Soil was collected from a compost heap in a Mowbray suburban garden and a compost heap in a Plumstead suburban garden. The soil and ‘worm tea’ of a vermiculture farm from the same Mowbray suburban garden were also sampled. Using four different types of media (7H9, CZ, ISP2 and GOT) 135 isolates were putatively identified as actinobacteria based on colony morphology. These isolates were screened for antimycobacterial activity against the test bacterium Mycobacterium aurum A+. A Kribbella strain, isolated and identified by an intern in the lab, and a Micromonospora strain, isolated and identified during the authors Honours project, were also screened for antimycobacterial activity. Sixty-four (64) actinobacterial isolates displayed moderate antibiotic activity or higher (ZOI >1001 mm2 ) based on the standard overlay method. Kribbella strain SK5 displayed very strong antimycobacterial activity (3309 mm2 ). Forty (40) of the actinobacterial strains that exhibited moderate/strong/very strong antimycobacterial activity and/or had interesting morphological features were selected for genus identification via a standard nucleotide-nucleotide blastn analysis of their 16S rRNA gene sequences. Thirty-one (31) strains were identified as Streptomyces species, six strains were identified as Micromonospora species, one strain was identified as a Nocardia species, one strain was identified as a Kitasatospora species, and one strain was identified as a member of the genus Tsukamurella. These isolates were subjected to phylogenetic analysis using the partial 16S rRNA gene sequences. Based on analysis of the 16S rRNA gene sequences, Streptomyces strain PR10 was found to be the most interesting of the Streptomyces isolates and should be pursued as a novel species (99.7% sequence similarity to the top blastn hit and less than 98.8% sequence similarity from the third blastn hit onwards). Further analysis of the gyrase subunit B (gyrB) gene sequence of the Kitasatospora isolate (strain PR3) revealed that the isolate is more closely related to members of the genus Streptomyces. Further evidence to support the assignment of strain PR3 to the genus Streptomyces (rather than Kitasatospora) is that it has two Streptomyces-specific gyrB gene indels signatures. Tsukamurella strain G4 was noted for characterisation as a novel species. The potential for seven isolates to produce ansamycin, glycopeptide, non-ribosomal peptide, and/or TypeII polyketide antibiotics was determined by detection of antibiotic biosynthetic gene clusters using PCR. Strain M27 demonstrated the potential to produce all the aforementioned antibiotics. Strain Y10 demonstrated the potential to produce a non-ribosomal peptide antibiotic. Strains PR10, PR28, PR47 and UK1 demonstrated the potential to produce Type-II polyketide and non-ribosomal peptide antibiotics. The PCR products were sequenced and analysed via blastn to compare them to the known antibiotic biosynthetic gene sequences in the GenBank database. The non-ribosomal peptide synthetase (NRPS) A domain sequences were analysed using the NRPSpredictor2 software to identify the A domain substrate specificity Solvent extraction was done on the broth cultures of Streptomyces strains PR3, UK1 and Y30 and Kribbella strain SK5 to isolate the antimycobacterial compounds. It was found that the cell mass extract of the three Streptomyces isolates had active compounds against M. aurum A+. The culture broth extract of the Kribbella isolate was found to have an active compound against M. aurum A+ and Staphylococcus aureus ATCC 25923. One-dimensional and two-dimensional TLC of the culture broth extract from strain SK5 revealed that a single compound was active against M. aurum A+ and S. aureus ATCC 25923. Nocardamine was purified from the culture broth extract of strain SK5 by Mr Kojo Acquah (PhD student, Department of Chemistry, University of Cape Town). In a side-by-side spot bioautography analysis of the purified nocardamine and the strain SK5 culture broth extract, it was found that the active compound in the culture broth extract was not nocardamine, because nocardamine only had activity against M. aurum A+ while the culture broth extract had activity against M. aurum A+ and S. aureus ATCC 25923. Using the polyphasic taxonomic approach, Kribbella strain SK5 was tentatively characterised as a novel species, for which the name Kribbella stellenboschensis sp. nov. is proposed. The closest phylogenetic relatives were identified as the type strains of Kribbella aluminosa, Kribbella karoonensis, Kribbella pittospori, Kribbella shriazensis, ‘Kribbella sindirgiensis’ and ‘Kribbella soli’. Genetic distances of 0.030 and 0.016 were calculated for ‘K. soli’ and ‘Kribbella sindirgiensis’, respectively, for the concatenated gene sequence of five housekeeping genes (gyrB, rpoB, recA, relA, and atpD). Thus, DNA-DNA hybridisation (DDH) will need to be carried out to confirm that strain SK5 is a separate species. Phenotypic differences were observed between strain SK5 and all the type strains of the most closely related species. Chemotaxonomically, strain SK5 possessed the key characters definitive of the genus Kribbella: i) MK9(H4) as the major menaquinone; ii) LL-diaminopimelic acid as the diagnostic diamino acid; iii) anteiso-C15:0 and iso-C16:0 as the major fatty acids (>10%); and iv) phosphatidylcholine in the polar lipid profile.

