Thesis (Ph. D.)--University of Wisconsin--Madison, 1953. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 57-58).

Thesis (doctoral)--University of Stockholm, 1983. / Bibliography: p. 36-39.

No description available.

Le cycle de Krebs occupe une place centrale dans le métabolisme cellulaire et est le point de jonction de nombreuses voies essentielles. Depuis le début des années 2000, un lien a été démontré entre l’apparition de certains cancers et des mutations affectant des gènes codant pour des enzymes du cycle de Krebs, i.e., la succinate déshydrogénase, la fumarase ou les iso-enzymes 1 et 2 de l'isocitrate déshydrogénase (IDH). Les mutations des gènes IDH sont présentes dans 15 à 20 % des leucémies myéloïdes aigues (LAM) et jusqu'à 80 % dans certains gliomes. Ces mutations affectent le site actif des enzymes et elles induisent une néo-fonction enzymatique qui se traduit par la production et l'accumulation d'un oncométabolite : le stéréoisomère D du 2-hydroxyglutarate (D-2-HG) responsable de dérégulations énergétiques et épigénétiques au sein de la cellule. Afin de mieux comprendre les mécanismes mis en jeu entre ces anomalies et la pathologie humaine, mon travail de thèse a impliqué le développement de différentes méthodes analytiques : - tout d'abord une méthode robuste de séparation et de quantification des stéréoisomères D et L par dérivation chirale du 2-HG, ceci en GC tandem MS, - également par GC tandem MS, des méthodes métabolomiques ciblées à haute spécificité pour l'analyse de plus de 120 composés d'intérêt clinique, - des méthodes analytiques à haute résolution et non-ciblées (masse exacte; n=360 composés) adaptées à l'étude de cellules, - et des méthodes d'étude de flux métaboliques sur culture cellulaire basées sur l'analyse des dérivés de traceurs marqués aux isotopes stables. Le développement de ces méthodes m'a permis d'obtenir les résultats suivants. J'ai démontré l'importance du D-2-HG comme marqueur de la présence de mutations IDH1/2 dans une large cohorte de patients leucémiques, à la fois pour le diagnostic et pour le suivi des patients sous traitement. Notre étude pilote a conduit à utiliser ce paramètre en pratique hospitalière courante dans le laboratoire de chimie analytique de l'institut Gustave Roussy (IGR; Villejuif). L'étude de profils métaboliques associés aux mutations affectant les enzymes IDH2 et succinate déshydrogénase nous a permis d'identifier des mécanismes compensatoires du dysfonctionnement du cycle de Krebs, par exemple la sur-activation de la pyruvate carboxylase. Nous avons par ailleurs montré que ces mécanismes ne sont que partiellement efficaces; ils pourraient ainsi servir de cibles thérapeutiques. Une mutation du gène IDH2 (R140Q) est retrouvée chez des patients atteint de LAM et chez des patients possédant une acidurie D-2-hydroxyglutarique, maladie héréditaire du métabolisme extrêmement rare. Un inhibiteur spécifique de l'enzyme IDH2 possédant la mutation R140Q est actuellement testé comme traitement dans un essai clinique à l'IGR pour les patients leucémiques. Nous avons étudié les effets de ce composé sur des fibroblastes de notre patient atteint d'acidurie D-2-hydroxyglutarique. Nous avons confirmé ses effets sur l'enzyme IDH et observé des effets secondaires sur le métabolisme des lipides et du cycle de Krebs, à la fois dans les fibroblastes témoin et du patient. Cet inhibiteur étant connu pour avoir des effets sur la différenciation cellulaire, nos résultats pourraient permettre d'expliquer les mécanismes impliqués. Ce travail a apporté de nouveaux outils pour l'exploration des maladies métaboliques traditionnelles ainsi que de certains types de cancers, et il met en avant de nouvelles illustrations de la puissance de l'approche métabolique pour identifier des points d'intervention et de surveillance thérapeutique personnalisée des patients ("théranostique"). / The Krebs cycle has a central role in cellular metabolism and is at the junction of many essential pathways. Since 2000, a link has been shown between the development of particular cancers and mutations affecting genes coding for several Krebs cycle enzymes, i.e., succinate dehydrogenase, fumarase or iso-enzymes 1 and 2 of the isocitrate dehydrogenase (IDH). The IDH mutations are found in 15 to 20 % of acute myeloid leukemias and up to 80% of specific gliomas. These mutations affect the enzyme active site and are responsible for an neomorphic activity that is the production and accumulation of a putative oncometabolite : the D stereoisomer of the 2-hydroxyglutarate (D-2-HG) which is linked to energetic and epigenetic deregulations in the cell. To better understand the mechanisms between these abnormalities and human pathology, my PhD work involved the development of different analytical tools : - First of all, a robust method of separation and quantification of the stereoisomers D and L by chiral derivatization of the 2-HG, in tandem mass spectrometry, - GC tandem MS was also used to develop targeted metabolomic methods with high specificity for the analysis of more than 120 compounds of clinical interest, - An analytical non-targeted method using high mass resolution (exact mass; n=360 compounds) adapted to the study of fibroblast cells, - and finally, methods for the study of metabolic flux in culture cell based on derivatives of stable labeled tracers. The development of these methods led to the following results. I highlight the importance of the D-2-HG as a biomarker of the presence of IDH1/2 mutations in a large cohort of leukemic patients, for the diagnostic and the follow-up of patients under treatment. Our pilot study was the starting point for routine usage of this test in the clinical setting at the Institut Gustave Roussy (IGR; Villejuif). The study of metabolic profiles related to the mutations affecting IDH enzymes and succinate dehydrogenase allowed us to identify compensatory mechanisms of the dysfunction of the Krebs cycle, notably, the overactivation of pyruvate carboxylase. Moreover, we have shown that because these mechanisms are only partially efficient; they have potential to provide therapeutic targets. An IDH2(R140Q) mutation is shared between patients with AML and patients with D-2-hydroxyglutaric aciduria, a very rare hereditary disease of the metabolism. A specific inhibitor of the IDH2 enzyme mutant for R140Q is currently used in a clinical trial at the IGR institute. We studied the effects of this compound in fibroblasts of our aciduria patient. We confirmed the expected effect in the IDH enzyme and also observed moderate off-target effects concerning the lipid and the Krebs cycle metabolism, both in control and patient fibroblasts. Because this inhibitor is known to have effects in the cellular differentiation, our results could explain the underlying mechanisms. This work provides new tools for the exploration of traditional inherited metabolic diseases, as well as particular cancers, and illustrates the power of the metabolic approach to identify therapeutic targets and for the personalized monitoring of patients ("theranostics").

Le cycle de Krebs occupe une place centrale dans le métabolisme cellulaire et est le point de jonction de nombreuses voies essentielles. Depuis le début des années 2000, un lien a été démontré entre l’apparition de certains cancers et des mutations affectant des gènes codant pour des enzymes du cycle de Krebs, i.e., la succinate déshydrogénase, la fumarase ou les iso-enzymes 1 et 2 de l'isocitrate déshydrogénase (IDH). Les mutations des gènes IDH sont présentes dans 15 à 20 % des leucémies myéloïdes aigues (LAM) et jusqu'à 80 % dans certains gliomes. Ces mutations affectent le site actif des enzymes et elles induisent une néo-fonction enzymatique qui se traduit par la production et l'accumulation d'un oncométabolite : le stéréoisomère D du 2-hydroxyglutarate (D-2-HG) responsable de dérégulations énergétiques et épigénétiques au sein de la cellule. Afin de mieux comprendre les mécanismes mis en jeu entre ces anomalies et la pathologie humaine, mon travail de thèse a impliqué le développement de différentes méthodes analytiques : - tout d'abord une méthode robuste de séparation et de quantification des stéréoisomères D et L par dérivation chirale du 2-HG, ceci en GC tandem MS, - également par GC tandem MS, des méthodes métabolomiques ciblées à haute spécificité pour l'analyse de plus de 120 composés d'intérêt clinique, - des méthodes analytiques à haute résolution et non-ciblées (masse exacte; n=360 composés) adaptées à l'étude de cellules, - et des méthodes d'étude de flux métaboliques sur culture cellulaire basées sur l'analyse des dérivés de traceurs marqués aux isotopes stables. Le développement de ces méthodes m'a permis d'obtenir les résultats suivants. J'ai démontré l'importance du D-2-HG comme marqueur de la présence de mutations IDH1/2 dans une large cohorte de patients leucémiques, à la fois pour le diagnostic et pour le suivi des patients sous traitement. Notre étude pilote a conduit à utiliser ce paramètre en pratique hospitalière courante dans le laboratoire de chimie analytique de l'institut Gustave Roussy (IGR; Villejuif). L'étude de profils métaboliques associés aux mutations affectant les enzymes IDH2 et succinate déshydrogénase nous a permis d'identifier des mécanismes compensatoires du dysfonctionnement du cycle de Krebs, par exemple la sur-activation de la pyruvate carboxylase. Nous avons par ailleurs montré que ces mécanismes ne sont que partiellement efficaces; ils pourraient ainsi servir de cibles thérapeutiques. Une mutation du gène IDH2 (R140Q) est retrouvée chez des patients atteint de LAM et chez des patients possédant une acidurie D-2-hydroxyglutarique, maladie héréditaire du métabolisme extrêmement rare. Un inhibiteur spécifique de l'enzyme IDH2 possédant la mutation R140Q est actuellement testé comme traitement dans un essai clinique à l'IGR pour les patients leucémiques. Nous avons étudié les effets de ce composé sur des fibroblastes de notre patient atteint d'acidurie D-2-hydroxyglutarique. Nous avons confirmé ses effets sur l'enzyme IDH et observé des effets secondaires sur le métabolisme des lipides et du cycle de Krebs, à la fois dans les fibroblastes témoin et du patient. Cet inhibiteur étant connu pour avoir des effets sur la différenciation cellulaire, nos résultats pourraient permettre d'expliquer les mécanismes impliqués. Ce travail a apporté de nouveaux outils pour l'exploration des maladies métaboliques traditionnelles ainsi que de certains types de cancers, et il met en avant de nouvelles illustrations de la puissance de l'approche métabolique pour identifier des points d'intervention et de surveillance thérapeutique personnalisée des patients ("théranostique"). / The Krebs cycle has a central role in cellular metabolism and is at the junction of many essential pathways. Since 2000, a link has been shown between the development of particular cancers and mutations affecting genes coding for several Krebs cycle enzymes, i.e., succinate dehydrogenase, fumarase or iso-enzymes 1 and 2 of the isocitrate dehydrogenase (IDH). The IDH mutations are found in 15 to 20 % of acute myeloid leukemias and up to 80% of specific gliomas. These mutations affect the enzyme active site and are responsible for an neomorphic activity that is the production and accumulation of a putative oncometabolite : the D stereoisomer of the 2-hydroxyglutarate (D-2-HG) which is linked to energetic and epigenetic deregulations in the cell. To better understand the mechanisms between these abnormalities and human pathology, my PhD work involved the development of different analytical tools : - First of all, a robust method of separation and quantification of the stereoisomers D and L by chiral derivatization of the 2-HG, in tandem mass spectrometry, - GC tandem MS was also used to develop targeted metabolomic methods with high specificity for the analysis of more than 120 compounds of clinical interest, - An analytical non-targeted method using high mass resolution (exact mass; n=360 compounds) adapted to the study of fibroblast cells, - and finally, methods for the study of metabolic flux in culture cell based on derivatives of stable labeled tracers. The development of these methods led to the following results. I highlight the importance of the D-2-HG as a biomarker of the presence of IDH1/2 mutations in a large cohort of leukemic patients, for the diagnostic and the follow-up of patients under treatment. Our pilot study was the starting point for routine usage of this test in the clinical setting at the Institut Gustave Roussy (IGR; Villejuif). The study of metabolic profiles related to the mutations affecting IDH enzymes and succinate dehydrogenase allowed us to identify compensatory mechanisms of the dysfunction of the Krebs cycle, notably, the overactivation of pyruvate carboxylase. Moreover, we have shown that because these mechanisms are only partially efficient; they have potential to provide therapeutic targets. An IDH2(R140Q) mutation is shared between patients with AML and patients with D-2-hydroxyglutaric aciduria, a very rare hereditary disease of the metabolism. A specific inhibitor of the IDH2 enzyme mutant for R140Q is currently used in a clinical trial at the IGR institute. We studied the effects of this compound in fibroblasts of our aciduria patient. We confirmed the expected effect in the IDH enzyme and also observed moderate off-target effects concerning the lipid and the Krebs cycle metabolism, both in control and patient fibroblasts. Because this inhibitor is known to have effects in the cellular differentiation, our results could explain the underlying mechanisms. This work provides new tools for the exploration of traditional inherited metabolic diseases, as well as particular cancers, and illustrates the power of the metabolic approach to identify therapeutic targets and for the personalized monitoring of patients ("theranostics").

Le cycle de Krebs occupe une place centrale dans le métabolisme cellulaire et est le point de jonction de nombreuses voies essentielles. Depuis le début des années 2000, un lien a été démontré entre l’apparition de certains cancers et des mutations affectant des gènes codant pour des enzymes du cycle de Krebs, i.e., la succinate déshydrogénase, la fumarase ou les iso-enzymes 1 et 2 de l'isocitrate déshydrogénase (IDH). Les mutations des gènes IDH sont présentes dans 15 à 20 % des leucémies myéloïdes aigues (LAM) et jusqu'à 80 % dans certains gliomes. Ces mutations affectent le site actif des enzymes et elles induisent une néo-fonction enzymatique qui se traduit par la production et l'accumulation d'un oncométabolite : le stéréoisomère D du 2-hydroxyglutarate (D-2-HG) responsable de dérégulations énergétiques et épigénétiques au sein de la cellule. Afin de mieux comprendre les mécanismes mis en jeu entre ces anomalies et la pathologie humaine, mon travail de thèse a impliqué le développement de différentes méthodes analytiques : - tout d'abord une méthode robuste de séparation et de quantification des stéréoisomères D et L par dérivation chirale du 2-HG, ceci en GC tandem MS, - également par GC tandem MS, des méthodes métabolomiques ciblées à haute spécificité pour l'analyse de plus de 120 composés d'intérêt clinique, - des méthodes analytiques à haute résolution et non-ciblées (masse exacte; n=360 composés) adaptées à l'étude de cellules, - et des méthodes d'étude de flux métaboliques sur culture cellulaire basées sur l'analyse des dérivés de traceurs marqués aux isotopes stables. Le développement de ces méthodes m'a permis d'obtenir les résultats suivants. J'ai démontré l'importance du D-2-HG comme marqueur de la présence de mutations IDH1/2 dans une large cohorte de patients leucémiques, à la fois pour le diagnostic et pour le suivi des patients sous traitement. Notre étude pilote a conduit à utiliser ce paramètre en pratique hospitalière courante dans le laboratoire de chimie analytique de l'institut Gustave Roussy (IGR; Villejuif). L'étude de profils métaboliques associés aux mutations affectant les enzymes IDH2 et succinate déshydrogénase nous a permis d'identifier des mécanismes compensatoires du dysfonctionnement du cycle de Krebs, par exemple la sur-activation de la pyruvate carboxylase. Nous avons par ailleurs montré que ces mécanismes ne sont que partiellement efficaces; ils pourraient ainsi servir de cibles thérapeutiques. Une mutation du gène IDH2 (R140Q) est retrouvée chez des patients atteint de LAM et chez des patients possédant une acidurie D-2-hydroxyglutarique, maladie héréditaire du métabolisme extrêmement rare. Un inhibiteur spécifique de l'enzyme IDH2 possédant la mutation R140Q est actuellement testé comme traitement dans un essai clinique à l'IGR pour les patients leucémiques. Nous avons étudié les effets de ce composé sur des fibroblastes de notre patient atteint d'acidurie D-2-hydroxyglutarique. Nous avons confirmé ses effets sur l'enzyme IDH et observé des effets secondaires sur le métabolisme des lipides et du cycle de Krebs, à la fois dans les fibroblastes témoin et du patient. Cet inhibiteur étant connu pour avoir des effets sur la différenciation cellulaire, nos résultats pourraient permettre d'expliquer les mécanismes impliqués. Ce travail a apporté de nouveaux outils pour l'exploration des maladies métaboliques traditionnelles ainsi que de certains types de cancers, et il met en avant de nouvelles illustrations de la puissance de l'approche métabolique pour identifier des points d'intervention et de surveillance thérapeutique personnalisée des patients ("théranostique"). / The Krebs cycle has a central role in cellular metabolism and is at the junction of many essential pathways. Since 2000, a link has been shown between the development of particular cancers and mutations affecting genes coding for several Krebs cycle enzymes, i.e., succinate dehydrogenase, fumarase or iso-enzymes 1 and 2 of the isocitrate dehydrogenase (IDH). The IDH mutations are found in 15 to 20 % of acute myeloid leukemias and up to 80% of specific gliomas. These mutations affect the enzyme active site and are responsible for an neomorphic activity that is the production and accumulation of a putative oncometabolite : the D stereoisomer of the 2-hydroxyglutarate (D-2-HG) which is linked to energetic and epigenetic deregulations in the cell. To better understand the mechanisms between these abnormalities and human pathology, my PhD work involved the development of different analytical tools : - First of all, a robust method of separation and quantification of the stereoisomers D and L by chiral derivatization of the 2-HG, in tandem mass spectrometry, - GC tandem MS was also used to develop targeted metabolomic methods with high specificity for the analysis of more than 120 compounds of clinical interest, - An analytical non-targeted method using high mass resolution (exact mass; n=360 compounds) adapted to the study of fibroblast cells, - and finally, methods for the study of metabolic flux in culture cell based on derivatives of stable labeled tracers. The development of these methods led to the following results. I highlight the importance of the D-2-HG as a biomarker of the presence of IDH1/2 mutations in a large cohort of leukemic patients, for the diagnostic and the follow-up of patients under treatment. Our pilot study was the starting point for routine usage of this test in the clinical setting at the Institut Gustave Roussy (IGR; Villejuif). The study of metabolic profiles related to the mutations affecting IDH enzymes and succinate dehydrogenase allowed us to identify compensatory mechanisms of the dysfunction of the Krebs cycle, notably, the overactivation of pyruvate carboxylase. Moreover, we have shown that because these mechanisms are only partially efficient; they have potential to provide therapeutic targets. An IDH2(R140Q) mutation is shared between patients with AML and patients with D-2-hydroxyglutaric aciduria, a very rare hereditary disease of the metabolism. A specific inhibitor of the IDH2 enzyme mutant for R140Q is currently used in a clinical trial at the IGR institute. We studied the effects of this compound in fibroblasts of our aciduria patient. We confirmed the expected effect in the IDH enzyme and also observed moderate off-target effects concerning the lipid and the Krebs cycle metabolism, both in control and patient fibroblasts. Because this inhibitor is known to have effects in the cellular differentiation, our results could explain the underlying mechanisms. This work provides new tools for the exploration of traditional inherited metabolic diseases, as well as particular cancers, and illustrates the power of the metabolic approach to identify therapeutic targets and for the personalized monitoring of patients ("theranostics").

Le processus conduisant à la prolifération et différenciation des cellules souches hématopoïétiques (CSH) en cellules de toutes les lignées sanguines s’appelle l’hématopoïèse. Bien que l'engagement des CSH soit régi par les cytokines, les facteurs de transcription, les modificateurs épigénétiques et la niche des CSH, notre groupe a constaté que leur engagement vers la lignée érythroïde dépendait aussi du métabolisme de la glutamine. La glutaminolyse contribue à la biosynthèse des nucléotides de novo ainsi qu’à la production de l'alpha-kétoglutarate (αKG), intermédiaire métabolique du cycle TCA (Oburoglu et al. 2014). Il est cependant important de noter que la différenciation érythroïde est un processus unique, où chaque cellule fille est structurellement et fonctionnellement différente de sa cellule mère. Chaque division définit un stade de différenciation précis avec un dernier cycle de division produisant un réticulocyte énucléé. Ainsi, nous avons émis l'hypothèse que les réseaux métaboliques mobilisés dans les progéniteurs érythroïdes changent en fonction du stade de différenciation et que ces réseaux régulent la transition des progéniteurs d'un stade à l'autre.Au cours de ma thèse, j’ai caractérisé les états métaboliques associés aux différents stades de différenciation des progéniteurs érythroïdes. Nous avons ainsi montré qu'aux stades précoces de différenciation érythroïde, avant la différenciation terminale, les progéniteurs hématopoïétiques présentent une activité métabolique accrue avec un niveau de phosphorylation oxydative (OXPHOS) plus élevé. Ces données sont en corrélation avec l'augmentation de la génération de l’αKG à ces stades de différenciation. De plus, nous avons constaté une augmentation de l’OXPHOS de ces progéniteurs en présence d’αKG exogène. Cependant, la différenciation terminale des précurseurs érythroïdes, caractérisée par la perte de la masse mitochondriale et de leur potentiel membranaire, est associée à une diminution du niveau d'OXPHOS. Ainsi, l'administration exogène d’αKG, a fortement atténué la différenciation érythroïde terminale et l'énucléation, sans affecter la différenciation des pro-érythroblastes. Inversement, un antagoniste de l’αKG (diméthyloxalylglycine, DMOG) n'a pas altéré la différenciation terminale ou l'énucléation, malgré l'abrogation de l'OXPHOS dans les érythroblastes.Ces données suggèrent que la production d’αKG et sa contribution à l’OXPHOS perturbent l'énucléation des globules rouges. C'est pourquoi, dans le but de réduire les niveaux intracellulaires d’αKG, nous avons inhibé l’expression de l'isocitrate déshydrogénase I (IDH1), enzyme cytosolique catalysant la conversion de l'isocitrate en αKG. Cependant, comme IDH1 peut catalyser les réactions dans les deux sens, la diminution de son expression pourrait également augmenter les niveaux d’αKG. En effet, nous avons constaté que le knockdown d'IDH1 entraînait une forte atténuation de la différenciation terminale et de l'énucléation des précurseurs érythroïdes. Cet effet est probablement dû à un déséquilibre de la disponibilité des substrats ; ainsi l’administration ectopique de l’αKG ainsi que du citrate renforce l’altération de la différenciation terminale des précurseurs érythroïdes IDH1-/- ainsi que leur énucléation. Cette étude identifie donc un rôle crucial pour le métabolite αKG dans la régulation de la fonction mitochondriale et de l’OXPHOS, processus qui sont une condition sine qua non pour la différenciation des précurseurs érythroïdes au stade proérythroblaste. Nous montrons en outre que la suppression d’OXPHOS et la catalyse d’intermédiaires du TCA, substrats d’IDH1, sont requis pour les phases terminales de la différenciation érythroïde et l'énucléation.En conclusion, les résultats obtenus au cours de ma thèse mettent en évidence la nature dynamique des réseaux métaboliques qui régulent la progression des précurseurs érythroïdes tout au long des différents stades de la différenciation érythroïde. / Hematopoiesis is the process whereby hematopoietic stem cells (HSCs) proliferate and differentiate to all blood cell lineages. While HSC commitment is known to be regulated by cytokines, transcription factors, epigenetic modifiers and the HSC niche, our group found that specification of HSCs to the red cell lineage is dependent on glutamine metabolism. Glutaminolysis contributes to de novo nucleotide biosynthesis and to the generation of the alpha-ketoglutarate (αKG) TCA cycle metabolite (Oburoglu et al. 2014). Importantly though, erythroid differentiation is a unique process as each daughter cell is structurally and functionally different from its parent cell. Each division defines a stage of differentiation with the final division cycle resulting in the production of an enucleated reticulocyte which further matures to a biconcave erythrocyte. Thus, we hypothesized that progenitor metabolic networks change as a function of the erythroid differentiation stage and moreover, that they regulate the transition of progenitors from one stage of differentiation to the next.During my PhD, I assessed the metabolic alterations that occur as a function of the erythroid differentiation stage. We showed that at early stages of human red cell development, prior to terminal differentiation, hematopoietic progenitors exhibited an increased metabolic activity with a significantly higher level of oxidative phosphorylation (OXPHOS). This correlated with the increased generation of αKG and indeed, we found that ectopic αKG directly augmented OXPHOS in these progenitors. However, the terminal differentiation of erythroid precursors, characterized by the loss of mitochondrial mass and membrane potential, was associated with a decreased level of OXPHOS. Notably, ectopic αKG, which did not alter pro-erythroblast erythroid differentiation, severely attenuated terminal differentiation and enucleation. Conversely, an αKG antagonist (dimethyloxalyl glycine, DMOG) did not negatively impact on terminal differentiation or enucleation despite abrogating OXPHOS in erythroblasts.These data suggested that the production of αKG and its subsequent contribution to oxidative phosphorylation perturb red cell enucleation. We therefore downregulated isocitrate dehydrogenase I (IDH1), the cytosolic enzyme that catalyzes the conversion of isocitrate to αKG, by an shRNA approach in an attempt to decrease αKG levels. However, because IDH1 can catalyze both the forward and reverse reactions, its downregulation could also increase αKG levels. Indeed, we found that IDH1 knockdown resulted in a severe attenuation of terminal erythroid differentiation and enucleation. This effect was likely due to an imbalance in substrate availability––both ectopic αKG as well as citrate further decreased polychromatic to orthochromatic erythroblast differentiation and the subsequent enucleation of IDH1-knockdown erythroid precursors. Thus, the present study identifies a crucial role for the αKG metabolite in regulating mitochondrial function and oxidative phosphorylation, processes that are a sine qua non for erythroid precursors at the pro-erythroblast stage. We further show that terminal erythroid differentiation and enucleation requires OXPHOS suppression and the IDH1-mediated enzymatic catalysis of its TCA substrates.To conclude, the results generated during my PhD highlight the dynamic nature of the metabolic networks that regulate the progression of erythroid precursors through the distinct stages of erythroid differentiation.

Epigenetic control of gene expression by histone post-translational modifications (PTMs) is a complex process regulated by proteins that can 'read', 'write' or 'erase' these PTMs. The histone lysine demethylase (KDM) family of epigenetic enzymes remove methyl modifications from lysines on histone tails. The Jumonji C domain (JmjC) family is the largest family of KDMs. Investigating the scope and mechanisms of the JmjC KDMs is of interest for understanding the diverse functions of the JmjC KDMs in vivo, as well as for the application of the basic science to medicinal chemistry design. The work described in this thesis aimed to biochemically investigate the inhibitor and substrate diversity of the JmjC KDMs, it led to the identification of new inhibitors and substrates and revealed a potential combinatorial dependence between adjacent histone PTMs. Structure-activity relationship studies gave rise to an n-octyl ester form of IOX1 with improved cellular potency and selectivity towards the KDM4 subfamily. This compound should find utility as a basis for the development of JmjC inhibitors and as a tool compound for biological studies. The rest of this thesis focused on the biochemical investigations of potential substrates and inhibitors for KDM3A, a JmjC demethylase with varied physiological functions. Kinetic characterisation of reported KDM3A substrates was used as the basis for evaluations of novel substrates and inhibitors. Further studies found TCA cycle intermediates to be moderate co-substrate competitive inhibitors of KDM3A. Biochemical investigations were carried out to study potential protein-protein interactions of KDM3A with intraflagellar transport proteins (IFTs), non-histone proteins involved in the formation of sperm flagellum. Work then addressed the exploration of novel in vitro substrates for KDM3 (KDM3A and JMJD1C) mediated catalysis, including: methylated arginines, lysine analogues, acetylated and formylated lysines. KDM3A, and other JmjC KDMs, were found to catalyse novel arginine demethylation reaction in vitro. Knowledge gained from studies with unnatural lysine analogues was utilised to search for additional novel PTM substrates for KDM3A. These results constitute the first evidence of JmjC KDM catalysed hydroxylation of an Nε-acetyllysine residue. The H3 K4me3 position seems to be required for acetyllysine substrate recognition, implying a combinatorial effect between PTMs. Preliminary results provide evidence that JMJD1C, a KDM3 protein previously reported to be inactive, may catalyse deacetylation in vitro. An additional novel reaction, observed with both KDM3A and JMJD1C, is deformylation of N^ε-formyllysine residues on histone H3 fragment peptides. Interestingly, H3 K4 methylation was also observed to enhance the apparent deformylation of both KDM3A and JMJD1C catalysed reactions. Overall, findings in this thesis suggest that the catalytic activity of JmjC KDMs extends beyond lysine demethylation. In a cellular context, members of the KDM3 subfamily might provide a regulatory link between methylation and acylation marks. Such a link will further highlight the complex relationships between histone PTMs and the epigenetic enzymes that regulate them. The observed dependency of H3 K9 catalysis on H3 K4 methylation adds another layer of complexity to the epigenetic regulation by histone PTMs.

