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.

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.

Soil salinization is prominent in agricultural land and has detrimental effects on plant growth and yield. Salt imposes both an osmotic and ionic stress on plants, and the aim of this study was to investigate the molecular basis of the plant response to the ionic component of salinity stress. To do so, transcriptome profiling using microarrays was performed at two developmental stages (two and four weeks post-germination) exposing plants to either NaCl (which imposes an osmotic and ionic stress) or iso-osmolar sorbitol (which imposes osmotic stress only). Clear transcriptomic differences in the plant response to NaCl and sorbitol were observed, allowing the identification of genes that may be involved specifically in the response to the ionic component of salinity stress. Differences in salt-responsive gene expression were also observed between early and later development as well as root and shoot tissues, indicating that there may be both developmental and tissue specific responses to NaCl. ‘Response to auxin’ was a highly enriched gene ontology term associated with genes that are significantly induced specifically in response to ionic stress, suggesting a potential role for this plant growth hormone in ionic stress tolerance. This hypothesis was supported by mass spectrometry analysis which demonstrated that active IAA levels show a significantly greater increase in response to NaCl than to sorbitol, demonstrating an ionic specific auxin response. In planta quantification and spatial distribution of IAA was further analysed in salt stressed plants using two auxin reporter lines, DR5::GUS and DII::VENUS. These analyses showed that IAA levels increased in the shoot and root tip in response to NaCl. Also, local IAA maxima distributed along the primary root of salt stressed seedlings were decreased compared to the untreated control, which may explain the decrease in lateral root number and primary root bending observed in salt stressed plants. There are several different pathways in the tryptophandependent auxin biosynthesis process, each with unique biosynthetic intermediates and ratelimiting enzymes. For this reason, changes in the levels of the different IAA biosynthetic intermediates were analysed in NaCl and sorbitol stressed Arabidopsis to identify a predominant pathway responsible for increasing IAA during ionic stress. The changes in IAN (the IAA biosynthetic intermediate of the IAOx pathway) in response to NaCl and sorbitol mirrored those observed for IAA. Additionally, mRNA levels of Nitrilase 2 (which converts IAN to IAA) were significantly increased in response to NaCl but not sorbitol. Taken together, these results suggest that IAA biosynthesis is increased in response to NaCl via the hydrolysis of IAN to IAA by NIT2. In agreement with this, a Nit2 overexpressing line (35s::Nit2) displayed both improved survival and growth in the presence of NaCl. Furthermore, the Nit2 overexpressor had increased IAA but deceased IAN compared to wild type plants in response to salt stress. Together these results indicate that Nit2 is likely involved in the salt stress response through altering IAA levels in planta. GH3.12 and ILL6, two genes involved in IAA storage and/or degradation via hydrolysis and conjugation to amino acids, were also differentially expressed in response to NaCl at both developmental stages tested. However, mutants for ill6 and gh3.12 were not altered in their salt tolerance, suggesting that IAA storage and/or degradation may not play an important role in modulating active IAA levels in response to salt stress. Finally, preliminary evidence was obtained that the expression of sorghum Nit2 homologs is also increased in response to salt stress, implying that auxin might play a role in salt tolerance across a phylogenetically broad range of plants, making this a potential route to improve salt tolerance in crop plants. Overall, this study provides novel information indicating that IAA levels are altered specifically in response to the ionic component of salinity stress which may contribute to the altered plant growth observed under these conditions, as well as the identification of an auxin-related candidate gene which can improve salt tolerance in plants when overexpressed.

Bibliography: pages 117-130. / Protection of plants from pathogens and pests by introduced microorganisms provides an alternative to environmentally hazardous chemical pesticides and fungicides. Plant associated, free living, non-pathogenic bacteria found in the rhizosphere and phyllo plane have been the emphasis of research on biological control· agents. These regions however pose problems of competition between introduced microorganisms and native microflora and environmental extremes. Endophytic bacteria, present in the interior regions of healthy plants, offer a solution to these problems. Since little work has been done to date with endophytes, the work reported in this thesis comprises a novel approach to achieving biological control of a plant pest and pathogens .An endophytic strain of Pseudomonas fluorescens was isolated from micro propagated apple plantlets and shown to be present in the roots of beans at a level of 1.2 x 10⁵ CFU/g fresh weight 10 days after introduction. Generation of spontaneous rifampicin resistant mutants of this strain resulted in P. fluorescens Rifl. Isolates of P. fluorescens and Aeromonas caviae were recovered from the interior regions of surface sterilized bean seeds. In addition, two sugarcane endophytes, Acetobacter diazotrophicus and Herbaspirillum seropedicae, which has also been isolated from rice, maize and sorghum, as well as an endophytic Citrobacter sp. of pine seeds and bean plants were used in this work. The gene coding for the major chitinase of S. marcescens, chiA, was cloned under the control of the tac promoter by PCR amplification. The gene contained the endogenous Shine Dalgarno sequence 7 bases upstream of the ATG start codon in the plasmid pTC33. The plasmid ptacchiA had previously been constructed with a distance of 20 bases between the ribosome binding site of this vector and the A TG start codon of the gene. Gene expression of chiA carried on pTC33 was shown to be approximately 16-foldhigher than that of ptacchiA. This clearly illustrated that the distance between the Shine Dalgarno sequence and ATG start codon was critical and that the shorter distance of 7 bases significantly increased the translation efficiency of chiA. The first and second generation tacchiA cassettes from ptacchiA and pTC33 respectively were cloned into the broad host range plasmids pKT240, pDER405 and pML122 and into the integration vector pJfF350.These plasmids were introduced into the endophytes P.fluorescens Rift, H. seropedicae and Citrobacter sp.AJl by conjugative transfer and electroporation and were shown to express the gene at varying levels. pKTCl and pMTC33 carrying the 1 and 2nd generation tacchiA cassettes on pKT240 and pML122respectively were not stably maintained in P. fluorescens Rift or H. seropedicae.

The mesophilic Ruminococcus flavefaciens FD-1 (NCDO 2215) is a Gram-positive obligate anaerobic bacterium. The aim of this thesis was to clone, sequence and analyze a cellodextrinase gene (celA) and a carboxymethylcellulase gene (celE), and study their regulation and induction at the transcriptional level. The sequence of the celA gene from FD-1 was determined and the amino acid sequence of the CelA enzyme (336 amino acid residues) deduced. It showed 40% identity with endoglucanase C of Clostridium thermocellum and 27.4% identity with endoglucanase 3 of Fibrobacter succinogenes. These three enzymes are grouped into subfamily "A3". The ATG start codon of celA is preceded by a GAGG sequence, predicted to be a ribosome binding site. The derived amino acid sequence corresponded to a protein of Mᵣ 38686. SDS-PAGE analysis of in vitro and in vivo translational products showed that CelA has a molecular mass of ca 39 kDa and was secreted into the Escherichia coli periplasmic space. Although CelA has activity on carboxymethylcellulose, further study on the enzyme showed that it degraded cellopentaose and other cellodextrins to predominantly cellobiose. Thus CelA is a cellodextrinase. It also has high activity against p-nitrophenyl-β-D-cellobioside. A gene, expressing a protein with both carboxymethyl cellulase and xylanase activity, was cloned from R. flavefaciens FD-1 using an E. coli/Bacillus subtilis shuttle vector, pEBl. The 3.6 kb DNA insert on the plasmid pWFl, which carried the gene, celE, contained an open reading frame of 963 bp encoding 320 amino acid residues with a caculated Mᵣ of 35937. Homology analysis showed 11.6% identity and 55.3% similarity with the N-terminal catalytic region of the cellulase gene of alkalophilic Bacillus sp. strain 1139. In order to obtain expression in E. coli, the gene had to be transcribed from the lambda Pᵣ promoter. To determine whether cellulase genes of R. flavefaciens FD-1 were regulated at the level of transcription, celA and celE were used as probes against RNA isolated from R. flavefaciens FD-1 grown on cellobiose, cellulose or cellotriose. Transcription of both genes was induced when cellulose was added to cells growing in cellobiose. This induction continued after cellulose depletion and after cell division had ceased. Transcription of both genes was also induced by cellotriose although not to the same extent as by cellulose. This suggests that cellotriose and possibly ether dextrins may act as key inducers to trlgger celA and eels gene expression in R. flavefaciens FD-1.

