Chairman: Professor G.G.S. Dutton Periodate Oxidation Products of Some Partially Methylated Hexoses and Hexitols Complete periodate oxidation of 2,3-di-O-methyl-D-glucitol and 2,3-di-O-methyl-D-mannitol produced 2,3-di-O-methyl-L-threose and 2,3-di-0-methyl-D-erythrose respectively. Reduction with sodium borohydride produced the corresponding tetritols, 2,3-di-O-methyl-L-threitol and 2,3-di-0-methyl-erythritol. Incomplete periodate oxidation of the two hexitols lead to the formation of large amounts of the respective pentoses, 3,4-di-0-methyl-L-xylose and 3,4-di-O-methyl-D-arabinose. Attempts to prepare 4-0-methyl-D-threose from methyl 6-0-methyl-αβ-D-galactofuranoside and 2-0-methyl-D-erythrose from methyl 4-0-methyl-α-D-mannopyranoside by periodate oxidation were only partially successful. Although the oxidation proceeded smoothly, the cleavage of the oxidized product could not be readily effected with mineral acid, methanolysis or mercaptolysis without extensive degradation taking place. Reduction of the aldehydes generated by periodate oxidation allowed the facile preparation of the corresponding tetritols. A scheme to correlate the original aldehyde structure to the reduced tetritol is proposed. The structure of an unknown component in the hydrolysis of methyl 2,3-di-0-methyl-α-D-glucopyranoside in the presence of 2,3-di-0-methyl-D-glucitol was shown to be 1,4-anhydro-2,3-di-0-methyl-D-glucitol. The paper chromatographic characteristics of the prepared tetroses, tetritols and pentoses in the two most commonly used solvent systems have been recorded. / Science, Faculty of / Chemistry, Department of / Graduate

Thesis (MSc)--University of Stellenbosch, 2005. / Bibliography. Also available via the Internet.

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Saccharomyces cerevisiae is confronted with continually changing and diverse nutrient conditions. As such, it has adapted to utilize both a wide variety of nutrient sources and to preferentially use the most nutrient-rich source to obtain a competitive advantage. The transcription, intracellular trafficking and protein turnover of nutrient transporters, including the hexose transporter proteins (Hxts), are strictly regulated in response to nutrient conditions. The low affinity hexose transporter Hxt3p is highly expressed and localizes to the plasma membrane during growth in abundant glucose where it plays a major role in the transport of this sugar. However, following a shift to ethanol as a sole carbon source, Hxt3p is endocytosed and targeted to the vacuole for degradation while its expression is also repressed. In contrast, the high affinity hexose transporter Hxt7p is actively expressed and functional in the plasma membrane when glucose is limiting and nitrogen is abundant. Upon nitrogen starvation or rapamycin treatment, HXT7 transcription decreases and the protein is targeted for degradation. The mechanisms that govern these regulatory steps are poorly understood. The Vid and Gid proteins, several of which compose the Vid30 complex (Vid30c), facilitate the nutrient-dependent degradation of the gluconeogenic enzymes FBPase and Mdh2p when glucose-starved cells are replenished with glucose. Here we show that components of the Vid30c are needed for the ethanol-induced turnover of Hxt3p and the rapamycin or nitrogen starvation-induced degradation of Hxt7p. In addition, we demonstrate that the signals for the ethanol-induced turnover of Hxt3p and the rapamycin-induced turnover of Hxt7p converge on the Vid30c upstream of the Ras/cAMP/PKA pathway, ultimately controlling the degradation of both these hexose transporters. Finally, we provide evidence that the Vid30c controls the localization of the Ras GEF Cdc25p and may therefore directly regulate the activity of the Ras/cAMP/PKA pathway.

O fungo filamentoso Trichoderma reesei é caracteristicamente reconhecido pela produção de celulases e hemicelulases, que lhe permitem utilizar uma ampla variedade de polissacarídeos como fonte de carbono. Neste trabalho, descrevemos a caracterização de dois genes de T. reesei, Trhxt1 e Trhxt2, que codificam proteínas com alta similaridade a transportadores de glicose de vários microorganismos. Os dois genes foram identificados em um banco de dados de ESTs de T. reesei. A análise computacional de Trhxt1 e Trhxt2 indica que ambos fazem parte da major facilitator superfamily (MFS), apresentando, tipicamente, 12 segmentos transmembrânicos. A expressão de Trhxt1 ocorre apenas em baixos níveis de glicose(≈ 100 µmol 1-1), enquanto a de Trhxt2 parece ocorrer de forma constitutiva, independentemente da fonte de carbono. Em baixas concentrações de oxigênio, a expressão de Trhxt1 é induzida e a de Trhxt2, reprimida. O sistema de transporte em T. reesei apresenta um componente de afinidade muito alta por glicose (Km ≈ 20 µmol 1-1) semelhante ao de outros fungos filamentosos. Dados sobre o transporte de glicose em uma cepa mutante ΔTrhxt1 indicam que o gene Trhxt1 está envolvido com o transporte em baixos níveis de glicose (≤ 100 µmol 1-1) que correspondem, provavelmente, aos valores encontrados no solo, o habitat natural de T. reesei.. Interessantemente, a indução do sistema de celulases de T. reesei por celulose está retardada no mutante ΔTrhxt1, o que sugere a importância do transporte de glicose na expressão dos genes das celulases. Finalmente, além de descrever os primeiros genes de transportadores de glicose em T. reesei, esperamos que este trabalho possa contribuir para o preenchimento de uma lacuna em relação ao transporte de açúcares em fungos filamentosos em geral. / The filamentous fungus Trichoderma reesei is a natural soil inhabitant capable of metabolizing a vast number of polysaccharide substrates. In this work, we describe two genes of T. reesei, named Trhxt1 and Trhxt2, which code for proteins with significant similarities to glucose transporters from other fungi. These genes were identified in an EST database of T. reesei. Sequence analysis of TrHXT1 and TrHXT2 revealed 12 putative transmembrane domains and several other characteristic motifs found in members of the major facilitator superfamily (MFS). Trhxt1 is transcriptionally induced only by low levels of glucose(≈ 100 µmol 1-1), while Trhxt2 expressionis independent of both glucose concentration and carbon source. We also show that Trhxt1 expression is enhanced when cells are exposured to low oxygen levels; in contrast, Trhxt2 expression seems to be repressed at these conditions. Glucose transport in T. reesei is apparently mediated by a multicomponent uptake system, in which the high-affiníty component has a Km of approximately 20 µmol 1-1. This low Km value is similar to the values reported for glucose uptake by other filamentous fungi. Kinetics of glucose transport in a T. reesei ΔTrhxt1 strain suggests that Trhxt1 is involved in glucose uptake in conditions of low glucose (≤ 100 µmol 1-1), which are most probably found in the soil, a low-nutrient environment. Interestingly, índuction ofthe T. reesei cellulase system by cellulose ís significantly delayed in the ΔTrhxt1 mutant, suggesting that glucose transport may be important to the mechanisms of expression of the cellulase genes. Finally, we hope that this work may be helpful to provide a better understanding of sugar uptake in filamentous fungi, for which there is little information available.

O fungo filamentoso Trichoderma reesei é caracteristicamente reconhecido pela produção de celulases e hemicelulases, que lhe permitem utilizar uma ampla variedade de polissacarídeos como fonte de carbono. Neste trabalho, descrevemos a caracterização de dois genes de T. reesei, Trhxt1 e Trhxt2, que codificam proteínas com alta similaridade a transportadores de glicose de vários microorganismos. Os dois genes foram identificados em um banco de dados de ESTs de T. reesei. A análise computacional de Trhxt1 e Trhxt2 indica que ambos fazem parte da major facilitator superfamily (MFS), apresentando, tipicamente, 12 segmentos transmembrânicos. A expressão de Trhxt1 ocorre apenas em baixos níveis de glicose(≈ 100 µmol 1-1), enquanto a de Trhxt2 parece ocorrer de forma constitutiva, independentemente da fonte de carbono. Em baixas concentrações de oxigênio, a expressão de Trhxt1 é induzida e a de Trhxt2, reprimida. O sistema de transporte em T. reesei apresenta um componente de afinidade muito alta por glicose (Km ≈ 20 µmol 1-1) semelhante ao de outros fungos filamentosos. Dados sobre o transporte de glicose em uma cepa mutante ΔTrhxt1 indicam que o gene Trhxt1 está envolvido com o transporte em baixos níveis de glicose (≤ 100 µmol 1-1) que correspondem, provavelmente, aos valores encontrados no solo, o habitat natural de T. reesei.. Interessantemente, a indução do sistema de celulases de T. reesei por celulose está retardada no mutante ΔTrhxt1, o que sugere a importância do transporte de glicose na expressão dos genes das celulases. Finalmente, além de descrever os primeiros genes de transportadores de glicose em T. reesei, esperamos que este trabalho possa contribuir para o preenchimento de uma lacuna em relação ao transporte de açúcares em fungos filamentosos em geral. / The filamentous fungus Trichoderma reesei is a natural soil inhabitant capable of metabolizing a vast number of polysaccharide substrates. In this work, we describe two genes of T. reesei, named Trhxt1 and Trhxt2, which code for proteins with significant similarities to glucose transporters from other fungi. These genes were identified in an EST database of T. reesei. Sequence analysis of TrHXT1 and TrHXT2 revealed 12 putative transmembrane domains and several other characteristic motifs found in members of the major facilitator superfamily (MFS). Trhxt1 is transcriptionally induced only by low levels of glucose(≈ 100 µmol 1-1), while Trhxt2 expressionis independent of both glucose concentration and carbon source. We also show that Trhxt1 expression is enhanced when cells are exposured to low oxygen levels; in contrast, Trhxt2 expression seems to be repressed at these conditions. Glucose transport in T. reesei is apparently mediated by a multicomponent uptake system, in which the high-affiníty component has a Km of approximately 20 µmol 1-1. This low Km value is similar to the values reported for glucose uptake by other filamentous fungi. Kinetics of glucose transport in a T. reesei ΔTrhxt1 strain suggests that Trhxt1 is involved in glucose uptake in conditions of low glucose (≤ 100 µmol 1-1), which are most probably found in the soil, a low-nutrient environment. Interestingly, índuction ofthe T. reesei cellulase system by cellulose ís significantly delayed in the ΔTrhxt1 mutant, suggesting that glucose transport may be important to the mechanisms of expression of the cellulase genes. Finally, we hope that this work may be helpful to provide a better understanding of sugar uptake in filamentous fungi, for which there is little information available.

Trypanosoma brucei is an extracellular kineotoplastid parasite that causes human African trypanosomiasis (HAT), also known as sleeping sickness. As trypanosomes undergo vector to host transition, heavy transcriptional adaptation such as metabolic shift to glycolysis and upregulated endocytosis occurs. Specifically, glycolysis in the infectious stage becomes the sole source of energy production; thus, the glucose transport mechanism in T. brucei provides one of the most promising therapeutic targets for development of new drugs to treat HAT. Despite an established trypanosome hexose transporter (THT) model for glucose transport across the plasma membrane, there remains gaps in the detailed mechanism of glucose transport especially as it relates to glucose transport across the glycosomal membrane. Using 2-NBDG, a fluorescent glucose analog, we measured glucose uptake rates in the presence of small molecule inhibitors and by using RNA interference (RNAi) to knockdown key proteins to investigate the mechanism of glucose transport in trypanosomes. We have confirmed a direct role of THT in glucose transport of BSF trypanosomes; however, in our investigations, we observed an unexpected ATP-dependence on glucose transport in live trypanosomes, which initiated further study where we focused on the role of endocytosis as an ATP-coupled bulk glucose transport mechanism. Experimental approaches that inhibited endocytosis reduced the observed glucose uptake rate confirming a role for endocytosis-coupled glucose transport in BSF trypanosomes. We provide evidence for an endocytosis-coupled glucose transport mechanism in BSF trypanosomes as an additional and important mechanism that functions in parallel with the established THT model.

Vineyards in New Zealand suffer bird damage caused by several avian species, including blackbirds and silvereyes. The introduced European Blackbird takes whole grapes which reduces yield. The self-introduced Australasian Silvereye pecks on grapes, leaving them on the vine to be further attacked by fungi and bacteria, and the subsequent off-odours can cause grapes to be refused by the winery or to suffer a price-reduction. Bird control methods remain primitive and largely ineffective during the long ripening period of wine grapes. An ecologically sound method to manage and reduce bird pressure requires deeper understanding of why some birds eat grapes, especially since grapes are not particularly nutritious. This work investigated the extent to which blackbirds and silvereyes are attracted by various compounds in ripening grapes. Since in natural grapes these compounds develop and change simultaneously, I developed an artificial grape in which a single parameter could be investigated. Artificial grapes (and sometimes nectar) were presented on a bird feeder table and the responses of birds to hexose sugars, the aromas 2-3-isobutylmethoxypyrazine and geraniol, tartaric and malic acids, grape tannins, and purple and green colour were recorded on timelapse video and analysed.

Thesis (MSc (Genetics. Plant Biotechnology))--University of Stellenbosch, 2005. / Sucrose accumulation, marked by a continuous cycle of synthesis and degradation, is characterised by a shift of carbon away from the insoluble matter and respiratory intermediates into sucrose. Despite this shift, a significant proportion of carbon is returned to these pools by hexose-phosphate: triose-phosphate cycling and/or sucrose cycling. Little is known about the magnitude and behaviour of these cycles in sugarcane. Contradictory reports on the relationship between these two cycles have led to the evaluation of the link between the hexose-phosphate: triose-phosphate- and sucrose cycle. In addition, it still needs to be tested whether these cycles could significantly influence carbon partitioning within sugarcane internodal tissue. In this work, a comprehensive metabolic profile was constructed for sugarcane internodal tissue by gas chromatography-mass spectrometry (GC-MS) in order to determine the steady state levels of a broad range of primary metabolites that are involved in these cycles. The power of GC-MS was illustrated by the detection of raffinose, maltose, ribose, xylitol, inositol, galactose, arabinose and quinic acid, which was quantified for the first time in sugarcane internodal tissue. Analyses were not solely based on the prevailing metabolite levels, but also on the interactions between these metabolites. Thus, in a complementary approach the metabolic flux between the two substrate cycles was assessed by 13C nuclear magnetic resonance (NMR). Analyses of transgenic sugarcane clones with 45-95% reduced cytosolic pyrophosphate: D-fructose-6-phosphate 1-phosphotransferase (PFP, EC 2.7.1.90) activity displayed no visual phenotypic change, but significant changes were evident in in vivo metabolite levels. Sucrose concentrations increased six and three-fold in young and maturing internodal tissue, respectively. Reduced PFP activity also resulted in an eight-fold increase in the hexose-phosphate: triose-phosphate ratio in the transgenic immature internodes. In addition, the hexose-phosphate: triose-phosphate cycling decreased in the immature internodes of the transgenic lines if compared to the immature control internode. However, there was no significant difference between the hexose-phosphate: triose-phosphate cycling in the mature internodal tissue of the transgenic and the control lines. This illustrated that PFP mediates hexose-phosphate: triose-phosphate cycling in immature sugarcane internodal tissue. Unpredictably, reduced PFP activity led to a ten-fold increase in sucrose cycling in the transgenic immature internodes. The combination of metabolite profiling and flux distribution measurements demonstrated that the fluxes through the sucrose and the hexose-phosphate pools were not co-regulated in sugarcane internodal tissue. From these observations a model was constructed that implicates higher sucrose cycling as a consequence of increased sucrose concentrations.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21837号 / 農博第2350号 / 新制||農||1069(附属図書館) / 学位論文||H31||N5209(農学部図書室) / 京都大学大学院農学研究科地域環境科学専攻 / (主査)教授 田中 千尋, 教授 本田 与一, 准教授 刑部 正博 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM