As operárias de Apis mellifera realizam diferentes tarefas de acordo com a sua idade (polietismo etário), sendo que operárias jovens cuidam da cria e quando mais velhas forrageiam. Entretanto, tal transição não segue uma cronologia fixa, mas se ajusta às necessidades da colônia, especialmente as condições nutricionais, tanto da colônia quanto dos indivíduos. Nesse sentido, abordamos a questão de como o metabolismo oxidativo no corpo gorduroso das operárias, i.e., o centro do metabolismo intermediário destes insetos, está relacionado com alterações da função das operárias na colônia e como o mesmo responde a um estresse nutritivo imposto individualmente em duas fases, na fase larval e na fase adulta. Como métodos para avaliar a atividade mitocondrial e o estado redox das células do corpo gorduroso utilizamos um sistema de respirometria de alta resolução para medir o consumo de O2, e realizamos ensaios bioquímicos para quantificar a geração de espécies reativas de oxigênio (EROs), os níveis de óxido nítrico, o número de unidades mitocondriais, e eventuais danos oxidativos resultantes. Ademais, por meio de PCR quantitativa medimos a expressão relativa de genes ligados à biogênese mitocondrial, ao sistema antioxidante e à via de sinalização por hipóxia. Na comparação destes parâmetros entre operárias nutridoras e forrageiras avaliamos separadamente os três principais compartimentos do corpo (cabeça, tórax e abdômen) uma vez que esses estão diferencialmente relacionados a tais funções. Para os tecidos da cabeça observamos que as amostras de nutridoras apresentaram maior consumo de O2, associado a uma maior produção de EROs e expressão elevada dos genes da via de hipóxia, enquanto forrageiras apresentaram maiores quantidades de transcritos dos genes do sistema antioxidantes e menores níveis de danos oxidativos. Para os tecidos do tórax observamos nas forrageiras uma maior capacidade de produção de ATP, acompanhada de uma elevada expressão de genes codificadores de enzimas do sistema antioxidante e menores níveis de danos oxidativos. Com relação aos tecidos abdominais, as abelhas nutridoras apresentaram maior atividade mitocondrial, enquanto as amostras de forrageiras apresentaram maior número de unidades mitocondriais, elevados níveis EROs e também uma expressão elevada dos genes do sistema antioxidante e da via de hipóxia. Assim, os dados revelaram uma clara mudança na atividade mitocondrial e nos padrões redox teciduais associados a esta transição de polietismo etário. Também notamos diferenças nestes parâmetros para cada compartimento do corpo analisado, refletindo uma demanda energética diferencial em cada tecido. A segunda parte do projeto teve como objetivo avaliar os mesmos parâmetros redox em uma situação de restrição alimentar em três situações distintas. O primeiro grupo experimental foi constituído de larvas que sofreram restrição calórica por umperíodo de 10 horas. O segundo grupo foi composto por abelhas recém emergidas que sofreram ou não tal restrição calórica no período larval. O terceiro grupo consistiu de abelhas que sofreram ou não restrição calórica no período larval, e após emergirem foram mantidas em caixas até atingirem a idade de 7 dias, quando foram submetidos novamente a uma restrição calórica. Para o primeiro grupo, as larvas, verificamos que após o período de restrição calórica os genes relacionados com a biogênese mitocondrial e o sistema antioxidante apresentaram níveis de expressão menores comparadas com larvas controle. Observamos também que larvas que sofreram restrição calórica apresentaram menor consumo de O2 e menores níveis de EROs. Para o segundo grupo, as abelhas recém emergidas, não observamos nenhuma diferença na expressão de genes mitocondriais e do sistema antioxidante entre as que sofreram ou não restrição calórica na fase larval, demostrando uma recuperação nestes parâmetros após o período de metamorfose. No terceiro grupo, as abelhas de 7 dias foram submetidas a uma nova restrição calórica para observar se indivíduos que passaram por a situação de estresse no período larval apresentariam uma resposta mais imediata a um estresse nutricional na fase adulta. Os resultados do terceiro grupo revelaram que abelhas que haviam sido expostas a uma situação prévia de restrição calórica, apresentaram uma redução dos transcritos de genes ligados a biogênese mitocondrial e do sistema antioxidante quando comparados ao grupo que sofreu restrição calórica apenas na fase adulta. Em conjunto nossos dados sugerem que a restrição calórica é capaz de diminuir o metabolismo oxidativo e que existe uma resposta aparentemente adaptativa em indivíduos adultos que passaram por essa situação previamente durante o desenvolvimento larval. Assim, esses resultados podem servir para direcionar estudos futuros sobre a relação entre restrição calórica, metabolismo oxidativo e longevidade nesses insetos sociais. / Apis mellifera workers perform different tasks according to their age (age polyethism), where young workers care for the brood and older ones become foragers. However, this a transition does not follow a fixed chronology, but is fitted to the needs of the colony, especially the nutritional conditions of both, the colony and the individual bee. In this sense, we address the question of how oxidative metabolism in the fat body of the workers, e.g., the center of the intermediary metabolism of these insects, is related to changes in the function of the workers in the colony, and how these parameters responds to a nutritional stress imposed individually in two stages, in the larval and adult phase. For evaluating mitochondrial activity and the redox status of fat body cells we used a high resolution respirometry system to measure O2 consumption, and we performed biochemical assays to measure the generation of reactive oxygen species (ROS), levels of nitric oxide, the number of mitochondrial units, and eventual oxidative damages. Furthermore, using quantitative PCR, we measured the relative expression of genes linked to mitochondrial biogenesis, to the antioxidant system, and to the hypoxia signaling pathway. In the comparison of these parameters between nurse and forager workers, we evaluated separately the three main body compartments (head, thorax and abdomen), since these are differentially related to these functions. For the head tissues, we observed that nurses showed higher O2 consumption, associated with higher ROS production and elevated expression of hypoxia pathway genes, while foragers presented higher amounts of transcripts of the antioxidant system genes and lower levels of oxidative damages. For the thorax tissues we found in the foragers a greater capacity of ATP production, accompanied by a higher expression of genes encoding enzymes of the antioxidant system and lower levels of oxidative damage. With respect to the abdominal tissues, nurse bees presented higher mitochondrial activity, while foragers had a higher number of mitochondrial units, elevated ROS levels, and also a higher expression of genes related to the antioxidant system and hypoxia pathway. Thus, these data revealed a clear change in mitochondrial activity and redox tissue patterns associated with this transition of age polyethism. We also noticed differences in these parameters for each of the analyzed body compartment, reflecting a differential energy demand in each tissue. The second part of the project aimed to evaluate the same redox parameters in a food restriction condition in three distinct situations. The first experimental group consisted of larvae that experienced caloric restriction for a 10 hour period. The second group consisted of newly emerged bees that had or not experienced such a caloric restriction in the larval period. The third group consisted of bees that had experienced or not caloric restriction during the larval period, and after emergence were kept in boxes until reaching 7 days old, when they were resubmitted to another caloricrestriction. For the first group, the larvae, we found that after the period of caloric restriction, genes related to mitochondrial biogenesis and the antioxidant system presented lower levels of expression compared to control larvae. We also observed that larvae that had experienced caloric restriction presented lower O2 consumption and lower ROS levels. For the second group, newly emerged bees, we did not observe any differences in the expression of mitochondrial genes and the antioxidant system among those individuals that had experienced or not caloric restriction in the larval phase, indicating a recovery in these parameters after the period of metamorphosis. In the third group, the 7-day-old bees had experienced a new caloric restriction to see whether individuals that had been in a stressful situation during larval period would respond more readily to nutritional stress in adulthood. The results for this group showed that bees that had been exposed to a previous caloric restriction presented a reduction in the transcript levels of genes related to mitochondrial biogenesis and the antioxidant system when compared to the group that experienced caloric restriction only in adulthood. Taken together, our data suggest that caloric restriction is able to diminish the oxidative metabolism, and that there is a seemingly adaptive response in adult individuals that had previously experienced this situation during larval development. Thus, these results can serve to direct future studies on the relationship between caloric restriction, oxidative metabolism and longevity in these social insects.

Les mutations germinales activatrices de la voie RAS sont responsables de maladies rares regroupées sous le nom de RASopathies : le Syndrome de Noonan, le Syndrome de Noonan avec de Multiples Lentigines, la Neurofibromatose de type 1, le Syndrome de Malformations Capillaires et Malformations Artério-Veinseuses, le Syndrome Cardio-Facio-Cutané, le Syndrome de Legius et le Syndrome de Costello. Cette thèse s’intéresse au syndrome de Costello causé par une mutation hétérozygote de novo du gène HRAS. Ce syndrome est révélé dans les premiers mois de la vie et se caractérise par un retard de croissance postnatal, des traits du visage épais, un déficit intellectuel, des anomalies cutanées, ainsi qu’une prédisposition à développer des tumeurs. De plus, les patients atteints du syndrome de Costello développent une cardiomyopathie hypertrophique, de l’hypertension, une hypotonie et une myopathie d'origine moléculaire inconnue. En lien avec une association de malade et le service de génétique du CHU de Bordeaux, nous avons mené une exploration des anomalies protéomiques dans les tissus d’une souris modèle du syndrome de Costello ainsi que dans des fibroblastes de patients et des cellules modèles exprimant les formes mutées de HRASG12S et HRASG12A. Cette analyse globale et sans a priori a révélé des altérations au niveau du métabolisme énergétique et plus particulièrement de la composition des mitochondries. Le déficit fonctionnel des mitochondries, centrale énergétique du corps humain, a été caractérisé par des approches de biochimie, de bioénergétique et de biologie cellulaire. De plus, l’analyse des données ‘omiques’ a permis de suggérer une nouvelle hypothèse dans la physiopathologie du syndrome de Costello. Cette hypothèse considère l’implication d’un micro-ARN, le miR-221* dans l’inhibition du métabolisme oxydatif. Les analyses génétiques réalisées sur les cellules de patients et les cellules modèles ont démontré l’inhibition de l’expression de la protéine AMPK, un régulateur majeur du métabolisme mitochondrial, par le miR-221* sous le contrôle de HRASG12S et HRASG12A. Ces découvertes ont permis d’élaborer une stratégie thérapeutique visant à réduire la cardiomyopathie dans le syndrome de Costello. Les analyses précliniques effectuées sur les modèles cellulaires et le modèle murin ont permis d’évaluer l’efficacité d’une stimulation pharmacologique du métabolisme mitochondrial. Cette thèse révèle donc l’implication des mitochondries dans le syndrome de Costello et l’analyse moléculaire réalisée propose une série de données ‘Omiques’ qui permettront de progresser dans la compréhension de cette maladie rare. / Germline activating mutations of the RAS pathway are responsible for rare diseases grouped under the name of RASopathies: Noonan Syndrome, Noonan Syndrome with multiple Lentigines, Type 1-neurofibromatosis, Capillaries malformations and arteriovenous malformations syndrome, Cardio-Facio-Cutaneous Syndrome, Legius Syndrome and Costello Syndrome. This Ph.D thesis focuses on Costello syndrome that is caused by a heterozygous de novo mutation of the HRAS gene. This syndrome is revealed in the first months of life and is characterized by postnatal growth retardation, thick facial features, intellectual deficit, skin abnormalities, and a predisposition to developing tumors. In addition, patients with Costello syndrome develop hypertrophic cardiomyopathy, hypertension, hypotonia and myopathy of unknown molecular origin. In connection with a patients association and the genetics department of Bordeaux University Hospital, we conducted an exploration of proteomic abnormalities in the tissues of a mouse model of the Costello syndrome as well as in patients’ fibroblasts and cell models expressing mutated forms of HRASG12S and HRASG12A. This global and unbiased analysis revealed alterations in energy metabolism and more particularly in the composition of mitochondria. The functional deficiency of mitochondria, energy plants of the human body, has been characterized by biochemistry, bioenergetics and cell biology approaches. In addition, the 'omic' analysis of Costello syndrome suggested a new pathophysiology hypothesis that considered the involvement of a microRNA, miR-221* in the alteration of oxidative metabolism. Functional genetic analyzes performed on patient cells and cell models demonstrated the inhibition of the expression of the major mitochondrial metabolism regulator AMPK protein by miR-221* under the control of HRASG12S and HRASG12A. These findings led to the development of a preclinical therapeutic strategy to reduce cardiomyopathy in Costello syndrome. Preclinical investigations performed on the cellular models and the murine model made it possible to evaluate the efficacy of a pharmacological stimulation of mitochondrial metabolism. This thesis thus reveals the involvement of mitochondria in Costello syndrome and the molecular analysis carried out makes available a series of 'Omics' data that will allow progress in the understanding of this rare disease.

A restrição calórica (RC) estende a expectativa de vida de muitos organismos por mecanismos ainda em estudo. Entre os vários efeitos fisiológicos da RC encontra-se o aumento na biogênese mitocondrial, dependente de óxido nítrico (NO•), sintetizado pela enzima óxido nítrico sintase endotelial (eNOS). Um dos indutores fisiológicos mais potentes da eNOS é a insulina, cujos níveis plasmáticos são consideravelmente reduzidos nos organismos em RC. O objetivo deste trabalho foi investigar os mecanismos associados ao aumento da sinalização por NO• durante a RC in vivo e in vitro, e as conseqüências celulares do aumento de massa mitocondrial no que diz respeito à longevidade e capacidade respiratória celulares. Submetemos camundongos Swiss fêmeas à RC de 40% e observamos um considerável aumento tecido-específico na fosforilação basal de Akt e eNOS em músculo esquelético, tecido adiposo visceral e cérebro, os quais também apresentaram maior massa mitocondrial. A associação entre a sinalização por insulina, NO• e biogênese mitocondrial foi adicionalmente confirmada em um grupo de camundongos tratados com o desacoplador mitocondrial dinitrofenol (DNP), que também reduz a insulinemia e aumenta a longevidade em camundongos. Para o estudo mecanístico deste fenômeno, usamos soros de ratos Sprague-Dawley submetidos à RC de 40% ou alimentados ad libitum (AL) em cultura celular de células vasculares da musculatura lisa (VSMC), reproduzindo um protocolo descrito para RC in vitro. O uso do soro RC aumentou a fosforilação do receptor de insulina e Akt, a expressão de eNOS e nNOS (forma neural da NOS) e a fosforilação de eNOS, o que se refletiu em maior liberação de nitrito (NO2) no meio de cultura. Inibindo-se a Akt, todos os efeitos promovidos pela RC na sinalização por NO• foram revertidos. Ao se imunoprecipitar do soro a adiponectina, citocina conhecida por aumentar a sensibilidade à insulina, aumentada durante a RC, os efeitos do soro RC na via de sinalização de insulina foram abolidos e, conseqüentemente, os efeitos na sinalização por •NO foram prevenidos. Neurônios de células granulosas de cerebelo, que não expressam eNOS, apenas nNOS, foram cultivados com os soros AL ou RC, e também apresentaram considerável aumento na sinalização por •NO. Estas alterações induziram a biogênese mitocondrial e capacidade respiratória, e foram associadas à maior longevidade celular. Os mesmos efeitos mitocondriais foram observados em células secretoras de insulina, INS1, entretanto a secreção de insulina em resposta à glicose tornou-se inibida, por um mecanismo desconhecido, porém associado a reduzidos níveis intracelulares de espécies oxidantes, moléculas-chave para a secreção de insulina; e à alteração da morfologia mitocondrial, provavelmente devido à maior expressão de mitofusina-2 (Mfn-2). Ao se nocautear a Mfn-2, houve um aumento na geração de EROs e as células em RC passaram a secretar insulina a níveis comparáveis aos das células controle. Concluímos que durante a RC a maior sensibilidade à insulina aumenta a atividade de eNOS, via Akt, associada à maior biogênese mitocondrial. A adiponectina é uma molécula-central nestes eventos. A expressão de nNOS também é afetada, por mecanismos desconhecidos. O aumento de biogênese mitocondrial eleva a capacidade respiratória celular e impacta positivamente a longevidade in vitro. A alteração da morfologia mitocondrial associa-se a alterações na produção de oxidantes intracelulares e mudanças na secreção de insulina. / Calorie restriction (RC) is known to extend the lifespan in many organisms, and its mechanisms of action are still under investigation. Enhanced mitochondrial biogenesis driven by nitric oxide (•NO), synthesized by the endothelial nitric oxide synthase (eNOS), is proposed to be a CR central effect. Insulin is one of the most potent physiological activators of eNOS. However, plasmatic insulin levels are dramatically reduced in organisms under CR. The goal of this work was uncover the mechanisms associated with enhanced •NO signaling during CR, in vivo and in vitro, as well as the cellular consequences of increased mitochondrial mass, regarding lifespan and reserve respiratory capability. Female Swiss mice were submitted to 40% of CR. A tissue-specific (skeletal muscle, abdominal adipose tissue and brain) increment in basal Akt and eNOS phosphorylation, which was related to enhanced mitochondrial biogenesis, was observed. Indeed, this association was also verified in tissues from mice treated with low doses of a mitochondrial uncoupler, dinitrophenol (DNP). To unveil the mechanism behind the insulin signaling effects on •NO levels, serum from Sprague-Dawley rats submmited to 40% of CR was used to culture in VSMC cells, an in vitro CR protocol. CR sera enhanced insulin receptor (IR) and Akt phosphorylation, as well as nitrite (NO2-) accumulation in the culture media, the expression of eNOS and nNOS (neural NOS isoform) and eNOS phosphorylation. The effects of CR sera were reversed by Akt inhibition. The immunoprecipitation of serum adiponectin, a cytokine known to improve peripheral insulin sensitivity, also reversed the CR serum effects on insulin and •NO signaling. Cerebellar neurons, which do not express eNOS, just nNOS, were also cultured with CR or AL serum and also presented striking increments in •NO signaling, associated with mitochondrial biogenesis, increased reserve respiratory capability and lifespan extension. The mitochondrial effects promoted by CR were also observed in insulin secreting cells (INS1). However, under the CR condition, insulin secretion stimulated by glucose was impaired. The likely explanations are reduced mitochondrial reactive oxygen species (ROS) generation, or the alteration in mitochondrial morphology, associated, in our model, with enhanced mitofusin-2 expression (Mfn-2). In cells which the Mfn-2 was knocked down, insulin secretion in CR and AL groups was responsive to glucose at the same level, and the intracellular oxidants levels were much higher. Overall, CR improves •NO signaling due to enhanced insulin sensitivity, through Akt, and results in mitochondrial biogenesis. Adiponectin is a key molecule in this phenomenon. Increments in mitochondrial mass enhance the cellular reserve respiratory capability and lifespan. Mitochondrial morphology alterations are associated with possible decreases in ROS generation and impaired insulin release, maintained the low levels of plasmatic insulin.

Das COP9 Signalosom ist ein hochkonservierter Proteinkomplex bestehend, aus acht Untereinheiten. In der vorgelegten Promotionsarbeit konnte ein bislang unbekannter Regulationsmechanismus der Biogenese des COP9 Signalosoms identifiziert werden. Die siRNA-vermittelte Reduktion der CSN1-Expression führte zu einer Reduktion der Expression aller CSN-Untereinheiten. Die Transfektion von His-CSN1 in siCSN1-Zellen induzierte die CSN-Neusynthese und ferner einen Anstieg der c-Myc- und STAT1 Expression. Durch die Stimulation der Zellen mit IFN alpha bzw. IFN gamma konnte die de novo Synthese des CSN-Komplexes induziert werden. Die siRNA-vermittelte Inhibition von STAT1, c-Myc bzw. Lin28B führte ebenso wie die Behandlung der Zellen mit AG9 bzw. AG490, pharmakologischen Inhibitoren der JAK-Kinasen, zu einer Reduktion der Proteinexpression der CSN-Untereinheiten. Dabei standen signifikanten Veränderungen auf der Proteinebene geringfügige Änderungen auf der mRNA-Ebene gegenüber. Daher wurde ein post-transkriptioneller Mechanismus zur Regulation der Expression der CSN-Untereinheiten unter Beteiligung von miRNAs postuliert. Diese Regulation wird vermutlich durch die Aktivität von c-Myc und Lin28B verstärkt. Dies stellt einen neuen, bislang unbekannten Mechanismus für die Regulation der Biogenese des COP9 Signalosoms, vermutlich über den c-Myc/Lin28B/let-7-Weg, dar. Die Co-Transfektion der siCSN1-Zellen mit spezifischen komplementären Inhibitoren dieser miRNAs führte zu einer Induktion der Proteinexpression der CSN-Untereinheiten. Die Transfektion von let-7 miRNA-Mimics bewirkte eine Reduktion der Expression der CSN-Untereinheiten in den siCSN1-Zellen. Ferner konnten im Rahmen dieser Arbeit mittels der miRBase Sanger Datenbank und der Software MicroInspector Bindestellen für let-7 miRNAs an den mRNAs der CSN- und der proteasomalen Lid-Untereinheiten identifiziert werden. Der gezeigte Regulationsmechanismus könnte auch für die Biogenese des proteasomalen Lids von Bedeutung sein. / The COP9 signalosome is a highly conserved protein complex composed of eight subunits. In this study a novel, regulatory mechanism of CSN biogenesis was identified. We used stable transfected siCSN1 cells in which the protein and the mRNA expression of CSN subuntis were downregulated. Transfection of His-CSN1 in those siCSN1 cells led to the induction of the de novo Synthesis of the whole CSN complex. In addition the expression of the transcription factors STAT1 and c-Myc was elevated. The cells were treated with IFN alpha or IFN gamma, respectively. This resulted in the induction of the CSN de novo synthesis. Moreover, the siRNA-mediated inhibition of STAT1, c-Myc, Lin28B as well as treatment with the pharmacological inhibitors AG9 or AG490 led to a reduced protein expression of the analysed CSN subunits. We found that in all experiments there was a significant change on protein level in contrast to a marginal change on the RNA level. Based on our study we hypothesized that the CSN biogenesis ist regulated post-transcriptionally by miRNAs. The participation of miRNAs in the regulation of CSN biogenesis was further analysed. The siCSN1 cells were transfected with complementary hairpin inhibitors of let-7 miRNAs, leading to an induction of the CSN synthesis. The transfection of let-7 miRNA-mimics, which enhance the impact of miRNA on target mRNAs, resulted in an decrease of the CSN expression. These findings prove the involvement of miRNAs in CSN biogenesis, presumably via the c-Myc/Lin28B/let-7 pathway. Furthermore, using miRBase Sanger database and MicroInspector software, potential binding sites for let-7 miRNAs were detected within the mRNA sequences of CSN subunits as well as of subunits of the proteasomal lid. Therefore, there is evidence to suggest that this mechanism is crucial for the regulation of the biogenesis of the proteasomal lid as well.

Der obligat intrazelluläre Parasit Toxoplasma gondii ist der Erreger der Toxoplasmose, und dient zugleich als wichtiger Modellorganismus für weitere Human- und Tierpathogene, wie z.B. Plasmodium oder Eimeria. Die Vermehrung von T. gondii erfordert eine effiziente Biosynthese von Phospholipiden für die Herstellung neuer Membranen, was durch die de novo Synthese durch den Parasiten, und/oder den Import von Lipiden aus der umgebenden Wirtszelle gewährleistet werden kann. Während der Parasit zahlreiche Möglichkeiten für Synthese oder Import von PtdEtn und PtdSer verwendet, scheint die Biosynthese des abundantesten Membranlipids PtdCho auschließlich über den CDP-Cholin Weg zu erfolgen. Dieser erstreckt sich in T. gondii über 3 zelluläre Kompartimente, mit einer cytosolischen Cholin-Kinase (TgCK), einer im Zellkern lokalisierenden Cholin-Cytidylyltransferase (TgCCT) und einer Cholin-Phosphotransferase (TgCPT) im ER. Anders als die substrat-spezifische Ethanolamin-Kinase (TgEK), kann TgCK neben Cholin außerdem Ethanolamin phosphorylieren. TgCK zeigt eine geringe Affinität zu Cholin (Km ~0.77 mM), während eine verkürzte TgCK (TgCKS), welcher eine als Signalpeptid vorhergesagte N-terminale Sequenz (20 Aminosäuren) fehlt, eine etwa 3-fach höhere Aktivität aufweist (Km ~0.26 mM). Während jedoch die Wildtyp-TgCK cytosolische Cluster in Toxoplasma bildet, zeigt die verkürzte TgCK eine gleichmäßigere cytosolische Lokalisierung. Wir schlussfolgern daraus, dass der hydrophobe N-Terminus nicht notwendig ist für eine funktionale TgCK, sondern eine strukturelle Funktion bei der Protein-Lokalisierung hat. Eine konitionelle Mutante, in welcher der TgCK Promoter gegen den Tetracyclin-regulierbaren Promoter pTetO7Sag4 ausgetauscht wurde (Deltatgcki), zeigt erstaunlicherweise normales Wachstum und PtdCho Biosynthese. Die TgCK Aktivität und die daraus resultierende PtdCho Synthese sind nur zu ~30% regulierbar. Unsere Ergebnisse deuten auf die Verwendung eines alternativen Startcodons bzw. Promoters hin, welcher zur Expression einer verkürzten (~53-kDa) aber vermutlich aktiven Cholin Kinase führt, wodurch der Verlust der TgCK (~70-kDa) kompensiert wird. Der konditionelle Knockout von TgCCT, dem regulatorischen Enzym des CDP-Cholin Wegs, hatte einen 50%igen Wachstumsdefekt zur Folge. Diese Studie zeigt eine erstaunliche Flexibilität des Parasiten bezüglich seiner Membranzusammensetzung, und bestätigt zugleich die Annahme, dass PtdCho nicht von der Wirtszelle importiert werden kann. Diese Anpassungsfähigkeit stellt einen möglichen Faktor dar, der es T. gondii erlaubt sich in einem breiten Spektrum von Wirten zu vermehren. / Toxoplasma gondii is an obligate intracellular apicomplexan parasite that causes life-threatening disease in neonates and in immunocompromised people. Successful replication of Toxoplasma requires substantial membrane biogenesis, which must be satisfied irrespective of the host-cell milieu. Like in other eukaryotes, the two most abundant phospholipids in the T. gondii membrane are phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn). Bioinformatics and precursor labeling analyses confirm their synthesis via the CDP-choline and CDP-ethanolamine pathway, respectively. This work shows that the 3-step CDP-choline pathway, involving the activities of TgCK, TgCCT and TgCPT, localizes to the cytosol, nucleus and ER membrane, respectively. The initial reaction is catalyzed by a dual-specificity choline kinase (TgCK, ~70-kDa), capable of phosphorylating choline as well as ethanolamine. The purified full-length TgCK displayed a low affinity for choline (Km ~0.77 mM). TgCK harbors a unique N-terminal hydrophobic peptide that is required for the formation of enzyme oligomers in the parasite cytosol but not for activity. The displacement of the TgCK promoter in a conditional mutant of T. gondii (deltatgcki) attenuated the enzyme expression by ~80%. Unexpectedly, the ?tgcki mutant was not impaired in intracellular growth, and exhibited a normal PtdCho biogenesis. To recompense for the loss of full-length TgCK, the mutant appears to make use of an alternative promoter and/or start codon, resulting in the expression of a shorter but active TgCK isoform identified by the anti-TgCK antiserum, which correlated with its persistent choline kinase activity. Accordingly, the ?tgcki showed an expected incorporation of choline into PtdCho, and susceptibility to dimethylethanolamine (a choline analog). Interestingly, the conditional mutant displayed a regular growth in off state despite a 25% decline in PtdCho content, which suggests a compositional flexibility in T. gondii membranes and insignificant salvage of host-derived PtdCho. The two-step conditional mutagenesis of TgCCT, which caused a reduced growth rate to about 50%, further substantiated this finding. The enzymatic activity of TgCCT and its role in PtdCho synthesis remain to be proven, however. Taken together, the results demonstrate that the CDP-route is likely essential in T. gondii. The competitive inhibition of choline kinase to block the parasite replication appears a potential therapeutic application.The work also reveals a remarkably adaptable membrane biogenesis in T. gondii, which may underly the evolution of Toxoplasma as a promiscuous pathogen.

La biogenèse des ribosomes matures et fonctionnels est notamment dépendante de petites particules ribonucléoprotéiques composées d’ARN et de protéines, les snoRNP (small nucleolar RiboNucleoProteins). Celles-ci sont subdivisées en deux familles : les snoRNP à boîtes C/D et les snoRNP à boîtes H/ACA. Ces deux classes de snoRNP catalysent des modifications chimiques, respectivement de 2’-O-méthylation et de pseudouridylation, sur des positions spécifiques des ARN ribosomiques (ARNr), ou sont impliquées dans des clivages du long ARNr précurseur. Les snoRNP à boîtes C/D sont composées d’un snoARN à boîtes C/D servant de guide pour cibler la position à modifier, et d’un jeu invariant de quatre protéines : Snu13p/SNU13, Nop1p/Fibrillarine, Nop56p/NOP56 et Nop58p/NOP58 (levure/Homme). Ces snoRNP sont produites par la cellule grâce à la présence de plusieurs complexes protéiques constituant une machinerie pour leur assemblage. Outre plusieurs facteurs protéiques déjà connus dans la biogenèse de snoRNP à boîtes C/D comme les protéines Rsa1p/NUFIP, Hit1p/ZNHIT3 et les protéines du complexe R2TP, d’autres protéines pourraient compléter cette machinerie. Parmi ces facteurs additionnels, la protéine Bcd1p/ZNHIT6, pour Box C/D snoRNA protein 1, est essentielle pour maintenir spécifiquement la stabilité in vivo des snoARN à boîtes C/D, et des associations ont pu être identifiées entre Bcd1p/ZNHIT6 avec différents partenaires protéiques de la machinerie d’assemblage de ces particules. Toutefois, l’étape d’assemblage où Bcd1p/ZNHIT6 intervient et la fonction qu’elle y accomplit demeurent inconnues. L’utilisation d’outils in vivo et in vitro chez la levure S. cerevisiae et chez les mammifères nous ont permis de progresser dans la compréhension de la fonction de Bcd1p/ZNHIT6 dans l’assemblage des snoRNP à boîtes C/D. Bcd1p est un facteur d’assemblage recruté de manière co-transcriptionnelle sur les loci codant les snoARN à boîtes C/D et est requis pour le recrutement des complexes d’assemblage sur les snoARN en cours de transcription. Plus spécifiquement, Bcd1p affecte l’interaction de Nop58p avec le facteur d’assemblage Rsa1p, suggérant une fonction dans le recrutement de Nop58p dans une pré-snoRNP en cours d’assemblage. Ce travail a permis d’apporter des informations importantes permettant d’expliquer le caractère essentiel de Bcd1p dans la fonction et la biogenèse des snoRNP à boîtes C/D / Ribosome biogenesis is especially dependent on the action of small RNA/proteins complexes called small nucleolar ribonucleoproteins (snoRNPs). They are divided into two main families: the so-called box C/D snoRNPs and box H/ACA snoRNPs. Each category performs specific enzymatic processes, 2’-O-methylation and pseudouridylation, respectively, and induces target-specific chemical modification on rRNAs. Few snoRNPs are also essential for pre-rRNA processing. The box C/D snoRNPs are formed by the association of a box C/D snoRNA with a set of four invariant proteins: Snu13p/SNU13, Nop1p/Fibrillarine, Nop56p/NOP56 and Nop58p/NOP58 (yeast/Human). Biogenesis of these RNPs relies on the action of several proteins complexes which constitute a dedicated assembly machinery. Rsa1p/NUFIP, Hit1p/ZNHIT3, and components of the R2TP complex are the best characterized protein actors of this machinery. Additional protein factors probably participate in box C/D snoRNP biogenesis; Bcd1p/ZNHIT6 (Box C/D snoRNA protein 1) is such a candidate as it is essential for the in vivo stability of box C/D snoRNAs, and it was found associated with proteins involved in this machinery in yeast and Human. However, the mechanism governing the recruitment of this protein towards the biogenesis of box C/D snoRNP, and the step of the assembly process relying on the presence of Bcd1p are still unknown. In S. cerevisiae and Human, in vivo and in vitro tools allowed us to improve the understanding of the functions of Bcd1p/ZNHIT6 in box C/D snoRNP assembly. Bcd1p is an assembly factor that is recruited co-transcriptionally on box C/D snoRNA loci, and is required for the recruitment of assembly complexes on nascent snoRNAs. Bcd1p is important for Nop58p association with the assembly factor Rsa1p, which suggests that its primary function is to recruit Nop58p to nascent pre-snoRNPs. This work evidenced important information on the essential role of Bcd1p in C/D snoRNP biogenesis and function

La biogenèse des ribosomes est un processus complexe qui implique la production et l'assemblage de 4 ARN et d'environ 80 protéines. Chez l'Homme, la production des deux sous-unités ribosomiques débute dans le nucléole par la synthèse par l'ARN polymérase I d'un long transcrit contenant les séquences des ARN ribosomiques 5.8S, 18S et 25S, qui s'associe de manière co-transcriptionnelle à des protéines ribosomiques et à des facteurs d'assemblage. Le quatrième ARN ribosomique, l'ARNr 5S est transcrit séparément par l'ARN polymérase III, et s'associe avec les protéines ribosomiques Rpl5 et Rpl11 en dehors du ribosome. Ce sous-complexe, appelé particule 5S, est ensuite intégré au sein de la grande sous-unité. La particule 5S est également impliquée dans le contrôle de la prolifération cellulaire. En effet, en cas de dé-régulation de la biogenèse du ribosome, la particule 5S s'accumule dans le nucléoplasme et interagit directement avec l'ubiquitine-ligase MDM2, provoquant la stabilisation du suppresseur de tumeur p53. L'objectif principal de ma thèse est d'étudier le rôle des facteurs d'assemblage Rpf2 et Rrs1 dans la biogenèse du ribosome. Ces protéines assurent deux fonctions distinctes : elles sont requises pour l'association de la particule 5S avec la sous-unité pré-60S, et stimulent la transcription des ARNr par l'ARN polymérase I. Elles sont donc impliquées dans deux événements fondamentaux qui conditionnent les capacités de prolifération cellulaire. La combinaison d'études structurales par cristallographie aux rayons X, et d'études d'interactions protéine-ARN in vitro et in vivo, m'ont permis de mieux appréhender le rôle du complexe Rpf2/Rrs1 dans l'intégration de la particule 5S et dans la maturation de la grande sous-unité. J'ai également étudié le rôle du complexe Rpf2/Rrs1 dans la régulation de la transcription des ARNr, en caractérisant ses interactions avec la polymérase I. / Ribosome Biogenesis is a complex process that requires the production and the correct assembly of the 4 rRNA with more than 80 proteins. Ribosome biogenesis starts by the transcription of a pre-RNA precursor in the nucleolus. Three of the four ribosomal RNAs, the 5.8S, 18S, and 25S rRNAs, are cotranscribed as a single 35S precursor by polymerase I. This precursor is cotranscriptionally modified, folded, cleaved, and assembled with both ribosomal proteins and non-ribosomal factors to generate the mature ribosomes. The fourth rRNA, the 5S rRNA, is transcribed by RNA polymerase III and is assembled into the 5S particle, containing ribosomal proteins Rpl5 and Rpl11, prior to its incorporation into preribosomes. In mammals, the 5S RNP is also a central regulator of the homeostasis of the tumor suppressor p53 The main objective of my thesis was to understand the precise roles of the two assembly factors Rpf2 and Rrs1 in ribosome biogenesis. These proteins have two distinctive functions : Rpf2 and Rrs1 are required for the 5S particle incorporation into the large subunit, and stimulate the rRNA transciption by polymerase I. Using a combination of structural studies by X-Ray crystallography and biochemical approaches as in vitro and in vivo methods to study proteins-RNA interactions, I was able to uncover the function of the Rpf2/Rrs1 dimer in the maturation of the large subunit through the recruitment of the 5S particle. I also studied the function of Rpf2 and Rrs1 in the rRNA transcription regulation, by characterizing physical connection with polymerase I subunits.

Das 20S Proteasom spielt zusammen mit seinem 19S Regulator als 26S Proteasomkomplex eine zentrale Rolle beim Abbau von Proteinen in eukaryotischen Zellen. Dem 19S Regulator wird dabei die Funktion der Substraterkennung und -entfaltung sowie die Beteiligung an der Translokation der entfalteten Substrate zum katalytischen Zentrum zugeordnet. In dieser Arbeit konnte erstmals gezeigt werden, daß der 19S Regulator chaperonähnliche Eigenschaften besitzt, dadurch also durchaus die Entfaltung der Proteinsubstrate bewirken kann. Durch den 19S Regulator war das 26S Proteasom in der Lage, einen Teil denaturierter Citratsynthase, eines Modellsubstrats für die Untersuchung von Chaperonaktivitäten, ATP-abhängig zum nativen Zustand zurückzufalten. Desweiteren führte die Anwesenheit des 19S Regulators bzw. des 26S Proteasoms in Abwesenheit von ATP zu einer Aggregationshemmung denaturierter Citratsynthase. Auch konnte die direkte Interaktion zwischen der Citratsynthase und dem 26S Proteasom bzw. dem 19S Regulator durch Glyceroldichtegradientenzentrifugation gezeigt werden. Diese chaperonähnlichen Eigenschaften des 19S Regulators konnten dem aus sechs ATPasen und zwei nicht-ATPasen bestehenden Base-Subkomplex zugeordnet werden. Aufgrund der Wechselwirkungen zwischen dem 19S Regulator und dem 20S Proteasom und damit möglicherweise verbundenen Konformationsänderungen in den Komplexen, wurde postuliert, daß der 19S Regulator auch auf die Biogenese, also die Assemblierung und Reifung des 20S Proteasoms einen Einfluß haben könnte. Es konnte gezeigt werden, daß Mutationen in den 19S ATPasen zu einer Anreicherung der unprozessierten proteasomalen 20S Untereinheit beta5 bei erhöhter Temperatur führen. Die Ursache dieses Anreicherungseffektes konnte nicht aufgeklärt werden. Der Effekt läßt sich nicht auf eine Hochregulation der m-RNA-Synthese der beta5-Untereinheit zurückführen. Die Beteiligung des 19S Regulators an frühen Assemblierungsstadien des 20S Proteasoms ist aufgrund der Analyse der mit dem Maturierungsfaktor Ump1 im Komplex vorliegenden Proteine ebenfalls unwahrscheinlich. Eine Beteiligung des 19S Regulators an einem der letzten Schritte der 20S Proteasomenbiogenese, beispielsweise an der Katalyse der Prozessierung der beta-Untereinheiten, ist eher vorstellbar, konnte aber nicht eindeutig gezeigt werden. Auf die Prozessierung der beta-Untereinheiten hat aber auch die katalytische Aktivität der beta-Untereinheiten einen nicht unwesentlichen Einfluß. So werden die katalytisch aktiven Untereinheiten durch Autokatalyse zu ihrer aktiven Form prozessiert und bewirken die Prozessierung der katalytisch inaktiven Untereinheiten beta6 und beta7. Dies konnte so auch durch Inaktivierung der beta2i-Untereinheit bestätigt werden. Die Expression der inaktiven Maus-beta1iT1A-Untereinheit in humanen T2-Zellinien verhinderte ihre eigene vollständige Prozessierung, hatte aber auch Einfluß auf die Prozessierung von beta7 und von inaktiv exprimierten beta1i (Maus-beta1iT1A). / In eukaryotic cells the protein degrading proteasome/ubiquitin system is involved in a wide variety of regulatory processes. The 26S proteasome is composed of two subcomplexes, a proteolytic core (20S) and a regulatory complex (19S). It is proposed that the proteins of the 19S regulatory complex can recognize and unfold the substrates. Furthermore the RC participates in translocation of the substrates into the proteasomes inner chamber were peptide bond hydrolysis occurs. This work shows that the proteasome exhibits an ATPdependent chaperon-like activity on citrate synthase, a model substrat for chaperones. Human and yeast proteasomes stimulated the recovery of the native structure of citrate synthase in an ATPdependent manner. Furthermore the 19S complex was able to supress the aggregation of denatured citrate synthase. Glycerol gradient analysis indicated that proteasome facilitates the refolding of citrate synthase through the formation of citrate synthase-proteasome complexes as expected for a chaperon-like mechanism. The chaperonlike activity was mapped to the base of the 19S regulatory complex. The RC could be able to unfold protein substrates in the 26S proteasome by this activity. The crystal structure of S. cerevisiae 20S proteasome shows a closed gate to the proteasome interiors. The 19S regulatory complex may induce conformational changes not only to open the protease cavity but also to assit in beta-subunit processing during 20S proteasome biogenesis. To test this hypothesis some yeast 19S ATPase mutant strains were analyzed for defective 20S proteasome maturation by following the processing of beta5-subunit. This work has shown that some mutations in these ATPases led to an accumulation of the unprocessed proteasomal beta5-subunit at restricted temperature. This effect was not due to the upregulation of beta5 mRNA transcription. It is not very likely that proteins of the RC participate in early steps of proteasome biogenesis, since they could not be found in precurser intermediates containing the maturation factor Ump1p. However, they might be important for later assembly steps. During biogenesis five prosequence containing beta subunits have to be processed. This proceeds via a two-step mechanism involving autocatalytic or transcatalytic processing by neighbouring subunits. The proposed mechanism could be confirmed by inactivation of the beta2i subunit via substitution of the active site Threonin1 against Alanin (beta2iT1A). The inactivation blocked the autocatalysis of beta2i and influenced the processing of beta7 and that of inactivated beta1i (beta1iT1A).

Iberische und Eurasische Luchse zeigen einen saisonalen Monoöstrus. Nach der Ovulation findet man frisch gebildete (freshCL) und physiologisch persistierende Gelbkörper (corpora lutea, perCL). Funktionelle perCL verhindern eine Ovulation außerhalb der Zuchtsaison durch konstant erhöhte Progesteron-(P4)-Plasmawerte. Hauskatzen zeigen einen saisonalen Polyöstrus. Nach der Ovulation werden CL gebildet, deren Lebensspanne in Abhängigkeit von einer Trächtigkeit durch unterschiedliche P4-Plasmaprofile charakterisiert ist. Ziel der Dissertation war es, die Synthese und Rezeption von Steroiden und Prostaglandinen (PG) in CL von Feliden zu untersuchen, um potentiell luteotrophe und luteolytische Faktoren zu identifizieren. Während der Gelbkörperphase trächtiger und nicht-trächtiger Katzen weisen CL gleicher Histomorphologie, unabhängig vom Vorhandensein einer Trächtigkeit, ähnliche steroidogene Kapazitäten auf. Die Abnahme der CL-Funktion spiegelt sich im graduellen Verlust der Steroidbiogenese wider. Bei Luchsen ist die steroidogene Kapazität der perCL im Proöstrus herabgesetzt, aber im Metöstrus wieder verstärkt. Die steroidogene Kapazität ist demnach mit verschiedenen CL-Stadien und dem Reproduktionszyklus assoziiert. Die Synthese und Rezeption von PGE2 erfolgen bei Katze und Luchs unabhängig vom CL-Stadium und dem Reproduktionszyklus. Hohe Werte an luteotrophem PGE2 in perCL könnten für die funktionelle und strukturelle CL-Persistenz beim Luchs verantwortlich sein. Der feline CL ist zur Bindung von luteolytischem PGF2alpha fähig, jedoch ist die Kapazität zur Synthese begrenzt. Feliden weisen keine PGF2alpha-assoziierte luteale Regression in Abwesenheit einer Trächtigkeit auf. Allerdings wurden Höchstwerte an PGF2alpha in der Plazenta, wie auch im Plasma (PGFM), im letzten Trächtigkeitstrimester der Katze gemessen. Folglich ist die feline Plazenta zur Synthese von luteolytischem PGF2alpha fähig, welches die CL-Regression und Geburt am Ende der Trächtigkeit ermöglicht. / Iberian and Eurasian lynxes exhibit a seasonal monooestrus. After ovulation, freshly formed (freshCL) coexist with physiologically persistent luteal bodies (corpora lutea, perCL). Functional perCL prevent ovulation outside the breeding season through constantly elevated plasma progesterone (P4) levels. Domestic cats show a seasonal polyoestrus. After ovulation, CL are built with lifespans being characterised by different plasma P4 profiles dependent on pregnancy. The aim of the dissertation was to characterise the synthesis and reception of steroids and prostaglandins (PGs) in CL of felids to identify potential luteotrophic and luteolytic factors. During the luteal lifespan of pregnant and non-pregnant cats, CL of equal histomorphology exhibit similar steroidogenic capacities, irrespectively of an ongoing pregnancy. The functional demise of CL mirrors the gradual loss of steroid biogenesis. In lynxes, the steroidogenic capacity of perCL is limited at prooestrus, but is enhanced again during metoestrus. The steroidogenic capacity is thus associated with different CL stages and the reproductive cycle. The synthesis and reception of PGE2 in cat and lynx is independent on the CL stage and reproductive cycle. High levels of luteotrophic PGE2 in perCL might be responsible for the functional and structural CL persistence in lynxes. The feline CL is capable of binding luteolytic PGF2alpha; however, the capacity to synthesise PGF2alpha is limited. Felids show no PGF2alpha-associated luteal regression in the absence of pregnancy. Interestingly, peak levels of PGF2alpha in the placenta, as well as in plasma (PGFM), were measured during the last trimester of pregnancy in the cat. Therefore, the feline placenta is capable of synthesising luteolytic PGF2alpha, which enables CL regression and parturition at the end of pregnancy.

SoxR est un régulateur transcriptionnel à centre [2Fe-2S] qui induit une réponse adaptative permettant à E. coli de résister aux composés redox actifs, générateurs de stress superoxyde. En présence de composés redox actifs, le centre [2Fe-2S] de SoxR est oxydé ce qui lui permet d’activer l’expression du gène soxS codant pour un régulateur transcriptionnel activant l’expression d’une centaine de gènes. Parmi les gènes du régulon SoxRS on trouve ceux permettant de résister au superoxyde mais aussi aux antibiotiques. J’ai montré qu’en présence de phénazine méthosulfate (PMS), un composé redox actif, la machinerie de biogénèse des centres Fe-S utilisée pour la maturation de SoxR est différente suivant les conditions environnementales. En effet, en aérobie la maturation de SoxR est assurée par la machinerie SUF, alors qu’en anaérobie c’est la machinerie ISC qui intervient. J’ai également étudié l’importance de SoxR, et des machineries ISC et SUF, dans la résistance aux antibiotiques induite par la présence de PMS. J’ai montré qu’en présence de PMS, E. coli peut résister à la norfloxacine, par un mécanisme SoxR dépendent, et ceci quelque soit la machinerie de biogénèse des centres FeS présente. D’autre part, j’ai étudié l’impact des conditions environnementales, comme la teneur en CO2 dans l’atmosphère sur la capacité d’ E. coli à résister au stress oxydant. J’ai testé, expérimentalement les prédictions obtenues par un modèle d’équations différentielles permettant de simuler la concentration des ROS dans la cellule. J’ai montré que le CO2 a un effet de protection lors d’un stress au H2O2 probablement en capturant les HO• produits par la réaction de Fenton. / SoxR is a [2Fe-2S] cluster-containing transcriptional regulator that mounts the adaptive response allowing E. coli to tolerate superoxide-propagating compounds. When cells are exposed to redox cycling drugs the Fe-S cluster of SoxR undergoes a reversible univalent oxidation to yield the oxidized active protein. The only known target of SoxR is the soxS gene that is itself a transcriptional regulator activating the expression of more than 100 genes including those for superoxide and antibiotic resistance. I showed that the machinery used to mature SoxR under phenazine methosulfate (PMS) exposition, a redox cycling drug, was different depending on the environmental conditions used. In aerobiosis, the SUF machinery ensured SoxR maturation, while in anaerobiosis the ISC machinery was required. I also monitored the implication of SoxR, the ISC and SUF machineries, in antibiotic resistance induced by PMS exposition. I showed that E. coli can resist to norfloxacin under PMS exposition in a SoxR-dependent manner whatever the Fe-S cluster biogenesis machinery available. Last, I studied the impact of environmental conditions, such as atmospheric CO2 concentration, on the ability of E. coli to cope with oxidative stress. I have experimentally tested the predictions obtained by a mathematical model that simulates ROS dynamics. I showed that carbon dioxide has a protective effect on hydrogen peroxide stress likely by scavenging the radical hydroxyl produced by the Fenton reaction.