The evolution of a strikingly elongated and webbed FL in bats, which contrasts with a small, free-toed HL, has seen extensive research into bat wing development in an effort to determine the molecular mechanism driving limb development. A recent RNA-seq and ChIP-seq study carried out on M. natalensis showed differences in FL and HL activity for several genetic pathways known to be involved in bone formation during key bat development stages CS15-CS17. In this project the prediction made from the literature and the RNA-seq results was that the observed decreased Wnt/β-catenin signalling and increased BMP signalling in the bat FL may lead to elevated levels of Sox9 expression, and larger fields of mesenchymal condensations. This was tested by annotating Sox9 in the M. natalensis genome to further analyse the expression levels and associated ChIP-seq data. In addition the behaviour of condensing mesenchymal cells during bat and mouse limb development was observed by visualising the various stages of chondrogenesis, using H&E and PNA stains. In addition the RNA-seq study found 3000 genes to be differentially expressed. Thus, the project also set out to create an immortalised bat autopod cell line to facilitate future testing and predictions. The Sox9 gene was successfully annotated and revealed to not be differentially expressed between FL and HL as predicted. However downstream targets of Sox9 were further inspected as potential ideas for further investigation. The histological stains provided a set of data characterising mesenchymal condensation in both mouse and bat stages, revealing many interesting features such as the non-specific binding behaviours of PNA prior to digit formation. In addition, quantitative results demonstrated the bat FL digits are already longer than the HL digits at CS16. Cell line work established a working protocol for the storage, dissociation and plating of bat primary cells that retain their bat limb expression identity. Mouse cells were successfully immortalised and a cell line was established from a HL digit cell. This project has facilitated further studies in understanding extreme digit elongation in the bat FL autopod during development.

