Thesis (PhD(Agric) Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2009. / The main goal of this project was to elucidate the underlying genetic factors responsible for the different fermentation phenotypes and physiological adaptations of industrial wine yeast strains. To address this problem an ‘omic’ approach was pursued: Five industrial wine yeast strains, namely VIN13, EC1118, BM45, 285 and DV10, were subjected to transcriptional, proteomic and exometabolomic profiling during alcoholic fermentation in simulated wine-making conditions. The aim was to evaluate and integrate the various layers of data in order to obtain a clearer picture of the genetic regulation and metabolism of wine yeast strains under anaerobic fermentative conditions. The five strains were also characterized in terms of their adhesion/flocculation phenotypes, tolerance to various stresses and survival under conditions of nutrient starvation. Transcriptional profiles for the entire yeast genome were obtained for three crucial stages during fermentation, namely the exponential growth phase (day 2), early stationary phase (day 5) and late stationary phase (day 14). Analysis of changes in gene expression profiles during the course of fermentation provided valuable insights into the genetic changes that occur as the yeast adapt to changing conditions during fermentation. Comparison of differentially expressed transcripts between strains also enabled the identification of genetic factors responsible for differences in the metabolism of these strains, and paved the way for genetic engineering of strains with directed modifications in key areas. In particular, the integration of exo-metabolite profiles and gene expression data for the strains enabled the construction of statistical models with a strong predictive capability which was validated experimentally. Proteomic analysis enabled correlations to be made between relative transcript abundance and protein levels for approximately 450 gene and protein pairs per analysis. The alignment of transcriptome and proteome data was very accurate for interstrain comparisons. For intrastrain comparisons, there was almost no correlation between trends in protein and transcript levels, except in certain functional categories such as metabolism. The data also provide interesting insights into molecular evolutionary mechanisms that underlie the phenotypic diversity of wine yeast strains. Overall, the systems biology approach to the study of yeast metabolism during alcoholic fermentation opened up new avenues for hypothesis-driven research and targeted engineering strategies for the genetic enhancement/ modification of wine yeast for commercial applications.

Thesis (PhD (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Psychosocial stressors have frequently been associated with an increased risk for developing The contributions of the cholinergic (Lobeline) and opioid (Naltrexone) systems in place preference behaviour were determined by employing a post-methamphetamine pharmacological treatment strategy. These two treatments failed to reverse the methamphetamine-induced place preference. However, administration of the drugs did lead to alterations in striatal dopamine and serotonin levels which may infer beneficial effects against the biochemical alterations induced by methamphetamine. We used both 2-D gel-based proteomics and isobaric tagging for relative and absolute quantitation (iTRAQ) to identify proteins in the frontal cortex, and nucleus accumbens shell and core of rats that were subjected to maternal separation, methamphetamine or both regimes. The proteins were associated with cytoskeletal modifications, altered energy metabolism, degenerative processes, interruptions in normal neurotransmission and enhanced intracellular signalling. We found that more proteins were quantitatively expressed in rats that were exposed to maternal separation followed by methamphetamine treatment than those animals subjected to the individual interventions independently. Additional proteins recruited by the combination of MS followed by MA which remained unchanged with independent treatments included malate dehydrogenase, V-type proton ATPase subunit E1, beta-synuclein, brevican core protein, eukaryotic translation initiation factor 4H, histidine triad nucleotide binding protein 1 and stress-induced phosphoprotein in the nucleus accumbens shell subregion. Additional proteins recruited in the core subregion with the combination treatment included thymosin beta-4, calretinin, Arpp-21 protein, alpha-synuclein, ubiquitin carboxylterminal hydrolase isozyme L1, cytochrome c, brain acid soluble protein 1, prosaposin and stress-induced phosphoprotein 1. Although, on a behavioural level via the use of CPP we found that MS did not exacerbate the rewarding effects of MA, the proteomic data does infer a role played by early life stress by the recruitment of additional proteins. We therefore propose that the molecular mechanisms by which early adverse events predispose animals to the addictive state may involve a complex assembly of cellular processes within the brain. depression, anxiety or substance abuse in adult life. Animal studies have also suggested that stressful experiences may result in altered behavioural responses to drugs of abuse as evidenced by enhanced cocaine self-administration and psychostimulant-induced hyperlocomotor activity. The main aim of our study was to establish whether adversity early in life would render individuals more vulnerable to later drug usage. We adopted maternal separation as our animal model of early life adversity and treated these animals with methamphetamine during the adolescent stage of their life. A conditioned place preference (CPP) paradigm was subsequently used to determine the rewarding effects of methamphetamine. To obtain an understanding of the underlying molecular mechanisms of methamphetamine-induced behaviour, we measured neurochemical changes on a neuroendocrine, neurotrophic, neurotransmitter and proteome level. Firstly, we established that methamphetamine-induced place preference behaviour lasted for at least 2 weeks after the last methamphetamine administration. Contrary to expectation, this behaviour was not affected by prior exposure to maternal separation. However, rats subjected to maternal separation caused a decrease in apomorphine-induced locomotor behaviour in methamphetamine-treated rats. Maternal separation therefore preferentially affected the behavioural repertoire of the dorsal striatum rather than that of the ventral striatum. A general down regulation of neuroendocrine activity (ACTH and corticosterone levels) was observed in animals subjected to maternal separation or methamphetamine treatment, as well as those subjected to the combination of the two interventions. Increased concentrations of plasma prolactin levels in maternally separated as well as normally reared animals subjected to methamphetamine-CPP were found which suggested a reduction in dopamine inhibition. Maternal separation resulted in increased NGF levels in the ventral hippocampus of methamphetamine treated rats. This suggested that the ventral hippocampus may particularly be vulnerable to the effects of early life stress. The increased neurotrophin concentrations may reflect a compensatory response to stress and drug exposure. / AFRIKAANSE OPSOMMING: Psigososiale stressors word gereeld geassosieer met ‘n verhoogde risiko vir die ontwikkeling van depressie, angs en dwelm misbruik in volwassenheid. Diere studies het ook al bewys dat vroeë lewensstres in die vorm van moederlike skeiding lei tot veranderde gedrag teenoor dwelm misbruik. Hierdie veranderde gedrag veroorsaak deur moederlike skeiding sluit die verhoodge kokaïne toediening en psigostimulant geinduseerde verhoging in lokomotoriese aktiwiteit in. Die hoofdoel van die studie was om vas te stel of vroeë lewensstres mense meer vatbaar laat vir latere dwelm misbruik. ‘n Moederlike skeidings diere model was gebruik om vroeë lewensstres voor te stel and het verder hierdie diere behandel met metamfetamiene gedurende adolesensie. Die gekondisioneerde plek voorkeur model was gebruik om die euforiese / verslawende effekte van metamfetamiene te bepaal. Om die onderliggende molekulêre meganismes van metamfetamien geinduseerde gedrag te verstaan het ons neurochemiese veranderinge op ‘n neuroendokriene, neurotrofiese, neurotransmissie en proteinvlak vasgestel. Eerstens het ons was gestel dat metamfetamien geinduseerde plek voorkeur vir ten minste twee weke na die laaste metafetamien toediening voortduur. In teenoorstelling met verwagting, het moederlike skeiding nie metamfetamien geinduseerde plek voorkeur beinvloed nie, maar eerder apo-morfien geinduseerde lokomotoriese aktiwiteit geaffekteer. Moederlike skeiding stres het by voorkeur die gedrags funksie van die dorsale striatum beinvloed eerder as die ventrale gedragsfunksie. ‘n Algemene afregulering van neuroendokriene aktiwiteit was waargeneem (adrenokortikotrofiene en kortikosteroon vlakke) in diere wat aan moederlike skeiding of metafetamien behandeling sowel as die kombinasie behandeling blootgestel was. Verhoogde plasma prolaktien vlakke was gevind in moederlike skeidings rotte sowel as kontrole diere wat verder blootgestel is aan metamfetamien behandeling wat ‘n inhibisie van die dopamiene sisteem toon. Moederlike skeiding het ook ‘n verhooging in neurotrofiene (NGF) in die ventrale hippokampus van metamfetamien behandelde rotte veroorsaak. Hierdie bevinding stel voor dat die ventrale hippokampus veral vatbaar is vir die effekte van vroeë lewensstres. ‘n Verhoging in neurotrofien konsentrasies mag ‘n kompenserende teenslag van die brein wees teen stres en dwelm blootstelling. Die bydrae van die cholinergiese (Lobeline) en opiaat (Naltrexone) sisteme in plek voorkeur gedrag was bepaal deur farmaseutiese behandeling te volg na metamftemien toediening. Lobeline en naltrexone was egter nie suksesvol om die metamfetamien geinduseerde plek voorkeur te wysig nie. Alhoewel die toediening van die twee behandelings het tot veranderinge in neurotransmissie (dopamiene en serotoniene) gelei wat moontlik tot voordelige effekte teen die biochemiese veranderinge van metamfetamien kan lei. Om veranderinge op proteinvlak in die frontale korteks en nukleus akkumbens middel en buitenste subareas vas te stel het ons gebruik gemaak van twee-dimensie gel elektroforese en isobariese merkers vir relatiewe en absolute kwantifisering (iTRAQ) gevolg deur massa spektrofotometrie. Geindentifiseerde proteine was geassosieer met sitoskeletale modifikasies, veranderde energie metabolisme, afbrekende prosesse, onderbrekings met normale neurotransmissie en intrasellulêre seintransduksie. Meer proteine was beduidend in die diere wat aan beide moederlike skeiding en metamfetamien behandeling blootgestel was. Addisionele proteine wat deur die kombinasie behandeling geaffekteer is in die buitenste subarea van die nukleus akkumbens sluit ‘malate dehydrogenase’, ‘V-type proton ATPase subunit E1’, ‘beta-synuclein’, ‘brevican core protein’, ‘eukaryotic translation initiation factor 4H’, ‘histidine triad nucleotide binding protein 1’ en ‘stress-induced phosphoprotein’ in. Additionele proteine geaffekteer in die middelste subarea van die nukleus akkumbens sluit ‘thymosin beta-4’, ‘calretinin’, ‘Arpp-21 protein’, ‘alpha-synuclein’, ‘ubiquitin carboxylterminal hydrolase isozyme L1’, ‘cytochrome c’, ‘brain acid soluble protein 1’, ‘prosaposin’ en ‘stress-induced phosphoprotein 1’ in. Vanuit ‘n gedrags benadering deur die gebruik van metamfetamien geinduseerde plek voorkeur het moederlike skeiding nie diere meer vatbaar gemaak vir die effekte van metamfetamien nie, maar die protein data wys wel dat vroeë lewens stres ‘n rol speel deur dat meer proteine geaffekteer word deur die kombinasie van moederlike skeiding gevolg deur later metamfetamien toediening. Ons stel voor dat die molekulêre meganismes waardeur vroeë lewensstres diere meer vatbaar maak vir die verslawende effekte van stimulante behels ‘n komplekse samestelling van sellulêre prosesse in die brein.

published_or_final_version / abstract / Molecular Biology / Master / Master of Philosophy

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Biological Sciences / Master / Master of Philosophy

published_or_final_version / Biological Sciences / Master / Master of Philosophy

published_or_final_version / Biological Sciences / Master / Master of Philosophy

Proteomics is the science of understanding the dynamic protein content of an organism's cells (its proteome), which is one of the largest current challenges in biology. Computational proteomics is an active research area that involves in-silico methods for the analysis of high-throughput protein identification data. Current methods are based on a technology called tandem mass spectrometry (MS/MS) and suffer from low coverage and accuracy, reliably identifying only 20-40% of the proteome. This dissertation addresses recall, precision, speed and scalability of computational proteomics experiments. This research goes beyond the traditional paradigm of analyzing MS/MS experiments in isolation, instead learning priors of protein presence from the joint analysis of various systems biology data sources. This integrative `systems' approach to protein identification is very effective, as demonstrated by two new methods. The first, MSNet, introduces a social model for protein identification and leverages functional dependencies from genome-scale, probabilistic, gene functional networks. The second, MSPresso, learns a gene expression prior from a joint analysis of mRNA and proteomics experiments on similar samples. These two sources of prior information result in more accurate estimates of protein presence, and increase protein recall by as much as 30% in complex samples, while also increasing precision. A comprehensive suite of benchmarking datasets is introduced for evaluation in yeast. Methods to assess statistical significance in the absence of ground truth are also introduced and employed whenever applicable. This dissertation also describes a database indexing solution to improve speed and scalability of protein identification experiments. The method, MSFound, customizes a metric-space database index and its associated approximate k-nearest-neighbor search algorithm with a semi-metric distance designed to match noisy spectra. MSFound achieves an order of magnitude speedup over traditional spectra database searches while maintaining scalability. / text

Although molybdenum blue solutions have been known for more than twocenturies, an understanding of their chemical nature is only beginning to emerge.This dissertation aimed at elucidating the structural nature of the polydisperse,nanoscopic components in the solution phases and the solid states of partiallyreduced polyoxomolybdate (Mo-POM). The study offered at least fourcontributions to the area: (1) a rational protocol for the molecular recognition ofMo-POM with de novo organic hosts. (2) demonstration of kinetic precipitation ofa dynamic mixture of polyoxomolybdates and application of the technique to thestudy of the dynamic mixture by TEM (3) characterization of the Mo-POMnanostructures by an unusual combination of complementary analyticaltechniques. (4) a general approach for the synthesis of crown-ethers-containingtripodal molecules.The molecular recognition of Mo-POM with designer tripodal hexaminetris-crown ethers opened a window to the solution phase structures of Mo-POMnanoscopic components. Studies with a series of structurally analogous hostsprobed the relationship between the structure of the molecular host and theformation of nanostructures.An unusual combination of complementary analytical protocols: flow fieldflowfractionation, electron microscopy (transmission and scanning), andinductively coupled plasma – emission spectroscopy, was used to monitor thesolution-phase evolution of Mo-POM nanostructures. The crystallization – drivenformation of keplerate Mo-POM and solution-phase evolution of structurallyrelated nanoscopic species were apparent in the self-assembling process ofpartially reduced Mo-POM.