In the current work, the photophysics and photochemistry of the phototoxic red fluorescent protein (RFP) KillerRed was investigated. KillerRed's phototoxicity makes it useful for studying oxidative stress on cell physiology and for cell killing in photodynamic therapy. Spectroscopic probes were used to show that the phototoxicity of KillerRed stems primarily from a type I photosensitization mechanism producing radicals. The production of radicals was supported by electron paramagnetic resonance (EPR) studies, where a long-lived radical was observed in KillerRed and two other RFPs (mRFP and DsRed) following excitation. Transient absorption spectroscopy, various other spectroscopic techniques, and the published crystal structure of KillerRed indicate that the long-filled water channel is likely responsible for the increased phototoxicity of KillerRed. In the blue fluorescent protein (BFP) mKalama1, some of the same techniques were applied to understand the photophysics and photochemistry on the timescale ranging from femtoseconds to seconds. Transient absorption spectroscopy and previously published results demonstrate that two-photon excitation of mKalama1 likely results in the formation of a radical cation and solvated electrons. This may explain the blinking behavior which has been observed on the single molecule level for many fluorescent proteins, the identity of which has remained elusive. It was also shown that the chromophore, while neutral in the ground state, does not exhibit excited-state proton transfer (ESPT) during its nanosecond excited-state lifetime; however, the chromophore undergoes a deprotonation in the ground state after electronic relaxation. This work plays a key role in our understanding of fluorescent proteins and will help pave the way to developing new ones. The research on the BFPs was extended to improve them for cellular imaging. This was accomplished by identification of dark states in the BFPs which are longer in wavelength than the collected fluorescence. Using dual lasers, it was shown that these dark states could be optically depleted, thereby increasing the overall fluorescence without enhancing the background fluorescence. Rational site-directed mutagenesis was carried out on the BFPs and the mutants were screened for fluorescence enhancement. These proteins were then analyzed using transient absorption spectroscopy to elucidate the identity of the dark state(s) used for fluorescence enhancement.

Contrary to what many scholars hold, a case can be made for human population reduction (the practice of artificially decreasing the number of human beings on the Earth). Robin Attfield's, Paul Taylor's, Arne Næss's and J. Baird Callicott's theories are considered for justifying human population reduction; however, only Næss's actually justifies reduction. Another argument for human population reduction is developed, based on rectification: humans have unjustly harmed other living entities and themselves, and they should provide rectification for the harm done. Human population reduction is a way in which this rectification can be given.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Damping-off of rooibos (Aspalathus linearis), which is an important indigenous crop in South Africa, causes serious losses in rooibos nurseries and is caused by a complex of pathogens of which oomycetes, mainly Pythium, are an important component. The management of damping-off in organic rooibos nurseries is problematic, since phenylamide fungicides may not be used. Therefore, alternative management strategies such as rotation crops, compost and biological control agents, must be investigated. The management of damping-off requires knowledge, which currently is lacking, of the Pythium species involved, and their pathogenicity towards rooibos and two nursery rotation crops (lupin and oats). Pythium species identification can be difficult since the genus is complex and consists of more than 120 species. Species identification is, however, greatly facilitated by analyses of the internal transcribed spacer (ITS) regions. These regions, have also been used to divide the genus into 11 phylogenetic clades (A to K), with some clades, such as clade G, still being poorly characterised. The first aim of the study was to characterize 12 Pythium clade G isolates that were obtained from damped-off rooibos seedlings, along with six known clade G species. Subsequently, oligonucleotides were designed for differentiating two rooibos associated groups that may represent new taxons, for future use in DNA macro-array analyses. Phylogenetic analyses of the ITS region and a combined phylogeny of four gene regions (ITS, -tubulin and, COX1 and COX2 [cytochrome c oxidase subunits I and II]) identified five sub-clades within Pythium clade G. The rooibos isolates formed two groups, Rooibos group I (RB I) and II (RB II) that clustered into two groups within sub-clade 1 with good support (64%-89% bootstrap, 1.00 probability). The Pythium RB I isolates had P. iwayamai as its nearest neighbour, and may represent a new species. The Pythium RB II isolates had P. canariense and P. violae as their closest relatives and may, along with other isolates contained in the RB II sub-clade, represent several new species. Morphological analyses of the rooibos isolates were inconclusive, since the isolates all contained similar morphological characteristics that did not correspond to the description of known Pythium species. The Pythium RB I and II isolates were all non-pathogenic toward rooibos, lupin and oats seedlings. For each of the two rooibos groups, one newly developed oligonucleotide was able to differentiate the isolates from clade G reference isolates using DNA macro-array analyses. The second aim of the study was to determine the oomycetes species associated with rooibos in nurseries and in a native rooibos site, and their pathogenicity towards rooibos and two nursery rotation crops (lupin and oats). Since some isolates were shown to be nonpathogenic, another aim was to determine whether these isolates, along with the previously characterised non-pathogenic Pythium RB I and RB II isolates, could suppress pathogenic oomycetes. Characterisation of isolates from 19 nurseries and one native rooibos site revealed the presence of five Pythium species (P. acanthicum, P. irregulare, P. mamillatum, P. myriotylum, and P. pyrilobum) and Phytophthora cinnamomi. In nurseries, P. irregulare was the most common species (81%) followed by P. myriotylum (14%). Similarly, P. irregulare was also the most prevalent species (57%) in native rooibos, but P. pyrilobum (26%) was second most prevalent. Pathogenicity studies on rooibos showed that all species, except P. acanthicum, were highly virulent causing 100% damping-off. On lupin, P. acanthicum was also the only non-pathogenic species, with the other species being less virulent on lupin than on rooibos. Only P. irregulare, P. myriotylum, and P. pyrilobum were pathogenic towards oats, and were also less virulent on oats than on rooibos. On lupin and oats, not all off the isolates from a specific species was pathogenic. Non-pathogenic Pythium species (P. acanthicum, Pythium RB I and II) was only effective at suppressing disease on the less susceptible crops of lupin and oats, but not on rooibos. The third aim of the study was to investigate the management of rooibos damping-off using two composts (A and B), and composts combined with non-pathogenic Pythium species. Evaluation of the suppression by composts of Ph. cinnamomi and 29 Pythium isolates, which represented the four pathogenic Pythium rooibos species, showed that both composts were able to suppress some, but not all of the pathogenic Pythium isolates. Both composts were very effective at, and the highest percentage control was achieved, with suppression of Ph. cinnamomi. Most isolates of P. mamillatum and P. pyrilobum were suppressed by composts, whereas most P. irregulare (> 62%) and P. myriotylum (>50%) isolates were not suppressed. Non-pathogenic Pythium species combined with either of the two composts were able to significantly reduce damping-off caused by P. irregulare or a combination of pathogenic species (P. irregulare, P. mamillatum, P. myriotylum, P. pyrilobum, and Ph. cinnamomi), compared to than when only the pathogens were present. In the absence of non-pathogenic species, neither of the composts was able to suppress the aforementioned pathogenic isolates. This study has improved our knowledge of the oomycete species that are involved in rooibos damping-off, and has identified possible management strategies for use in organic nurseries. Several oomycete species are involved in causing damping-off and their differential virulence, and responses to being suppressed by composts, will require the use of integrated management strategies. Management strategies that showed promise include the combined use of compost and non-pathogenic Pythium taxons. The use of oats, which is susceptible to fewer oomycete isolates than rooibos, could also be valuable as a rotation crop. Altogether, knowledge obtained in this study can be used to (i) optimize integrated management strategies for organic nurseries, (ii) elucidate the mechanisms involved in disease suppression and (ii) develop molecular techniques, such as DNA macro-arrays and quantitative PCR (qPCR) for the rapid assessment of the species involved, and the quantification of inoculum in nursery soils. / AFRIKAANSE OPSOMMING: Omvalsiekte van rooibos (Aspalathus linearis), wat ‘n belangrike inheemse gewas in Suid-Afrika is, veroorsaak ernstige verliese in rooiboskwekerye, en word deur ‘n kompleks van patogene veroorsaak, waarvan oömysete, hoofsaaklik Pythium, ’n belangrike komponent is. Die bestuur van omvalsiekte in organiese rooiboskwekerye is problematies, aangesien fenielamied fungisiedes nie gebruik mag word nie. Alternatiewe bestuurstrategieë, soos rotasie-gewasse, kompos en biologiese beheer-agente, moet dus ondersoek word. Die bestuur van omvalsiekte vereis kennis, wat tans ontbreek, naamlik die Pythium spesies wat betrokke is, hul patogenisiteit teenoor rooibos, en twee kwekery rotasie-gewasse (lupiene en hawer). Pythium spesie-identifikasie kan moeilik wees aangesien die genus kompleks is en uit meer as 120 spesies bestaan. Spesie-identifikasie word egter grootliks vergemaklik deur analise van die interne getranskribeerde spasieerder (ITS) areas. Hierdie areas is ook gebruik om die genus in 11 filogenetiese “clades” (A tot K) te verdeel, met sommige “clades”, soos “clade” G, wat steeds swak gekarakteriseer is. Die eerste doelwit van die studie was om 12 Pythium “clade” G isolate te karakteriseer, wat vanaf omvalsiekte rooibossaailinge verkry is, tesame met ses bekende “clade” G spesies. Gevolglik is oligonukleotiede ontwerp ten einde twee rooibosgeassosieerde groepe, wat nuwe taksons kan verteenwoordig, te onderskei, en vir toekomstige gebruik in DNS makro-“array” analise. Filogenetiese analise van die ITS area en ‘n gekombineerde filogenie van vier geen-areas (ITS, -tubulien en, COX1 en COX2 [sitokroom c oksidase sub-eenhede I en II]) het vyf sub-“clades” binne Pythium “clade” G geïdentifiseer. Die rooibos isolate het twee groepe gevorm, Rooibos groep I (RB I) en II (RB II) wat twee groepe binne sub-“clade” 1 gevorm het, met goeie ondersteuning (64%-89% “bootstrap”, 1.00 waarskynlikheid). Die Pythium RB I isolate het P. iwayamai as sy naaste verwant, en mag ‘n nuwe spesie verteenwoordig. Die Pythium RB II isolate het P. canariense en P. violae as hul naaste verwante en mag, tesame met ander isolate wat in die RB II sub-“clade” ingesluit word, verskeie nuwe spesies verteenwoordig. Morfologiese analise van die rooibos-isolate was onbeslis, aangesien die isolate almal soortgelyke morfologiese kenmerke bevat het, wat nie met die beskrywing van bekende Pythium spesies ooreengestem het nie. Die Pythium RB I en II isolate was almal nie-patogenies teenoor rooibos-, lupien- en hawersaailinge. Vir elk van die twee rooibosgroepe, was een nuut-ontwikkelde oligonukleotied in staat om die isolate van “clade” G verwysingsisolate te differensieer, deur die gebruik van DNS makro-“array” analise. Die tweede doelwit van die studie was om die oömysete spesies wat met rooibos in kwekerye en in ‘n inheemse rooibos-area geassosieer word, te bepaal, en hul patogenisiteit teenoor rooibos en twee kwekery rotasie-gewasse (lupien en hawer). Aangesien van die isolate nie-patogenies was, was ’n ander doelwit om te bepaal of hierdie isolate, tesame met die voorheen gekarakteriseerde nie-patogeniese Pythium RB I en RB II isolate, patogeniese oömysete kan onderdruk. Karakterisering van isolate van 19 kwekerye en een inheemse rooibos-area, het op die teenwoordigheid van vyf Pythium spesies (P. acanthicum, P. irregulare, P. mamillatum, P. myriotylum, en P. pyrilobum) en Phytophthora cinnamomi gedui. P. irregulare was die mees algemene spesie (81%) in kwekerye, gevolg deur P. myriotylum (14%). Soortgelyk was P. irregulare ook die mees algemene spesie (57%) in inheemse rooibos, maar P. pyrilobum (26%) was tweede mees algemeen. Patogenisiteitstudies op rooibos het getoon dat alle spesies, behalwe P. acanthicum, hoogs virulent was en 100% omvalsiekte veroorsaak het. Op lupien was P. acanthicum ook die enigste nie-patogeniese spesie, terwyl die ander spesies minder virulent op lupien as op rooibos was. Slegs P. irregulare, P. myriotylum en P. pyrilobum was patogenies teenoor hawer, en was ook minder virulent op hawer as op rooibos. Op lupien en hawer was nie alle isolate van ‘n spesifieke spesie patogenies nie. Nie-patogeniese Pythium spesies (P. acanthicum, Pythium RB I en II) was slegs effektief om siekte op die minder vatbare gewasse, lupien en hawer, te onderdruk, maar nie op rooibos nie. Die derde doelwit van die studie was om die bestuur van rooibos omvalsiekte te ondersoek, deur die gebruik van twee tipes kompos (A en B), en kompos gekombineer met nie-patogeniese Pythium spesies. Evaluasie van die onderdrukking deur kompos van Ph. cinnamomi en 29 Pythium isolate, wat die vier patogeniese Pythium rooibosspesies verteenwoordig het, het getoon dat beide tipes kompos in staat was om sommige, maar nie al die patogeniese Pythium isolate, te onderdruk nie. Beide tipes kompos was baie effektief, en die hoogste persentasie beheer was met die onderdrukking van Ph. cinnamomi verkry. Meeste isolate van P. mamillatum en P. pyrilobum is deur kompos onderdruk, terwyl meeste P. irregulare (> 62%) en P. myriotylum (>50%) isolate nie onderdruk is nie. Nie-patogeniese Pythium spesies, in kombinasie met enige van die twee tipes kompos, was in staat om betekenisvol omvalsiekte veroorsaak deur P. irregulare, of in ’n kombinasie met patogeniese spesies (P. irregulare, P. mamillatum, P. myriotylum, P. pyrilobum, en Ph. cinnamomi), te verminder, in vergelyking met wanneer slegs die patogene aanwesig was. In die afwesigheid van nie-patogeniese spesies, was nie een van die tipes kompos in staat om die voorafgenoemde patogeniese isolate te onderdruk nie. Hierdie studie het ons kennis rakende die oömysete spesies betrokke in rooibos omvalsiekte verbeter, en het moontlike bestuurstrategieë geïdentifiseer wat in organiese kwekerye gebruik kan word. Verskeie oömysete spesies is betrokke in die oorsaak van omvalsiekte, en hul verskille in virulensie, en reaksies op onderdrukking deur kompos, sal die gebruik van geïntegreerde bestuurstrategieë vereis. Bestuurstrategieë wat belofte toon, sluit die gekombineerde gebruik van kompos en nie-patogeniese Pythium taksons in. Die gebruik van hawer, wat vir minder oömysete isolate as rooibos vatbaar is, kan ook waardevol as ‘n rotasie-gewas wees. Tesame, kan kennis wat in die studie opgedoen is gebruik word om (i) geïntegreerde bestuurstrategieë vir organiese kwekerye te optimaliseer, (ii) die meganismes betrokke in siekte-onderdrukking te bepaal, en (iii) molekulêre tegnieke, soos DNS makro- “arrays” en kwantitatiewe PKR (qPKR) te ontwikkel vir die vinnige bepaling van die spesies betrokke, en die kwantifisering van inokulum in kwekery-gronde.

Thesis (MScAgric (Agronomy)--Stellenbosch University, 2008. / Honeybush (Cyclopia spp. Fabaceae) is indigenous to the fynbos botanical biome of the Eastern and Western Cape of South Africa. The increase in the international demand for honeybush tea for health benefits, concern over exploitation of wild populations and the lack of published agronomic information necessitated this study to evaluate different aspects of honeybush propagation. The main objectives of this study were to evaluate the effect of species and cutting position on rooting of cuttings of Cyclopia species using different rooting hormones, to evaluate the effect of an organic plant fertilizer and cutting position on growth and establishment of rooted cuttings and to study the influence of different seed pre-treatments on germination of Cyclopia species. Terminal and sub-terminal cuttings of C. intermedia and C. genistoides treated with different rooting hormones were rooted under day/night temperature controlled glasshouse conditions. Intermittent mist was used as means of moisture supply to the cuttings for 45-60 seconds daily every 30 minutes. C. genistoides rooted significantly better compared to C. intermedia as measured by rooting percentage, number of roots per cutting, length of longest root and mean root length during the summer season. The cutting position had a significant effect on rooting of the cuttings in summer compared to winter and spring season. The interactive effect of species, treatment and cutting position resulted into 86% of rooting in summer from the terminal cuttings of C. genistoides, while only 4% was recorded as the highest rooting percentage in both winter and spring seasons. The highest number of roots and the greatest root length per cutting were obtained with 2 and 4 g L-1 IBA from terminal cuttings of C. genistoides and these hormone concentrations were not significantly different to each other. To evaluate the effect of an organic plant fertilizer and cutting position on plant growth and establishment, rooted cuttings of two Cyclopia species (C. intermedia and C. genistoides) from two cutting positions (terminal and sub-terminal) were transferred to pots (576 cm3) and treated with Nitrosol fertilizer at application rates of 3.33 ml.L-1, 1.67 ml.L-1 and 0 ml.L-1 (control). Cyclopia plantlets were uniformly inoculated once with a symbiotic Rhizobium bacteria to improve the formation of nodules. Nitrosol® at 3.33 ml.L-1 significantly affected fresh and dry plant weight, fresh and dry root weight, number of shoots and nodules per plant compared to either 1.67 ml.L-1 or the control. Relative to species, C. genistoides performed better in terms of fresh and dry plant weight, fresh and dry root weight, and number of shoots and nodules per plant compared to C. intermedia. The origin of the cutting position did not significantly affect the above mentioned parameters. Plant mineral analysis revealed that most of the essential elements increased with increasing Nitrosol® application rates, with C. genistoides having higher levels of mineral elements than C. intermedia. This could be an indication of the differences between the two species in terms of nutrient uptake, utilization and distribution within the plant tissues. In the germination studies, seeds obtained from different seed sources of Cyclopia species were subjected to different pre-sowing treatments. Seed treatments were sulphuric acid (95%), hot water (100°C), water with smoke paper disk, and demineralised water (control). The study revealed that all the treatments had a significant effect on germination with the exception of eight year old seeds obtained from C. subternata (seed source two). Although hot water treatment improved germination compared to smoked paper disk and the control, seeds treated with hot water degenerated rapidly. The highest overall germination (77.33%) was found with one year old seeds compared to other seed sources older than one year. Although smoked paper disks generally did not improve germination compared to the control, in one year old seeds from seed source one, this treatment greatly influenced germination, suggesting that seed age might have influenced germination of these seeds. In terms of germination rate, germination generally started after four days in most treatments.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Alien invasive species are a global concern not only threating biodiversity, but also negatively impacting regional economies. Consequently research aimed at understanding the invasion process is crucial for management of invasive alien species to reduce these impacts. Identifying accurate natal ranges is the key first step for efficient alien plant management, especially biological control. Such knowledge may assist in selecting host-specific biological control agents and help prevent non-target impact on native species. A case in point is the genus Cardiospermum of which species have been extensively moved around the globe with two species now being possible widespread invaders (C. grandiflorum and C. halicacabum), and a third species (C. corindum) having significant invasion potential. However, in some regions the native statuses of these species are not clear, hampering management. In order to minimize potential non-target impacts it is prudent to determine the relationships between Cardiospermum taxa in unknown native ranges prior to the release of biological control agents. With this thesis I aim to review available literature on the genus Cardiospermum, determine potential spread to suitable habitats globally for selected taxa, resolve unknown native ranges in southern Africa, and investigate rapid speciation of an endemic Namib Desert species of balloon vine. Through exhaustive searches for available literature on the genus Cardiospermum I report on the biology and ecology of selected species within the genus, with special focus on the most widespread species. Specifically, using species distribution modelling I investigate the potential spread of C. halicacabum, C. corindum and C. grandiflorum globally and assess the accuracy with which this can be determined using known native ranges to predict current alien ranges. Results indicate that these species have significant potential to spread, though highlighting that species distribution modelling over-fit predicted suitable ranges when using native range data alone. To resolve uncertain native ranges of selected species within the genus I reconstructed a dated multi-gene phylogeny and chloroplast haplotype network to investigate relationships within and among Cardiospermum species. I conclude that C. halicacabum is non-native in southern Africa due to polyphyletic relationships among accessions, while C. corindum is native due to natural long distance dispersal as indicated by a single monophyletic southern African clade. The close relationship observed between C. corindum and the African species, C. pechuelii, lead to a population-level genetic investigation of these two taxa. For this, I constructed a multi-gene phylogeny, chloroplast haplotype network and conducted population genetic diversity analyses which all indicated that the African arid adapted taxa, C. pechuelii, endemic to the Namib Desert of Namibia, evolved from C. corindum following long distance dispersal from South America. This study firstly provides insights into the ecology and biology of Cardiospermum, warning against further introduction due to potential spread. Secondly I resolved the unknown native statutes of Cardiospermum species in southern Africa and, lastly, illustrated the origin of African C. pechuelii. My research findings have major implications for on-going biological research against C. grandiflorum in South Africa and cautions strongly against the release of already-identified biological control agents due to their potential non-target impacts. / AFRIKAANSE OPSOMMING: Indringerspesies is 'n wêreldwye probleem as gevolg van hul direkte bedreiging van biodiversiteit asook hul negatiewe impakte op streeksekonomieë. Gevolglik is navorsing aangaande die indringingsproses van uiterste belang om doeltreffende beheer van indringerspesies te verseker. Die bepaling van die inheemse herkoms van indringerpopulasies is die eerste belangrike stap tot doeltreffende beheer, veral biologiese beheer. Hierdie informasie kan help met die identifisering van gasheer-spesifieke biologiese agente en om potensiële nie-teiken impakte op inheemse plant spesies verhoed. ‘n Voorbeeld is ballon rankplante in die genus Cardiospermum, waarvan verskeie spesies wêreldwyd verspreid is, met die gevolg dat minstens twee spesies (C. halicacabum en C. grandiflorum) nou moontlike wydverspreide indringers is en ‘n derde spesie (C. corindum) ook potensiaal toon as ‘n indringerspesie. In sommige streke is die in- of uitheemse statusse van hierdie spesies onbekend en belemmer gevolglik hul doeltreffende beheer. Met hierdie tesis streef ek om die genus Cardiospermum te hersien en potensiële verspreiding van geselekteerde spesies na geskikte habitatte te bepaal, om onbekende inheemse streke binne Suider-Afrika op te los en laastens, om die herkoms van ‘n ware Afrika spesie (C. pechuelii) te ondersoek. Na deeglike ondersoek van beskikbare literatuur doen ek verslag omtrent die biologie en ekologie van spesies binne Cardiospermum, met spesiale fokus op die mees wydverspreidste spesies. Deur gebruik te maak van spesies verspreidings modelle bestudeer ek die potensiaal van verdere verspreiding wêreldwyd vir C. halicacabum, C. corindum en C. grandiflorum en rapporteer ook omtrent die akkuraatheid van hierdie tegniek. Resultate toon dat alle spesies aansienlike potensiaal toon om verder te versprei, maar beklemtoon ook dat hierdie metode inheemse streek data “oor-aanpas”. Om enige onsekerhede aangaande inheemse streke op te los, het ek ‘n gedateerde multi-geen filogenie en ‘n chloroplas haplotipe netwerk opgetrek om die verhoudings binne en tussen geselekteerde Cardiospermum spesies te bepaal. Ek kom tot gevolgtrekking dat C. halicacabum nie inheems in Suider-Afrika is nie, as gevolg van polifiletiese verhoudings, en dat C. corindum inheems is en moontlik ook ‘n voorbeeld van natuurlike langafstand verspreiding, soos aangedui deur ‘n enkele monofiletiese groep. Die naverwante filogetiese verhouding tussen C. corindum en C. pechuelii het daartoe gelei dat ek die herkoms van C. pechuelii in Afrika ondersoek het. ‘n Multi-geen filogenie, chloroplas haplotipe netwerk en populasie genetiese diversiteit analises het aangedui dat C. pechuelii endemies is tot die Namib Woestyn van Namibië, en die produk is van lang afstand verspreiding van C. corindum vanaf Suid-Amerika. Hierdie studie gee eerstens insig tot die ekologie en biologie van Cardiospermum en waarsku teen verdere verspreiding. Tweedens dui my studie ook die opgeloste inheemse streke in Suider-Afrika aan van sekere Cardiospermum spesies, en laastens, illustreer ek die herkoms van C. pehuelii. My navorsing het baie belangrike gevolge vir die huidige biologiese beheer program teen C. grandiflorum in Suid-Afrika en waarsku teen die vrystelling van reeds geïndentifiseerde biologiese beheer agente weens potensiële nie-teiken impakte op inheemse spesies.

Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Mycoplasma nasistruthionis sp. nov. str. Ms03 (Ms03) is one of three Mycoplasma species that were identified from ostriches. Mycoplasmas infections have been implicated in ostrich chick mortalities, growth retardation and downgrading of ostrich carcasses. Currently there is no vaccine available for the treatment of mycoplasmosis in ostriches. This study investigated the development of DNA vaccines against Ms03 infections in ostriches. To this end, the Ms03 genome was sequenced and annotated. The vaccine candidate gene, oppA, was identified within the genome sequence and characterized before DNA vaccines containing the oppA were developed and tested. The genome of Ms03 was sequenced and the resulting 172 contigs were annotated. This dissertation presents the first Ms03 draft genome and annotation which contributed to the understanding of Ms03 as a miniature genetically independent organism. In Ms03, genome replication, cell division, RNA transcription, protein translation and glycolysis resemble that of the closely related Mycoplasma synoviae 53. Purine and pyrimidine metabolism was incomplete and de novo synthesis thereof was not possible. Amino acid synthesis in Ms03 was mostly absent and only the genes that convert aspartate to asparagine and glycine to serine were found. More importers than exporters were annotated owing to the lack of synthesis pathways in Ms03, which is typical for mycoplasmas that have parasitic life styles. Two oligopeptide permease (opp) operons were annotated within the Ms03 genome. The potential of the oppA as a vaccine candidate gene was evaluated by investigating the need for a substrate-binding domain (OppA) as part of the OppBCDF transporter within Mycoplasma species. An oppA homologue could be identified for each oppBCDF operon in all species and therefore must play an essential role in oligopeptide transport. All mycoplasmas (except for hemoplasma) had one, two or three opp operons that could be divided into three types (Type A, B and C). Each type had unique InterPro and MEME domains and motifs which together with the phylogenetic analysis suggest unique roles in their survival under different conditions. Ms03 had a Type A and a Type B opp operon, the Type A oppA was used as vaccine candidate gene. The Type A oppA was cloned and site-directed mutagenesis was used for codon correction before the mutated gene was sub-cloned into three DNA vaccine vectors. The three DNA vaccines (pCI-neo_oppA, VR1012_oppA and VR1020_oppA) were used to vaccinate ostriches and the OppA-antibody response was analysed by ELISA. The VR1020_oppA and pCI-neo_oppA constructs elicited a primary immune response in ostriches, indicating that the OppA protein was expressed in vivo and was immunogenic. This can therefore be viewed as the first step in the development of a DNA vaccine for the control of mycoplasma infections in ostriches. / AFRIKAANSE OPSOMMING: Mycoplasma nasistruthionis sp. nov. str. Ms03 (Ms03) is een van drie mikoplasma spesies wat volstruise infekteer. Mikoplasma-infeksies in volstruise veroorsaak kuiken vrektes, vertraagde groei en afgradering van volstruis karkasse. Daar is tans geen geregistreerde mikoplasma entstof beskikbaar vir gebruik in volstruise nie. Hierdie studie het die ontwikkeling van DNS-entstowwe teen Ms03-infeksies in volstruise ondersoek. Vir hierdie doel was die Ms03-genoomvolgorde bepaal en geannoteer. Die entstofkandidaat-geen, oppA, was geïdentifiseer in die genoomvolgorde en gekarakteriseer voordat DNS-entstowwe (wat die oppA-geen bevat) ontwikkel en getoets is. Die Ms03-genoomvolgorde was bepaal en die gegenereerde 172 aaneenlopende volgordes was geannoteer. Hierdie proefskrif bied die eerste voorlopige volgorde en annotering van die Ms03-genoom wat bygedra het tot die kennis van Ms03 as 'n miniatuur geneties onafhanklike organisme. Genoom-replikasie, seldeling, RNS-transkripsie, proteïen-translasie en glikolise in Ms03 stem ooreen met dié prosesse in die naverwante Mycoplasma synoviae 53. Die purien en pirimidien metabolisme was onvolledig en de novo sintese daarvan was nie moontlik in Ms03 nie. Aminosuursintese in Ms03 was meestal afwesig en net die gene wat aspartaat omskep na asparagien en glisien na serien was gevind in die annoteerde genoom. Meer invoerders as uitvoerders was geannoteer, wat dui op die gebrek aan sintesepadweë in Ms03. Dit is tipies van mikoplasmas wat ‘n parasitiese lewensstyle het. Twee oligopeptied-permeases (opp) operons was gevind in die Ms03-genoom. Die potensiaal van die oppA-geen as 'n entstofkandidaat-geen was geëvalueer deur die behoefte van 'n substraatbindingsdomein (OppA) as deel van die OppBCDF-vervoerder binne mikoplasma spesies te ondersoek. 'n Homoloog van die oppA-geen kon geïdentifiseer word vir elke oppBCDF-operon in al die spesies en behoort daarom 'n noodsaaklike rol te speel in die vervoer van oligopeptiede. Alle mikoplasmas (behalwe vir hemoplasmas) het een, twee of drie opp-operons, wat verdeel kan word in drie tipes (Tipe A, B en C). Elke tipe het unieke InterPro en MEME domeine en motiewe wat saam met die filogenetiese ontleding daarop dui dat hulle unieke rolle in oorlewing onder verskillende omstandighede speel. Ms03 het 'n Tipe A en Tipe B opp-operon, die Tipe A oppA is gebruik as entstofkandidaat-geen. Die Tipe A oppA-geen was gekloneer en teikengerigte-mutagenese was gebruik vir kodonregstellings voordat die gemuteerde geen in drie DNS-entstof vektore gesubkloneer was. Die drie DNS-entstowwe (pCI-neo_oppA, VR1012_oppA en VR1020_oppA) was gebruik om volstruise in te ent en die OppA-teenliggaamsreaksie was geanaliseer deur ELISA. Inenting met die VR1020_oppA en pCI-neo_oppA entstowwe het tot 'n primêre immuniteitsreaksie in volstruise gelei. Dit dui daarop dat die OppA proteïen in vivo uitgedruk en immunogenies was. Dit kan beskou word as die eerste stap in die ontwikkeling van 'n DNS-entstof vir die beheer van mikoplasma-infeksies in volstruise.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: I investigated the impacts of the invasive wattle species (Acacia mearnsii, A. dealbata, A. decurrens), on the ecological function and productivity of rangelands in South Africa and their ability to sustain livestock production. More specifically, this study set out to: (1) assess grazing areas at a national scale; (2) identify evidence of progressive impacts of these species on livestock production across a selection of magisterial districts; (3) determine the effects of A. mearnsii density on growth form dominance of indigenous plant species, and highlight how this translates into impacts in forage quality and quantity; (4) determine the effects of A. mearnsii invasion on soil resources and conditions (key determinates of ecological function) required to support grazing production; and finally (5) determine to effects that clearing operations have had on the provision of grazing resources. My results indicate that the level of overlap between wattle invasion and areas with a high grazing potential, at the national scale, is relatively low (2.9%). However, at a magisterial district scale, areas with a high grazing potential have a moderate (approximately 35.1% high grazing potential area under wattle invasion for highest invaded district) level of invasion. Scattered and moderate levels of invasion currently dominate or characterise these species invasion patterns. Both light and dense A. mearnsii invasions reduce grazing capacity. Dense invasion had the most severe effect reducing grazing capacity from 2 ha, required to support one large stock unit, to 8 ha. The clearing of this species was found to improve grazing capacity within 5 years. The reduction in grazing capacity following invasion was largely due to reduced basal cover and herbaceous biomass. The clearing of invaded sites was found to increase both basal cover and herbaceous biomass to pre-invasion levels. Acacia mearnsii invasion was found to affect certain soil properties. Under an A. mearnsii canopy, plant litter, carbon content of the soil and nitrogen concentrations were all found to be elevated. Overall, this study demonstrates that Acacia invasions have a significant effect on grazing resources at a local or site scale, and that for the eastern region of South Africa, these effects are ultimately scaled up to a district level. This is likely to have important financial and human well-being repercussions. However clearing and restoration programs are clearly able to reverse or mitigate these effects. This study therefore lends support to or complements other ecosystem service provision arguments for clearing alien invasive species. / AFRIKAANSE OPSOMMING: Die impak van die indringer- wattelbome spesies ( A. mearnsii , A. dealbata , A. decurrens) , op die ekologiese funksie en produktiwiteit van weivelde in Suid-Afrika en hul vermoë om vee produksie te onderhou was ondersoek. Meer spesifiek, is hierdie studie uiteengesit om: (1) ondersoek in te stel na die mate van oorvleueling tussen indringing deur wattelbome spesies en belangrike weidingsgebiede op 'n nasionale skaal; (2) bewyse te verskaf van die progressiewe impak van hierdie spesies op vee-produksie oor 'n seleksie van politieke distrikte, (3) die gevolge van A. mearnsii digtheid op groei vorm en oorheersing van inheemse plante te wys, en hoe dit voer kwaliteit en kwantiteit affekteer; (4) die gevolge van A. mearnsii indringing op grond hulpbronne sowel as die kondisie (sleutel bepalende faktore van ekologiese funksie) wat benodig word om weiding produksie te ondersteun vas te stel, en uiteindelik (5) om die gevolge van skoonmaak bedrywighede op die voorsiening van weiding hulpbronne te bepaaal. My resultate dui daarop dat die vlak van oorvleueling tussen die wattelbome indringing en gebiede met 'n hoë weiding potensiaal, by die nasionale skaal, relatief laag is (2’9 %). Maar op 'n landdrosdistrik skaal, het gebiede met 'n hoë weiding potensiaal 'n matige (ongeveer 35.1 % weiding potensiaal area onder wattelbome indringing vir die hoogste binnegevalde distrik ) vlak van indringing. Verspreide en matige vlakke van indringing oorheers tans of kenmerk hierdie spesies se indringings patrone. Beide lig en digte A. mearnsii besettings verminder dravermoë. Digte inval het die mees ernstige uitwerking en verminder dravermoë van 2 ha, wat nodig is om een grootvee-eenheid te ondersteun na 8 ha. Bevindinge wys dat die verwydering van hierdie spesies weiding kapasiteit kan verbeter binne 'n tydperk van 5 jaar. Die afname in dravermoë na indringing is grootliks te wyte aan die afname in basale bedekking en kruidagtige biomassa. Na die skoonmaak van indringer persele is bevind dat beide basale bedekking en biomassa weer verhoog tot voor- indringings vlakke. Acacia mearnsii indringing beïnvloed sekere grondeienskappe. Onder A. mearnsii blaredak, is alle blaarafval, koolstof-inhoud van die grond en stikstof konsentrasies verhef. In die algeheel, toon hierdie studie dat die Acacia indringings 'n beduidende uitwerking op weidings hulpbronne by 'n plaaslike of perseel skaal het, en vir die oostelike streek van Suid-Afrika, is hierdie effekte ook uiteindelik vertaalbaar tot 'n distrik vlak. Dit sal waarskynlik belangrike finansiële en menslike welsyn gevolge hê, maar die skoonmaak en herstel programme is duidelik daartoe instaat om die gevolge te keer of te verminder. Hierdie studie leen dus ondersteuning aan of komplimenteer ander ekostelsel dienslewering argumente (soos dié van die Werk vir Water program) vir die verwydering van indringerspesies.

Thesis (PhD (Botany and Zoology))--Stellenbosch University, 2008. / ENGLISH ABSTRACT: The worldwide movement of biota is of substantial concern for the conservation of biodiversity. The movement of species takes place at three different scales. These are translocations of indigenous species within their natural distribution ranges, the translocation of species outside their natural distribution ranges within geopolitical boundaries (i.e. extralimital introductions) and the translocation of species outside geopolitical boundaries and their natural distribution ranges (i.e. extraregional introductions). Ungulates are extensively translocated at each of these scales and each scale of translocation is expected to have different impacts on biodiversity. Ungulates are translocated for conservation purposes such as reintroducing species to places where they have previously gone extinct and to mitigate inbreeding in small, isolated populations. Ungulates are also extensively translocated for economic and recreational reasons, such as for sport hunting. Translocations for sport hunting include indigenous, extralimital and extraregional species. Concerns for translocations of indigenous species are largely for the loss of genetic diversity through the mixing of genetically distinct populations, and concerns for extralimital and extraregional translocations are for impacts on indigenous biodiversity such as through herbivory, competition, hybridization and disease transmission. This thesis investigates the extent of ungulate introductions globally and at a finer resolution in South Africa. It investigates the pathways, drivers and impacts of ungulate introductions and it also investigates the use of surrogates for genetic distinctiveness for advising the translocation of indigenous ungulates. The study finds that ungulate translocations have been extensive and have lead to the homogenization of ungulate assemblages in countries globally and at a quarter-degree grid-cell resolution in South Africa. Zoos were identified as a potential introduction pathway for extraregional ungulates globally and in South Africa extraregional introductions have made ungulate assemblages more different; whereas large numbers of extralimital introductions have made ungulate assemblages more similar. The homogenization of ungulate species in South Africa has increased with time, due to increased numbers of translocations, particularly of extralimital species. In South Africa translocations have most recently been made to high-income areas with high human population density and high livestock density; whereas in the 1960s ungulates were introduced to areas species poor for indigenous ungulates and marginal for livestock. In South Africa, long distance translocations of indigenous species extralimitally has resulted in extensive range expansions of a magnitude greater than predicted range changes as a result of predicted climate change. When the use of surrogates of genetic distinctiveness for advising translocations was investigated for Africa, the East African rift valley was found to be important in delineating genetic distinctiveness and translocations across this feature should be prevented. Major rivers in Africa also showed potential for delineating genetic distinctiveness in ungulates, but relevant phylogeographic data are needed to confirm this. Sufficient evidence for the impacts of non-indigenous ungulates on biodiversity both in South Africa and globally is lacking despite substantial concern for their impacts. It is suggested that to demonstrate the impacts of non-indigenous ungulates exclosure and enclosure experiments should be used and population declines in indigenous species should be shown. / AFRIKAANSE OPSOMMING: Verskuiwing van plante en diere wêreldwyd lei tot groot kommer in gevolge die bewaring van biodiversiteit. Verskuiwings vind op drie skale plaas, naamlik, die verskuiwing van inheemse spesies binne hulle natuurlike verspreidingsgebied, die verskuiwing van spesies buite hul natuurlike verspreidingsgebied maar binne landsgrense (d.w.s. buite limiet verskuiwings), en die verskuiwing van spesies buite hul natuurlike verspreidingsgebied en buite landsgrense (d.w.s. buite grondgebied verskuiwings). Grootskaalse verskuiwing van hoefdiere vind op al drie skale plaas. Verskuiwing op elk van die skale sal na verwagting biodiversiteit verskillend beïnvloed. Hoefdiere word ook vir bewaringsdoeleindes verskuif, bv. die herbevolking van ‘n gebied waar die spesies uitgesterf het, asook om genetiese probleme wat gepaardgaan met klein bevolkingsgroottes te vermy. Hoefdiere word ook verskuif vir ekonomiese- en ontspannings redes, o.a. vir sportjag, en sluit verskuiwings op al drie skale in. Kommer oor verskuiwings binne ‘n spesies se verspreidingsgebied rus hoofsaaklik op die verlies van genetiese diversiteit a.g.v. die vermenging van vorig genetiese eiensoortige bevolkings, terwyl op die ander twee skale kommer hoofsaaklik gebaseer is op die impakte van kompetisie, interteling, predasie, herbivoor effek, en die verspreiding van parasiete op inheemse biodiversiteit. Hierdie proefskrif ondersoek die omvang van hoefdier verskuiwings globaal asook in Suid Afrika. Die verskuiwingsweë, oorsake en impakte van verskuiwings word hier ondersoek, asook die maatstawwe van genetiese eiensoortigheid om advies oor verskuiwings te verskaf. Die bevindings toon op ekstensiewe verskuiwing van hoefdiere wat eenvormigheid van hoefdier spesiesamestellings wêreldwyd bevorder het, sowel as op ‘n kwartgraad skaal in Suid Afrika. Dieretuine is geïdentifiseer as ‘n potensiële bron van buite grondgebied verskuiwings. In Suid Afrika het buite grondgebied verskuiwings hoefdier spesiesamestellings minder eenvormig gemaak, terwyl buite limiet verskuiwings hoefdier samestellings binne Suid Afrika meer eenvormig gemaak het. Eenvormigheid in hoefdier spesiesamestellings het met tyd in Suid Afrika toegeneem as gevolg van ‘n toename in veral buite limiet verskuiwings. In Suid Afrika is verskuiwings mees onlangs na hoë-inkomste gebiede met hoë menslike bevolkingsen veëdigthede gemaak. In die sestiger jare is hoefdiere egter verskuif na gebiede waar min inheemse hoefdiere voorgekom het en wat marginaal was vir veëboerdery. In Suid Afrika het langafstand buite limiet verskuiwings versoorsaak dat die verspreidingsgebiede van sekere hoefdier spesies groter is as wat die geval sal wees met voorspelde klimaatsveranderinge. Waneer surogaat inligting gebruik word om genetiese verskille tussen bevolkings te identifiseer om verskuiwings te adviseer, word die Oos Afrika Rift vallei geïdentifiseer as ‘n belangrike breuk tussen bevolkings. Verskuiwings oor die vallei moet verkieslik nie plaasvind nie. Groot riviere in Afrika speel ‘n soortgelyke rol, maar verdere genetiese inligting is nodig om dit te bevestig. Alhoewel kommer oor die impak van verskuiwings groot en teoreties verdedigbaar is, bestaan daar te min konkrete bewyse vir die impakte in Suid Afrika. Daar word voorgestel dat manipuleringseksperimente uitgevoer moet word om impakte te demonstreer, en dat aandag veral gegee moet word aan die demonstrasie van impakte op inheemse spesies.

The structure of direct-current microdischarges is investigated using a detailed two-dimensional multi-species continuum model. Microdischarges are directcurrent discharges that operate at a relatively high pressure of about 100 Torr and geometric dimensions in the 10-100 micrometer range. Our motivation for the study of microdischarges comes from a potential application of these devices in microthrusters for small satellite propulsion. The Micro Plasma Thruster (MPT) concept consists of a direct-current microdischarge in a geometry comprising a constant area flow section followed by a diverging exit nozzle. A detailed description of the plasma dynamics inside the MPT including power deposition, ionization, coupling of the plasma phenomena with high-speed flow, and propulsion system performance is reported in this study. A two-dimensional model is developed as part of this study. The model consists of a plasma module coupled to a flow module and is solved on a hybrid unstructured mesh framework. The plasma module provides a self-consistent, multispecies, multi-temperature description of the microdischarge phenomena while the flow module provides a description of the low Reynolds number compressible flow through the system. The plasma module solves conservation equations for plasma species continuity and electron energy, and Poisson’s equation for the self-consistent electric field. The flow module solves mass, bulk gas momentum and energy equations. The coupling of energy from the electrostatic field to the plasma species is modeled by the Joule heating term which appears in the electron and heavy species energy equations. Discretization of the Joule heating term on unstructured meshes requires special attention. We propose a new robust method for the numerical discretization of the Joule heating term on such meshes using a cell-centered, finite volume approach. A prototypical microhollow cathode discharge (MHCD) is studied to guide and validate the modeling effort for theMPT. Computational results for the impedance characteristics as well as electrodynamic and chemical features of the discharge are reported and compared to experimental results. At low current (< 0.1 mA), the plasma activity is localized inside the cylindrical hollow region of the discharge operating in the so-called “abnormal regime”. For larger currents, the discharge expands over the outer flat surface of the cathode and operates in the “normal regime”. Transient relaxation oscillations are predicted in the plasma properties for intermediate discharge currents ranging from 0.1 mA to 0.3 mA; a phenomenon that is reported in experiments. The MPT, in its present configuration, is found to operate as an electrothermal, rather than as an electrostatic thruster. A significant increase in specific impulse, compared to the cold gas micronozzle, is obtained from the power deposition into the expanding gas. For a discharge voltage of 750 V, a power input of 650 mW, and an argon mass flow rate of 5 sccm, the specific impulse of the device is increased by a factor of 1.5 to a value of 74 s. The microdischarge remains mostly confined inside the micronozzle and operates in an abnormal regime. Gas heating, primarily due to ion Joule heating, is found to have a strong influence on the overall discharge behavior. The study provides crucial understanding to aid in the design of direct-current microdischarge based thrusters. / text

Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and superoxide (O2 • −) have been shown to serve as messengers in biological signal transduction, and many prokaryotic and eukaryotic proteins are now known to have their function controlled via ROS-mediated oxidation reactions occurring on critical cysteine residues. The tumour-suppressor protein p53 is involved in the regulation of a diverse range of cellular processes including apoptosis, differentiation, senescence, DNArepair, cell-cycle arrest, autophagy, glycolysis and oxidative stress. However, little is understood about the specific molecular mechanisms that allow p53 to discriminate between these various different functions. p53 is a multiple cysteine-containing protein and there is mounting evidence to suggest that redox-modification of p53 Cys residues participate in control of its biological activity. Furthermore, p53 activity has been linked to intracellular ROS levels. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) offers superior mass resolving power and mass measurement accuracy, which is beneficial for the study of intact proteins and the characterisation of their posttranslational modifications (PTMs). The primary goal of the work described in this thesis was to employ FT-ICR mass spectrometry to investigate the molecular details of p53 redox-regulation. The relative reactivity of each of the ten cysteine residues in the DNA-binding core domain of recombinant human p53 was characterised by treatment with the Cys-alkylating reagent N-ethylmaleimide (NEM) under various conditions. A combination of top-down and middle-down FT-ICR MS was used to unambiguously identify Cys182 and Cys277 as sites of preferential alkylation. These results were confirmed by site-directed mutagenesis. Interestingly, Cys182 and Cys277 have previously been implicated in p53 redox-regulation. Alkylation beyond these two residues was found to trigger rapid alkylation of the remaining Cys residues, presumably accompanied by protein unfolding. These observations have implications for the re-activation of mutant p53 with Cys-targeting compounds which result in the death of cancer-cells. Furthermore, the molecular interaction between p53 and the ROS hydrogen peroxide was investigated. p53 was found to form two disulfide bonds upon treatment with H2O2. An enrichment strategy was developed to purify oxidised p53 and top-down FT-ICR mass spectrometry revealed unambiguously that Cys176, 182, 238 and 242 were the oxidised residues. Interestingly, Cys176, 238 and 242 are Zn2+- binding residues suggesting that p53 contains a zinc-redox switch. The mechanism of H2O2 oxidation was investigated, and revealed that oxidation via an alternative pathway results in indiscriminate over-oxidation of p53. Moreover, Cys176, 238 or 242 was shown to act as a nucleophile, and the intracellular antioxidant glutathione (GSH) did not prevent oxidation of the Zn2+-binding Cys residues, providing further evidence for a role in p53 redox-regulation. This study has revealed hitherto unknown details regarding the chemistry of cysteine residues within the important tumour-suppressor protein p53. Furthermore, the analytical power of FT-ICR MS for the study of multiple Cys-containing proteins has been very clearly demonstrated.