Call number: LD2668 .T4 1985 L43 / Master of Science

Call number: LD2668 .T4 1985 W36 / Master of Science

Thesis (DPhil)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: In this dissertation, I grapple with the problem of global drug resistance and moral responsibility which, as far as I am aware, has so far not been presented as a topic of ethical inquiry. It represents a conundrum involving three major factors: microbial adaptation and change, human social factors and environmental changes. Drug resistance is a phenomenon in which certain microorganisms, when exposed to antimicrobial agents, may acquire the beneficial trait of drug resistance which ensures a better potential for their survival. The acquired trait of drug resistance I argue renders such microorganisms 'supra-natural '. Supra-natural is a term I coin for entities that have been imposed upon nature by human design; they do not follow the natural evolutionary processes of adaptation and change. Drug resistance is classified as an emerging infectious disease. Human social factors and environmental change (particularly population growth, density and consumerist practices) enhance the rise of emerging infectious diseases. Through such increasing destructive practices, stress is placed on the environment. Environmental stress facilitates the rise of new and old infectious diseases and the spread of drug resistant supra-natural microorganisms. Thus, our ability to treat successfully illnesses and injuries in humans, animals and plants is increasingly impaired. Morally, we are responsible for the problem of global drug resistance. Drug resistant microorganisms exist in nature and concerning this, we can do nothing. At best, we can only try to control the problem using prudential measures. The problem of global drug resistance represents both a biomedical ethical and an environmental ethical issue. Is there a way out of the human-nature debate? Through Bryan Norton's enlightened anthropocentrism, I identify the ways in which his thesis may be applied to the problem of human and environmental concerns and show its applicability in broadening the parameters of biomedical ethics education to include environmental concerns. / AFRIKAANSE OPSOMMING: In hierdie proefskrif bespreek ek die probleem van die verskynsel dat mikroorganismes op 'n globale skaal weerstand begin bied teen mediese middels (globale middel-weerstandigheid) en die morele verantwoordelikheid wat dit oproep - 'n probleem wat, na my beste wete, nog nooit aangebied is as 'n tema van etiesfilosofiese ondersoek nie. Dit verteenwoordig 'n kompleks van drie belangrike oorwegings: mikrobiese aanpassings en veranderinge, menslike sosiale faktore, en omgewingsveranderinge. Middel-weerstandigheid is 'n verskynsel waarin sekere mikro-organismes, wanneer hulle blootgestel word aan antimikrobiese agente, die (vir hulself) voordelige kenmerk kan bekom van weerstandigheid teen die middel; iets wat 'n beter potensiaal vir hul eie oorlewing verseker. Hierdie bekomde kenmerk (middel-weerstandigheid) maak, volgens my argument, sulke mikro-organismes 'supra-natuurlik'. Supra-natuurlik is 'n term wat ek munt vir entiteite wat aan die natuur blootgestel is as gevolg van menslike ontwerp; hulle volg nie die natuurlike evolusionêre prosesse van adaptasie en verandering nie. Middel-weerstandigheid word geklassifiseer as 'n opkomende aansteeklike siekte. Menslike sosiale faktore en omgewingsveranderinge (veral bevolkingsgroei, -digtheid and verbruikerspraktyke ) vergroot die opkoms van aansteeklike siektes. Deur sodanige toenemende destruktiewe praktyke word stres geplaas op die omgewing. Omgewingstres fasiliteer die opkoms van nuwe en ou aansteeklike siektes asook die verspreiding van weerstandige supra-natuurlike mikro-organismes. Ons vermoë om siektes en beserings van mense suksesvol te behandel, word gevolglik toenemend ondermyn. Moreel gesproke is ons verantwoordelik vir die probleem van globale middelweerstandigheid. Middel-weerstandige mikro-organismes bestaan in die natuur, en aan daardie feit as sodanig kan ons niks doen nie. Ons kan, ten beste, probeer om die probleem te beheer deur middel van verstandige maatreëls. Die probleem van globale middel-weerstandigheid verteenwoordig sowel 'n biomedies-etiese as 'n omgewingsetiese kwessie. Is daar 'n uitweg uit die mens-natuur debat? Ek identifiseer, met 'n beroep op Bryan Norton se swak antroposentrisme, maniere waarop sy tese toegepas sou kon word op die probleem van menslike en omgewingsoorgwegings Ek wys ook op die toepaslikheid daarvan vir die verbreding van die parameters van biomediese etiek-opvoeding ten einde omgewingsoorwegings deel van 19.te maak.

Thesis (MSc)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: Plant biomass is potentially an inexhaustible source of bioenergy. To be more useful in an industrialised context, conversion to liquid biofuel is necessary, which could provide the motor vehicle market with energy. To enable fermentation of both hexose and pentose sugars present in plant biomass, many researchers have introduced eukaryotic D-xylose utilisation metabolic pathways into S. cerevisiae as these yeasts cannot utilise D-xylose. The aim of this study was to increase D-xylose utilisation and lower the xylitol production found with the eukaryotic pathway, thus redirecting carbon to the increased production of ethanol. In order to reduce xylitol yield a two-fold approach was followed. Firstly S. cerevisiae transformed with eukaryotic XR and XDH genes were subjected to random mutagenesis and selection for improved D-xylose utilisation. Unfortunately no mutant superior to the parental strain with respect to D-xylose utilisation, lowered xylitol production and improved ethanol production was obtained. Subsequently a bacterial xylose isomerase (XI) gene was introduced into S. cerevisiae. Bacterial xylose isomerase converts D-xylose to xylulose in a single step, while eukaryotic pathways produce the intermediate xylitol. The chosen gene encodes for a putative xylose isomerase gene (xylA) from the bacterium Bacteroides thetaiotaomicron, which has not previously been transformed into yeast. When the native xylA was expressed in E. coli and S. cerevisiae no XI activity was found, nor growth on D-xylose sustained. Lack of activity was surmised to be due to an amino acid modification, or possibly due to a vastly different codon bias in yeast compared to the Bacteroides strain. Northern analysis revealed that no D-xylose transcript was formed. A synthetic D-xylose isomerase gene (SXI) based on the B. thetaiotaomicron XI amino acid sequence, but optimised for S. cerevisiae codon bias, was designed and manufactured. S. cerevisiae transformed with the synthetic gene showed sustained, non-pseudohyphal growth on D-xylose as sole carbon source, both on solid and liquid medium. This ability to utilise D-xylose represents a significant step for recombinant S. cerevisiae to potentially ferment D-xylose for bioethanol. / AFRIKAANSE OPSOMMING: Plant biomassa is potensieel ‘n onuitputlike bron van bio-energie. Om in die huidige industriële konteks van groter nut te wees, en die motor-industrie met energie te voorsien, is omskakeling na ‘n vloeistof-energievorm nodig. Om die fermentasie van beide heksoses en pentoses teenwoordig in plantbiomassa te bewerkstellig, het verskillende navorsingspanne eukariotiese D-xilose-afbraak metabolise weë na S. cerevisiae oorgedra om dié gis die vermoë te gee om D-xilose af te breek. Die doel van hierdie studie was om D-xilose-verbruik in geneties gemodifiseerde S. cerevisiae te verhoog en die hoeveelheid xilitol wat met die eukariotiese sisteem verkry word, te verminder om ‘n hoë etanol opbrengs te handhaaf. Twee moontlikhede is ondersoek om die xilitol opbrengs te verminder. Eerstens is ‘n rekombinante S. cerevisiae met die xilose reduktase (XR) en xilitol dehidrogenase (XDH) gene aan nie-spesifieke mutagenese onderwerp en vir verbeterde D-xilose verbruik geselekteer. Ongelukkig kon geen mutante wat beter as die oorspronklike ras D-xilose kon gebruik, en etanol produseer met relatief min xilitol opbrengs, gevind word nie. Daarna is ‘n bakteriese D-xilose-afbraak geen na S. cerevisiae oorgedra. Bakteriese xilose isomerases skakel D-xilose om na xilulose in ‘n enkele stap, terwyl die eukariotiese paaie die tussenganger xilitol produseer. Die gekose xylA geen wat vir xilose isomerase (XI) van die bakterium Bacteriodes thetaotaomicron kodeer, is vir die eerste keer in gis getransformeer. Toe die natuurlike xylA geen In E. coli en S. cerevisiae uitgedruk is, is geen XI-aktiwiteit of volhoubare groei op D-xilose waargeneem nie. Die tekort aan aktiwiteit is aan 'n aminosuurverandering, of aan die groot verskil tussen kodonkeuse (“codon bias”) in gis teenoor die Bacteroides ras toegeskryf. Noordkladanaliese het bepaal dat geen mRNA spesifiek tot die XI-geen geproduseer is nie. Die xilose isomerase geen van B. thetaiomicron is toe sinteties ontwerp, met die DNA-volgorde vir die S. cerevisiae kodonkeuse geoptimiseer. S. cerevisiae wat met die sintetiese geen (SXI) getransformeer is, het aanhoudende, nie-pseudohife groei op D-xilose as enigste koolstofbron op beide soliede en in vloeibare medium getoon. Die vermoë om D-xilose te verbruik verteenwoordig ‘n betekenisvolle stap tot die fermentasie van D-xilose na etanol met geneties gemodifiseerde S. cerevisiae.

published_or_final_version / Physiology / Master / Master of Philosophy

published_or_final_version / abstract / Microbiology / Master / Master of Philosophy

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

The heat shock response is a highly conserved genetic mechanism which is induced by a wide range of environmental stimuli. Although intensively studied in both prokaryotes and eukaryotes, no regulatory mechanism has been identified by which the environmental stimuli affect expression of the heat shock genes. In addition, although many inducers of the heat shock response are known to cause DNA damage, the role of heat shock in repair of DNA damage remains unclear. Mutants of Escherichia coli defective in the recA, uvrA, and xthA genes are more sensitive to heat than wild type. However, these mutants are able to develop thermotolerance, suggesting that thermotolerance is an inducible response capable of repairing heat-induced DNA damage independent of recA, uvrA, and xthA. Thermotolerance itself is shown to depend on the dnaK gene, directly linking the E. coli heat shock response to thermotolerance. In addition, the dnaK mutant is sensitive to heat and H₂O₂, but not to UV suggesting that the DnaK protein may function to protect cells from the specific DNA damage caused by heat and H₂O₂. An E. coli grpE mutant was found to be substantially more resistant to 50°C heat treatment than wild type. However, grpE⁻ cells have the same H₂O₂ and UV sensitivity as wild type. This implies that the conditions, for which a grpE mutation is beneficial, are unique to heat exposure and are not caused by H₂O₂ or UV exposure. Furthermore, heat shock protein synthesis occurs sooner in the grpE mutant than in wild type, indicating that the grpE gene product of E. coli may act as a negative regulator of the heat shock response. An adenyl cyclase deletion mutant of E. coli (cya) failed to exhibit a heat shock response even after 30 min. at 42°C. Furthermore, a presumptive cyclic AMP receptor protein (CRP) binding site exists within the promoter region of the E. coli htpR gene. Together, these results suggest that the cya gene may regulate the heat shock response, through cyclic AMP, by directly affecting the level of expression of the heat shock sigma factor.

