A hipoxantina, principal oxipurina envolvida na via de salvação das purinas no cérebro, acumula-se na doença de Lesch-Nyhan, um erro inato do metabolismo das purinas. Os sintomas clínicos manifestam-se precocemente na vida dos pacientes, incluindo alterações motoras e cognitivas, retardo mental e automutilação. Embora os mecanismos subjacentes da disfunção cerebral na doença de Lesch-Nyhan sejam pouco compreendidos, o acúmulo de hipoxantina parece contribuir para os danos neurológicos. O objetivo deste estudo foi investigar os efeitos da administração intraestriatal de hipoxantina em ratos infantis e adultos jovens submetidos à cirurgia estereotáxica. Neste estudo, analisamos primeiramente o efeito da hipoxantina sobre os parâmetros neuroinflamatórios e oxidativos em estriados de ratos infantis e adultos jovens. Foram avaliados também alguns parâmetros neuroenergéticos. Ratos Wistar de 21 e 60 dias de vida foram submetidos à cirurgia estereotáxica e foram divididos em dois grupos: controle (infusão de solução salina 0,9%) e hipoxantina (10 IJM). A administração intraestriatal de hipoxantina aumentou os níveis de IL-6 e TNF-a e o imunoconteúdo da subunidade NF-kB I p65 nuclear em estriado de ambas as idades de ratos. A ativação microglial e astrocitária foram observadas pelo aumento do imunoconteúdo de lba1 e GFAP, respectivamente, no estriado de ratos de 21 dias. Todos os parâmetros oxidativos foram alterados, sugerindo uma forte relação neurotóxica de hipoxantina e estresse oxidativo. Em ratos de 60 dias de vida, a hipoxantina aumentou a atividade da succinato desidrogenase e do complexo 11 e diminuiu a atividade da citocromo c oxidase e seu imunoconteúdo. A injeção de hipoxantina diminuiu a porcentagem marcação de membrana mitocondrial e aumentou marcação de potencial mitocondrial. A hipoxantina também diminuiu o número de células vivas e aumentou o número de células apoptóticas. Em ratos de 21 dias de vida, a hipoxantina alterou alguns parâmetros do metabolismo energético e diminui a atividade de Na+,K+-ATPase, provavelmente por danos às proteínas, visto pela redução de conteúdo de sulfidrilas. Nossos achados mostram que a administração de hipoxantina alterou parâmetros neuroinflamatórios e neuroenergéticos, possivelmente por meio de desequilíbrio oxidativo, sugerindo que esses processos podem estar envolvidos, pelo menos em parte, com os distúrbios neurológicos encontrados em pacientes com doença de Lesch-Nyhan. / Hypoxanthine, the major oxypurine metabolite involved in purine's salvage pathway in the brain, is accumulated in Lesch-Nyhan disease, an in bom errar of metabolism of purine. The clinicai symptoms manifest early in the patients' lives, including motor and cognitiva alterations, mental retardation and self-mutilation. Although the underlying mechanisms of brain dysfunction in Lesch-Nyhan disease are poorly understood, the accumulation of hypoxanthine appears to contribute to neurological damage. The purpose of this study was to investigate the effects of hypoxanthine intrastriatal administration in infant and young adult rats submitted to stereotactic surgery. We firstly analyzed the effect of hypoxanthine on neuroinflammatory and oxidative parameters in striatum of infant and young adult rats. We also evaluated some neuroenergetic parameters. Wistar rats of 21 and 60 days of life underwent stereotactic surgery and were divided into two groups: contrai (infusion of saline 0.9%) and hypoxanthine (10 IJM). lntrastriatal administration of hypoxanthine increased IL- 6 and TNF-a leveis and nuclear immunocontent of NF-KB/p65 subunit in striatum of rats of both ages. Microglial and astrocyte activation was seen by the increase in lba1 and GFAP immunocontent, respectively, in striatum of infant rats. Ali oxidative parameters were altered, suggesting a strong neurotoxic hypoxanthine role on oxidative stress. In 60-day-old rats hypoxanthine increased succinate dehydrogenase and complex 11 activities and diminished cytochrome c oxidase activity and immunocontent. Hypoxanthine injection decreased the percentage of cells with mitochondrial membrana label and increased mitochondrial mass potential labeling. Hypoxanthine also diminished the number of live cells and increased the number of apoptotic cells. In 21-days-old rats hypoxanthine altered some energy metabolism parameters and decreased Na•,K•-ATPase activity probably by protein damage, seen in the reduction of sulfhydryl content. Our findings show that the administration of hypoxanthine altered neuroinflammatory and neuroenergetic parameters, possibly through oxidative imbalance, suggesting that these processes may be involved, at least in part, with the neurological disorders found in patients with Lesch-Nyhan disease.

Les membranes plasmiques et les membranes du trans-Golgi des cellules eucaryotes présentent une asymétrie des lipides qui les composent, avec les aminophospholipides (APLs : phosphatidylsérine et phosphatidyléthanolamine) enrichis dans le feuillet cytosolique. La dissipation de cette asymétrie est impliquée dans de nombreux processus (patho)physiologiques. Plusieurs études suggèrent que les ATPases P4 sont les candidats les plus probables pour le transport des APLs et le maintien de leur distribution asymétrique ; leur délétion dans la levure inhibe le trafic membranaire. En outre, des études ont montré que les ATPases P4 interagissaient avec les protéines de la famille CDC50 ; cette interaction est essentielle pour l’adressage et peut-être aussi la fonction des ATPases P4. Afin de contribuer à la compréhension du mécanisme de transport des lipides par les ATPases P4, l’objectif de ce travail a été de mettre au point la co-expression fonctionnelle, dans la levure, de l’ATPase P4 Drs2p et de sa protéine partenaire Cdc50p. Nous avons obtenu une fraction membranaire enrichie à 3% avec la protéine Drs2p, majoritairement en complexe avec Cdc50p. L’étude fonctionnelle du complexe nous a permis de mettre en évidence un rôle crucial du phosphatidylinositol-4-phosphate (PI(4)P), un important régulateur du trafic membranaire, au cours d’une étape particulière du cycle catalytique. Nous avons également développé un protocole de purification sur résine streptavidine du complexe Drs2p/Cdc50p. Enfin, comme un site potentiel d’interaction avec le PI(4)P est présent sur l’extrémité C-terminale de Drs2p, nous avons engendré différentes constructions de Drs2p, dans lesquelles une partie de l’extrémité C-terminale a été délétée ; dans une autre construction, l’extrémité N-terminale a également été délétée. Notre travail ouvre la voie à la caractérisation fonctionnelle et structurale détaillée du complexe Drs2p/Cdc50p, et à l’étude du rôle du transport de lipides dans le trafic membranaire. / Trans-Golgi membranes and plasma membranes of eukaryotic cells are asymmetric, with their cytosolic leaflet enriched in aminophospholipids (APLs: phosphatidylserine and phosphatidylethanolamine). Dissipation of this asymmetry is involved in many (patho)physiological processes. P4 ATPases are prime candidates for APL transport and for maintaining asymmetry across membranes. In addition, yeast deleted for P4 ATPases display membrane trafficking defects. Besides, CDC50 proteins have been shown to interact physically with P4 type ATPases, and this interaction is important for addressing the complex to the right destination, and possibly also for its function. To gain insight into the molecular mechanism of lipid transport by P4 ATPases, the goal of my thesis was to develop the co-expression, in yeast, of a functional P4 ATPase, Drs2p, together with its partner, Cdc50p. The strategy we developed allowed us to obtain a membrane fraction enriched in Drs2p (~3%), mainly in complex with Cdc50p. Functional characterization of the complex identified phosphatidylinositol-4-phosphate (PI4P), a major regulator of membrane trafficking, as a crucial component for rapid completion of the Drs2p/Cdc50p catalytic cycle. We also purified the complex in one step on streptavidin beads. Finally, we started investigating the potential auto-inhibitory roles of the C-terminus (as the C-terminus of Drs2p contains a PI4P binding site) and the N-terminus of Drs2p, by expressing various truncated versions of Drs2p. Our work sets the stage for detailed functional and structural characterization of the Drs2p/Cdc50p complex and its role in membrane traffic.

Accurate smoltification and disease management in Atlantic salmon (Salmo salar) are key issues for the aquaculture industry. Due to their anadromous lifecycle the transfer of salmon from fresh water (FW) to seawater (SW) is crucial to their survival; too early can cause mortality, too late can cause desmoltification and long-term health problems. Both scenarios can increase susceptibility to four viral diseases: Salmon alphavirus (SAV), Infectious salmon anemia virus (ISAV), orthoreovirus (PRV), and Piscine myocarditis virus (PMCV). They all show similar clinical and histopathological symptoms and can easily spread throughout farms. Understanding the initial innate immune response to these viruses may provide biomarkers that could help identify and monitor infections. An in house and onsite Na+/K+ ATPase (NKA) qRT-PCR assay was developed for the salmon biomarker ATPase to test smoltification readiness in salmon smolts. Tested against NKA enzymatic assays it showed a similar success rate over 3 years: NKA qRT-PCR (57%), NKA activity assay (60%). Onsite tests confirmed that the ATPase mRNA transcript is a useful biomarker for smoltification detection. An in-lab and mobile multiplex qRT-PCR assay was developed for detection of SAV, PRV and PMCV. The analytical sensitivity of the SAV (86.5% SE 0.11), PRV (90.94%, SE 0.09) and PMCV (100.46%, SE 0.19) assays was 102 copies for PMCV and 103 for SAV and PRV. Initial results suggest individual assays could be run on site at farms. Addition of an internal control, probit analysis and viral positive tests are still required for multiplex assay integration. Salmon erythrocytes were infected with ISAV, SAV and Poly I:C to investigate whether they induce and up-regulate innate immune response genes. All genes were expressed at low levels in all parameters investigated including non-infected control erythrocytes. These findings suggest erythrocytes act as an initial buffer to viral infections and may help stimulate the innate immune response.

Saccharomyces cerevisiae teve seu seqüenciamento genômico finalizado em 1996, sendo composto de cerca de 6600 ORF\'s ( \"open reading frames\", quadros abertos de leitura) das quais aproximadamente 20% estão anotadas como não caracterizadas e de função molecular desconhecida. Com o objetivo de caracterizar algumas dessas ORF\'s relacionadas aos processos antioxidantes, escolhemos a proteína Yjl077cp. Através de nossos experimentos foi possível relacionar a proteína Yjl077cp com a resposta antioxidante, pois a linhagem mutante para o gene YJL077C apresentou letalidade quando exposta por 24 horas a uma concentração de 3mM de peróxido de hidrogênio (H2O2) . Testes de tolerância ao cobre, demonstraram que esse metal na concentração de 10mM, adicionado diretamente ao meio de cultura YPD, após um período de 4 horas de incubação afeta a cinética de crescimento, e após 24 horas de incubação, essa concentração é letal para a linhagem mutante. Observamos que a adição de CuSO4, causa modificação de pH do meio de cultura YPD, foram realizados experimentos para que verificássemos o efeito da acidificação do meio de cultura na viabilidade de Δics3, através de experimentos de tolerância em meio YPD ácido (pH 4.0) e com pH corrigido com adição de CuSO4. De acordo com os resultados obtidos, o efeito da letalidade de 10 mM de CuSO4 observado sem correção de pH, não ocorre quando em meio de cultura com o pH corrigido (pH6.0), demonstrando que, a letalidade da linhagem Δics3 é causada pelo acréscimo do metal e a mudança de pH (ácido, pH4.0). Testes de ICP-AES detectaram uma maior absorção intracelular de cobre na linhagem mutante Δics3, em condições de pH 4.0, quando comparada a absorção desse mesmo metal em condições de pH 6.0, demonstrando que grandes quantidades de cobre, estão sendo incorporadas para o meio intracelular, devido a acidificação do meio, sugerindo o envolvimento de Δics3, às ações das VATP-ases. Em conjunto esses resultados demonstram que o gene ICS3 é importante para a manutenção da viabilidade celular de S. cerevisiae crescida em meio de cultura com pH ácido e em condições de altas concentrações de CuSO4. O objetivo do presente trabalho foi entender a relação de ICS3 na resposta antioxidante e sensibilidade ao cobre através de experimentos de tolerância e em meio YPD ácido (pH 4.5) e com pH corrigido após adição de CuSO4. / Saccharomyces cerevisiae had its genome sequencing completed in 1996, consisting of about 6600 ORF\'s (\"open reading frames\" open reading frames) of which approximately 20% are annotated as uncharacterized and unknown molecular function. In order to characterize some of these ORF\'s related to antioxidants processes, we chose Yjl077cp protein . Through our experiments it was possible to relate the Yjl077cp protein with the antioxidant response, because the mutant strain to YJL077C gene showed lethality when exposed for 24 hours at a concentration of 3 mM hydrogen peroxide (H2O2). Copper tolerance tests demonstrated that this metal at concentration 10mM added directly to the medium YPD culture after a 4-hour period of incubation affects the growth kinetics, and after 24 hours of incubation, these concentration is lethal to mutant strain. We found that the addition of CuSO4, causes pH modification in the YPD culture medium, experiments were conducted to we check the effect of acidification of the culture medium on the viability of Δics3 through experiments of acid tolerance in YPD medium (pH 4.0) and pH adjusted with addition of CuSO4. According to the results, the effect of mortality of 10 mM CuSO4 observed without pH correction, does not occur when the culture medium with the Ph adjusted (pH6.0), demonstrating that the lethality of the strain Δics3 is caused by adding the metal and the change of pH (acid, pH4.0). ICP-OES tests detected greater intracellular uptake of copper in the mutant strain l1ics3 under conditions of pH 4.0 when compared to absorption of the same metal under conditions of pH 6.0, demonstrating that large amounts of copper, are being incorporated into the intracellular medium due to acidification of the medium, suggesting the involvement of Δics3 to the actions of the VATP-ases. Together, these results demonstrate that ICS3 gene is important for maintaining cell viability of S. cerevisiae grown in culture medium at acidic pH, and under conditions of high concentrations of CuSO4. The objective of this study was to understand the relationship ICS3 in antioxidant response and sensitivity to copper through experiments tolerance and YPD medium acid (pH 4.5) and fixed pH after addition of CuSO4.

Symmetric cell division in gram-negative bacteria is essential for generating two equal-sized daughter cells, each containing cellular material crucial for growth and future replication. The Min system, comprised of proteins MinC, MinD and MinE, is particularly important for this process since its deletion leads to minicells incapable of further replication. This thesis focuses on the interactions involving MinE that are important for allowing cell division at the mid-cell and for directing the dynamic localization of MinD that is observed in vivo. Previous experiments have shown that the MinE protein contains an N-terminal region that is required to stimulate MinD-catalyzed ATP hydrolysis in the Min protein interaction cycle. However, MinD-binding residues in MinE identified by in vitro MinD ATPase assays were subsequently found to be buried in the hydrophobic dimeric interface in the MinE structure, raising the possibility that these residues are not directly involved in the interaction. To address this issue, the ability of N-terminal MinE peptides to stimulate MinD activity was studied to determine the role of these residues in MinD activation. Our results implied that MinE likely undergoes a change in conformation or oligomerization state before binding MinD. In addition we performed circular dichroism spectroscopy of MinE. The data suggest that direct interactions between MinE and the lipid membrane can lead to conformational changes in MinE. Using NMR spectroscopy in an attempt to observe this structure change, different membrane-mimetic environments were tested. However the results strongly suggest that structural studies on the membrane-bound state of MinE will pose significant challenges. Taken together, the results in this thesis open the door for further exploration of the interactions involving MinE in order to gain a better understanding of the dynamic localization patterns formed by these proteins in vivo.

Major Histocompatibility Complex Class-II (MHC-II) molecules are critical regulators of adaptive immunity that present extracellular antigens required to activate CD4+ T cells. MHC-II are regulated at the level of transcription by master regulator, the Class II Transactivator (CIITA), whose association with the MHC-II promoter is necessary to initiate transcription. Recently, much research focused on novel mechanisms of transcriptional regulation of critical genes like MHC-II and CIITA; findings that the macromolecular complex of the 26S-proteasome is involved in transcription have been perhaps the most exciting as they impart novel functions to a well studied system. Proteasome is a multi-subunit complex composed of a 20S-core particle capped by a 19S-regulatory particle. The 19S contains six ATPases which are required for transcription initiation and elongation. We demonstrate that 19S ATPase-S6a inducibly associates with CIITA promoters. Decreased expression of S6a negatively impacts recruitment of the transcription factors STAT-1 and IRF-1 to the CIITA due to significant loss in histone H3 and H4 acetylation. S6a is robustly recruited to CIITA coding regions, where S6a binding coordinates with that of RNA polymerase II. RNAi mediated S6a knockdown significantly diminishes recruitment of Pol II and P-TEF-b components to CIITA coding regions, indicating S6a plays important roles in transcriptional elongation. Our research is focused on the ways in which accessibility to and transcription of DNA is regulated. While cancers are frequently linked to dysregulated gene expression, contribution of epigenetics to cancers remains unknown. To achieve metastatic ability, tumors alter gene expression to escape host immunosurveilance. MHC-II and CIITA expression are significantly downregulated in highly metastatic MDA-MB-435 breast cancer cells. This suppression correlates with elevated levels of the silencing modification H3K27me3 at CIITA and a significant reduction in Pol II recruitment. We observe elevated binding of the histone methyltransferase to CIITApIV and demonstrate this enzyme is a master regulator of CIITA gene expression. EZH2 knockdown results in significant increases in CIITA and MHC-II transcript levels in metastatic cells. In sum, transcriptional regulation by the 19S-proteasome and histone modifying enzymes represents novel mechanisms of control of mammalian gene expression and present novel therapeutic targets for manipulating MHC expression in disease.

Mediante diferentes técnicas histoquímicas, se analizan los tipos de fibras de la musculatura esquelética (Mm. semitendinoso y flexor carporradial) de perros adultos y cachorros (0-65días). Tomando el músculo semitendinoso como patrón, se establecen los criterios para la correcta identificación de las distintas fibras. En base a la actividad mATPasa se reconocen 4 tipos diferentes, que denominamos: I, IIA, IIp y IIC. Con idéntica metodología y criterios de interpretación, se procede al estudio el M. flexor carporradial. Secciones transversales completas de este músculo obtenidas en criosotato a -20ºC, fueron analizadas desde el punto de vista histoquímico, procediéndose al estudio de los tipos de fibras y distribución de las mismas. Los resultados obtenidos indican que el M. flexor carporradial presenta dos cabezas, radial y cubital, cuya funcionalidad puede ser diferente. La cabeza radial se integra casi de forma exclusiva por fibras tipo I, mientras la cabeza cubital muestra una distribución en mosaico con presencia de fibras I, IIA y IIC. Las fibras IIp no fueron identificadas en ninguna de las dos cabezas. Los resultados parecen sugerir que el M. flexor carporradial del perro no solo participa en la protracción del miembro torácico (flexión del carpo), sino que desarrolla un importante papel en el aplomo del mismo. Los estudios llevados a cabo durante el periodo postnatal (0-65días), confirman la aparición gradual y progresiva de las fibras identificadas en animales adultos, aunque en distintos periodos de tiempo según el músculo y/o cabezas. Las técnicas de m-ATPasa permiten formular una nomenclatura específica para las fibras durante el desarrollo, que puede ser tomada como referencia para futuros estudios en estos periodos. / By jeans of different histochemical techniques, fibre types of skeletal muscle (Mm. semitendinosus and flexor carpi radialis) in adults and young dogs (0-65 days) were analysed. Histochemical criteria to the right identification of the different fibres were established by using semiteninosus muscle as control. According to their m-ATPase activity, we observed four different fibre types designated as I, IIA, IIp and IIC. To study the flexor carpi radialis muscle the same techniques and histochemical criteria were followed too. Cryostat whole cross serial sections were histochemically analysed, and the fibre types as well as their distribution studied. The results showed that flexor carpi radialis muscle has two heads, radial and cubital, probably with different functionality. Radial head was almost exclusively constituted by type I fibres; on the other hand, cubitl head exhibited mosaic pattern distribution, containing I, IIA and IIC fibres. None of the heads showed IIp fibre types. These findings suggest that flexor carpi radialis muscle in dog not only participates in the protraction of the forelimb (flexes carpal joint) but also takes an important part in its own aplomb. This study which was carried out throughout postnatal period (0-65 days) confirmed that occurrence of the different fibre types is gradual and progressive in time depending on muscle and / or head portion. mATPase techniques allow us the formulation of an specific nomenclature for the fibres during their development thus, it is recommended for further studies of muscle development.

La famille RecQ des ADN hélicases est impliquée dans la maintenance de la stabilité génomique. Nous nous sommes intéressés, au cours de ce travail, au rôle du doigt de zinc du domaine RecQ Ct de ces hélicases. Les résultats indiquent que le motif à doigt de zinc est impliqué dans la fixation à l'ADN et le repliement correct de la protéine RecQ. Nous avons montré que le domaine hélicase de RECQ5 possède une activité intrinsèque qui tend à favoriser l'hybridation d'ADN et que le doigt de zinc de RECQ5(3 régule la fixation à l'ADN, l'activité ATPase, le déroulement de l'ADN, l'hybridation de l'ADN et l'échange des brins d'ADN. II semble que la présence du doigt de zinc est essentielle pour les activités ATPase et hélicase des hélicases RecQ, mais pas pour l'activité d'hybridation d'ADN. Notre travail sur les hélicase RecQ de Bacillus subtilis soutient aussi cette conclusion.

Le chloroplaste est un organite spécifique de la cellule végétale. Il est délimité par une double membrane ou enveloppe qui renferme de nombreux systèmes de transports d'ions et de métabolites essentiels au fonctionnement du chloroplaste et de la cellule végétale. A ce jour, seuls quelques transporteurs de métaux associés à l'enveloppe des plastes ont été identifiés : un transporteur de fer, un transporteur de magnésium ainsi que deux ATPases à cuivre de type P1B : HMA1 et PAA1. PAA1 représenterait la voie principale d'import du cuivre dans le chloroplaste, notamment pour alimenter la photosynthèse. HMA1 constituerait une voie additionnelle d'import du cuivre, voie essentielle pour répondre à un stress oxydatif qui apparaît en particulier lorsque la plante est cultivée en lumière forte. Afin de mieux comprendre les rôles respectifs de ces deux ATPases dans la régulation de l'homéostasie du cuivre chloroplastique, deux approches complémentaires ont été développées : - une approche in planta visant à produire de nouvelles lignées affectées dans l'expression de l'une (mutant paa1 surexprimant HMA1) ou de ces deux ATPases (double mutant hma1/paa1), puis à identifier des conditions (stress lumineux, stress salin, excès de métaux) révélant le rôle essentiel de ces ATPases ou induisant une réponse transcriptionnelle différente des gènes codant ces ATPases ou d'autres acteurs liés à l'homéostasie du cuivre... Les résultats obtenus montrent que la fonction de HMA1, connue pour être essentielle lors d'un stress lumineux, est aussi requise lorsque la plante subit un stress salin. Ces résultats confortent le rôle de HMA1 dans la délivrance du cuivre à la superoxyde dismutase (cuivre/zinc) du chloroplaste. D'autre part, nous avons montré qu'en condition de culture photoautotrophe, le cuivre permet de supprimer partiellement la photosensibilité du mutant hma1, validant ainsi l'implication de HMA1 dans l'homéostasie du cuivre. Nous avons aussi démontré que les fonctions de HMA1 et PAA1 ne sont pas redondantes. Le cuivre importé par ces deux ATPases est probablement délivré à des protéines cibles par des voies différentes. Enfin, nous avons montré qu'il existe une troisième voie d'import de cuivre dans le chloroplaste, voie encore non caractérisée. - une approche in vitro visant à produire ces deux ATPases HMA1 et PAA1 dans le système hétérologue Lactococcus lactis afin de déterminer leurs spécificités ioniques et leurs caractéristiques biochimiques. Le système d'expression procaryote L. lactis a été mis en place au laboratoire et s'avère parfaitement adapté à la production de protéines membranaires de plantes. Ce système a permis de produire plusieurs protéines membranaires d'Arabidopsis, dont HMA1 et PAA1, en quantités compatibles avec des analyses biochimiques. Nous avons déterminé les conditions de solubilisation et de purification de ces deux protéines. Nous n'avons pas pu mesurer d'activité ATPase associée à ces protéines. En revanche, nos données indiquent que ces deux protéines recombinantes peuvent lier l'un de leur substrat ; l'ATP. Nous avons aussi pu démontrer que PAA1 peut lier du cuivre sous forme 1+ et sous forme 2+. Au bilan, ces données suggèrent que le contrôle de l'homéostasie du cuivre chloroplastique requiert plusieurs systèmes de transport indépendants et une régulation fine de ces voies d'import de cuivre afin d'alimenter les besoins liés à la photosynthèse et les besoins liés aux mécanismes de résistance aux stress oxydatifs.

L'adaptabilité des cellules aux changements environnementaux repose sur leur capacité à mettre en place des complexes enzymatiques qui régulent leurs différentes voies métaboliques. Ces complexes protéiques nécessitent des cofacteurs (hème et métaux) comme le cuivre. En effet, le cuivre est un oligoélément essentiel pour la survie des organismes vivants, il est notamment présent dans des complexes tels que la cytochrome c oxydase de la chaine respiratoire aérobie. Cependant, l'excès de Cu est aussi néfaste pour la cellule. Il génère du stress oxydatif responsable de divers dommages cellulaires. Il est donc nécessaire aux cellules de contrôler la prise en charge du cuivre afin d'assurer la régulation de sa concentration intracellulaire. Dans ce travail, nous avons étudié le système de tolérance au cuivre chez Rubrivivax gelatinosus. J'ai identifié plusieurs gènes impliqués dans ce système, un de ces gènes code pour un transporteur de cuivre CopA (homologue aux ATP7A/ATP7B de l'homme). Les autres gènes codent pour : un régulateur de ce système sensible aux variations du cuivre CopR et deux protéines solubles (CopI et CopJ) fortement induites dans des conditions d'excès de cuivre. J'ai également montré que malgré l'homologie de séquence entre les pompes à cuivre CopA et CtpA (identifiées auparavant au laboratoire) ; elles remplissent deux rôles physiologiques différents dans la cellule. CopA est vitale pour la tolérance au Cu alors que CtpA a un rôle dans l'insertion du Cu au sein des cuproprotéines. Par ailleurs, j'ai montré qu'en absence de CopA, la toxicité du cuivre est plus importante dans les conditions de croissance en microaérobie ou en photosynthèse anaérobe. En effet, l'excès du Cu affecte la voie de biosynthèse des porphyrines (hèmes et bactériochlorophylles) reflété par l'accumulation d'un intermédiaire de cette voie, la coproporphyrine III. Cette accumulation résulte de l'effet de l'excès du cuivre sur la coproporphyrine III oxydase (HemN) probablement en déstabilisant son cluster [4Fe-4S]. Ces résultats sont soutenus par le fait que l'effet toxique du cuivre sur les tétrapyrolles n'est pas observé à forte aération en raison de la substitution de HemN par une enzyme à centre bimétalliques HemF. Cette dernière tolère et utilise l'oxygène, notre étude suggère qu'elle tolère aussi le cuivre. Les résultats obtenus sur les cibles du cuivre en anaérobie au cours de cette étude ainsi que celle publiés par Macomber (Macomber and Imlay 2009), suggèrent que les métaux ont pu éventuellement jouer un rôle dans l'émergence des enzymes à clusters bimétalliques lors du passage à une atmosphère oxygénique permettant une adaptation à l'augmentation de la toxicité de certains métaux. L'ensemble des résultats obtenus à partir de l'analyse des différents mutants au cours de cette étude sur la tolérance au cuivre chez R. gelatinosus me permet de proposer un modèle d'efflux du cuivre différent de celui d'E. coli. Je propose d'attribuer un rôle crucial aux protéines solubles CopI et CopJ qui fixeraient probablement le cuivre grâce aux nombreux résidus histidines et méthionines. Leurs rôles seraient soit de séquestrer le cuivre au niveau du périplasme, soit de l'oxyder ou encore de l'excréter vers un système d'efflux de la membrane externe afin de le chasser de la cellule.