Bibliography: pages 199-213. / The infectivity of the replicative form (RF) DNAs of MSV-Kom, MSV-Set and PanSV-Kar contained in the plasm ids pKom500, pSet100 and pPS100 was established by agroinoculating susceptible Jubilee sweetcorn with partial homodimeric Agrobacterium tumefaciens (C58C1) clones of RF-DNAs. Biological characteristics typical of Mastreviruses; such as, the appearance and leafhopper transmissibility of streak symptoms on infected plants, the presence of 18x30nm geminate particles in electron micrographs of leaf-dip preparations, and the presence of single-stranded and double-stranded DNA in Southern blot tests of infected plant DNA extracts, indicated that the RF-DNAs in pKom500, pSet100and PanSV-Kar represent the entire genomes of MSV-Kom, MSV-Set and PanSV-Karrespectively. The complete nucleotide (nt) sequence of the genome of MSV-Set was determined and characterised, and compared with those of MSV-Kom and PanSV-Kar. The genome sizes of MSV-Kom, MSV-Set and PanSV-Kar are 2701, 2690 and 2705 nt respectively, and all share Mastreviral genomic features. Phylogenetic analyses on the nt sequences and the putative amino acid sequences of the movement, coat and replication-associated proteins (MP, CP and Rep respectively) indicate that MSV-Set is grouped with, yet distinct from the MSV group of viruses isolated from maize. MSV-Set shares a 78% nt sequence identity with MSV-Kom which shares a >96% nt sequence identity with other MSVs. The PanSV-Kar genome shares a 60% nt sequence identity with the MSV group and89% with the Kenyan PanSV-Ken. PanSV-Kar causes mild non-persistent streak in Jubilee sweetcorn. MSV-Kom (previously isolated from maize in Komatipoort, Mpumalanga) and MSV-Set (previously isolated from a Setaria species in Mt. Edgecombe, Kwazulu/Natal) have different pathogenicities, and have overlapping, but non-identical, host ranges. Leafhopper transmission tests determined that MSV-Kom and MSV-Set generally cause severe and moderate streak in maize cultivars, or mild and severe streak in wheat cultivars respectively.

Drought is the primary challenge facing agricultural productivity and food sustainability productivity in Africa and many parts of the world. Very few higher plants, including crop plants, can survive periods of extended water loss. However, a small group of angiosperms, termed resurrection plants are able to lose up to 95% of their cellular water content in vegetative tissue upon extended periods of water, remain in an air-dry for months to years and regain full metabolic activity in the same tissue upon re-watering. The aim of this study was to investigate the changes in acyl chain composition within the major glycerophospholipids (in total and chloroplast suspension) and galactolipids (chloroplast suspension) at different stages of dehydration and rehydration treatments in the resurrection plant Xerophyta humilis (Bak) Dur and Schniz. This was done in order to ascertain their roles during acquisition of desiccation tolerance in X. humilis. The galactolipids and glycerophospholipids acyl chain compositions were determined by multiple reaction monitoring (MRM) mass spectrometry. The glycerophospholipids profiles from total leaves and roots lipid extracts showed an increased representation of unsaturated molecular species such as 18:3/18:3 (in leaves) and 18:3/18:2 (in leaves and roots) during dehydration, with a decrease in saturated and mono-saturated molecular species such as 16:0/16:0. Rehydration was associated with the opposite trend. Furthermore, increased representation of molecular species with unusual fatty acids were observed during dehydration, with the odd-numbered fatty acids such as 15:0, 17:0 and 19:0 increasing during dehydration and decreasing during re-watering. On the other hand, the 23:0 and 25:0 fatty acids decreased during dehydration and increased upon rehydration. Within the chloroplast profiles, the galactolipids (i.e. monogalactosyldiacylglycerolipids and digalactosyldiacylglycerolipids) showed maintenance of 18:3/18:3 and 18:3/18:2 molecular species during dehydration and rehydration. The phosphatidylglycerolipids profiles in the chloroplast, together with the uncommon chloroplastic glycerophospholipids such as phosphatidylethanolamine, phosphatidylcholine and phosphatidylinositol also showed an increase in the unsaturated molecular species during dehydration and decrease upon rehydration. Taken together, our data suggest that water deficit in X. humilis roots and leaves induce fatty acid unsaturation, as well as production of uncommon fatty acids. These unsaturated fatty acids may aid in maintaining membrane integrity during dehydration. This study shows that changes in lipid composition are part of the desiccation tolerance strategies used by X. humilis.