Orientador: Antonio Condino Neto / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-12T09:56:00Z (GMT). No. of bitstreams: 1 Blanco_MarcosLuengo_D.pdf: 1688728 bytes, checksum: fc6f41e84fcfcc3055bd8f9334a45c46 (MD5) Previous issue date: 2008 / Resumo: Neste trabalho, estudou-se o papel funcional de fator nuclear kappa B (NF-?B), sobre a regulação do gene CYBB e, conseqüentemente, a influência que esse fator exerce sobre o desenvolvimento do sistema NADPH oxidase humano. Células mielocíticas da linhagem U937 transformadas com um vetor carregando o gene repressor de, NF-?B (I-?Ba-S32A/ S36A) demonstraram uma redução na liberação de ânion superóxido e uma diminuição nos níveis de expressão do gene CYBB comparado ao grupo controle de células U937 selvagens. Em paralelo, os mesmos parâmetros foram avaliados em linfócitos B de pacientes com Displasia Anidrótica Ectodérmica com Imunodeficiênca (DAE-ID) transformadas com vírus de Epstein-Barr (EBV), (EBV-DAE-ID?B S32I e DAE-ID NEMO/IKK? X420W), e células de um paciente portador de Doença Granulomatosa Crônica (DGC), com mutação ligada ao X (EBV-DGC-X91°). Esses experimentos também mostraram os mesmos efeitos inibidores da atividade NADPH oxidase, assim como a redução dos níveis de expressão do gene CYBB. Os ensaios de EMSA supershift (anti-p50 humano) demonstraram ausência da ligação do complexo protéico à seqüência consenso para NF-?B 5' do gene do CYBB, confirmando o bloqueio da translocação deste fator para o interior do compartimento nuclear das células U937 transformadas e das células EBV-DAE-ID?B S32I e DAE-ID NEMO/IKK? X420W. Diante destes resultados, conclui-se que o NF-?B é necessário para a ativação da expressão do gene CYBB e conseqüentemente para o desenvolvimento do sistema NADPH oxidase de fagócitos humanos. / Abstract: This work investigated the functional role of nuclear factor-? B (NF-?B) in respiratory burst activity and in expression of the human phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase gene CYBB. U937 cells with a stably transfected repressor of NF-?B (I-?Ba-S32A/ S36A) demonstrated significantly lower superoxide release and lower CYBB gene expression compared with control U937 cells. We further tested Epstein-Barr virus (EBV)-transformed B cells from patients with anhidrotic ectodermal dysplasia with immunodeficiency (EDAID), an inherited disorder of NF-?B function (DAE-IDI?B S32I e DAE-ID NEMO/IKK? X420W). Superoxide release and CYBB gene expression by EDA-ID cells were significantly decreased compared with healthy cells and similar to cells from patients with X-linked chronic granulomatous disease (X91° CGD). Gel shift assays demonstrated loss of recombinant human p50 binding to a NF-?B site 5' to the CYBB gene inU937 cells treated with NF-?B inhibitors, repressor-transfected U937 cells, and EDA-ID patients' cells. These studies show that NF-?B is necessary for CYBB gene expression and activation of the phagocyte NADPH oxidase in this model system. / Doutorado / Doutor em Farmacologia

Acreditava-se inicialmente que a produção de espécies reativas de oxigênio (EROs) estava associada apenas aos danos oxidativos e efeitos deletérios às células. Atualmente, evidências sugerem que as EROs desempenham papel benéfico e estão associadas às adaptações estruturais e funcionais das células, por meio de regulação de vias de sinalizações celulares. Nas células musculares, sabe-se que sua função é dependente do estado redox das mesmas. De fato a produção exacerbada destas EROs é um fator limitante da contração muscular, no entanto, um ambiente celular reduzido também afeta negativamente a função muscular. Além disso, adaptações ao exercício físico parecem ser reguladas por vias de sinalizações sensíveis a oxidação por EROs. A NADPH oxidase é um importante complexo enzimático produtor de EROs no músculo esquelético (ME) e considerada como principal fonte de EROs no citosol durante a contração. Além disso, as proteínas envolvidas na contração muscular são sensíveis e reguladas dependente do estado redox celular, e, a NADPH oxidase esta localizada, aparentemente de forma estratégica, próxima a estas proteínas. Desta forma, tornou-se pertinente o estudo da inibição da NADPH oxidase, com apocinina in vivo, em adaptações ao treinamento físico intervalado intenso (TFII), uma vez que esta enzima tem sua atividade aumentada em estudos de contração muscular in vitro. Para investigar o efeito do TFII associado à administração de apocinina sobre as adaptações estruturais, funcionais e redox do músculo esquelético, foram utilizados ratos wistar (3 meses de idade) distribuídos aleatoriamente em 4 grupos: controle sedentário (CS), controle treinado (CT), apocinina sedentário (AS), e apocinina treinado (AT). O protocolo de TFII foi de corrida em esteira rolante durante 2 meses (1h, 5x/sem) com intensidade intervalada (3min a 60% VO2máx e 4min a 85% VO2máx) em inclinação de 20°. O tratamento com a apocinina (30 mg/kg/dia) foi por gavagem durante 2 meses. Foram avaliadas nos músculos sóleo e EDL, as medidas de capilarização, área de secção transversa (AST), distribuição de tipos de fibra, atividades de enzimas antioxidantes: superóxido dismutase (SOD) e catalase (CAT), o estado redox pela razão GSH:GSSG, e lesões oxidativas pelas concentrações de hidroperóxidos lipídicos e proteínas carboniladas. Os resultados demonstraram, que no músculo sóleo, o TFII não alterou a atividade da NADPH oxidase, mas aumentou a capilarização (82%), a atividade da SOD (47%) e a razão GSH:GSSG (52%), e diminuiu a atividade da CAT (-38%). No músculo EDL, o TFII aumentou as atividades das enzimas NADPH oxidase (141%), SOD (36%) e CAT (88%), bem como a capilarização (50%) e mudanças de tipos de fibras. Com isso observou-se que a apocinina não teve efeito sobre a função, estrutura e estado redox do ME de ratos sedentários. No entanto, a apocinina inibiu as adaptações induzidas pelo TFII em ambos os músculos (sóleo e EDL). O TFII aumentou a atividade da NADPH oxidase apenas no músculo EDL mostrando comportamentos diferentes das atividades desta enzima, em resposta a este tipo de treino, entre os músculos de características oxidativas e glicolíticas. Sendo assim, a NADPH oxidase parece participar das vias sinalizadoras para as adaptações induzidas pelo TFII apenas nos músculos glicolíticos. Diante desses resultados, conclui-se que músculos glicolíticos e oxidativos podem ter vias de sinalizações diferentes para as adaptações do ME ao exercício. Isto reforça e também explica a importância da intensidade e duração do exercício em respostas adaptativas, uma vez que estas variáveis influenciam o estado redox e também desencadeiam adaptações diferentes no ME. Futuramente, informações do estado redox muscular podem ser usadas para melhorar a especialização do treinamento físico de atletas / Initially it is believed, the production of reactive oxygen species (ROS) was associated just with oxidative damage and harmful effects on cells. Currently, evidence suggests that ROS play beneficial role and are associated with structural and functional adaptations of the cells by means of regulating cellular signaling pathways. In muscle cells, it is known that its function is dependent on the redox state. In fact, the exacerbated production of ROS is a limiting factor of muscle contraction, however, a reduced cellular environment also adversely affects the muscle function. In addition, adaptations to exercise seems to be regulated by signaling pathways sensitive to oxidation by ROS. The NADPH oxidase is an important enzymatic complex producer of ROS in skeletal muscle (SM) and considered as the main source of ROS in the cytosol during contraction. Besides, the proteins involved in muscle contraction are sensitive and controlled by the cellular redox state. Furthermore, NADPH oxidase is located, apparently in a strategic way, next to these proteins. Thus, it has become relevant to the study, in vivo, the inhibition of NADPH oxidase with apocynin on adaptations to high intense interval training (HIIT), since this enzyme activity has been increased in studies of muscle contraction in vitro. To investigate the effect of HIIT associated with the administration of apocynin on the structural and functional adaptations and the redox state of skeletal muscle, Wistar rats (3 months old) were randomly distributed into 4 groups: sedentary control, trained control, sedentary apocynin, and trained apocynin. The HIIT protocol consisted of treadmill running during two months (1h, 5x / week) with intensity intervals (3min 60% VO2max and 4 min at 85% VO2max) in a inclination of 20 degrees, and the apocynin treatment (30 mg / kg / day) was by gavage during 2 months. Were evaluated in soleus and EDL muscles, the capillarity, cross-sectional area (CSA), the distribution of fiber types, activities of antioxidant enzymes: superoxide dismutase (SOD) and catalase (CAT), the redox state by GSH: GSSG ratio, and oxidative damage by concentrations of hydroperoxides lipid and protein carbonyls levels. The results showed that in the soleus muscle, the HIIT did not increase the NADPH oxidase activity, but increased capillarity (82%), the activity of SOD (47%) and the ratio GSH: GSSG (52%), but decreased CAT activity (-38%). In EDL muscle, the HIFF increased the activity of the NADPH oxidase enzyme (141%), SOD (36%) and CAT (88%), and the capillarity (50%) and the change of fiber types. Thus it was observed that apocynin had no effect on the function, structure and redox state of SM of sedentary rats. However, the apocynin inhibited adaptations HIIT induced in both muscles (soleus and EDL). The HIIT increased the activity of NADPH oxidase only in the EDL muscle showing different behaviors of the activity of this enzyme in response to this type of training, between the oxidative and glycolytic muscles. Therefore, NADPH oxidase appears to participate in the signaling pathways for adjustments HIIT induced only in the glycolytic muscles. Given these results, it is concluded that glycolytic and oxidative muscles may have different pathways for the adjustments to the SM to exercise. This reinforces and also explains the importance of the intensity and duration of exercise in adaptive responses, since these variables influence the redox state and also trigger different adjustments in SM. In the future, muscle redox status information could even be used to improve the expertise of physical training of athletes

O mutante redA de Dictyostelium discoideum, obtido por inativação gênica aleatória, tem crescimento aparentemente normal, porém seu ciclo de desenvolvimento não progride além do estágio de agregados compactos. Neste trabalho relatamos a caracterização deste mutante, cujo gene defeituoso codifica a enzima NADPH citocromo P450 redutase (NCPR). O principal papel desta enzima é transportar elétrons do NADPH para as várias isoformas do citocromo P450. Um cDNA de 2094 pb que codifica a NCPR de D. discoideum (DdNCPR) foi isolado através da varredura de uma biblioteca de cDNA com uma sonda derivada de um fragmento do gene inativado no mutante redA. A análise da seqüência de aminoácidos deduzida do cDNA DdNCPR revelou que esta codifica uma proteína de 631 aminoácidos com 31% de identidade e 50% de similaridade com a NCPR humana. Verificamos o acúmulo do mRNA da DdNCPR durante fase de crescimento mas durante as fases iniciais do desenvolvimento ocorre significativa diminuição em seus níveis até a formação dos agregados compactos onde o mRNA da NCPR não é detectável. Demonstramos que o gene que codifica a NCPR aparentemente está presente em uma única cópia no genoma de Dictyostelium. Ademais, a análise de outras linhagens mutantes nocautes do gene da NCPR confirmaram que a inativação deste gene está diretamente relacionada ao fenótipo exibido pelo mutante redA-. Contudo, é provável que um ou mais produtos gênicos possam complementar a ausência desta enzima, uma vez que nem a linhagem redA nem as outras linhagens nocautes do gene da NCPR apresentaram alteração na taxa de crescimento e, em algumas circunstâncias experimentais, não exibiram qualquer alteração no ciclo de desenvolvimento. Nossos resultados sugerem, ainda que o bloqueio do desenvolvimento eventualmente observado no mutante redA pode ser devido a um provável papel da NCPR no metabolismo de DIF-1 (fator indutor de diferenciação-1), que parece desempenhar um papel primordial no controle da diferenciação de células pré-talo e células pré-esporo durante o desenvolvimento de D. discoideum. / The Dictyostelium discoideum redA mutant, obtained by random gene inactivation, exhibits normal growth but has its developmental cycle impaired at tight mound stage. In this study we describe the characterization of this mutant whose defective gene encodes the enzyme NADPH cytochrome P450 reductase (NCPR). NCPR is known to play an essential role in the transfer of reducing equivalents from NADPH to various cytochrome P450 isoforms. We isolated a 2094 bp cDNA that encodes D. discoideum NCPR (DdNCPR) by screening a cDNA library using as probe the mutated gene fragment rescued from redA cells. Analysis of the deduced aminoacid sequence of DdNCPR cDNA shows that it encodes a 631 aminoacid protein with 31% of identity and 50% of similarity with human NCPR. Northern blot analysis showed that DdNCPR mRNA levels is maximum during growth phase and decreases at early stages of the development. After slug stage this mRNA is not detectable. D. discoideum has a single copy of NCPR gene and, as shown by analysis of other NCPR knockout mutants, inactivation of this gene is strongly correlated to the redA phenotype. However, redA, as well as the other NCPR knockout strains, do have growth alterations and in some circumstances they do not show the described developmental defects. Thus, it is possible that one or more proteins be able to compensate for the lack of NCPR in these mutants. Our results also suggest that the redA developmental phenotype might play a role of NCPR on the metabolism of DIF-1, a prime candidate for controlling prestalk and prespore cell differentiation during D. discoideum development.

Processos celulares que governam as NADPH oxidases vasculares em condições patológicas não estão claros ainda. Como os processos redox são parte intrínseca da resposta da célula ao estresse, temos investigado se o estresse oxidativo pode convergir com outros tipos de estresse via Nox(es). No presente estudo, focamos na inibição do proteasoma como uma condição relevante de estresse. A incubação de células musculares lisas com concentrações não apoptóticas de inibidores do proteasoma, MG132 e lactacistina, promoveu aumento na produção basal de superóxido e na atividade da NADPH oxidase, diminuição da atividade da SOD e da razão GSH/GSSG. Por outro lado, a inibição do proteasoma diminui a atividade da Nox após estímulo com Angiotensina II ou Tunicamicina, conhecido estressor do retículo endoplasmático. Em condições basais, MG132 induz a expressão de mRNA da Nox1, entretanto o aumento de Nox1 induzido por Angiotensina II foi diminuído na presença de MG132. O mesmo efeito ocorre com a indução de Nox4 pela Tunicamicina, que nesse caso foi drasticamente reduzida na presença de MG132. Além disso, tanto Angiotensina II quanto Tunicamicina induziram a atividade lítica do proteasoma 20S. A seguir, investigamos as conseqüências fisiológicas do MG132 na sinalização do estresse do RE, uma conhecida resposta mediada por Nox4. Células vasculares incubadas com MG132 induzem a expressão de marcadores do estresse do RE, GRP78 e XBP1, e também os marcadores mais tardios ATF4 e o próapoptótico CHOP/GADD153. Resultados similares ocorrem também com a Tunicamicina. Entretanto, a co-incubação de Tunicamicina e MG132 diminui e a sinalização do estresse do RE. AKT e p38 MAPK foram ativados por MG132, possivelmente como resposta ao estresse induzido pela inibição do proteasoma. Assim, a inibição do proteasoma bloqueia a NADPH oxidase, com aumento da atividade basal e expressão da Nox1 versus forte inibição da ativação e expressão da Nox4 frente ao estímulo. A inibição da Nox4 associa-se e pode contribuir para a inibição pelo MG132 da sinalização do estresse do RE. Portanto, o proteasoma parece exercer papel na integração de estresses celulares envolvendo a NADPH oxidase. A inibição do proteasoma pode ter papel na terapia de doenças associadas a estresse do RE. / Cellular processes governing vascular Nox family NADPH oxidases in disease conditions are unclear. Since redox processes are intrinsic to cell stress response, we asked whether oxidative stress merges with other types of stress via Nox(es). We focused on proteasome inhibition as a relevant stress condition. Vascular smooth muscle cells (VSMC) incubation with non-apoptotic concentrations of proteasome inhibitors MG132 or lactacystin promoted increased baseline superoxide generation (HPLC/DHE products) and NADPH oxidase activity, decreased SOD activity and GSH/GSSG ratio. Conversely, proteasome inhibitors decreased by Nox response to Angiotensin II (AngII) and abrogated Nox response to endoplasmic reticulum (ER) stressor tunicamycin. With MG132, basal Nox1 mRNA levels were increased, while Nox1 response to AngII was blunted. Moreover, MG132 abolished Nox4 mRNA levels TN-induced. Both AngII and TN (at 2 and 4 hs) promoted increased 20S proteasome lytic activity. We next assessed physiological consequences of MG132 in ER stress signaling, a known Nox4- mediated response. VSMC incubation with MG132 alone enhanced expression of the ER stress markers Grp78 and XBP1 and late markers such as ATF4 and proapoptotic CHOP/GADD153. Similar results occurred with the known ER stressor TN. However, co-incubation of TN and MG132 decreased Grp78, Grp94 and CHOP/GADD153, indicating that proteasome inhibition interrupts ER stress. AKT and p38 are activated by MG132 as response to stress and recover to survival. Thus, proteasome inhibition disrupts NADPH oxidase, with increased baseline activity and Nox1 expression vs. strong inhibition of stimulated Nox1 and Nox4 activation/expression. The later effect may underlie MG132-mediated inhibition of ER stress signaling. (Support: FAPESP, CNPq Milênio Redoxoma)

Des études épidémiologiques ont démontré que l’exposition aux pesticides tels que le chlordécone augmente le risque du cancer, en particulier de la prostate. Il avait été précédemment montré au laboratoire que le chlordécone possède des propriétés pro-angiogéniques impliquant la libération de NO et la production de VEGF suite à l’activation du récepteur aux oestrogènes de type alpha (ERα). Les processus angiogéniques pouvant impliquer les espèces réactives de l’oxygène (EROs), produites notamment par la mitochondrie, l’objectif de ce travail a été d’évaluer la contribution de la biogenèse mitochondriale et du stress oxydatif dans l’angiogenèse induite par le chlordécone.Les résultats obtenus montrent que la biogenèse mitochondriale n’est pas essentielle pour la réponse angiogénique du chlordécone puisqu’elle n’est retrouvée que pour de forte concentration de chlordécone. Les mécanismes mis en jeu ont été identifiés au niveau des cellules endothéliales humaines. Ils impliquent une régulation spatio temporellede la production des EROs impliquant dans un premier temps la NADPH oxydase, elle même capable de stimuler la production mitochondriale d’EROs via la voie impliquant la NO synthase. Le chlordécone serait par ailleurs capable de favoriser la localisation périnucléaire des EROs afin de favoriser la production de VEGF. L’ensemble de ces effets implique le récepteur aux oestrogènes. Ce travail a donc permis d’identifier les mécanismes cellulaires impliqués dans la modulation de l’angiogenèse par le chlordécone. Ces mécanismes moléculaires pourraient contribuer à identifier de nouvelles cibles permettant de réguler les processus angiogéniques et la tumorigenèse induites par ce toxique. / Epidemiological studies report that exposure to pesticides like chlordecone increases risk of prostate cancer and tumorigenesis. We have reported recently that the pro-angiogenic effect of chlordecone involving NO release and VEGF production is mediated through activation of α isoform of the estrogen receptor (ERα). Since mitochondria and ROS have been implicated inthe process of angiogenesis, this study aims to determine the contribution of mitochondrial biogenesisand oxidative stress in chlordecone-induce dangiogenesis. Firstly, our results indicate that mitochondrial biogenesis is not essential for chlordecone angiogenic response. We also identified the molecular mechanism involved; chlordecone induces endothelial cells angiogenesis by a spatiotemporal regulation of ROS production involving NADPH oxidase then mitochondrial O2 -via a NO sensitive pathways through activation of ERα.These findings propose that a molecular mechanism may partly explain the epidemiological evidence implicating chlordecone as risk factor of prostatic cancer.