Este trabalho descreve o planejamento, desenvolvimento e validação de um modelo de software educacional. O aplicativo é um ambiente multimídia de ensino e aprendizagem do Metabolismo dos Glicídios e o Ciclo de Krebs, denominado e-Metabolismo: Glicídios e contém um jogo de seqüência para o ensino de Bioquímica, denominado Diagrama Metabólico Dinâmico Virtual. O estudo de teorias pedagógicas e a experiência em aulas com os alunos do curso de medicina da Fundação Faculdade Federal de Ciências Médicas de Porto Alegre apontou a necessidade de mudanças no ensino de Bioquímica com uso das novas tecnologias de informação e comunicação. A justificativa do uso de um jogo virtual como método de ensino tem por base os resultados obtidos com o uso de um jogo de seqüência lógica em tabuleiro, na Disciplina de Bioquímica. O desenvolvimento do e-Metabolismo: Glicídios, tendo como referência a prática pedagógica baseada na epistemologia genética Jean Piaget, incluiu no seu planejamento a escolha de ferramenta de programação para permitir a interação do usuário (aluno) com o ambiente. O produto utiliza amplamente recursos de multimídia e pode ser disponibilizado num servidor ou em forma de CD-ROM. O ambiente virtual possibilita a interação do aluno com o ambiente e com colegas e professores através de ferramentas como, por exemplo, acesso a e-mails, chats, fóruns, mapas conceituais e diário de bordo. Instrumentos de avaliação de software foram estudados e aplicados com alunos de Disciplinas de Bioquímica no sentido de validar o software e-Metabolismo tanto no que se refere aos aspectos técnicos como a aprendizagem do conteúdo pelos alunos. Experiências com o uso do software foram, primeiramente, realizadas com alunos do curso de Medicina da FFFCMPA e depois com alunos de outros cursos. O primeiro grupo de alunos que avaliaram o e-Metabolismo foi formado pelos monitores da Disciplina. Mapas conceituais, testes escritos e avaliação dos registros deixados pelos usuários no próprio software foram utilizados como instrumentos de avaliação do conhecimento dos alunos. O grau de satisfação com o uso do método de estudo, foi avaliado por um questionário, cujas respostas foram analisadas e categorizadas. Os resultados obtidos indicam que o ambiente apresenta interface de fácil acesso, desperta o interesse, possibilita ao aluno escolher de que maneira quer fazer o seu estudo sem prejuízos no seu desempenho e facilita o estudo, sendo, portanto, considerado válido como instrumento educacional. Por se tratar de um ambiente dinâmico, deve ser constantemente atualizado, e a versão atual contém as modificações sugeridas por professores e alunos, facilitando o uso na Internet e o acompanhamento do aluno. / This work describes the planing, the development and the validation of a game-like educational software. This multimedia ambient was designed for the study of carbohydrates metabolic pathways and the Krebs's Cycle, called e-Metabolism: carbohydrates, and contains the sequential game, called Virtual Dynamic Metabolic Diagram. The study of pedagogical theories and experiments in classroom with medicine students of the “Fundação Faculdade Federal de Ciências Médicas de Porto Alegre”, pointed the necessity of changes in Biochemistry courses, involving new technologies of information and communication. The use of a game-like software as a tool for teaching is based on experiments related to the use of tray games at Biochemistry courses. The development of the e-Metabolism took as a reference the integrationists’ pedagogical practice, based on Jean Piaget's concepts, related to genetic epistemology and constructivism, yet allowing the professors to choose the teaching method they wish to use. This product integrates multimedia resources extensively, and can be used in computer networks or in the format of a CD-ROM. In the virtual environment students will be able to interact with the environment as well as with classmates and professors through such tools as chats, forums, concept maps and notepads. Software ’s evaluation Instruments were studied and applied with undergraduate students of Biochemistry classes in the way to value the eMetabolism software in its technical aspects and student’s content learning aspects. Conceptual maps, written tests and evaluation of user’s registers realized with this software where used as evaluation instruments of students knowledge. The level of satisfaction was evaluated by a questionnaire, which answers had been analyzed and categorized. The results show that the e-Metabolism is easy to use, awakes the interest and facilitates the study, improving the student performance and can be considered a valid educational instrument. Since this is a dynamic ambient and is constantly actualized, the current version contains the changes suggested by teachers and students, making easier to use it at the Internet and to do a better analysis of the student’s learning.