Su veikliojo dumblo suspensijomis, gautomis iš Vilniaus miesto, Nemenčinės ir Utenos miesto nuotekų valyklų buvo vykdomi tyrimai, kuriais siekta ištirti nuotekose esančių teršalų poveikį veikliojo dumblo mikroorganizmams ir nustatyti indikatorines rūšis šio poveikio įvertinimui. Tyrimai atlikti su šiais teršalais: 3,5 – dichlorfenoliu (3,5 – DCP), etanoliu ir valgomąja druska (NaCl). Veikliojo dumblo suspensijos buvo analizuojamos mikrobiologiškai, skaičiuojami mikroorganizmų rūšys ir individų skaičius (ind./ml). Per tyrimą buvo atlikta 16 eksperimentų ir nustatytos mikrobiologinė nuotekų valyklų sudėtys, iš kurių buvo gautos veikliojo dumblo suspensijos. Buvo įvertinti teršalų poveikiai veikliojo dumblo mikroorganizmų bendrijoms. Prie mažų (2,5 ppm, 5 ppm ir 7,5 ppm) 3,5 – DCP koncentracijų veikliojo dumblo mikroorganizmai adaptavosi, o didelės (15 ppm ir 20 ppm) koncentracijos naikina veikliojo dumblo mikroorganizmus. Etanolis veikliojo dumblo mikroorganizmų bendrijas veikia skatinančiai, kadangi auga veikliojo dumblo koncentracija ir bakterijų monokolonijų skaičius. Mažos (3 g/l, 5 g/l ir 7 g/l) NaCl koncentracijos skatina veikliojo dumblo mikroorganizmų bendrijas, prie 10 g/l dumblo organizmai adaptuojasi, o prie didelės (15 g/l) NaCl koncentracijos mikroorganizmų skaičius mažėjo. Taip pat nustatytos jautriausios mikroorganizmų rūšys (Acineria uncinata, Aspidisca costata, A. lynceus, Vorticella spp., Ptelomonas sp., plikosios amebos ir bakterijos, sudarančios... [toliau žr. visą tekstą] / The investigations of active sludge taken from Vilnius, Utena and Nemenčinė wastewater treatment plants was carries out. The main goal was investigate the effects of wastewater pollutants on communities of microorganisms taken from activated sludge and to identify indicator species of this impact assessment. Investigations were carried out with these pollutants: 3,5 - dichlorophenol (3,5 - DCP), ethanol, and salt (NaCl). Active sludge was analyzed microbiologically, number of species and number of individuals (ind. / ml) were counted. In this study 16 experiments was carried out and microbiological composition of wastewater treatment plants was estimated. Results showed that activated sludge microorganisms adapted to low (2.5 ppm, 5 ppm and 7.5 ppm) concentrations of 3,5 – DCP, but high (15 ppm and 20 ppm) concentrations reduced abundance of microorganisms. Ethanol stimulated community of activated sludge, because concentration of activated sludge and number of bacterial colonies increased. Low concentrations (3 g/l, 5 g/l ir 7 g/l) of NaCl also stimulated abundance of active sludge community, microorganisms adapted to the concentration of 10 g/l, and number of microorganisms decreased when concentration of NaCl was high (15 g/ l). Acineria uncinata, Aspidisca costata, A. lynceus, Vorticella spp., Ptelomonas sp., gymnamoebae and bacteria (which makes clusters) – this was the most sensitive species of microorganism in activated sludge. A. costata resisted quite high (even 10... [to full text]

Bovine mastitis is recognised worldwide as the most important and costly disease affecting dairy cattle. The reduction of herd mastitis rates is crucially needed to improve animal welfare and profitability, and lessen the reliance on antibiotics. Single nucleotide polymorphisms (SNPs) within genes that have a critical role in the innate immune response, such as Toll-like receptors (TLRs) and the chemokine receptors CXCR1 and CXCR2, could impact on establishment and progression of intramammary infection, and therefore influence an animal’s susceptibility to disease. The genetic selection of animals with favourable TLR and CXCR1/2 mutations, with no impact on production traits, could be incorporated into dairy breeding programmes. In order to investigate any associations with clinical mastitis (CM) incidence and milk quality and quantity, this study identified and analysed SNPs alongside actual CM and production data from a Holstein-Friesian herd. This revealed 46 SNPs, 9 of which are novel, within boTLR1/4/5, boCD14, boCXCR1 and boCXCR2; selected SNPs were then tested for association with CM. This is the first report of boTLR1 SNPs and a non-coding boCXCR1 SNP that associate significantly with susceptibility to CM. Favourable linkage of reduced CM with increased milk fat and protein was observed, indicating selection for these markers would not be detrimental to milk quality. Furthermore, this study provides evidence that some of these SNPs underpin functional variation in bovine TLR1 and CXCR1, and possibly underlie an immunological mechanism for disease susceptibility. SNPs in boTLR1 and boCXCR1 were significantly associated with impaired transcript levels in milk somatic cells. In addition boTLR1 SNPs associated with impaired cytokine responses from cell populations when exposed to ligand or heat-killed mastitis-causing bacteria. The potential impact of boTLR1 variation on the immune response to Staphylococcus aureus is demonstrated, and this has implications for boTLR1-mediated immune responses to other pathogens.