Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: The Cape Floristic Region (CFR) is situated at the southern tip of Africa and possesses a flora that is unique amongst the floras of the rest of the world, both in terms of its incredibly high species richness, and its high levels of endemism. Proteaceae, the family to which Serruria belongs, is widely distributed amongst the landmasses of the southern hemisphere, with its centres of diversity occurring in Australia and southern Africa. Previous molecular and morphological analyses performed on the South African subfamily Proteoideae have shown Serruria, a CFR endemic, to form a well-supported monophyletic group. Based upon the strong monophyly of Serruria, DNA sequence data were collected for 53 of the 55 species from the plastid (rps16 intron, atpB-rbcL intergenic spacer, trnL-F region and psbA-trnH intergenic spacer) and nuclear (internal transcribed spacer region or ITS) genomes in order to investigate evolutionary relationships within the genus. Spatalla taxa were used as the outgroup. Both parsimony and Bayesian analyses were carried out on each of these data sets. The resulting trees were reasonably well resolved. All the Serruria taxa grouped together in a well-supported clade, except for S. f1ava, which emerged well within the Serruria clade in the analyses of the nuclear genome, but outside the clade in the plastid analyses. It was therefore proposed that this taxon represents a hybrid. Apart from this case, there was widespread agreement between the trees reconstructed using data from the two genomes. The plastid and nuclear data were therefore combined in order to analyse the data sets together. The molecular data does not support most of the groupings proposed by previous authors based on morphological data. Additionally, in some cases, multiple representatives of species do not group together. These specimens probably do not represent monophyletic taxa. Current ideas about relationships within Serruria are based predominantly on floral characters, and it is suggested that pollinator pressures have led to plasticity in the floral characters. Consequently, it is evident from this study that relationships within Serruria need to be re-examined in order to determine the patterns of evolution within the genus. / AFRIKAANSE OPSOMMING: Die Kaapse Floristiese Streek is aan die suiderpunt van Afrika geleë, en beskik oor 'n unieke flora relatief tot ander wêreldfloras, beide ten opsigte van die ongelooflike hoë spesie diversiteit en die hoë vlakke van endemisme. Proteaceae, die familie waaraan Serruria behoort, kom wydverspreid tussen die vastelande van die Suidelike Halfrond voor, en het diversiteitsentrums in Australië en suider Afrika. Vorige molekulêre sowel as morfologiese analises wat op die Suid-Afrikaanse subfamilie Proteoideae uitgevoer is, dui aan dat Serruria (wat endemies is tot die Kaapse Floristiese Streek) 'n goed ondersteunde monofiletiese groep is. Gebaseer op die sterk monofilie van Serruria, is DNA-volgorde-data vir 53 van die 55 spesies vanuit die plastied (rps16 intron, atpB-rbcL intergeniese spasie, trnL-F area en psbA-trnH intergeniese spasie) en kern (intern getranskribeerde spasie area, ook ITS genoem) ingewin om die evolusionêre verwantskappe binne die genus te ondersoek. Spatalla is as die buitegroep gebruik. Beide parsimonie en Bayesian analises is op elk van hierdie datastelle uitgevoer. Die resulterende bome het redelike hoë resolusie getroon. AI die Serruria-taxa het in 'n goed ondersteunde klade saam gegroepeer, behalwe vir S. f1ava, wat binne die Serruria klade val vir die kern genoom, maar buite die klade vir die plastied analise. Dit is dus voorgestel dat hierdie taxon as 'n hibried beskou mag word. Behalwe vir hierdie geval, was daar wydverspreide ooreenstemming tussen die bome wat verkry is vanaf data van die twee genome. Die plastied- en kern-data is derhalwe gekombineer om die datastelle saam te kan analiseer. Die molekulêre data ondersteun nie die meerderheid van morfologiese groeperings wat deur verskeie outeurs voorgestel is nie. Verder, in sommige gevalle, groepeer verskillende monsters van dieselfde spesies nie bymekaar nie. Dit is derhalwe voorgestel dat hierdia taxa nie monofileties is nie. Huidige idees omtrent die verwantskappe binne Serruria is grotendeels op blommorfologiese kenmerke gebaseer, en dit word voorgestel dat bestuiwing-druk gelei het tot plastisiteit van die blommorfologiese kenmerke. Verskille tussen die bome wat uit plastied- en kern-data gerekonstrueer is word aan vroeëre hibridisasie gebeure toegeskryf. Op grond van hierdie studie is dit duidelik dat die verhoudings binne Serruria verder ondersoek moet word om die patrone van evolusie binne die genus te bepaal.

P. aerguinosa is a serious human bacterial pathogen. This thesis describes attempts to use structural biology to identify new starting points for drugs against P. aerguinosa .A number of fragment-based screening techniques were used in order to identify potential inhibitors to P. aerguinosa RmlA protein, the first enzyme in the L-Rhamnose pathway. A 500 “Rule of 3” Fragment Library (Maybridge) was investigated. The first approach was the application of Differential Scanning Fluorimetry (DSF) approach to detect ligands that bind and stabilize RmlA protein. The stabilisation of RmlA was determined by thermal unfolding in the presence of each of the 500 compounds. 21 of those compounds were found to increase the protein stability. The library was then screened by NMR spectroscopy for binding to RmlA. Two techniques were evaluated STD and WaterLOGSY. 106 compounds gave positive results in both NMR experiments. These hits were then tested by a simple STD competition binding with dTTP, a natural RmlA substrate, in order to identify those binding at the active or allosteric site. 21 out of the 106 compounds were observed to compete with dTTP. The results were compared to the results of the DSF screening. Compounds that tested positive in the dTTP competition binding STD experiment and in the DSF screening were tested for their ability to inhibit RmlA in a biological assay. A coupled enzyme assay was used to monitor RmlA activity. Only one compound, 3-pyridin-3-ylaniline, showed significant inhibition of the enzyme activity. The PA1645 protein from P. aerguinosa has been identified as essential. The protein was overexpressed, purified and crystallised. Data were collected at Diamond on beamline IO3 and phases were determined by S-SAD at a wavelength of 1.6Å. Final coordinates have been deposited in the protein data bank under entry code 2XU8. The structure has 3 molecules in the asymmetric unit. There is some ambiguity as to the validity of the proposed trimeric arrangement, with results from solution and crystal disagreeing. Fragment-based screening approach has been applied to RmlA protein, using the DSF technique, a number of ligand-based NMR experiments and a coupled enzyme biological assay. 3-pyridin-3-ylaniline was the only compound that showed significant inhibition of the enzyme activity. The structure of PA1645 from P. aerguinosa has been solved. This work will help to design new drugs to combat multi-drug resistant P. aerguinosa and MTB.

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Anatomy / Master / Master of Philosophy

published_or_final_version / Ecology and Biodiversity / Master / Master of Philosophy

published_or_final_version / abstract / Pathology / Master / Master of Philosophy

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy