed for the identification of more than 100 serotypes, resulting in the development of new, effective vaccines. However, in recent years, it has become challenging to identify serotypes, due to mutations in the capsular gene cluster of atypical isolates. The encoded capsules have been shown to cross react with anti-sera from other pneumococcal serogroups. We, therefore, aimed to phenotypically and genotypically characterise a subset of isolates collected from a nationwide surveillance program, exhibiting atypical serotyping results. Methods: A convenient sample of 68 atypical isolates were selected for the purpose of this study. Pneumococci were isolated and characterized using conventional microbiological laboratory techniques. Confirmed pneumococci were then subjected to antimicrobial susceptibility testing using the Kirby Bauer disk diffusion method and serotyped using the Quellung method. Genotypic characterization was conducted by analysing whole-genome sequencing data to reveal the in-silico serotype, multi-locus sequence type profiles, virulence factors, and antibiotic-resistance genes present, including whole genome sequencing-inferred minimum inhibitory concentration where applicable. Analysis of the capsular loci for single nucleotide polymorphism mutations were also conducted. Nuclear magnetic resonance was used to characterise the pneumococcal polysaccharide capsule. Results: Of all the pneumococci included in this study, 21% (14/68), 7% (5/68), 6% (4/68), 1% (1/68) of the isolates were resistant to tetracycline, chloramphenicol, azithromycin, and cefuroxime, respectively. Only 11% of the isolates were resistant to more than one drug. A total of 24 Sequence types (ST) were identified amongst the isolates. Ten of the isolates were identified as novel STs, and were thus assigned a unique ST. Among the 14 known STs, 12 have been known to be associated with invasive encapsulated pneumococci. ST9813 was the most prevalent ST. All isolates belonged to serogroup 35 (35A, 35B, 35C and 35F). The majority of isolates were primarily identified as serotype 35B, 72% (49/68) vs 59% (40/68) for the Quellung reaction and whole genome sequencing, respectively. Analysis of the capsular loci revealed the presence of 146 non-synonymous mutations in the capsule encoding biosynthetic genes. Nuclear magnetic resonance data was inconclusive, although electron microscopy revealed the presence of the polysaccharide capsule. Conclusions: Comparative genomics analysis revealed single nucleotide polymorphism mutations in the capsular biosynthetic genes, that may alter the production of the polysaccharide capsule; resulting in atypical behaviour observed within these strains. A high concordance rate between phenotypic and molecular tests was observed, and the utilization of both methods allowed for a detailed investigation of atypical pneumococcal isolates. There is a need for continued surveillance of atypical strains and optimisation of pneumococcal capsular extraction protocols, to further elucidate the chemical structure of these novel atypical strains of pneumococci.

