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Les Troubles du Spectre Autistique (TSA) sont caractérisés par deux principaux symptômes : des difficultés de communication sociale et des comportements stéréotypés et intérêts restreints. Les TSA touchent 5 fois plus les garçons que les filles et une augmentation de la prévalence exponentielle et continue a été observée aux États-Unis ces dernières décennies. Cette augmentation ne peut s’expliquer par les facteurs génétiques à eux seuls qui ne représentent que 5 à 15% des cas de TSA. Il est donc indispensable d’identifier de potentiels facteurs de risque environnementaux des TSA. Le but de ce travail est d’étudier différents facteurs environnementaux potentiellement modifiables dans le développement de phénotypes autistiques dans différents modèles précliniques des TSA. Les objectifs spécifiques sont : (i) caractériser les effets neurocomportementaux provoqués par une exposition périnatale simultanée à 5 perturbateurs endocriniens parmi les plus prévalent dans notre environnement quotidien (DEHP, DBP, DiNP, BDE-47, BDE-99) à de faibles doses pertinentes pour l’exposition humaine, (ii) identifier les effets neurocomportementaux associés à une altération périconceptionnelle du microbiote maternelle (iii) déterminer les effets neurocomportementaux associés à une altération périconceptionnelle du métabolisme monocarboné. Les résultats présentés dans cette thèse démontrent le potentiel de chacun de ces facteurs environnementaux d’altérer le développement cérébral fœtal. Chaque condition expérimentale a provoqué l’apparition de traits autistiques chez les rats, avec des spécificités comportementales pour chaque exposition développementale. Des déficits d’interactions sociales ont été observés dans chaque situation expérimentale, associés soit à de l’anxiété, de l’hyperactivité, des altérations d’intégration sensorimotrice, et/ou des stéréotypies. Cela nous force à considérer les TSA comme une pathologie aux multiples facettes où l’hétérogénéité des tableaux cliniques est représentative de l’hétérogénéité des causes possibles. La multitude des interactions environnementales courantes possibles avec l’épigénome pourrait être à la base de la grande diversité observée dans la sévérité des symptômes et / ou des comorbidités des TSA. Ce travail ouvre des perspectives futures de prévention ciblée des TSA fondées sur de potentielles modifications de l’environnement comme la réduction de l’exposition aux perturbateurs endocriniens, ou des supplémentations en donneurs monocarbonés (e.g. acide folique) et/ou probiotiques.

Toxoplasma gondii ist ein weit verbreiteter einzelliger Parasit, der fast alle warmblütigen Organismen infizieren kann. Asexuelle Fortpflanzung des Parasiten in seiner Wirtszelle wird durch aufeinanderfolgende lytische Zyklen erreicht, was die Bereitstellung einer signifikanten Menge an Energie und Biomasse erforderlich macht. Diese Arbeit zeigt, dass Glukose und Glutamin die beiden wichtigsten physiologischen Nährstoffe für die Synthese von Makromolekülen (ATP, Nukleinsäure, Proteine und Lipide) in T. gondii sind. Die Verfügbarkeit einer der beiden Kohlenstoffquellen reicht aus, um das Überleben des Parasiten sicherzustellen. Der Parasit kann durch Erhöhen des Flusses von Glutamin-abstammendem Kohlenstoff durch den TCA-Zyklus und durch gleichzeitige Aktivierung der Gluconeogenese, eine stetige Biogenese von ATP und Biomasse zur Wirtszellinvasion und Replikation gewährleisten, bzw. der genetischen Deletion des Glukosetransporters entgegenwirken. Der Wachstumsdefekt in der Glykolyse-Mutante wird durch eine kompromittierte Synthese von Lipiden verursacht, die durch Glutamin nicht ausgeglichen werden kann. Die Zugabe von exogenem Acetat kann diesen Wachstumsdefekt allerdings kompensieren. In dieser Arbeit konnten darüber hinaus zwei unterschiedliche Phosphoenolpyruvat-Carboxykinase (PEPCK) Enzyme im Parasiten identifiziert werden, von denen eines im Mitochondrium lokalisiert ist (TgPEPCKmt), während das andere Protein nicht in Tachyzoiten (TgPEPCKnet) exprimiert wird. Parasiten mit intakter Glykolyse können die Deletion von TgPEPCKnet, als auch die genetische Deletion von TgPEPCKmt tolerieren, was ihre Redundanz für das Überleben der Tachyzoiten zeigt. TgPEPCKnet kann auch in der Glykolyse-defizienten Mutante deletiert werden, während TgPEPCKmt für das Überleben des Parasiten in dieser Mutante essentiell ist. Dies zeigte sich durch ein konditionelles Knockdown von TgPEPCKmt, das zu einer Inhibierung des Wachstums des Parasiten führte. / Toxoplasma gondii is a widespread protozoan parasite, infecting nearly all warm-blooded organisms. Asexual reproduction of the parasite within its host cells is achieved by consecutive lytic cycles, which necessitates biogenesis of significant energy and biomass. This work shows that glucose and glutamine are the two major physiologically important nutrients used for the synthesis of macromolecules (ATP, nucleic acid, proteins and lipids) in T. gondii, and either of them is sufficient to ensure the parasite survival. The parasite can counteract genetic ablation of its glucose transporter by increasing the flux of glutamine-derived carbon through the TCA cycle and by concurrently activating gluconeogenesis, which guarantee a continued biogenesis of ATP and biomass for host-cell invasion and parasite replication, respectively. Growth defect in the glycolysis-impaired mutant is caused by a compromised synthesis of lipids, which cannot be counterbalanced by glutamine, but can be restored by acetate. Consistently, supplementation of parasite cultures with exogenous acetate can amend the lytic cycle of the glucose transport mutant. Furthermore, this work revealed two discrete phosphoenolpyruvate carboxykinase (PEPCK) enzymes in the parasite, one of which resides in the mitochondrion (TgPEPCKmt), whereas the other protein is not expressed in tachyzoites (TgPEPCKnet). Parasites with an intact glycolysis can tolerate genetic deletions of TgPEPCKmt as well as of TgPEPCKnet, indicating their nonessential roles for the tachyzoite survival. TgPEPCKnet can also be ablated in glycolysis-deficient mutant, whereas TgPEPCKmt is refractory to deletion. In accord, the lytic cycle of a conditional mutant of TgPEPCKmt in the glycolysis-impaired strain was aborted upon induced repression of the mitochondrial isoform, demonstrating its essential role for the glucose-independent survival of tachyzoites.

Der Citratzyklus (TCA) ist einer der bedeutendsten Stoffwechselwege für alle lebenden Organismen. Trotz der zentralen Rolle dieses Prozesses im Pflanzenmetabolismus ist er nur relativ wenig untersucht worden. In dieser Arbeit berichte ich über die Produktion und die funktionale Analyse von Tomatenpflanzen (Solanum lycopersicum), die unabhängig eine leicht eingeschränkte Aktivität der mitochondrialen Citrat-Synthase (CS) und zweier Isocitrat-dehydrogenasen (mitochondriale NAD-IDH und cytosolische NADP-ICDH) zeigen. Die transgene Pflanzen wiesen mehrheitlich keine erkennbare Veränderung eines Wachstumphänotyps auf. Obwohl die photosyntetische Leistung keine Änderungen gezeigt hatte, war die mitochondriale Respiration gestiegen, begleitet von einem reduzierten Kohlenstoff-fluss durch den Citratzyklus. Darüber hinaus waren die CS Pflanzen charakterisiert durch wesentliche Änderungen im Blattmetabolismus, einschließlich eines eingeschränkten Niveaus des photosynthetischen Pigments und Zwischenprodukten des Citratzyklus zusammen mit einer Akkumulation von Nitraten, verschiedenen Aminosäuren und Stärken. Zusammengefasst deuten diese Ergebnisse auf eine Einschränkung der Nitrat-Aufnahme hin. Das mit Hilfe von TOM1 Mikroarrays und quantitativer RT-PCR durchgeführte Transcript-profiling hat gezeigt, dass die fehlende Aktivität der mitochondrialen CS teilweise von einer gestiegenen, peroxisomalen CS Isoform ausgeglichen wird. Die metabolische Verschiebung ergab eine Verstärkung der photorespiratorischen Leistung, die vermutlich eine ausgleichende Rolle in der Produktion organischer Säuren und der Wiederherstellung der Redox-Balance spielt. Interessantenweise war die metabolische Antwort von Blättern auf Stickstoffmangel in NADP-ICDH Pflanzen dramatischer als in NAD-IDH Pflanzen, was darauf hindeutet, dass die cytosolische Isoform der Hauptlieferant von 2-Oxoglutarat im Tomatenmetabolismus sein könnte. / Although the TCA cycle is a respiratory metabolic pathway of central importance for all living organisms, relatively few molecular physiological studies of plants were performed to date. Here, I report the generation and functional analysis of tomato plants (Solanum lycopersicum) independently displaying mildly limited activity of mitochondrial citrate synthase (CS) and two isocitrate dehydrogenases, namely mitochondrial NAD-IDH and cytosolic NADP-ICDH. The transgenic plants revealed minor phenotypic alterations. Although the leaf photosynthetic performance was largely unaltered, the changes in mitochondrial respiration and carbon flux through the TCA cycle were observed. Moreover, the plants were characterized by significant modifications in the leaf metabolic content and in maximal catalytic activities of several enzymes involved in primary C and N metabolism. These results hint towards limitations in nitrate assimilation pathway. The transcript profiling performed by utilizing TOM1 microarrays and quantitative RT-PCR approach revealed that the deficiency in mitochondrial CS activity was partially compensated by up-regulation of peroxisomal CS isoform. The limitations in the activities of isocitrate dehydrogenases resulted in up-regulation of the photorespiratory pathway, which presumably played a compensatory role in supporting organic acid production and re-establishing redox balance in the transgenic leaves. Interestingly, the leaf metabolic response towards nitrogen starvation conditions was far more dramatic in NADP-ICDH transgenic plants than NAD-IDH plants, hinting that the cytosolic isoform may be the major 2-oxoglutarate supplier in tomato metabolism.

Paraburkholderia phytofirmans souche PsJN est une bactérie endophyte capable d’induire une promotion de croissance de la vigne et de lui conférer une tolérance vis-à-vis des basses températures. Puisque la bactérie PsJN induit également une tolérance de la vigne contre la pourriture grise causée par le champignon Botrytis cinerea, l’objectif de ce travail était de caractériser les mécanismes de la résistance induite par P. phytofirmans suite à une infection par B. cinerea chez la vigne.Nos résultats indiquent que l’inoculation des racines de vitroplants par la bactérie PsJN entraîne des modifications du métabolisme phénolique au niveau des plantules bactérisées. En effet, des tanins d’origine gallique et ellagique sont accumulés au niveau des racines en réponse à PsJN alors qu’une accumulation de lignine a été observée dans la tige et d’anthocyanes dans les feuilles. De même, PsJN induit une résistance contre B. cinerea en potentialisant l'expression de gènes de défense dépendants de l’acide salicylique et de l’acide jasmonique dans les feuilles de vigne. Parallèlement, une accumulation de callose et d’H2O2 a été observée ainsi qu’une meilleure mobilisation des glucides dans les plantules bactérisées, corrélée à une réduction des dommages de l’activité photosynthétique.Suite aux résultats obtenus avec le modèle vitroplant, nous avons optimisé la bactérisation des boutures fructifère et évaluer l’impact de cette bactérie sur la résistance des inflorescences contre B. cinerea. Les résultats présentés dans ce travail permettent d’envisager l’utilisation de la bactérie PsJN comme agent de lutte biologique. / Paraburkholderia phytofirmans strain PsJN is an endophytic beneficial bacterium able to promote the growth of grapevine and to induce the plant resistance to biotic and abiotic stresses. Indeed, the interaction between PsJN and grapevine confers to the grapevine a protection towards cold stress (4 °C), by reducing damages on the photosynthetic system and modulating the carbon metabolism. The PsJN strain also induces a protection against gray mold caused by Botrytis cinerea. In this context, the aim of this work was to better characterize this P. phytofirmans strain PsJN-induced resistance in grapevine against B. cinerea.Our results indicate that the inoculation of in vitro-plantlets roots with PsJN altered the phenolic metabolism in bacterized-plantlets. Gallic and ellagic derived tannins are clearly accumulated in roots of bacterized-plantlets whereas an accumulation of lignin in stems and anthocyanins in leaves was observed. We also showed that PsJN induces a protection against B. cinerea by priming the expression of genes involved in salicylic acid and jasmonic acid pathways. In parallel, an accumulation of callose and H2O2, as well as a better mobilization of carbohydrates in bacterized plantlets were observed. In addition to the defense mechanisms setting up by plants, we demonstrated that PsJN reduces in vitro and in vivo development of B. cinerea, in a dose-dependent manner.Following results obtained with the in vitro-plantlets model, we optimized the protocol of bacterization of grapevine fruiting cuttings by the PsJN strain, and evaluate the impact of the bacteria on to induce the resistance of inflorescences to B. cinerea. Our results provide new insights for the use of PsJN and suggest its commercial use as a biological control agent.

Numerous studies have investigated how bulk lipid metabolism is influenced in obesity and in particular how the composition of triglycerides found in the cytosol change with increased adipocyte expansion. However, in part reflecting the analytical challenge the composition of cell membranes, and in particular glycerophospholipids, an important membrane component, have been seldom investigated. Cell membrane components contribute to a variety of cellular processes including maintaining organelle functionality, providing an optimized environment for numerous proteins and providing important pools for metabolites, such as choline for one-carbon metabolism and S-adenosylmethionine for DNA methylation. Here, I have conducted a comprehensive lipidomic and transcriptomic study of white adipose tissue in mice that become obese either through genetic modification (ob/ob genotype), diet (high-fat diet) or a combination of the two across the life course. Specifically, I demonstrated that the changes in triglyceride metabolism that dominate the overall lipid composition of white adipose tissue were distinct from the compositional changes of glycerophospholipids. These latter lipids became more unsaturated to maintain the fluidity and normal function of the membrane in the initiation of obesity but then turned saturated after long-term administration of HFD and aging. This suggests that while triglycerides within the adipose tissue may be a relatively inert store of lipids, the compositional changes occur in cell membranes with more far-reaching functional consequences in both obesity and aging. The two-phase change of phospholipids can be correlated well with transcriptional and one-carbon metabolic changes within the adipocytes. The transcriptomic study demonstrated that the lipid metabolic pathways regulated by the peroxisome, AMPK, insulin and PPARγ signaling were activated in the initiation of obesity but inhibited in the adipose tissue of old ob/ob mice along with up-regulated inflammation pathways. The brown and white adipose tissue of PPARα-knock-out mice were also studied by lipidomic tools to get a deeper understanding of the effect of the peroxisome and PPAR system on adipose tissue and lipid metabolism during obesity. Most of the lipids were increased and became more saturated and shorter in adipose tissues of PPARα null mice, which is in good accordance with the results of the former animal study. In conclusion, my work using different rodent models and multi-omics techniques demonstrated a protective metabolic mechanism activated in the initiation but impaired at the end of the processes of obesity and aging, which could be an explanation of the similarity of obesity and aging in terms of high incidence of the metabolic syndrome and related diseases.

Submitted by Maria Beatriz Vieira (mbeatriz.vieira@gmail.com) on 2017-06-22T16:51:34Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) tese_junior_borella.pdf: 3753354 bytes, checksum: 6f336478c32b30a019b543871b0367e6 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-06-22T20:38:33Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) tese_junior_borella.pdf: 3753354 bytes, checksum: 6f336478c32b30a019b543871b0367e6 (MD5) / Made available in DSpace on 2017-06-22T20:38:33Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) tese_junior_borella.pdf: 3753354 bytes, checksum: 6f336478c32b30a019b543871b0367e6 (MD5) Previous issue date: 2015-05-06 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / O alagamento é um fator ambiental comum que causa deficiência de oxigênio às plantas, levando a uma inibição da respiração e redução do status energético celular, desencadeando uma série de mudanças no metabolismo do carbono e do nitrogênio. Além disso, alterações no fluxo de elétrons da cadeia de transporte de elétrons mitocondrial e cloroplastídica levam a produção de espécies reativas de oxigênio (EROs) que podem ocasionar vários danos ao metabolismo celular. No entanto, a aplicação exógena de nitrato tem sido reportada por promover efeitos benéficos em muitas espécies de plantas sob condições de hipóxia. Embora muitos estudos tenham sido envidados com soja, pouco se sabe a respeito das alterações metabólicas primárias do carbono e do nitrogênio que permitem diferenciar genótipos contrastantes ao alagamento e os efeitos no sistema antioxidante pela aplicação exógena de nitrato nas plantas. Considerando o exposto, os objetivos deste trabalho foram: I – avaliar mudanças no metabolismo do carbono e do nitrogênio e sua relação com a enzima alanina aminotransferase (AlaAT) em genótipos de soja nodulada; II – verificar possíveis efeitos benéficos no metabolismo antioxidante em plantas cultivadas na presença de nitrato (plantas não-noduladas) e na ausência de nitrato (plantas noduladas). Para isso, dois experimentos foram conduzidos em casa de vegetação com plantas de soja (Glycine max (L.) Merril) sob condições naturais de luz e temperatura. Experimento I: plantas noduladas de soja, nutridas na ausência de N mineral (Fundacep 53 RR – tolerante e BRS Macota – sensível) foram cultivadas em vermiculita e transferidas para sistema hidropônico, no estádio reprodutivo R2. O sistema radicular das plantas foi submetido à hipóxia pelo borbulhamento de nitrogênio gasoso na solução nutritiva diluída 1/3 da concentração normal, por 24 e 72 h. Para recuperação, após 72 h de hipóxia as plantas retornaram para vermiculita por 24 e 72 h. Foram avaliados, em raízes e nódulos, metabólitos fermentativos e ácidos orgânicos (GC-MS), aminoácidos (HPLC), expressão relativa dos genes (RT-PCR) e atividade da enzima AlaAT. Fundacep 53 RR acumulou mais teores de piruvato e lactato que BRS Macota e embora a composição de aminoácidos não tenha diferido entre os genótipos, foi observado uma ligação entre a glicólise e o ciclo dos ácidos tricarboxílicos via indução dos genes e atividade da AlaAT, que, posteriormente, levou ao acúmulo de succinato em raízes de Fundacep 53 RR, podendo aumentar o ganho energético em relação à BRS Macota sob hipóxia. Experimento II: A condução experimental adotada foi semelhante ao experimento I, no entanto conduzido com plantas noduladas e não-noduladas (nutridas com nitrato) de soja, de ambos os genótipos. Foi avaliado o sistema antioxidante em raízes e folhas através da atividade das enzimas superóxido dismutase (SOD), ascorbato peroxidase (APX), catalase (CAT), glutationa redutase (GR), guaiacol peroxidase (GPOD) e glutationa S-transferase (GST), o conteúdo de ascorbato reduzido e ascorbato total, bem como conteúdo de superóxido (O2), peróxido de hidrogênio (H2O2) e peroxidação de lipídeos. O sistema antioxidante foi fortemente induzido nas raízes das plantas nutridas com nitrato de ambos os genótipos, com elevada atividade de SOD, APX, CAT, GR e GPOD, bem como o aumento do conteúdo de ascorbado reduzido e total e diminuição da produção de EROs em condições de hipóxia e de recuperação, enquanto que nas folhas de plantas noduladas e não-noduladas foi observado um ligeiro aumento nos componentes enzimáticos e não enzimáticos antioxidantes. O nitrato exerce efeitos benéficos em plantas de soja em condições de hipóxia e consequentemente na recuperação por induzir o sistema antioxidante nas raízes, permitindo modular os possíveis danos oxidativos causados pela produção de EROs, além de poder prolongar a tolerância dessas plantas. / Waterlogging is a common environmental stress which causes oxygen deprivation in plants leading to an inhibition of the mitochondrial respiration. It leads to a reduction of cellular energy status triggering changes at different levels of carbon and nitrogen metabolism. In addition, it leads to electron scape from the mitochondrial and chloroplast electron transport chain, producing reactive oxygen species (ROS) which cause severe oxidative damage to cells. However, exogenous nitrate supply has been reported to promoting beneficial effects in several plant species under hypoxic conditions. Although many studies have been carried out with soybean, a little is known about the primary metabolic changes in carbon and nitrogen metabolism, which may differ between tolerant and sensitive plant genotypes in response to waterlogging and the effects on antioxidant system in nitrate-supplied plants in comparison to non-nitrate-supplied plants. Thus, the aims of this study were: I – to evaluate the hypoxia-induced alterations of carbon and nitrogen metabolism and its relation with alanine aminotransferase (AlaAT) enzyme in nodulated soybean genotypes; II – to verify possible beneficial effects on antioxidant metabolism in nitrate-supplied plants (non-nodulated plants) in comparison to plants growing in absence of nitrate (nodulated plants). For that, two experiments were carried out in greenhouse under natural light and temperature conditions. Experiment I: Nodulated soybean plants (Fundacep 53 RR – tolerant and BRS Macota – sensitive) were grown in vermiculite and transferred to hydroponic system at reproductive stage. Root system was subjected to hypoxia by flushing N2 gas into the solution for 24 or 72 h. For the recovery, after 72 h in hypoxia, plants returned to normoxic conditions by transferring back to vermiculite for 24 and 72 h. Root and nodule organic acids and amino acids were analysed by gas chromatography-mass spectrometry and high-performance liquid chromatography, respectively. Relative expression of AlaAT (qRT-PCR) and AlaAT activity were also verified in both genotypes. Plants of Fundacep 53 RR and BRS Macota genotypes responded distinctly upon hypoxia. Fundacep 53 RR presented higher pyruvate and lactate accumulation than BRS Macota, which is indicative of higher glycolytic and fermentation rates in root tissues. Furthermore, Fundacep 53 RR responds more effectively to the recovery by restoring pre-hypoxic levels of the metabolites. Although the amino acid composition did not differ between the genotypes, there was a clear link between glycolysis and the TCA via increase of gene expression and activity of AlaAT enzyme by leading a succinate accumulation in Fundacep 53 RR, wich represents a metabolic advantage compared to BRS Macota under hypoxic stress. Experiment II: was carried out in a similar way of Experiment I, however with plants growing in presence (non-nodulated) and absence (nodulated) of nitrate, for both soybean genotypes. Superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), guayacol peroxidase (GPOD) and glutathione S-transferase (GST) enzymes; reduced ascorbate and ascorbate redox state; superoxide content (O2•-), hydrogen peroxide (H2O2) and lipid peroxidation were analysed in roots and leaves of both soybean genotypes. Antioxidative system was strongly induced in roots of nitrate-supplied plants of both genotypes, with high activity of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR) and guayacol peroxidase (GPOD), as well as increased ascorbate reduced and ascorbate redox state and decreased ROS production under hypoxia and recovery, while in leaves of nodulated and non-nodulated plants a slight increase on antioxidant system was observed. Furthermore, the results did not show tolerance differences between the genotypes. Nitrate exerts beneficial effects in soybean plants under hypoxic conditions and consequent recovery by inducing the antioxidant system manly in roots, to cope possible oxidative damage caused by ROS production and also can postpone the effects of hypoxia in both genotypes.

Apparues il y a environ trois milliards d'années, les cyanobactéries ont façonné notre planète, en produisant l’atmosphère oxygénique. De nos jours, les cyanobactéries sont les organismes photosynthétiques les plus abondants dans notre environnement, elles assurent environ 30 à 40% de la production d'O2, et de la consommation du CO2 par les océans et constituent le premier maillon de la chaîne alimentaire. A part la photosynthèse, leur métabolisme est encore très mal connu. Ainsi, pour mieux comprendre le métabolisme cyanobactérien et proposer des stratégies de reprogrammation, il est primordial de développer des méthodes analytiques permettant l’étude globale de leur métabolisme en réponse à des variations de conditions environnementales et de stress. La cyanobactérie modèle Synechocystis PCC6803 convient parfaitement à ce type d’analyse. En effet, Synechocystis est un unicellulaire, hétérotrophe facultative capable de se développer en eau douce ou saumâtre et à un pH alcalin. Synechocystis possède un petit génome d’environ 4.0 Mb entièrement séquencé et facilement manipulable grâce aux outils développés au laboratoire. Son génome prédit l'existence d'un métabolisme carboné complexe mais encore peu étudié. Mon travail de thèse est centré sur cette analyse par la combinaison de deux approches, la génomique fonctionnelle et la métabolomique. Durant ma thèse en collaboration avec le LEMM dirigé par Christophe Junot iBiTec-S/SPI, j’ai développé un protocole d’extraction des métabolites de Synechocystis, ainsi qu’une méthode d’analyse métabolomique par couplage de la chromatographie liquide à la spectrométrie de masse LTQ-Orbitrap à haute résolution. L’application de cette nouvelle méthode analytique m’a permis d’étudier l’influence de la lumière et du glucose sur le métabolisme de Synechocystis. Ainsi, j’ai montré que Synechocystis cultivée en présence du glucose reprogramme fortement son métabolisme. Parmi les résultats très intéressants, j’ai montré que le glucose engendre un stress oxydant. Chez tous les organismes, une forte activité du métabolisme carboné entraîne la production de métabolites toxiques tels que le méthyglyoxal (MG). Le MG modifie irréversiblement de nombreuses bio-molécules. Dans le cadre de ma thèse, j’ai commencé à m’intéresser à l'effet du MG sur la physiologie et le métabolisme de Synechocystis. J'ai construit 25 mutants KO pour les gènes de la glycolyse et du métabolisme du glycérol permettant de moduler la concentration intracellulaire de MG et également les gènes impliqués dans les voies de détoxication du MG dont celle dépendante de la synthèse du GSH (la voie des glyoxalases). J’ai pu montrer que les gènes responsables de la synthèse du GSH sont essentiels à la viabilité cellulaire. Je suis parvenu toutefois à obtenir un mutant déplété de gshB et ne produisant plus de GSH à un niveau détectable. En faisant une analyse métabolomique approfondie, j’ai mis en évidence pour la première fois que Synechocystis était capable produire deux tripeptides non-thiolés analogues structuraux du GSH; l’acide ophthalmique et l’acide norophthalmique identifiés jusqu’à présent uniquement chez les mammifères. La comparaison des métabolomes de culture de souches sauvage, ou dépletées en gshA, gshB ou ggt, a permis de montré que ces analogues sont synthétisés par les mêmes enzymes que le GSH à savoir GshA et GshB. Par ailleurs, une autre molécule anti-oxydante dont la synthèse est connue chez quelques champignons et qui s’accumule chez l’Homme par l’apport alimentaire a également été observée. / Cyanobacteria are fascinating microorganisms. They are among the oldest life forms, regarded as the progenitors of the oxygenic photosynthesis and plant chloroplast. Furthermore, cyanobacteria have evolved as the largest and most diverse groups of bacteria in colonizing most marine and fresh waters, as well as soils. An important reason for the hardness of cyanobacteria is their successful combination of effective metabolic pathways driven by their efficient photosynthesis that uses nature's most abundant resources, solar energy, water and CO2, to produce a large part of the Planet's oxygenic atmosphere and organic assimilates for the food chain. Hence, cyanobacteria are receiving a growing attention because of their potential for the carbon-neutral production of biofuels and bioplastics. To better understand cyanobacteria and turn their biotechnological potentials into an industrial reality, we need to develop robust protocols for global analysis of their metabolism and its responses to environmental stresses. The model cyanobacterium Synechocystis PCC6803 is well suited for this purpose. Synechocystis is a basic organism, i.e. unicellular, which grows well (i) in fresh- and marine-waters; (ii) in the presence of glucose that can compensate for the absence of light; and (iii) at high pH that prevents microbial contaminations. Furthermore, Synechocystis harbors a small sequenced genome (about 4.0 Mb), which can be easily manipulated. In the present work, we developed a robust protocol for metabolome analyses of Synechocystis, using liquid chromatography (LC) for metabolite separation, coupled to a LTQ-Orbitrap mass spectrometer that provides high sensitivity and resolution, accurate mass measurements, and structural informations with MS/MS or sequential MSn experiments that facilitate metabolite identification. Consequently, we applied the PFPP-LC/MS method to analyze the metabolome of Synechocystis growing under various conditions of light and glucose, which strongly influence cell growth. We found that glucose increases glucose storage and catabolism, while it decreases the Calvin-Benson cycle that consumes photosynthetic electrons for CO2 assimilation. Depending on light and glucose availabilities, this global metabolic reprogramming can generate an oxidative stress, likely through the recombination of the glucose-spared electrons with the photosynthetic oxygen thereby producing toxic reactive oxygen species. Furthermore, we studied the metabolism of an endogenous toxic the méthylglyoxal and its main catabolic pathway going through the glyoxalases system glutathione dependent.

La réplication de l’ADN est une fonction cellulaire responsable de la duplication du matériel génétique. Elle est assurée par un complexe protéique appelé réplisome. Ce processus est hautement régulé en fonction des conditions de croissance cellulaire. Durant cette thèse je me suis intéressé principalement au contrôle de la réplication par le Métabolisme Central Carboné (MCC) et, dans une moindre mesure, au fonctionnement du réplisome chez la bactérie modèle Bacillus subtilis. J’ai analysé la réplication de l’ADN dans des mutants métaboliques, par deux techniques ; la QPCR et la cytométrie en flux. Mes analyses révèlent que la réplication de l’ADN est dérégulée dans des cellules mutées dans les cinq dernières réactions de la glycolyse et dans celles affectées dans des réactions connectant cette petite région du métabolisme aux autres réactions du MCC (haut de la glycolyse, voie des pentoses phosphate et cycle de Krebs) et au milieu extérieur (voies overflow qui éliminent les métabolites du MCC produits en excès). J’ai constaté que dans ces mutants la réplication commence plutôt et dure plus longtemps que dans une souche sauvage. L’ensemble de ces résultats montre que les réactions situées au cœur du MCC sont importantes pour assurer un bon contrôle temporel de la réplication. J’ai aussi établi que le ppGpp, une petite molécule fonctionnant comme une alarmone de l’état nutritionnelle des cellules, ne joue pas un rôle déterminant dans le contrôle de la réplication par le métabolisme dans des cellules à l’état d’équilibre. L’ensemble de nos connaissances actuelles sur les réplisomes repose essentiellement sur les données accumulées à partir de la dissection du réplisome de la bactérie modèle Escherichia coli et des phages T4 et T7. Chez Bacillus subtilis, deuxième modèle bactérien le mieux connu et représentant des Gram+ à faible GC%, il existe deux ADN polymérases essentielles à la réplication : PolC et DnaE. Nous avons montré que DnaE, comme PolC, fait partie du réplisome. Nos études fournissent une explication moléculaire à la spécialisation de DnaE dans la synthèse du brin d’ADN discontinu. En conclusion, nos résultats montrent que les réplisomes bactériens ont beaucoup plus évolué qu’attendu tant dans leur composition protéique que dans leur organisation et leur fonctionnement. Ils montrent également, et pour la première fois, que le contrôle temporel de la réplication dépend de réactions situées au cœur du MCC chez B. subtilis. Ces données et d’autres de la littérature suggèrent que cette propriété pourrait être universelle et pourrait jouer un rôle important dans la carcinogenèse. / DNA replication is a central cellular function for the duplication of the genetic material. A protein complex that is called replisome carries out this function. The process of replication is highly regulated with respect to cell growth conditions. During my thesis I was primarily interested in the control of replication by the central carbon metabolism (CCM) and to a lesser extent, to the functioning of the replisome in the bacterium Bacillus subtilis. The thesis studied the DNA replication in metabolic mutants by employing two techniques; QPCR and flow cytometry. The analyses showed that DNA replication is deregulated in cells that carry the following mutations: First, cells with mutations in the last 5 reactions of glycolysis. Second, cells with mutations in the reactions that connect the last part of glycolysis to the other parts of CCM (upper part of glycolysis pathway, pentose phosphate and Krebs cycle). Third, cells mutated in the overflow genes (channels that eliminate overflow metabolites produced in excess in CCM). The results demonstrate that in these mutants the replication begins and lasts longer than in the wild strain. All of these results show that the reactions that are centrally located to the CCM are important to ensure a correct control of replication timing. I also found that the ppGpp, a small molecule that functions as an alarmone of nutritional state in the cells, does not play a decisive role in the control of replication by metabolism in cells in steady state. The current knowledge of replisomes is mainly based on accumulated data from the dissection of the replisome of the model bacterium Escherichia coli and the phages T4 and T7. Bacillus subtilis is the second well studied bacterial model, a representative of Gram+ low GC%, it carries –unlike E. coli- two essential DNA polymerases for replication: PolC and DnaE. The thesis showed that DnaE as PolC form a part of the replisome in B. subtilis and provide a molecular explanation to the specialization of DnaE in the synthesis of the DNA lagging strand. In conclusion, the results show that there is much more diversity in the protein composition, organization and functioning of replisomes in bacteria than it is expected. In addition, the thesis concluded for the first time that the temporal control of replication depends on reactions located in the heart of CCM in B. subtilis. This property, in combination with other data from the literature, suggests that it could be universal and play an important role in carcinogenesis.

Thesis (MSc (Genetics. Institute for Plant Biotechnology)--Stellenbosch University, 2008. / Strawberry (Fragaria x ananassa) fruit quality is largely determined by the relative amounts of sugars and organic acids present, as well as soluble solid content. This study had three components: 1) Characterisation of cytosolic carbohydrate metabolism and carbon partitioning to sugars and organic acids in two commercial varieties, 2) analysis of transgenic strawberry fruit with increased pyrophosphate: D-fructose-6-phosphate 1-phosphotransferase (PFP) activity and 3) analysis of transgenic strawberry fruit with increased ß-fructosidase (invertase) activity in either cytosol or apoplast. Analyses of transgenic strawberry may inform similar attempts in grape berries. Festival and Ventana, two popular commercial strawberry cultivars in South Africa, were fairly similar with respect to sugar and organic acid content. Twelve cytosolic enzymes were investigated. Temporal differences in maximum catalytic activity were observed for invertase, PFP, pyruvate kinase and ADP-glucose pyrophosphorylase (AGPase). Invertase, PFP and AGPase activity also differed between the cultivars. One enzyme, SuSy, could not be analysed effectively, due to the purification method employed. These analyses established methodology for the analysis of transgenic berries. Constructs were designed to constituitively express Giardia lamblia PFP (GL-PFP), or to express Saccharomyces cerevisiae invertase (SCI) in a fruit-specific manner. A second invertase construct was designed to target SCI to the apoplast. Strawberry (cv. Selekta) was transformed and the presence of each transgene confirmed by PCR. Untransformed Selekta was used as control in both transgenic studies. Transgenic lines were selected based on GL-PFP activity in leaves and total PFP activity in ripe fruit. Sugar and organic acid content of ripe berries with high PFP activity was determined. Although berries displayed marked changes in sugar composition, the total sugar content was similar to controls, in all except one line. Organic acid content was decreased, leading to a clear reduction in organic acid-to-sugar ratio. This points to a gluconeogenic role for PFP in strawberry fruit. Transgenic berries were screened for SCI activity. Berries containing untargeted SCI exhibited total invertase activity similar to controls and were not analysed further. Berries with apoplasttargeted SCI displayed three-fold increases in invertase activity compared to controls. Total sugar content was reduced and exhibited reduced sucrose content relative to hexoses. Despite the effect of increased invertase activity on metabolites, maximum catalytic activity of enzymes involved in cytosolic sucrose, hexose and organic acid metabolism were unchanged. Transgenic plants selected in these studies were subsequently vegetatively replicated and future work will include immature fruit.