Este trabalho descreve o planejamento, desenvolvimento e validação de um modelo de software educacional. O aplicativo é um ambiente multimídia de ensino e aprendizagem do Metabolismo dos Glicídios e o Ciclo de Krebs, denominado e-Metabolismo: Glicídios e contém um jogo de seqüência para o ensino de Bioquímica, denominado Diagrama Metabólico Dinâmico Virtual. O estudo de teorias pedagógicas e a experiência em aulas com os alunos do curso de medicina da Fundação Faculdade Federal de Ciências Médicas de Porto Alegre apontou a necessidade de mudanças no ensino de Bioquímica com uso das novas tecnologias de informação e comunicação. A justificativa do uso de um jogo virtual como método de ensino tem por base os resultados obtidos com o uso de um jogo de seqüência lógica em tabuleiro, na Disciplina de Bioquímica. O desenvolvimento do e-Metabolismo: Glicídios, tendo como referência a prática pedagógica baseada na epistemologia genética Jean Piaget, incluiu no seu planejamento a escolha de ferramenta de programação para permitir a interação do usuário (aluno) com o ambiente. O produto utiliza amplamente recursos de multimídia e pode ser disponibilizado num servidor ou em forma de CD-ROM. O ambiente virtual possibilita a interação do aluno com o ambiente e com colegas e professores através de ferramentas como, por exemplo, acesso a e-mails, chats, fóruns, mapas conceituais e diário de bordo. Instrumentos de avaliação de software foram estudados e aplicados com alunos de Disciplinas de Bioquímica no sentido de validar o software e-Metabolismo tanto no que se refere aos aspectos técnicos como a aprendizagem do conteúdo pelos alunos. Experiências com o uso do software foram, primeiramente, realizadas com alunos do curso de Medicina da FFFCMPA e depois com alunos de outros cursos. O primeiro grupo de alunos que avaliaram o e-Metabolismo foi formado pelos monitores da Disciplina. Mapas conceituais, testes escritos e avaliação dos registros deixados pelos usuários no próprio software foram utilizados como instrumentos de avaliação do conhecimento dos alunos. O grau de satisfação com o uso do método de estudo, foi avaliado por um questionário, cujas respostas foram analisadas e categorizadas. Os resultados obtidos indicam que o ambiente apresenta interface de fácil acesso, desperta o interesse, possibilita ao aluno escolher de que maneira quer fazer o seu estudo sem prejuízos no seu desempenho e facilita o estudo, sendo, portanto, considerado válido como instrumento educacional. Por se tratar de um ambiente dinâmico, deve ser constantemente atualizado, e a versão atual contém as modificações sugeridas por professores e alunos, facilitando o uso na Internet e o acompanhamento do aluno. / This work describes the planing, the development and the validation of a game-like educational software. This multimedia ambient was designed for the study of carbohydrates metabolic pathways and the Krebs's Cycle, called e-Metabolism: carbohydrates, and contains the sequential game, called Virtual Dynamic Metabolic Diagram. The study of pedagogical theories and experiments in classroom with medicine students of the “Fundação Faculdade Federal de Ciências Médicas de Porto Alegre”, pointed the necessity of changes in Biochemistry courses, involving new technologies of information and communication. The use of a game-like software as a tool for teaching is based on experiments related to the use of tray games at Biochemistry courses. The development of the e-Metabolism took as a reference the integrationists’ pedagogical practice, based on Jean Piaget's concepts, related to genetic epistemology and constructivism, yet allowing the professors to choose the teaching method they wish to use. This product integrates multimedia resources extensively, and can be used in computer networks or in the format of a CD-ROM. In the virtual environment students will be able to interact with the environment as well as with classmates and professors through such tools as chats, forums, concept maps and notepads. Software ’s evaluation Instruments were studied and applied with undergraduate students of Biochemistry classes in the way to value the eMetabolism software in its technical aspects and student’s content learning aspects. Conceptual maps, written tests and evaluation of user’s registers realized with this software where used as evaluation instruments of students knowledge. The level of satisfaction was evaluated by a questionnaire, which answers had been analyzed and categorized. The results show that the e-Metabolism is easy to use, awakes the interest and facilitates the study, improving the student performance and can be considered a valid educational instrument. Since this is a dynamic ambient and is constantly actualized, the current version contains the changes suggested by teachers and students, making easier to use it at the Internet and to do a better analysis of the student’s learning.