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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Effects of metal ions on the structural and biochemical properties of Trypanosomatid phosphoglycerate mutases

Fuad, Fazia Adyani Ahmad January 2012 (has links)
Flagellate protozoa from the order Trypanosomatida have developed a range of strategies to survive in their mammalian hosts. A consequence is that the glycolytic pathway has assumed an important role, especially in bloodstream-form Trypanosoma brucei, where it is essential as the sole producer of ATP. The seventh enzyme in the pathway, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGAM) is particularly attractive as a drug target because it shares no common properties with the corresponding enzyme in humans. This enzyme catalyses the conversion of 3PGA to 2PGA, with the requirement for metal ions to assist the catalytic function. In this study, two important biochemical and structural aspects of the enzyme were investigated: i) The in vitro and in vivo requirements for biologically relevant metal ions to support the activity of iPGAM, and ii) The ability of trypanosomatid iPGAM to exist in multiple conformations and oligomeric states in solution. The maximum activity of iPGAM in vitro requires Co2+, but this cannot be the case in vivo where ICP-OES analyses confirmed that Co2+ was essentially undetectable in T. brucei cytosolic fractions. The activity of iPGAM in vivo is therefore one of the lowest among the glycolytic enzymes. By contrast, Mg2+ and Zn2+ were found to be the most abundant metals in both cytosolic fractions and in purified bacterially expressed iPGAM. Our newly-developed multimode-plate reader discontinuous assay further revealed that of the biologically relevant metals, only Mg2+ can support iPGAM activity, but at less than 50% of the level of Co2+. By contrast, Zn2+ strongly inhibits iPGAM. This assay which was developed with minimal metal interference on the coupling enzymes, also showed that in solution, the ratio of the concentrations of 3PGA:2PGA (substrate:product) at equilibrium is not 1:1 as observed in the crystal structure, but is in fact 12:1, which may be due to the tighter binding of 2PGA to the enzyme. A series of biophysical analyses, notably by SEC-MALS showed that iPGAM from Leishmania mexicana, another trypanosomatid protozoan parasite exists in different forms and oligomeric states in solution, either as the closed-form monomer, openiii form monomer, or closed/open-form dimer which can be successfully separated by ion-exchange chromatography. The open-form LmiPGAM is particularly relevant for drug development, as the catalytic site in the closed-form structure is poorly inaccessible. Both virtual and high-throughput screening approaches were used to identify novel potential inhibitors. Out of a collection of 11 compounds tested at 1 mM, two showed substantial inhibition with 49% and 14% remaining activity. Taken together, the findings from this study demonstrated the potential of iPGAM to be a key modulator in controlling glycolytic flux in trypanosomes, and thus further validated it as an important drug target.
2

Regulation of glycolysis in Saccharomyces cerevisiae

Pearce, Amanda K. January 1999 (has links)
This thesis extends the work of Crimmins (1995) on the control of glycolytic flux in yeast by the enzymes 6-phosphofructo-1-kinase and pyruvate kinase (Pyk1p). This study also examines the influence of Pf1kp and Pyk1p upon yeast resistance to the weak acid preservative, benzoic acid. In <I>Saccharomyces cerevisiae</I>, Pyk1p is encoded by <I>PYK1</I>, and the α and β subunits of Pf1kp are encoded by <I>PFK1</I> and <I>PFK2</I>, respectively. To test the influence of these genes upon glycolytic control, an isogenic set of <I>S. cerevisiae</I> mutants were utilised in which <I>PYK1, PFK1</I> and <I>PFK2</I> expression is dependent on the <I>PGK1</I> promoter. Increased Pf1k levels had little effect upon rates of glucose utilisation or ethanol production during fermentative growth. However, overexpressing Pyk1p resulted in an increased growth rate and an increase in glycolytic flux. This suggests that Pyk1p, but not Pf1kp, exerts some degree of control over the glycolytic flux under these conditions. The effects of reducing Pf1kp and Pyk1p levels were also studied by placing <I>PYK1, PFK1</I> and <I>PFK2</I> under the control of the weak <I>PGK1Δuas</I> promoter. The double Pf1kp mutant showed no significant changes in doubling time, ethanol production or glucose consumption. However, a mutant with a 3-fold reduction ion Pyk1p levels displayed slower growth rates and reduced glycolytic flux. In addition, there was an imbalance in the carbon flow in this mutant, with reductions in ethanol and glycerol production evident, along with increased TCA cycle activity. Hence, while Pf1kp levels did not affect cell physiology significantly under the conditions studied, reduced Pyk1p levels seemed to disturb glycolytic flux and carbon flow. Decreased Pf1kp levels caused an increase in the sensitivity of yeast cells to benzoate, whereas the Pyk1p mutant was not affected. This confirmed that benzoic acid specifically inhibits Pf1kp rather than glycolysis in general.
3

An Analysis of Glycolytic Enzymes in the Cellular Response to Metal Toxicity

Shanmuganathan, Anupama 16 July 2009 (has links)
Metal toxicity is implicated in neurotoxicity, nephrotoxicity, aging and cancer. Protein oxidation resulting from oxidative stress is now known to be involved in metal toxicity. However, proteomic responses to metal induced oxidative stress have not been characterized. By using the yeast as a model, we characterized these changes occurring in response to sub-lethal doses of metals. Several proteins involved in protein synthesis, ribosome assembly decreased while antioxidant defenses, proteins involved in sulfur metabolism, and glutathione synthesis and ubiquitin increased following metal exposure. We also show that metals induced temporal and targeted protein oxidation independent of protein abundance. Among the targets were glycolytic enzymes and heat-shock proteins. As a consequence, glycolytic enzyme activities decreased whereas the levels and activities of the enzymes of the alternative pathway for glucose metabolism, pentose phosphate pathway (PPP) increased. True to prediction, we also found increased flow through the PPP as measured by elevated levels of NADPH and glutathione. NADPH and glutathione are crucial for maintaining the redox balance in the cell. Thus, rerouting of glucose metabolism into PPP is considered to be beneficial to the organism. Among the oxidation targets is a glycolytic protein, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) that is required for apoptosis in neuronal cells. We show that not only is GAPDH required for metal induced apoptosis in yeast but also the levels of GAPDH transcript and protein increase in the cytosol and the nucleus in an isoform specific fashion. Such changes strongly implicate the role of GAPDH in yeast apoptosis. This work provides evidence for the involvement of targeted protein oxidation in metal toxicity, shows the overlaps and differences in the mechanism of copper and cadmium toxicity, allows comprehension of how metabolic processes respond to metal stress and explores the potential of GAPDH as a sensor of oxidative stress and mediator for apoptosis.
4

Études de la réponse du métabolisme énergétique à la carence en fer dans les cultures cellulaires de Solanum tuberosum

Canelo Vivar, Marcela Paz 07 1900 (has links)
Le fer est un micronutriment important pour la croissance et le développement des plantes. Il agit comme cofacteur pour plusieurs enzymes et il est important pour des processus tels que la photosynthèse et la respiration. Souvent, le Fe dans le sol n’est pas bio-disponible pour la plante. Les plantes ont développé des stratégies pour solubiliser le Fe du sol pour le rendre disponible et assimilable pour elles. Il y a deux stratégies, la première est caractéristique des dicotylédones et la seconde est caractéristique des monocotylédones. Le modèle utilisé dans cette étude est une culture cellulaire de Solanum tuberosum. Une partie de la recherche effectuée a permis la mesure d’activité et d’expression relative de certaines enzymes impliquées dans le métabolisme énergétique et la fourniture de précurseurs pour la synthèse d’ADN : la Nucléoside diphosphate kinase, la Ribonucléotide reductase, la Glucose 6-phosphate déshydrogénase et la 6-Phosphogluconate déshydrogénase dans les cellules en présence ou en absence de Fe. Chez certains organismes, la déficience en Fe est associée à une perte de croissance qui est souvent liée à une diminution de la synthèse d’ADN. Chez les cultures de cellules de S. tuberosum, les résultats indiquent que la différence de biomasse observée entre les traitements n’est pas due à une variation de l’activité ou l’expression relative d’une de ces enzymes. En effet, aucune variation significative n’a été détectée entre les traitements (+/- Fe) pour l’activité ni l’expression relative de ces enzymes. Une autre partie de la recherche a permis d’évaluer l’activité des voies métaboliques impliquées dans la stratégie 1 utilisée par S. tuberosum. Cette stratégie consomme des métabolites énergétiques: de l’ATP pour solubiliser le Fe et du pouvoir réducteur (NAD(P)H), pour réduire le Fe3+ en Fe2+. Des études de flux métaboliques ont été faites afin d’étudier les remaniements du métabolisme carboné en déficience en Fe chez S. tuberosum. Ces études ont démontré une baisse du régime dans les différentes voies du métabolisme énergétique dans les cellules déficientes en Fe, notamment dans le flux glycolytique et le flux de C à travers la phosphoenolpyruvate carboxylase. En déficience de Fe il y aurait donc une dépression du métabolisme chez S. tuberosum qui permettrait à la cellule de ralentir son métabolisme pour maintenir sa vitalité. En plus des flux, les niveaux de pyridines nucléotides ont été mesurés puisque ceux-ci servent à réduire le Fe dans la stratégie 1. Les résultats démontrent des niveaux élevés des formes réduites de ces métabolites en déficience de Fe. L’ensemble des résultats obtenus indiquent qu’en déficience de Fe, il y a une baisse du métabolisme permettant à la cellule de s’adapter et survivre au stress. / Iron is an important micronutrient for plant growth and development. It participates as a cofactor for several enzymes and is important for processes such as photosynthesis and respiration. Often soil Fe is not bioavailable to the plant. Plants have developed strategies to solubilize the Fe in the soil to make it available and easy to assimilate. There are two strategies, the first is characteristic of dicotyledones and the second is characteristic of monocotyledones. The model used in these studies is a cell culture of Solanum tubersoum. A first part of the research involved the study of expression and activity of enzymes required in energy metabolism and the provision of precursors for DNA synthesis: Nucleoside dehydrogenase, Ribonucleotide reductase, Glucose 6-phohate dehydrogenase and 6-Phosphogluconate dehydrogenase. In several organisms, Fe deficiency induces a loss of biomass which is often associated with a decrease in DNA synthesis. In S. tuberosum cell cultures, the results indicate that the loss of biomass observed in Fe deficiency is not linked to a change in the activity or relative expression of these enzymes. Indeed, no significant changes were detected between treatments (+/- Fe) for activity or relative expression. In another part of the research, we evaluated the activity of the metabolism pathways involved in strategy 1, which is used by S. tuberosum. This strategy consumes energetic metabolites: ATP to solubilize Fe and reducing power (NAD(P)H) to reduce the Fe3+ to Fe2+. Metabolic flux studies were done to investigate the alterations of carbon metabolism during Fe deficiency in S. tuberosum. These studies demonstrated that in Fe deficient cells, there is a decrease in the fluxes of some pathways of energy metabolism. Particularly, in the glycolytic flux and the anaplerotic flux of PEPC. Under Fe deficiency there would be a depression of metabolism in S. tuberosum which would allow the cell to slow its metabolism to maintain its vitality. In addition to the fluxes, the levels of pyridine nucleotides were measured since they serve to reduce Fe in the strategy 1. The results show an increase in the reduced forms of these metabolites during Fe deficiency. All results together point out that during Fe deficiency the metabolism decreases, allowing the cell to survive and adapt to the stress.

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