Molecular farming is gaining traction as a cost-effective platform to produce recombinant proteins. Further improvements can be made, however, to increase overall yield especially for difficult to express proteins. In this study virus-derived silencing suppressors and replication elements were used with the aim of increasing expression and yield of enhanced green fluorescent protein (EGFP) and the Zika PrME polyprotein in Nicotiana benthamiana. A comparison of four viral silencing suppressor proteins was performed: these were tomato spotted wilt virus non-structural protein, NSs, tomato aspermy virus (TAV) 2b, tomato bushy stunt virus P19 and begomovirus alphasatellite Rep. Differences in EGFP expression in N. benthamiana due to the silencing suppression were determined using immunoblotting and fluorescence of EGFP. In addition, replication elements from three viruses (bean yellow dwarf virus [BeYDV], beak and feather disease virus [BFDV] and begomovirus alphasatellite) were assembled into novel plant expression vectors using GoldenBraid (GB) cloning technology and assessed using EGFP. Finally, the two approaches were combined in an attempt to express the Zika PrME polyprotein, which was assessed using immunoblotting. EGFP expression was found to be greatest in the presence of the TAV 2b protein and no difference in fluorescence intensity between the original BeYDV replicating plant expression vector and that constructed using GB could be detected; however, the GB assembly of the BFDV and alphasatellite plant expression vectors was unsuccessful. The TAV 2b combined with the BeYDV replicating elements were used for the expression of Zika PrME. The gene was successfully cloned into the replicating BeYDV vector and a vector that does not replicate (negative control). The PrME was not detected using anti-His tag immunoblotting despite optimisation for Agrobacterium infiltration density, harvest day post infiltration, signal peptides and buffers during extraction. In this study I demonstrated the following: that the TAV 2b protein out-performed all other silencing suppressors; that the GB cloning technology can be successfully applied in the development of novel plant expression vectors, although further optimisation is required for these and for Zika PrME expression. Further work in characterising the effect of silencing suppression on recombinant protein expression can be assessed using RT-qPCR to measure the effect on mRNA levels. In summary, these improvements in plant recombinant protein expression can be readily applied to large scale production of novel therapeutics and vaccines.

Common to orthodox seeds, desiccation tolerance (DT) is exceedingly rare in the vegetative tissues of modern angiosperms, being limited to a small number of "resurrection plants". While the molecular mechanisms of DT, as well as the transcription factors regulating the seed and vegetative DT programmes, have been identified, very little is known with regards to the role of regulatory noncoding RNAs (ncRNAs). To investigate the presence and roles of possible ncRNA players, RNA-Seq was performed on desiccating Xerophyta humilis leaves and a bioinformatic pipeline assembled to identify the potential decoy lncRNAs and miRNAs present. Interaction mapping was performed, identifying a number of small regulatory networks each regulating a small subset of the desiccation transcriptome. Predicted networks were screened for function related to DT and expression consistent with functional regulatory interactions. Of the predicted networks, two appear highly promising as potential regulators of key DT response genes. The results indicate that differentially expressed (DE) desiccation response ncRNAs are present in the vegetative tissues of X. humilis and likely play a key role in the regulation of DT. This suggests that ncRNAs appear to play a more important role in DT than previously thought, and may have facilitated the evolution of vegetative DT through reprogramming of seed DT programs in vegetative tissues.

Glucocorticoids (GCs) are steroid hormones widely prescribed to treat inflammatory disorders and are regarded as anti-inflammatory molecules. GCs classically induce the expression of antiinflammatory genes, while repressing pro-inflammatory genes via its endogenous cell receptor; the glucocorticoid receptor (GR). Emerging evidence, however, suggests that the mechanisms of GR action are more complex than previously assumed, with many reports of pro-inflammatory actions of GCs via the GR. While chronic exposure to GCs has been noted as anti-inflammatory, reports suggest that acute exposure can increase peripheral immune responses. Specifically, the GCs have been shown to positively regulate the innate immune response, which may be important in preventing the local, affected area from being immunocompromised. Furthermore, the GR can crosstalk with cell signalling pathways involved in pro-inflammatory responses, such as the TNFα pathway, to reciprocally modulate the expression of pro-inflammatory genes. The mechanisms behind the GR’s pro-inflammatory actions and crosstalk with inflammatory inducers are not well understood. GC’s pro-inflammatory actions are attributed to GC insensitivity in asthma patients. The insensitivity is attributed to long-term GC usage, and the increase in Th17 neutrophilic airway infiltration. A proposed hypothesis for the increase in neutrophils in the airways was that it was due to an increase in expression of chemokines by epithelial cells due to GC exposure. The pro-inflammatory, chemoattractant cytokine C-C motif chemokine ligand 20 (CCL20) has been previously shown to be induced by GCs and pro-inflammatory inducers in human bronchial cells, with positive modulation of their responses occurring with co-stimulation. The present study investigated whether the GC dexamethasone (dex) and the pro-inflammatory inducer TNFα could induce CCL20 expression in a variety of human epithelial cell lines, and a simian fibroblast cell line. Using Real-Time Quantitative Reverse Transcription PCR (qRT-PCR), it was confirmed that dex can induce CCL20 mRNA expression, and modulate the TNFα-induced expression in some, but not all cell lines. Moreover, in the HeLa cell line, there was an apparent synergistic response between dex and TNFα, and modulation of the CCL20 response was observed between dex and the pro-inflammatory inducers phorbol 12-myristate 13-acetate (PMA), interferon γ (IFNγ) and lipopolysaccharide (LPS). The GR was shown to be required for the GC induction and modulation of CCL20 mRNA expression. Using promoter-reporter assays, the results showed that the NFκB binding site was necessary for the activation by the pro-inflammatory inducers, but not dex, while the STAT binding region was necessary for the IFNγ activation. Interestingly, lack of the STAT binding site on the promoter-reporter construct caused IFNγ to have repressive effects on CCL20 activation. Stimulation of cells by the pro-inflammatory inducers in the presence or absence of dex had no effect on the total levels of the p65 subunit of NFκB, while dex did appear to cause GR turnover as expected. The results show that dex, via the GR, is able to crosstalk with different pro-inflammatory inducers to induce and potentiate CCL20 mRNA expression and promoter activation. The mechanisms of CCL20 induction and crosstalk with the GR may be different for each proinflammatory inducer, however. Regulation of CCL20 expression is complex, with many transcription factors converging on the promoter region to modulate its expression. This thesis shows that the NFκB binding site is important for the overall induction level of the promoter, however it is not necessary for the dex induced activation. The potentiation of the dex response by pro-inflammatory inducers may be due to the GR interacting with the AP-1 and C/EBP transcription factors, which have been shown to positively interact to increase gene expression. The potentiation of the dex response does not require the activation of NFκB, as IFNγ does not activate the transcription factor, yet can potentiate the dex response.

Myrothamnus flabellifolia (Myrothamnaceae) belongs to a group of ±300 angiosperm species known as “resurrection plants” that exhibit vegetative desiccation tolerance. They are able to survive dehydration to an air-dry state, tolerating up to 95% cellular water loss for a prolonged period of time followed by the rapid recovery of metabolism in the tissues within 24–72 h of rehydration. Prolonged cellular water loss is deleterious and is associated with the production of reactive oxygen species (ROS), which causes cellular degeneration, and ultimately, death. Resurrection plants have evolved various strategies to ameliorate this damage, including biochemical, ultrastructural, and anatomical modifications. Myrothamnus flabellifolia is widespread across southern Africa, and within its range it occurs in regions that experience high, moderate, and low rainfall; the low rainfall region also being associated with longer dry periods. Myrothamnus flabellifolia has historically been used for the treatment of chest infections, uterine pain, and gingivitis, and, more recently, has been shown to exhibit various phytochemical activities relating to the potential inhibition of diabetes, reverse transcriptases, and microbes. Previous studies have found M. flabellifolia extracts to contain high levels of polyphenolic compounds, which act as protectants against the ROS-induced damages caused by prolonged periods without moisture. However, a global assessment of the phenolic constituents, including anthocyanins, present in M. flabellifolia from across its geographic range is currently lacking. As the biosynthesis of compounds is likely to be subject to a fair amount of environmental control, an evaluation of the molecules present in this species from across its geographic range is warranted. Thus, in this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) metabolomics approach was used to screen for phenolic compounds, including anthocyanins, from leaf material sampled in the field from eight populations representing the western, southern, and eastern range of the species distribution. Putative phenolic compounds were identified based on their MSE spectra in the negative ionisation and positive ionisation (for anthocyanins) modes. Their potential roles in the ROS-scavenging capacity of this plant were also discussed. Using this information, multivariate statistics were used to compare the phenolic profiles of the different populations in order to ascertain whether plants from the different regions were associated with any particular phenolic signature, and this was also evaluated against a phylogenetic hypothesis for species relationships based on three non-coding chloroplastic markers. Additionally, a preliminary green chemistry-based extraction protocol using Natural Deep Eutectic Solvents was also used to further screen for phenolic compounds, and this was compared against the conventional organic solvent system. Several phenolic compounds not previously detected in M. flabellifolia were putatively identified, many of which, based on an assessment of the literature, are associated with high antioxidant activity. The phylogenetic analyses suggested that the Namibian plants are more highly diverged than the South African and Malawian plants. The metabolomics analysis corroborated the DNA analysis, in that the most differentially expressed ions in the Namibian population were able to discriminate these samples from both the Malawian and South African samples. While the phenolic profiles of the samples collected from the same countries were similar, there was reasonable withinpopulation variability in those collected from South Africa and Malawi. Conversely, the Namibian samples exhibited far less variability, suggesting that a particular suite of protective compounds may be required for survival in that comparatively drier region. A Natural Deep Eutectic Solvent-based system successfully targeted phenolics in M. flabellifolia and thus constitutes a potential future green chemistry solution for phytochemical investigations in medicinal plants.

The invention and development of Next Generation Sequencing has opened up new possibilities for exploring the genomes of non-model organisms. For this thesis, a diverse range of non-model species from both plants and animals were used to identify and answer questions of evolutionary interest in four case studies. In doing so, a wide assortment of methodologies were used and developed, taking full advantage of the versatility that whole genome sequencing can provide. The genome of the Natal Long Fingered Bat, Miniopterus natalensis, was assembled to investigate the genetic mechanisms responsible for the evolution of the bat wing. The assembled genome was required to facilitate RNA-seq and ChIP-seq analysis. In addition to the genome assembly and annotation, dN/dS analysis and lncRNA prediction were also conducted. This resulted in a high quality genome assembly with just over 24000 genes being annotated and 227 putative lncRNAs being identified. None of the genetic pathways highlighted by the RNA-seq analysis showed any elevated dN/dS signal, suggesting this was not the loci of evolutionary change. The Amboseli National Park in Kenya has a local population of Yellow baboons (Papio cynocephalus) that has recently come into contact and hybridised with a population of Olive baboons (Papio anubis). A genome assembly of P. cynocephalus was created and used to align low coverage sequencing from 45 baboons, including admixed individuals along with unadmixed individuals from each species. By identifying SNPs that were predictive of the species, hybrid individuals were confirmed and evidence for previous admixture events discovered, such as P. anubis SNPs already at fixation in the P. cynocephalus population at Amboseli. The Ruschioideae are a clade of plants that encompasses the prolific tribe, the Ruschieae, which is comprised of approximately 1500 recently diverged species. An exploratory analysis sequenced two Ruschieae genomes (Polymita steenbokensis and Faucaria felina) along with a sister taxon (Cleretum herrei) from a neighbouring tribe (Dorotheantheae). The three plants were compared to each other in order to try and identify any genetic signatures that could be influencing the rapid speciation. The two Ruschieae species were found to have increased levels of non-tandem duplication within the genome as well as on going transposable element activity when compared to C. herrei. Xerohpyta humilis is a desiccation tolerant plant. In order to further facilitate research into how this is possible, the genome was sequenced and assembled. Irregular data led to the discovery that the plant had a genome duplication as well as a large amount of somatic mutations in its genome. Further analysis confirmed that this pattern of somatic mutations was only present in plants that had undergone multiple cycles of desiccation and rehydration. These apparently disparate topics explored the possibilities and limitations for whole genome sequencing in the study of non-model organisms. Mechanisms of genetic change were examined at the genomic scale, from adaptation and hybridisation to various forms of duplication and mutation. In this way, a large variety of events responsible for the evolutionary change of genomes in plants and animals were analysed in a diverse set of systems.

Bibliography: pages 84-101. / Cellulose is the most abundant organic compound on earth and offers great potential as a source of renewable energy and other chemicals. Cellulases are being studied to elucidate the enzymatic degradation of cellulose. We are interested in the molecular mechanisms of microbial degradation of cellulose in the rumen. The long-term aim is the potential genetic modification of lignocellulolytic activities in the rumen. · Clostridium longisporum was obtained from Varel (1989) because oxygen-resistant endospores might be suitable "vectors" for the introduction of genetically modified enzyme systems into the rumen via animal feeds. It is a sporadically occurring rumen bacterium and its role in ruminal cellulolysis is unclear. The aim of this project was the initial characterization of cellulases produced by C. longisporum. The celA gene was obtained by screening a library of C. longisporum genomic DNA in Escherichia colifor clones expressing CMCase activity. Approximately 38 CMCase-positive clones were obtained and the plasmid pCM4 was isolated from the clone expressing the highest activity. Southern analysis indicated that another plasmid, pCM64, contained a larger insert including the insert of pCM4. A total of 3620 bp were sequenced and a 1548-bp open reading frame, termed celA, was found. This gene showed homology with other endo-B-1,4-glucanases from family 5 (Henrissat & Bairoch, 1993). Plasmid pCM64 was found to contain the whole celA gene encoding endoglucanase CelA, while pCM4 has a 5'-truncated gene, termed celM5', which encodes a fusion protein, CelMN', that was initiated from an ATG codon in the vector. Sequence analysis of celA revealed the presence of a type I cellulose-binding domain (Beguin & Aubert, 1994) at the COOH-terminus of CelA.

The upstream regulatory regions of numerous genes contain contiguous deoxyguanosine residues (G·C-rich sequences) which have been implicated in the regulation of gene expression, since they may involve alterations in their DNA structure, the binding of G-string factors and in some cases even the displacement of a nucleosome positioned over this area. A poly( dG).poly( de)-binding protein (suGF1) has previously been identified and purified on a small scale from embryonic nuclear extracts of the sea urchin Parechinus angulosus (1, 2). suGF1 binds in vitro to the H1-H4 intergenic region of the early histone gene battery, and the recognition site contains 11 contiguous Gs which are incorporated into a positioned nucleosome core in vitro. suGFI may be a member of a family of Gstring factors which could be involved in the developmental regulation of unrelated genes in various organisms. Prior to the commencement of this project no protein or DNA sequence information was available on the protein. The main objective of this thesis was to obtain the eDNA and the primary amino acid sequence for suGFI. Using this information, additional aims were to determine the developmental distribution of the protein and obtain insight into the molecular basis of the regulatory function of suGF 1 by analysis of the primary protein structure and expression of the eDNA.

Includes bibliographical references (pages 127-171). / The endocervical mucosae of the female reproductive tract (FRT) not only serve as a physical barrier against microbial infection, but they also express a wide variety of immune mediators. The endocervical epithelial cells express a distinct profile of immune-regulators, which is higher than vaginal and ectocervical epithelial cells. Constant cytokine production would ensure rapid responses to infections and maintenance of the sterility of the upper genital tract. However, overproduction of cytokines could inhibit normal reproductive processes and stimulate excess growth and cell proliferation. The synthetic progestins medroxyprogesterone acetate (MPA) and norethisterone (NET) and its derivatives (norethisterone enanthate (NET-EN); norethisterone acetate (NET-A)) are synthetic steroidal hormones designed to elicit progestational effects similar to those of the endogenous hormone progesterone (P4). They are extensively used as contraceptives and in hormone replacement therapy (HRT). Numerous studies, however, have reported that synthetic progestins affect immune function, increase the risk of sexually transmitted infections (STIs) and also change the morphology of the cervicovaginal mucosa. Despite these findings little is known about the molecular mechanisms of action of MPA and NET, in particular their differential effects on gene expression.