Hormonal contraceptives (HCs) and hormonal replacement therapies (HRTs) are widely used globally by women. However, the relative activity of some of the progestogens used in HCs and HRT has not been determined via their target steroid receptor (SR), the progesterone receptors and its isoforms (PR-A and PR-B). This thesis involves an in vitro investigation of some of the factors that affect PR activity and that may thus impact progestogen responses in women on HC or HRT. These include progestogen-specific effects via PR-A and PR-B, progestogen metabolism, the role of antiretroviral drugs (ARVs) and crosstalk with the glucocorticoid receptor (GR). The PR isoforms play important roles in multiple physiological processes, including cognition, regulation of inflammation, mitochondrial function, neurogenesis, female reproduction and disease. Due to the gender disparity of higher prevalence of HIV infection among women compared to same-aged men, there is increased interest in developing treatments designed to protect women from HIV infection. These include HIV pre-exposure prophylactics (PrEPs) and multipurpose prevention technologies (MPTs). Furthermore, many HIV -infected women are using combined antiretroviral therapy (cART) to treat AIDS as well as an HC to prevent unwanted pregnancy. Importantly, the effects of the combination of a progestogen with ARVs on the activity of the PR is unexplored. In addition, there are many studies which provide evidence that SRs interact and influence each other's activity. Therefore, the presence of the ubiquitously GR may also influence PR activity. To characterise the progestogen-induced activity of the PRs, dose-response analysis was performed using promoter-reporter genes and varying doses of progesterone (P4) and five widely-used progestogens namely; norethisterone (NET), etonogestrel (ETG), levonorgestrel (LNG), medroxyprogesterone acetate (MPA) and nestorone (NES), in parallel relative to the reference progestogen promegestone (R5020). The effects of experimental conditions and progestogen metabolism on PR activity were investigated in U2OS cells using two different transient transfection conditions to express PR-B. The effect of model system on PR activity was investigated by comparing the progestogen-induced PR-B responses obtained in U2OS cells to those obtained in MDA-MB-231 cells stably transfected with expression vectors for PR-A or PR-B. Progestogen-induced PR activity was also investigated on endogenous genes by quantitative real-time PCR (real-time qPCR) in the MDA-MB-231 PR-B-stably expressing (MDA-PR-B+) cells. Once the progestogen responses via the PR were characterised, PR-B activity in the presence of three ARVs namely; tenofovir disoproxil fumarate (TDF), dapivirine (DPV) and maraviroc (MVC), was investigated next using promoter-reporter assays and real-time qPCR in MDA-PR-B+ cells. The mechanism of ARV action on PR-B activity was investigated by competitive binding assays, as well as by determination of PR phosphorylation levels. The effect of the GR on progestogen-induced PR-B activity in terms of potency, efficacy and biocharacter was investigated using promoter-reporter assays, real-time qPCR on select endogenous genes and efficacy on multiple genes in a PCR array. Using GR-specific siRNA knockdown in MDA-PR-B+ cells, the progestogen-induced PR responses were compared for higher and lower GR levels. The mechanism of the effect of the GR on PR activity was further investigated by co-immunoprecipitation studies and the degree of PR phosphorylation in the presence of higher and lower GR levels was also compared. Results show that in the presence of the progestogens investigated, in vitro biological responses via PR-B can vary significantly in biocharacter and absolute values for efficacies and potencies. Progestogen-specific responses were found on the same synthetic promoter, as well as on the same endogenous gene in the same cell. These progestogen-specific responses negatively correlated with progestogen-specific metabolism in U2OS cells. Furthermore, the progestogen-specific responses also varied in a gene- and model system-specific manner. The majority of the progestogeninduced responses via PR-B were significantly more efficacious, but less potent that the progestogen-induced responses via PR-A, highlighting the importance of the relative expression of PR-B vs PR-A protein levels in determining the resultant progestogen-specific response. It was shown for the first time that the ARVs, MVC and TDF activated PR-B transcription in the absence of progestogens to result in increased transcription in promoter-reporter assays and increased mRNA for endogenous genes. MVC and TDF exhibited no direct binding to PR-B; however, novel data from this thesis showed increased PR-B phosphorylation at Ser294 with TDF but not MVC. DPV activated two PR-regulated genes in the absence of progestogens and competed for binding of P4 to PR-B, while resulting in no effect on PR-B phosphorylation. Using GR-specific siRNA, it was determined by promoter-reporter assays that the presence of the GR had an inhibitory effect on PR-B efficacy. This surprising observation was supported by the global and promoter-specific gene expression changes observed for some genes using a PCR array with and without GR knockdown. The mechanism by which the GR affects the PR-B activity was determined to be most likely by association with PR-B in the same protein complex, probably resulting in a significant decrease in PR-B phosphorylation. Taken together, the in vitro differences between progestogen actions via the PR suggest that the absolute values of the progestogen-induced efficacies and potencies are likely to vary in vivo in a progestogen-, promoter-, model system- and isoformspecific manner. While obtaining such data in vivo is not possible, the in vitro data from the present study show proof-of-concept of potential significant cell-, gene- and progestogen-specific PR-B effects, as well as PR isoform-specific effects. Additionally, this study shows that potential novel off-target immunomodulatory effects of MVC, TDF and DPV occur in vitro and these are most likely mediated by different mechanisms of PR-B activation. Lastly, this study shows that in cells where both the GR and PR-B are co-expressed, the resultant PR response is likely to be inhibited on select genes as a result of reduced PR-B phosphorylation. The extent to which the presence of the GR affected the determination of progestogen-, model system-, promoter- and isoform-specific responses of progestogens via the PR in this study was not investigated for all models and genes, but is likely to be an unavoidable confounding factor. However, since all cells that express the PR also express the GR in vivo to the best of the author's knowledge, characterising PR activity in the absence of detectable GR expression is unlikely to be physiologically relevant. Collectively these data suggest that multiple factors influence PR activity and these need to be taken into account, especially given the treatments available and under investigation, which are designed to specifically target the PR.

The South African abalone, Haliotis midae, is a commercially important shellfish that is farmed in the Western Cape. Histone H2A-derived peptides investigated from various organisms have been shown to act as antimicrobial peptides (AMPs) against a range of microbes. Therefore, the aim of the study was to determine whether the H. midae histone H2A acts as an AMP and thus plays a role in the innate immune system of this abalone. A peptide fragment corresponding to the first 42 amino acids of the N-terminus of histone H2A was examined in silico. This 4.63 kDa peptide, referred to as Midaesin, was found to possess a high proportion of hydrophobic and basic residues. Secondary structure prediction of Midaesin revealed the presence of an amphipathic α-helix. Alignment of the Midaesin peptide to other known histone H2A-derived AMPs revealed significant sequence similarity. Additionally, RT-qPCR of total cDNA isolated from cultured H. midae haemocytes exposed to heat-killed V. anguillarum for 1h showed that the histone H2A transcript was upregulated, implying a role in the immune response. A PCR-amplified DNA fragment coding for the Midaesin peptide was cloned into a bacterial expression vector and purified. Midaesin-containing peptides inhibited the growth of Staphylococcus aureus, Escherichia coli and Vibrio anguillarum at 10 µM in liquid growth inhibition assays. Scanning electron microscopy and fluorescent microscopy, employing the membrane impermeable dye propidium iodide, revealed membrane disruption of V. anguillarum cells exposed to 13 µM or 30 µM of the Midaesin-containing peptides for 30 minutes, respectively. Secondary structure analysis by circular dichroism indicated a shift in the secondary structure of the Midaesin-containing peptide upon incubation with V. anguillarum cells for 30 minutes with a trend towards more α-helical content. Taken together, the above indicates that histone H2A may be involved in the immune response of H. midae and that Midaesin has the potential to act as an AMP.

Bacterial fish diseases caused by yellow-pigmented, filamentous bacteria of the genus Flavobacterium are among those that lead to significant losses in the international aquaculture industry. An increasing number of Flavobacterium spp. have been isolated in association with diseased fish within the aquaculture industry. In salmonids, well known flavobacterial diseases include bacterial cold-water disease (F. psychrophilum), rainbow trout fry syndrome (F. psychrophilum), bacterial gill disease (F. branchiophilum, F. aquatile), and columnaris disease (F. columnare). Conventional diagnosis of Flavobacterium spp. is based on physico-chemical tests, but these tests are time-consuming and labor-intensive, and they are unable to distinguish between closely related species due to morphological similarities. Furthermore, little information exists on the diversity of fish associated Flavobacterium spp. from southern Africa. Recently, numerous, yellow-pigmented, filamentous bacteria were isolated from diseased rainbow trout farmed in South Africa and neighboring Lesotho. The aim of the present study was to elucidate the genotypic and phylogenetic diversity of the Flavobacterium spp. associated with these fish and to develop a new molecular system for rapid and accurate identification and differentiation of the isolates. In order to do this, the genotypic and phylogenetic diversity of ninety bacterial isolates, mostly yellow-pigmented, obtained from the gills, skin ulcers, liver, and kidneys of diseased Oncorhynchus mykiss was assessed following PCR and sequencing of the 16S rRNA gene. A BLAST search of the GenBank database revealed that 47 of the 16S rRNA gene sequences showed high similarity to several Flavobacterium spp.; 6 showed high similarity to Chryseobacterium spp., and 19 non-Flavobacterium isolates were identified, which included, amongst others, Hafnia spp. and Aeromonas spp. Fifteen isolates were excluded from further analysis due to poor DNA sequence data, whilst four isolates showed high similarity to uncultured bacteria and they were also excluded from further analysis. Isolate OM-46 was excluded from the phylogenetic analysis of Flavobacterium spp. since only the forward 16S rRNA sequence was available. Phylogenetic analysis based on the maximum likelihood method confirmed the allocation of 46 isolates as Flavobacterium spp., and 6 isolates as Chryseobacterium spp. Differential identification of the Flavobacterium spp. was achieved following PCR amplification of a hypervariable region of the 16S rRNA gene followed by high-resolution melt (HRM) analysis. Nine Flavobacterium isolates, presumed to be different species based on the phylogenetic analysis were identified and successfully differentiated using high resolution melt analysis. The present study has identified new Flavobacterium spp. (OM-13, OM-39, OM-51, OM-82, and OM84) from rainbow trout farmed in southern Africa and developed a tool that may be useful for the management of flavobacterial diseases worldwide.

This study aimed to examine the molecular basis of several aspects of plumage colouration in the black sparrowhawk (Accipiter melanoleucus), an African diurnal bird of prey. The adults of this species occur in two discrete morphs, light and dark. Light morph individuals were found to differ from dark morph individuals in the concentration of eumelanin in contour feathers from the breast feather tract, with light morph breast feathers containing no detectable eumelanin, while dark morph breast feathers contained similar amounts of eumelanin to the uniformly dark-coloured back feathers. No polymorphisms associated with morph were found in the coding regions of the melanogenesis genes melanocortin 1 receptor (MC1R), agouti signalling protein (ASIP), or proopiomelanocortin (POMC). However, the expression levels of several genes involved in melanogenesis differed between the two morphs, with light morph developing breast feathers showing significantly higher levels of expression of ASIP and lower expression of downstream melanogenesis genes tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), and microphthalmia-associated transcription factor (MITF) than the dark morph. The black sparrowhawk also shows distinct rufous juvenile plumage, which is a particularly common trait in raptors. The genetic basis of juvenile plumage was investigated, and eumelanin and pheomelanin levels were found to be tightly correlated in the breast and back contour feathers of juvenile black sparrowhawks. The amount of both eumelanin and pheomelanin produced was negatively correlated with expression of ASIP, as was expression of downstream melanogenesis genes TYR, MITF, and TYRP1, the same genes that appear to determine adult plumage morph. Pheomelanin levels were found to correlate with fault bar formation, and thus may reflect acute or chronic stress experienced during feather development, but not with condition or parental plumage morph. Finally, possible pleiotropic effects of morph were investigated. Dark morphs were found to have a lower haemoparasite infection intensity than light morphs, confirming previously published results, but differences in telomere length were not associated with parasite infection or morph in this species. However, sex was found to be significantly associated with telomere length, with males having longer telomeres than females.

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No description available.