Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Large-scale human-mediated movements of organisms promote the establishment of species outside their native ranges and a very small proportion of these species become invasive. Invasive species management typically assumes that introduced species are single, static evolutionary units that are genetically analogous to their native counterparts. However, studies have shown that native and introduced populations of a number of introduced plants differ vastly in their genetic composition. These differences may negatively affect the overall success of control and management programmes, particularly for species that are intra-specifically diverse. The influence of intra-specific diversity on the invasion process was tested in two widely exported tree species that are native to Western Australia, Acacia saligna (three subspecies) and Paraserianthes lophantha (two subspecies). Climate matching between the native and introduced range (using species distribution models, SDM) is widely used to forecast future invasion risks, however, it is unknown if SDMs can detect intra-specific niche differences in invasive plants. The SDMs I developed for the subspecies of A. saligna detected intra-specific differences within the native range, but did not predict the full invasive distribution in South Africa. Unsurprisingly, SDMs agreed with genetic analyses (based on nuclear microsatellites, nuclear DNA, and chloroplast DNA) and did not assign South African populations to any subspecies of A. saligna. South African populations were assigned to a novel genetic entity likely produced by human cultivation practices. A global phylogeny identified this cultivated genotype in introduced populations in eastern Australia and Portugal, while the remaining introduced populations differed markedly in their genetic composition. Overall, A. saligna‘s high intra-specific diversity and complex introduction history generated a variety of genetic patterns across the current global distribution of the taxon. Global populations of P. lophantha were processed using a similar approach to that used for A. saligna, and aimed to determine if the same pathways and modes of introduction produced analogous genetic patterns in a closely related species. Diverse arrays of genotypes were identified in introduced populations of P. lophantha, suggesting inconsistent sampling of a variety of native sources. Further work is however needed to clarify the morphological and genetic differences (if any) between the intra-specific entities, and identify exactly which P. lophantha subspecies were introduced outside of their native range, The variation in the global distribution of genetic diversity observed in A. saligna and P.lophantha demonstrated that intra-specific genetic variation, human usage, and the pathway and manner of introduction interact during several phases of the invasion process and collectively determine the introduced genetic patterns. The dissimilarity in the distribution of genotypes in both species suggests that they might not behave the same way throughout their introduced range. Consequently, management insights might not be transferrable between regions. More generally, my findings provide an important contribution to the debate whether (and how quickly) introduced and native populations should be treated as fundamentally different entities. / AFRIKAANSE OPSOMMING: Grootskaalse menslike verskuiwing van organismes bevorder die vestiging van spesies buite hul natuurlike voorkomsareas en 'n klein hoeveelheid van hierdie spesies word indringers. Tydens die bestuur van indringerspesies word dit tipies aanvaar dat ingevoerde indringerspesies enkele, statiese evolusionêre eenhede is wat analoog is aan hul inhmeemse eweknieë. Studies het egter getoon dat inheemse en uitheemse populasies van 'n aantal ingevoerde plante aansienlik verskil in hul genetiese samestelling. Hierdie verskille kan 'n negatiewe invloed op die algehele sukses van beheer- en bestuursprojekte hê, veral vir die spesies wat intra-spesifiek divers is. Die invloed van intra-spesifieke diversiteit op die indringingsproses is getoets aan twee boomspesies, inheems aan Wes-Australië, wat wyd uitgevoer word: Acacia saligna (drie subspesies) en Paraserianthes lophantha (twee subspesies). Vergelyking van klimaatstoestande tussen n spesie se in- en uitheemse voorkomsareas word wyd gebruik om toekomstige indringingsrisiko te voorspel. Dit was voor hierdie navorsing onduidelik of spesie verspreiding modelle (SVMs) intra-spesifieke nis-verskille in indringerplante kan uitwys. SVMs wat vir die subspesies van A. saligna ontwikkel is, kon intra-spesifieke verskille in Wes-Australië uitwys, maar het nie die volle verspreiding van die spesies in Suid-Afrika voorspel nie. Onverbasend, is geen Suid-Afrikaanse populasies deur genetiese analise (gebaseer op die kern mikrosatelliete, kern-DNS, en chloroplas-DNS) toegewys aan 'n subspesie van A. Saligna nie. Suid-Afrikaanse populasies het 'n nuwe genetiese entiteit wat waarskynlik gekweek is deur menslike verbouingspraktyke. 'n Globale filogenie het hierdie verboude genotipe in addisionele ingevoerde populasies in die ooste van Australië en Portugal geïdentifiseer. Mikrosatelliet genotipes van uitheemse populasies wêreldwyd in Oos-Australië, Israel, Italië, Nieu-Seeland, Portugal, Suid-Afrika, Spanje en die VSA verskil merkbaar in hul genetiese samestelling. A. saligna se hoë intra-spesifieke diversiteit en komplekse geskiedenis van invoer (wat verbouing, wye verspreiding en hoë ―propagule druk betrek), het 'n verskeidenheid van genetiese patrone oor die huidige globale verspreiding van die takson gegenereer. Om te bepaal of 'n globale uiteenlopende genetiese patroon binne nouverwante spesies bestaan, is globale bevolkings van Paraserianthes lophantha verwerk deur gebruik te maak van 'n soortgelyke benadering as wat vir A. saligna gebruik is. Globale populasies van beide studie-spesies bestaan uit 'n diverse verskeidenheid van genotipes. Resultate dui daarop dat P. lophantha van 'n verskeidenheid inheemse bronne ingevoer is. Om te identifiseer watter P. lophantha subspesies buite hul natuurlike voorkomsarea versprei is, word verdere werk benodig om die morfologiese en genetiese verskille (indien enige) tussen die intra-spesifieke entiteite vas te stel. In hierdie tesis het ek gewys dat intra-spesifieke genetiese variasie, menslike gebruik en invoering-geskiedenis saam werk om genetiese patrone in uitheemse populasies te vorm. Verder het ek die waarde van die gebruik van verskillende molekulêre benaderings om indringing geskiedenis te verstaan, gedemonstreer. Die verskil in die verspreiding van die genotipes van A. saligna en P. lophantha dui daarop dat hulle moontlik nie op dieselfde manier dwarsdeur hul uitheemse verspreidingsarea mag optree nie. Bestuursinsigte mag gevolglik nie oordraagbaar wees tussen streke nie. Meer algemeen, bied my bevindings 'n belangrike bydrae tot die debat of (en hoe vinnig) inheemse en ingevoerde populasies behandel moet word as fundamenteel verskillende entiteite.

In this work, we examine the structure and function of two important human proteins. The first is human kallikrein 6 (hK6), which is a newly identified enzyme in the serine proteinase family that is expressed in the central nervous system. In chapter 2, the X-ray crystal structure of mature, active recombinant human kallikrein 6 at 1.75 Å is presented. This high resolution model provides the first three-dimensional view of one of the human kallikreins and one of only a few structures of serine proteinases predominantly expressed in the central nervous system. Enzymatic and X-ray data provide support for the characterization of human kallikrein 6 as a degradative proteinase with structural features more similar to trypsin than the regulatory kallikreins. In chapter 3, we have re-solved the structure of hK6 to a resolution of 1.56 Å. In addition, a detailed analysis of the preferred substrate specificity of hK6 at the positions P3, P2, P1′, P2′, and P3′ is undertaken using internally quenched fluorescent substrates based on a peptide background sequence of the identified autolysis region. Furthermore, the identified optimized substrate sequence is modeled into the 1.56 Å structure of human kallikrein 6 using docking in order to identify structural aspects of the protein responsible for this preference. The substrate specificity data show that human kallikrein 6 displays little discrimination for particular amino acids at the tested positions with the exception of P2′, where there is a pronounced preference for proline. The second protein studied in this work is human fibroblast growth factor-1 which is a member of the β-trefoil superfold. In chapter 4, a 1.10 Å atomic-resolution x-ray structure of human fibroblast growth factor 1, a member of the β-trefoil superfold, is reported. The FGF-1 structure exhibits numerous core packing defects detectable using a 1.0Å radius probe. In addition to contributing to the relatively low thermal stability of FGF-1, these defects may also permit domain motions within the structure. The availability of refined ADP's permits a translation/libration/ screw (TLS) analysis of putative rigid body domains. The observed rigid body motion in FGF-1 appears related to the ligand-binding functionalities. / Dissertation / PhD

Human acidic fibroblast growth factor (FGF-1) is a member of the £]-trefoil superfamily and exhibits a characteristic three-fold tertiary structure symmetry. However, evidence of this symmetry is not readily apparent at the level of the primary structure. This suggests that while selective pressures may exist to retain (or converge upon) a symmetric tertiary structure, other selective pressures have resulted in divergence of the primary structure during evolution. Using intra-chain and homologue sequence comparisons for 19 members of this family of proteins, we have designed mutants of FGF-1 that constrain a subset of core-packing residues to three-fold symmetry at the level of the primary structure. The consequences of these mutations upon structure, stability, folding and unfolding kinetics have been evaluated using a combination of x-ray crystallography, differential scanning calorimetry, isothermal equilibrium denaturation and stopped flow protein refolding/unfolding kinetics. An alternative core packing group has been introduced into FGF-1. The alternative core is very similar from the wild type (WT) core with regard to structure, stability, folding and unfolding kinetics. The remaining asymmetry within the protein core is related to asymmetry in the tertiary structure. The removal of tertiary structure asymmetry greatly increases protein stability and results in a conversion from three-state to a two-state folding pathway. The tertiary structure asymmetry is intimately linked to functional regions of the protein. Surprisingly, upon deletion of the functional insertions, the mutant protein is approximately 80 times more potent than the wild type form as determined by functional bioassays. The results show that the ƒÒ-trefoil superfold is compatible with a three-fold symmetric constraint upon the core region, as might be the case if the superfold arose as a result of gene duplication/fusion events. Furthermore, this new protein arrangement can form the basis of a structural "building block" that can greatly simplify the de novo design of ƒÒ-trefoil proteins by utilizing symmetric structural complementarity. This study implies that a symmetric architecture of the £]-trefoil fold is kinetically and thermodynamically ¡§fit¡¨. / Dissertation / PhD