Thiomicrospira crunogena, a deep-sea hydrothermal vent chemolithoautotroph, uses the Calvin-Bensen-Bassham cycle to fix carbon. To meet its biosynthetic needs for oxaloacetate, oxoglutarate, and succinyl-coA, one would expect that this obligately autotrophic Gammaproteobacterium would use a ‘wishbone’ version of the citric acid cycle (CAC) to synthesize the intermediates necessary for biosynthesis, instead of the fully oxidative version to minimize carbon loss as carbon dioxide. However, upon examination of its complete genome sequence, it became apparent that this organism did not fulfill this expectation. Instead of a wishbone pathway, T. crunogena appears to run a fully oxidative CAC. The cycle is ‘locked’ in the oxidative direction by replacement of the reversible enzyme malate dehydrogenase with malate: quinone oxidoreductase, which is capable only of operation in the oxidative direction. Furthermore, oxoglutarate decarboxylation is catalyzed by oxoglutarate: acceptor oxidoreductase. The presence of both oxidoreductases was confirmed via assays on T. crunogena cell extracts. To determine whether this peculiar CAC was novel, complete genome sequences of ~340 Proteobacteria were examined via BLAST and COG searches in the Integrated Microbial Genome database. Genes catalyzing steps in the CAC were collected from each organism and vetted for paralogs that had adopted an alternative, ‘non-CAC’ function through genome context and cluster analysis. Alignments were made with the remaining sequences and were verified by comparing them to curated alignments at Pfam database and examination of active site residues. Phylogenetic trees were constructed from these alignments, and instances of horizontal gene transfer were determined by comparison to a 16S tree. These analyses verified that the CAC in T. crunogena is indeed unique, as it does not resemble any of the canonical cycles of the six classes of proteobacteria. Furthermore, three steps of the nine in its CAC appear to be catalyzed by enzymes encoded by genes that are likely to have been acquired via horizontal gene transfer. The gene encoding citrate synthase, and perhaps aconitase, are most closely affiliated with those present in the Cyanobacteria, while those encoding oxoglutarate: acceptor oxidoreductase cluster among the Firmicutes, and malate: quinone oxidoreductase clusters with the Epsilonproteobacteria.

No description available.

Submitted by Aelson Maciera (aelsoncm@terra.com.br) on 2017-06-23T18:11:28Z No. of bitstreams: 1 DissJRFN.pdf: 1477527 bytes, checksum: cb09da4bfdd14f1e2c076f57d365b7a1 (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-06-28T08:18:41Z (GMT) No. of bitstreams: 1 DissJRFN.pdf: 1477527 bytes, checksum: cb09da4bfdd14f1e2c076f57d365b7a1 (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-06-28T08:18:52Z (GMT) No. of bitstreams: 1 DissJRFN.pdf: 1477527 bytes, checksum: cb09da4bfdd14f1e2c076f57d365b7a1 (MD5) / Made available in DSpace on 2017-06-28T08:24:37Z (GMT). No. of bitstreams: 1 DissJRFN.pdf: 1477527 bytes, checksum: cb09da4bfdd14f1e2c076f57d365b7a1 (MD5) Previous issue date: 2017-02-23 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / In recent years, the application of attenuated Salmonella spp. has been investigated for development of several biotechnological products, mainly vaccines. However, the implementation of industrial processes to obtain these products depends on the development of strategies for this microorganism high-cell density cultures (HCDC). One of the HCDC’s greatest challenges is overcoming Salmonella’s metabolic limitations, as it presents a high organic acids production (mainly acetic acid) that inhibits biomass formation. In this context, this work proposes two approaches to deal with this problem and implement Salmonella’s HCDC: studying the cultivation of S. typhimurium using glycerol as carbon source to reduce the generation of organic acids; studying the cultivation of a recombinant strain of S. typhimurium expressing the enzyme acetyl-CoA synthetase (ACS) from E. coli, for improved acetate assimilation). Initially, cultures were grown in agitated flasks in minimal media for two carbon sources (glucose or glycerol) for the wild-type and the recombinant strain. After the preliminary experiments, the recombinant strain was cultivated in bioreactor operated in batch mode with minimal medium formulated with glycerol, glucose or acetic acid as carbon source, to evaluate acetate production and assimilation. The wild-type strain was cultivated in a continuous-mode bioreactor on minimal medium with glycerol at D=0.10; 0.17 and 0.22 h-1 to evaluate the S. typhimurium glycerol metabolism. During the cultivations, samples were collected and analyzed by high-performance liquid chromatography to quantify the production of organic acids and substrate consumption. To quantify the concentration of biomass, optical density (600 nm) readings of culture broth and dry cell weight measurements were performed. Agitated flasks and batch cultivations results indicated that the acetate production is reduced in medium with glycerol for both strains, and that the genetically modified cells present a lower acetate accumulation phenotype compared to the wild-type. Continuous cultures of the wild-type strain showed no acetate accumulation for a 0.1 h-1 dilution rate. At rates of 0.17 h-1 and 0.22 h-1 acetate accumulation was observed, but acetate flux was 2-fold lower than the flux reached in chemostat with glucose-formulated medium. Simulations were performed with the STM_v1.0 model using as input data the glycerol and oxygen fluxes estimated from the experimental results. Good predictions were obtained for the biomass, CO2 and acetate fluxes at the higher dilution rates. The results suggest that fed-batch culture using glycerol as carbon source along with an exponential feed to maintain a 0.1 h-1 specific growth rate as a promising strategy to obtain high cellular concentrations of wild-type S. typhimurium. The efficient acetate uptake observed for the recombinant S. typhimurium cells may allow higher values of specific growth rate to be used for this strain, resulting in a higher productivity of biomass. / Nos últimos anos, a aplicação de linhagens atenuadas de Salmonella spp. vem sendo amplamente investigada para o desenvolvimento de diversos produtos biotecnológicos, principalmente vacinas. No entanto, a implementação de processos industriais para a obtenção destes produtos depende do desenvolvimento de estratégias para o cultivo em alta densidade celular (CADC) deste microrganismo. Para isso, um dos grandes desafios a ser superado se refere às limitações metabólicas da Salmonella, uma vez que esta apresenta elevada produção de ácidos orgânicos (principalmente ácido acético) que inibem a formação de biomassa. Neste contexto, este trabalho propõe duas abordagens para lidar com este problema e implementar CADC de Salmonella: estudar o crescimento de Salmonella typhimurium em glicerol avaliando seu metabolismo como uma fonte de carbono menos propensa à geração de ácidos orgânicos; e estudar a produção destes ácidos por uma cepa de S. typhimurium geneticamente modificada para diminuir o acúmulo de acetato (super-expressão do gene acs, de Escherichia coli, que codifica a enzima acetil-CoA sintetase (ACS) responsável pela assimilação de acetato). Inicialmente foram realizados cultivos em frascos agitados em meio mínimo para duas fontes de carbono (glicose ou glicerol), para a cepa selvagem e para a cepa recombinante. Após os experimentos preliminares, foram realizados cultivos em biorreator operado em modo batelada com a cepa modificada em meio mínimo formulado com glicerol, glicose ou ácido acético, como fonte de carbono, a fim de avaliar a produção e a assimilação de acetato. Foram realizados cultivos em biorreator em modo contínuo com a cepa selvagem em meio mínimo com glicerol com D=0,10; 0,17 e 0,22 h-1 para avaliar o metabolismo do glicerol pela S. typhimurium. Ao longo dos cultivos foram coletadas amostras do caldo e analisadas por cromatografia líquida de alta eficiência para quantificar a produção de ácidos orgânicos e o consumo de substrato. Para quantificar a biomassa produzida, foram realizadas medidas de densidade ótica (600 nm) e de massa seca. Os resultados dos cultivos em frascos agitados e das bateladas indicam que, para ambas as cepas, a produção de acetato é reduzida em meio formulado com glicerol, e que as células modificadas geneticamente apresentam um fenótipo de menor acúmulo de acetato comparadas à linhagem selvagem. Os cultivos contínuos realizados com a cepa selvagem mostraram que não houve acúmulo de acetato para a taxa de diluição de 0,1 h-1. Já nas taxas de 0,17 h-1 e 0,22 h-1, apesar de haver acúmulo, o fluxo de produção de acetato foi cerca de 2 vezes menor que o observado em quimiostato em meio formulado com glicose. Foram realizadas simulações com o modelo STM_v1.0, tendo como dados de entrada os fluxos de glicerol e de oxigênio estimados a partir dos dados experimentais. O modelo descreveu bem os fluxos de biomassa, CO2 e acetato para as taxas de diluição mais altas. Os resultados obtidos sugerem o cultivo em batelada alimentada com glicerol como fonte de carbono, e alimentação exponencial definida para manter a velocidade específica de crescimento em 0,1 h-1, como uma estratégia promissora para obter altas concentrações celulares de S. typhimurium selvagem. Para a S. typhimurium recombinante, devido à sua eficiente assimilação de acetato, valores ainda maiores de velocidade específica de crescimento poderiam ser impostos, com elevado aumento de produtividade em biomassa.

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.

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.

Submitted by Alison Vanceto (alison-vanceto@hotmail.com) on 2017-01-23T10:29:06Z No. of bitstreams: 1 TeseCRS.pdf: 3506039 bytes, checksum: 68f4c5fe1c6ae3672adcedf3450e2f31 (MD5) / Approved for entry into archive by Camila Passos (camilapassos@ufscar.br) on 2017-01-23T15:48:35Z (GMT) No. of bitstreams: 1 TeseCRS.pdf: 3506039 bytes, checksum: 68f4c5fe1c6ae3672adcedf3450e2f31 (MD5) / Approved for entry into archive by Camila Passos (camilapassos@ufscar.br) on 2017-01-23T15:48:42Z (GMT) No. of bitstreams: 1 TeseCRS.pdf: 3506039 bytes, checksum: 68f4c5fe1c6ae3672adcedf3450e2f31 (MD5) / Made available in DSpace on 2017-01-23T15:48:50Z (GMT). No. of bitstreams: 1 TeseCRS.pdf: 3506039 bytes, checksum: 68f4c5fe1c6ae3672adcedf3450e2f31 (MD5) Previous issue date: 2015-08-27 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / The genus Salmonella spp. has been extensively investigated because these bacteria are important pathogens that frequently cause severe diseases and gastrointestinal infections in humans and animals. Moreover, in recent years, Salmonella has called attention due to the excellent results in the production and in vivo delivery of various substances with potential application in Vaccinology. However, there is still little information available concerning aspects of its metabolism, which hampers both the development of new attenuated strains and the large-scale production of live cells and cellular components. Thus, this work aimed to study the S. typhimurium LT2 metabolism, using traditional and innovative approaches to investigate different carbon sources as well as different bioreactor operation modes and aeration conditions (aerobic and anaerobic). Results obtained in batch and chemostat cultivations indicated that S. typhimurium metabolism differs significantly from E. coli metabolism, closely related bacteria species with regard to the central carbon metabolism. The main difference observed between these bacteria was the high level of acetate production exhibited by S. typhimurium LT2 cells, which, differently from E. coli, occurred even at the lowest dilution rate evaluated. Currently, genome scale metabolic models are important tools for better understanding the phenotypic behavior of many organisms. Therefore the model STM_v1.0 reconstructed for S. typhimurium LT2 was evaluated, comparing experimental data, obtained in chemostat cultivations, with model predictions. Since this model was derived from E. coli model, the simulated results for biomass formation were overestimated and, consequently, predicted acetate fluxes were lower than those obtained experimentally. Therefore, to obtain experimental data useful to improve the model and to reach a better comprehension of S. typhimurium metabolism, the technique of metabolic flux analysis using isotopic labeled substrate was adopted, allowing determination of the fluxes for the main pathways of central carbon metabolism of Salmonella. This analysis revealed different preferred metabolic pathways depending on the specific growth rate. At the lowest dilution rate evaluated, D = 0.24 h-1, glucose was catabolized predominantly by the pentose phosphate and glycolysis pathways, while at the dilution rate of 0.48 h-1, the major pathway of glucose oxidation was Entner-Doudoroff. In addition, a relatively high flux through the citric acid cycle at the higher dilution rate studied was observed. / Bactérias do gênero Salmonella spp. são extensivamente estudadas por serem importantes patógenos, causando frequentemente graves doenças e infecções gastrointestinais em humanos e animais. Além disso, nos últimos anos, estas bactérias vêm ganhando um destaque ainda maior na área da biotecnologia por apresentarem ótimos resultados na produção e veiculação in vivo de diversas substâncias com fins vacinais. No entanto, ainda há poucas informações a respeito de seu metabolismo, dificultando tanto o desenvolvimento de novas linhagens atenuadas, como também a produção em larga escala de células vivas e de componentes celulares. Neste sentido, este trabalho se propôs a estudar o metabolismo de S. typhimurium LT2, utilizando inicialmente abordagens tradicionais para investigar seu comportamento na presença de diferentes fontes de carbono, em diferentes modos de operação de biorreator e de aeração (aeróbias e anaeróbias). Os resultados obtidos em cultivos em batelada e em quimiostatos evidenciaram que o metabolismo da S. typhimurium difere bastante do metabolismo da E. coli, espécies consideradas semelhantes com relação ao metabolismo do carbono central. A principal diferença observada entre essas duas bactérias foi a elevada produção de acetato pelas células de S. typhimurium LT2, mesmo em baixas velocidades de crescimento nas quais este metabólito não é produzido por diversas estirpes de E. coli. Atualmente, modelos metabólicos em escala genômica são ferramentas importantes para que o comportamento do fenótipo de diversos organismos sejam melhor compreendidos. Assim, avaliou-se o modelo STM_v1.0 reconstruído para S. typhimurium LT2, comparando-se dados obtidos experimentalmente, em quimiostatos, e os preditos pelo modelo. No entanto, como este modelo foi baseado no modelo da E. coli, os resultados simulados para produção de biomassa foram superestimados e, consequentemente, os fluxos de acetato foram inferiores aos obtidos experimentalmente. Sendo assim, para se obter dados experimentais úteis para aprimorar o modelo e para uma compreensão maior do metabolismo de S. typhimurium, utilizou-se a técnica de análise dos fluxos metabólicos com substrato isotopicamente marcado, permitindo a determinação dos fluxos das principais vias do metabolismo do carbono central da bactéria em estudo. Essa análise revelou diferenças na utilização das vias metabólicas em função da velocidade específica de crescimento, sendo que na menor taxa de diluição avaliada, D = 0,24 h-1, a glicose foi predominantemente catabolizada pelas vias pentose fosfato e glicólise, enquanto na taxa de diluição de 0,48 h-1, a via principal de oxidação da glicose foi a Entner- Doudoroff. Além disso, também observou-se um fluxo relativamente maior na via do ciclo do ácido cítrico na maior taxa de diluição estudada.

Die vorliegende Arbeit untersucht die Rolle des Metabolismus in der Entstehung und Progression des Hepatozellulären Karzinoms (HZK). Der Schwerpunkt der Studie liegt auf Veränderungen zentraler Stoffwechselwege, unter anderem der Glykolyse, der Gluconeogenese, des Citratzyklus und anderer Prozesse des Zellstoffwechsels. Umfassende Multiomikanalysen, wie etwa Proteomik, Metabolomik und gezielte Genomsequenzierung wurden angewandt, um in vivo die Mechanismen der HZK Entstehung zu verstehen. Es wurden zwei Systeme untersucht: das ASV-B Mausmodell und klinische Patientenproben. Die Kohorte bestehend aus Biopsien und Resektaten von 95 Patienten umfasste 47 Fälle von HZK und 48 Fälle ohne HZK. Das Proteom des Mausmodells und der Patientenkohorte zeigen eine deutliche Herabregulierung wesentlicher Energie bereitstellender Kreisläufe im HZK: Glykogenstoffwechsel, de novo Synthese von Glukose, Glutaminaufnahme in den Citratzyklus, des weiteren sind 60% der Enzyme des Citratzyklus, und des Transports von Pyruvat in Mitochondrien im HZK herabreguliert. In dieser Arbeit wurde ein Isoformenwechsel auf mehreren Ebenen des zentralen Kohlenstoffmetabolismus gezeigt. Sowohl das Mausmodell, als auch die Gewebeproben von HZK-Patienten weisen Isoformenwechsel der Phosphoglyzeratmutasen und der Pyruvatkinasen auf. Die Hauptmerkmale finden sich sowohl in Modellmäusen, als auch in Patienten, und stellen so einen universalen metabolomischen Fingerabdruck des HZK dar. Darüber hinaus demonstriert diese Studie, dass die Proteomanalyse von bioptischen Material ein aussagekräftiges und ausreichendes molekular-diagnostisches Instrument für die Krebsforschung ist: die Proteomanalyse von Lebermaterial erlaubt die Unterscheidung von Tumorgewebe und tumorfreien Proben und die Dokumentation des Krankheitsverlaufs. / The present work evaluates the role of metabolism in development and progression of hepatocellular carcinoma (HCC). This study focuses on changes of central metabolic pathways, including glycolysis, gluconeogenesis, tricarboxylic acid (TCA) cycle and other processes involved in cellular metabolism and known to be dysregulated during cancer formation. Comprehensive multiomics analyses, such as proteomics, metabolomics and targeted genome sequencing, were applied in order to better understand HCC developmental mechanisms in vivo. Two main systems were studied: the ASV-B mouse model and clinical samples from human patients. The human cohort was composed of biopsy and surgery material from 95 patients: 47 HCC and 48 non-HCC. Proteomic data from both mice and humans show a clear downregulation of the main energy-producing pathways in HCC. Glycogen metabolism, de novo glucose synthesis, glutamine uptake to the TCA cycle, approximately 60% of enzymes of TCA cycle, and transport of pyruvate to mitochondria are downregulated in HCC. An isoform switch at various levels of central carbon metabolism was demonstrated in this work. Both mice and humans with HCC reveal isoform switches at the level of phosphoglycerate mutases and pyruvate kinases. The key features are found in both mouse and human, showing a universal metabolic HCC fingerprint. This study also demonstrates that proteomic analysis of the bioptate material is a strong and sufficient molecular diagnostic tool for research in cancer: the proteomic analysis of liver material allows the distinction of tumor samples from non-tumor samples and also to track the level of disease progression. Targeted genome sequencing revealed that no clear distinction between cancer and precancerous conditions could be made exclusively from the mutation analysis. Human metabolomic data remains inconclusive, possibly due to the different sources of tissue samples.

Tuberkulose ist auch heute noch eine der bedrohlichsten Infektionskrankheiten weltweit, verantwortlich für über 1.5 Millionen Todesfälle jährlich. Diese „Erfolgsgeschichte“ ihres Erregers Mycobacterium tuberculosis ist dabei wesentlich durch einen extrem flexiblen Stoffwechsel bestimmt, der dem Bakterium das Wachstum unter den restriktiven Bedingungen der menschlichen Wirtszelle erlaubt. Diese Arbeit erkundet die Flexibilität des zentralen Kohlenstoffmetabolismus in Mykobakterien mit Hilfe mathematischer Modellierungsansätze, ergänzt durch die Integration von qualitativ hochwertigen experimentellen Daten. Ausgehend von einem Überblick über die metabolische Landschaft des zentralen Kohlenstoffmetabolismus, erhöht sich Schritt für Schritt die Detailtiefe bis hin zur genauen Analyse spezieller infektionsrelevanter metabolischer Wege. Die Verknüpfung des zentralen Kohlenstoffmetabolismus zu umgebenden Stoffwechsel- und Biosynthesewegen wird systematisch offen gelegt, als Voraussetzung für eine thermodynamische Charakterisierung des Systems, welche die Glykolyse als limitierenden Stoffwechselweg unter verschiedenen Wachstumsbedingungen charakterisiert. Basierend auf Protein- und Metabolitdaten im Fleißgleichgewicht, erlaubt eine neu vorgestellte Methode die Vorhersage regulatorischer Punkte für den metabolischen Übergang zwischen verschiedenen Kohlenstoffquellen. Abschließend wird mit Hilfe thermodynamisch-kinetischer Modellierung das Zusammenspiel zweier Stoffwechselwege mechanistisch erklärt, welche den robusten Abbau einer intrazellulären Kohlenstoffquelle ermöglichen. Durch die Entwicklung neuer Modellierungstechniken in Kombination mit hochauflösenden experimentellen Daten, trägt diese Arbeit zum besseren Verständnis der kontextabhängigen Flexibilität des mycobakteriellen Stoffwechsels bei, einem vielversprechenden Angriffspunkt für die Entwicklung neuer Medikamente gegen Tuberkulose. / Tuberculosis remains one of the major global health threats responsible for over 1.5 million deaths each year. This ’success story’ of the causative agent Mycobacterium tuberculosis is thereby closely linked to a flexible metabolism, allowing growth despite the restrictive conditions within the human host. In this thesis, the flexibility of the mycobacterial central carbon metabolism is explored by modeling approaches integrating high-quality experimental data. The analyses zoom in from a network based view to the detailed functionalities of individual, virulence relevant pathways. The interconnection of the central carbon metabolism to the remaining metabolic network is charted as a prerequisite to characterize its thermodynamic landscape, debunking glycolysis as bottleneck in different nutritional conditions. Based on steady state metabolomics and proteomics data, regulatory sites for the metabolic transition between different carbon sources are predicted by a novel method. Finally, the flexible interplay between two seemingly redundant pathways for the catabolism of an in vivo-like carbon source is explained mechanistically by means of thermodynamic-kinetic modeling. By employing novel modeling methods in combination with high-resolution experimental data, this work adds to the mechanistic understanding of the context dependent flexibility of mycobacterial metabolism, an important target for the development of novel drugs in the battle against tuberculosis.

The aim of this thesis was to develop stable-isotope steady-state metabolic flux analysis (MFA) based on ¹³C labeling to quantify intracellular fluxes of central carbon metabolism in plants. The experiments focus on the analysis of a heterotrophic cell suspension culture of Arabidopsis thaliana (L) Heynh. (ecotype Landsberg erecta). The first objective was to develop a robust methodology based on combining high quality steady-state stable labeling data, metabolic modeling and computational analysis. A comprehensive analysis of the factors that influence the outcome of MFA was undertaken and best practice established. This allowed a critical analysis of the subcellular compartmentation of carbohydrate oxidation in the cell culture. The second objective was to apply the methodology to nutritional perturbations of the cell suspension. A comparison of growth on different nitrogen sources revealed that transfer to an ammonium-free medium: (i) increased flux through the oxidative pentose phosphate pathway (oxPPP) by 10% relative to glucose utilisation; (ii) caused a substantial decrease in entry of carbon into the tricarboxylic acid cycle (TCA); and (iii) increased the carbon conversion efficiency from 55% to 69%. Although growth on nitrate alone might be expected to increase the demand for reductant, the cells responded by decreasing the assimilation of inorganic N. Cells were also grown in media containing different levels of inorganic phosphate (Pi). Comparison of the flux maps showed that decreasing Pi availability: (i) decreased flux through the oxPPP; (ii) increased the proportion of substrate fully oxidised by the TCA cycle; and (iii) decreased carbon conversion efficiency. These changes are consistent with redirection of metabolism away from biosynthesis towards cell maintenance as Pi is depleted. Although published genome-wide transcriptomic and metabolomic studies suggest that Pi starvation leads to the restructuring of carbon and nitrogen metabolism, the current analysis suggests that the impact on metabolic organisation is much less extreme.