Les conditions contraignantes provoquent la modification de l’état redox et la signalisation liée aux formes actives de l’oxygène (ROS), dont les concentrations sont régulées par des systèmes antioxydant complexes. On dénombre de plus en plus de processus qui sont affectés par la régulation redox, mais nous avons toujours des connaissances fragmentaires quant à l’importance des interactions centrales entre ROS et systèmes antioxydants pour la signalisation cellulaire chez les plantes. Cette étude a utilisé des approches de génétique classique et inverse chez l’espèce-modèle, Arabidopsis thaliana dans le but d’élucider les rôles des catalases et des systèmes NADPH-glutathion-ascorbate dans le métabolisme du H₂O₂ et la signalisation qui en dépend. Une analyse de mutants ADN-T pour les trois gènes codant la catalase a révélé que la mutation cat2, à la différence de cat1 et de cat3, a fortement affecté la croissance et le développement de la plante. Lorsqu’il était cultivé dans l’air, le mutant cat2 présentait une croissance réduite à la fois au niveau de la rosette et des racines, mais ces effets étaient absents lors de la culture des plantes sous un taux de CO₂ élevé, suggérant que la taille diminuée est causée, directement ou indirectement, par une capacité compromise de métaboliser le H₂O₂ produit par la photorespiration. Une étude de cat2 cultivé dans des photopériodes différentes a mis en évidence une forte influence de la période d’illumination sur la signalisation oxydative et ceci d’une manière qui est indépendante de l’intensité du stress. Lorsque cat2 est cultivé en jours longs, le stress oxydant induit la voie de l’acide salicylique (SA), provoquant des lésions visibles sur les feuilles. Cette réponse au stress oxydant est annulée dans un double mutant cat2 g6pd5, chez lequel l’expression d’une forme spécifique de la glucose-6-phosphate déshydrogénase (G6PDH) a également été inactivée. Une approche de génétique classique a permis d’identifier plusieurs gènes susceptibles d’être impliqués dans la régulation de la formation de lésions SA-dépendante dans ce double mutant. Afin d’explorer les rôles des monodéshydroascorbate réductases (MDHAR) spécifiques dans des conditions optimales et de stress, des mutants d’insertion pour plusieurs gènes codant la MDHAR ont été obtenus. Ces mutants présentaient un phénotype sauvage dans des conditions de culture optimales, mais à la suite de son introduction dans le fond cat2, l’un d’entre eux a fortement modifié l’induction de la voie SA par le stress oxydant. Pris dans leur ensemble, les résultats soulignent l’importance de CAT2 et permettent de dessiner un lien fonctionnel entre des G6PDH et MDHAR spécifiques dans les voies de signalisation oxydative chez Arabidopsis, lien qui pourrait s’expliquer par la production de NADPH par la G6PDH et son utilisation par la MDHAR. / Stress conditions lead to modified redox states and signaling linked to reactive oxygen species (ROS), whose cellular concentrations are regulated by complex antioxidative systems. While the list of processes subject to redox regulation continues to grow, our understanding of the importance of the core interactions between ROS and plant antioxidative systems in cell signaling remains very fragmentary. This work used forward and reverse genetics to analyze the roles of catalases and the NADPH-glutathione-ascorbate systems in H₂O₂ metabolism and related signaling in the model species, Arabidopsis thaliana. An analysis of T-DNA mutants for the three catalase-encoding genes revealed that cat2, but not cat1 or cat3, substantially impacted plant growth and development. While the cat2 mutant showed decreased shoot and root size when grown in air, both these effects were annulled by growth at high CO₂, suggesting that they were caused, directly or indirectly, by compromised capacity to metabolize photorespiratory H₂O₂. An analysis conducted in cat2 rosettes following growth in different photoperiods revealed that oxidative signaling is strongly influenced by day length in a manner that is independent of stress intensity. When cat2 is grown in long days, oxidative stress induces the salicylic acid (SA) pathway, leading to visible lesions on the leaves. This response to oxidative stress is annulled in cat2 g6pd5, which has additionally lost the function of a specific glucose-6-phosphate dehydrogenase (G6PDH). A forward genetics approach identified several genes that may be involved in regulating SA-dependent lesion formation in this double mutant. To explore the roles of specific monodehydroascorbate reductases (MDHAR) in optimal and stress conditions, insertion mutants for several MDHAR-encoding genes were obtained. While these mutants showed a wild-type phenotype in optimal growth conditions, one of them markedly altered induction of the SA pathway by oxidative stress when introduced into the cat2 background. Together, the results underline the importance of CAT2 and point to functional coupling between specific NADPH-producing G6PDH and NADPH-requiring MDHAR in oxidative stress signaling pathways in Arabidopsis.

Poldip2 est une protéine ubiquitaire initialement identifiée comme étant un partenaire de la sous unité p50 de la polymerase δ intervenant dans la réplication et la réparation de l’ADN. Depuis sa découverte en 2003, beaucoup d’autres partenaires et fonctions lui ont été attribués. Elle joue, entre autre, un rôle régulateur de l’isoforme NADPH oxydase NOX4. A ce jour, le mode d’action de cette régulation n’a pas encore été identifié. Seul fait connu est que Poldip2 augmente l’activité de NOX4 en s’associant au partenaire membranaire de NOX4, la protéine p22phox. L’association de p22phox à d’autres isoformes de NADPH Oxydase (NOX1, NOX2 ou NOX3) suggère une interaction possible entre ces derniers et Poldip2. Par ailleurs la co-localisation de NOX4 et NOX2 dans plusieurs types cellulaires, tels que les cellules du muscle lisse et l’endothélium mais également la coexpression de Poldip2 et NOX2 dans les artérioles rénale font de NOX2 un bon candidat pour l’étude de l’effet régulateur possible de Poldip2 de cet isoforme. Dans cette perspective, la protéine recombinante Poldip2 (rat) a été produite de manière hétérologue dans un système d’expression de levure P. pastoris. Grace à un vecteur d’expression spécifique de cette levure, Poldip2 est sécrété dans le milieu extracellulaire. La protéine a été purifiée à partir du milieu de culture. L’analyse de la séquence par spectrométrie de masse a permis de confirmer l’identité du Poldip2 recombinant produit par la levure. Après avoir caractérisé structuralement Poldip2 et confirmé sa capacité à augmenter l’activité de NOX4, nous avons étudié son effet sur NOX2 des phagocytes. De manière surprenante, nos études en système cell-free ont montré des propriétés inhibitrices de Poldip2 sur NOX2 avec des interactions privilégiées avec certaines des composantes du complexe oxydase. Nos résultats suggèrent que l’interaction de Poldip2 avec le complexe pourrait constituer une nouvelle voie de régulation de NOX2 en perturbant l’assemblage du complexe NADPH oxydase. / Poldip2 is an ubiquitous protein initially identified as a partner for polymerase δ p50 subunit and is involved in DNA replication and repairing. Since its discovery in 2003, many partners and functions has been assigned to it. Poldip2 is also involved in NADPH Oxidase NOX4 isoform regulation. The enhancement of NOX4 activity was attributed to Poldip2 interaction with the membrane partner p22phox. However the mechanism by which it regulates NOX4 has not been identified yet. The association of p22phox with other Nox isoform (NOX1, NOX2 or NOX3) questions the possible interaction of Poldip2 with NOX2. Furthermore the colocalization of NOX4 and NOX2 in several cell types as the smouth muscle cells and endothelium cells but also Poldip2 and NOX2 coexpression in renal arterioles, makes NOX2 a good candidate for studying the possible regulatory effect of Poldip2 on the NOX2 isoform. On this purpose the recombinant protein Poldip2 (rat) was produced in the yeast P. pastoris. Using a specific expression vector Poldip2 was secreted in the extracellular media. The protein was purified from culture media. Sequence analysis by mass spectrometry allowed to confirm the recombinant protein identity produced in yeast. After the structural characterization of poldip2 and the confirmation of its functionality on NOX4, the protein was used to study its effect on NOX2 phagocyte. Surprisingly our study on cell free assay shows that Poldip2 has inhibiting properties regarding NOX2 and interacts in a privileged manner with certain components of the NADPH Oxidase complex. Our result suggest that the interaction of Poldip2 and the complex might constitute a new regulation for NOX2 by disturbing the NADPH Oxidase assembly.

Les neutrophiles sont les leucocytes les plus nombreux et les premières cellules à arriver au site de l’infection où elles internalisent les pathogènes par phagocytose. Dès le début du processus, la NADPH oxydase s’assemble au phagosome où elle permet la production de formes réactives de l’oxygène contribuant ainsi à la destruction du pathogène. La sous-unité catalytique membranaire de la NADPH oxydase, Nox2, est donc présente à la coupe phagocytaire puis au phagosome. Le dessein de cette étude était de déterminer quelles sont les sources subcellulaires de la protéine Nox2, de savoir si la protéine s’accumule au phagosome et le cas échéant selon quelle cinétique. Dans le but de comprendre la dynamique de la protéine Nox2, la protéine d’échafaudage IQGAP1 qui est associée au cytosquelette a également été étudiée. Enfin l’étude de l’organisation spatiale de la protéine Nox2 à la synapse phagocytaire a également été abordé.En utilisant des cellules neutrophil-like (PLB-985) ainsi que des neutrophiles humains, notre étude a montré par immunofluorescence la présence de la protéine Nox2 dans des endosomes de recyclage ou dans des endosomes précoces. Lors de la phagocytose ils avoisinent le phagosome suggérant leur implication dans l’apport de la protéine Nox2 à la membrane de ce dernier. L’utilisation de cellules PLB-985 pour lesquelles l’expression de Nox2 a été supprimée puis réintroduite avec un transgène codant pour la protéine GFP-Nox2 montre que la sous-unité Nox2 s’accumule au phagosome pendant les vingt minutes suivant sa fermeture. Dans notre étude, la protéine IQGAP1 ne semble pas avoir d’effet sur la phagocytose ou sur la production de FRO par la NADPH oxydase. Enfin, grâce à une technique de microscopie super-résolution (le dSTORM) l’évolution du pattern de Nox2 dans la membrane a été évalué au cours du temps en phagocytose frustrée. En dix minutes, le nombre de clusters de protéine Nox2 augmente mais leur taille reste inchangée. / Neutrophils are the most numerous leukocytes and the first cells to arrive at the site of infection where they internalize pathogens by phagocytosis. From the beginning of the process, the NADPH oxidase is assembled at the phagosome, where it allows the production of reactive oxygen species (ROS), thus contributing to the destruction of the pathogen. The membrane bound catalytic subunit of the NADPH oxidase, Nox2, is therefore recruited at the phagocytic cup and then at the phagosome. The purpose of this study was to determine, which are the subcellular sources of the Nox2 protein, whether the protein accumulates at the phagosome and if so, according to which kinetics. In order to modify the dynamics of the Nox2 protein, the scaffold protein IQGAP1 that is associated with the cytoskeleton was also studied. Finally, the spatial organization of the Nox2 protein in the phagocytic synapse was also investigated.Using neutrophil-like cells (PLB-985) as well as human neutrophils, our study showed by immunofluorescence the presence of the Nox2 protein in recycled or early endosomes. During phagocytosis, they are close to the phagosome, suggesting their involvement in the contribution of the Nox2 protein to the phagosome membrane. The use of PLB-985 for which Nox2 expression has been suppressed and then reintroduced with a transgene encoding the GFP-Nox2 protein shows that the Nox2 subunit accumulates at the phagosome during the first twenty minutes after its closure. In our study, the protein IQGAP1 does not appear to have any effect on phagocytosis or on the production of ROS by NADPH oxidase. Finally, using super resolution microscopy (dSTORM) the evolution of the Nox2 pattern in the membrane has been evaluated over time in frustrated phagocytosis. Within ten minutes, the number of Nox2 protein clusters increases but their size remains unchanged.

L'élucidation des propriétés du réseau métabolique est fondamentale pour la compréhension du fonctionnement cellulaire et pour l'élaboration de stratégies d'ingénierie métabolique. L'objectif de cette thèse était de mieux comprendre la régulation du métabolisme du NADPH, un métabolite "hub" qui joue un rôle central dans de nombreux processus cellulaires, chez Saccharomyces cerevisiae en fermentation. Nous avons utilisé une démarche systématique couplant modélisation et approches multi-“omics” pour étudier de façon quantitative la réponse à une perturbation de la demande en NADPH. Un système expérimental original, basé sur l'expression d'une butanediol déshydrogénase modifiée NADPH-dépendante a été utilisé pour augmenter de façon contrôlée la demande en NADPH. L'utilisation de ce dispositif, le développement et l'utilisation d'un modèle stœchiométrique de la levure dédié à la fermentation ont permis de prédire la répartition des flux pour différents niveaux de perturbation. Ces analyses ont montré, en premier lieu, la très grande capacité de la levure à faire face à des demandes très importantes de NADPH représentant jusqu'à 40 fois la demande anabolique. Pour des demandes modérées (allant jusqu'à 20 fois la demande anabolique), la perturbation est principalement compensée par une augmentation du flux à travers la voie des pentoses phosphate (VPP) et à moindre titre à travers la voie acétate (Ald6p). Pour une forte demande en NADPH, correspondant à 40 fois la demande anabolique, le modèle prédit la saturation de la VPP ainsi que la mise en place du cycle glycérol-DHA, qui permet l'échange du NADH en NADPH. Des analyses fluxomique (13C), métabolomique et transcriptomique, ont permis de valider ces hypothèses et de les compléter. Nous avons mis en évidence différents niveaux de régulation selon l'intensité de la perturbation : pour les demandes modérées, les flux sont réajustés par un contrôle au niveau enzymatique ; pour de fortes demandes, un contrôle transcriptionnel de plusieurs gènes de la VPP ainsi que de certains gènes des voies de biosynthèse des acides aminés est observé, cet effet résultant probablement de la moindre disponibilité en NADPH. Dans l'ensemble, ce travail a apporté un nouvel éclairage sur les mécanismes impliqués dans l'homéostasie du NADPH et plus généralement dans l'équilibre redox intracellulaire. / The elucidation of the properties of metabolic network is essential to increase our understanding of cellular function and to design metabolic engineering strategies. The objective of this thesis was to better understand the regulation of the metabolism of NADPH, a “hub” metabolite which plays a central role in many cellular processes in Saccharomyces cerevisiae during fermentation. We used a systematic approach combining modeling and multi-“omics” analyses to study quantitatively the response to a perturbation of the NADPH demand. An original experimental system, based on the expression of a modified NADPH-dependent butanediol dehydrogenase was used to increase the demand for NADPH in a controlled manner. Through the use of this device and the development and use of a stoichiometric model of yeast dedicated to the fermentation, we predicted the flux distribution for different levels of perturbation. These experiments showed, first, the overwhelming ability of yeast to cope with very high NADPH demand, up to 40 times the anabolic demand. For a moderate level (up to 20 times the anabolic demand), the perturbation is mainly compensated by increased flux through the pentose phosphate pathway (PPP) and to a lesser extent through the acetate pathway (Ald6p). For a high NADPH demand, corresponding to 40 times the anabolic demand, the model predicts the saturation of the PPP as well as the operation of the glycerol-DHA cycle, which allows the exchange of NADH to NADPH. Fluxomics (13C), metabolomics and transcriptomics data were used to validate and to complement these hypotheses. We showed different levels of control depending on the intensity of the perturbation: for moderate demands, flux remodeling is mainly achieved by enzymatic control; for a high demand, a transcriptional control is observed for several genes of the PPP as well as some genes of the amino acids biosynthetic pathways, this latter effect being likely due to the low NADPH availability. Overall, this work has shed new light on the mechanisms governing NADPH homeostasis and more generally the intracellular redox balance.

INTRODUÇÃO: A infecção pelo vírus da hepatite C é a principal causa de doença hepática crônica que progride para cirrose e carcinoma hepatocelular. Diversos fatores relacionados ao vírus e ao hospedeiro determinam a progressão para formas mais graves de fibrose hepática. A produção de espécies reativas de oxigênio (EROS) promovendo o estresse oxidativo contribui significativamente na fibrogênese hepática e pode ser gerada de múltiplas fontes, incluindo a cadeia respiratória mitocondrial, o citocromo p4502E1, peroxissomos e as NADPH oxidases (NOXs) no fígado. O sistema da NADPH (nicotinamide adenine dinucleotide phospate) oxidase tem uma participação muito importante na geração de EROS induzidas pelo vírus da hepatite C (VHC). Por isso, no presente estudo, investigamos dois polimorfismos de um único nucleotídeo (SNPs) na região reguladora dos genes codificadores da subunidade catalisadora da NADPH oxidase 4 (NOX4) e sua subunidade regulatória p22phox (CYBA) e a associação deles com variáveis metabólicas e histológicas em pacientes com VHC. MÉTODOS: Cento e setenta e oito pacientes portadores do VHC e virgens de tratamento (49,3% sexo masculino; 65% VHC genótipo 1) foram analisados. Todos os pacientes tinham VHC RNA positivo e foram genotipados para os SNPs rs3017887 na NOX4 e -675 T -> A na CYBA usando-se primer e probes específicos. RESULTADOS: Nenhuma associação foi vista entre as frequências genotípicas dos SNPs da NOX4 e CYBA com marcadores de inflamação ou grau de fibrose hepática na população total estudada. A presença dos genótipos CA + AA do SNP da NOX4 SNP teve associação com menor concentração sérica da alanino aminotransferase (ALT) na população masculina (CA + AA = 72,23 ± 6,34 U/L versus CC = 100,22 ± 9,85; média ± SEM; P = 0,05). O genótipo TT do SNP da CYBA teve associação com menor concentração sérica da ALT na população masculina (TT = 84,01 ± 6,77 U/L versus TA + AA = 109,67 ± 18,37 U/L; média ± SEM; P = 0,047). Por outro lado, o alelo menor do SNP da NOX4 foi inversamente associado com a frequência de síndrome metabólica (SM) na população masculina (odds ratio (OR): 0,15; 95% intervalo de confiança (IC): 0,03 - 0,79; P = 0,025). CONCLUSÕES: Os resultados sugerem que os SNPs da NOX4 e CYBA avaliados no estudo não são marcadores genéticos diretos de fibrose hepática em pacientes portadores do VHC, entretanto o SNP rs3017887 da NOX4 poderia influir indiretamente na susceptibilidade da fibrose hepática devido a associação inversa com SM nos pacientes do sexo masculino. / BACKGROUND: The hepatitis C virus infection is the leading cause of chronic liver disease that progresses into cirrhosis and hepatocellular carcinoma. Several factors related to the virus and the host determine the progression to more severe forms of liver fibrosis. The production of reactive oxygen species (ROS) promoting oxidative stress contribute significantly in liver fibrogenesis and can be generated from multiple sources, including mitochondrial respiratory chain, cytochrome p4502E1, peroxisomes and NADPH oxidases (NOXS) in the liver. Given the important contribution of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system to the generation of reactive oxygen species induced by hepatitis C virus (HCV), we investigated in the present study, two single nucleotide polymorphisms (SNPs) in the putative regulatory region of the genes encoding NADPH oxidase 4 catalytic subunit (NOX4) and its regulatory subunit p22phox (CYBA) and their relation with metabolic and histological variables in patients with HCV. METHODS: One hundred seventy eight naïve HCV patients (49.3% male; 65% HCV genotype 1) with positive HCV RNA were genotyped using specific primers and fluorescent-labeled probes for SNPs rs3017887 in NOX4 and -675 T -> A in CYBA. RESULTS: No association was found between the genotype frequencies of NOX4 and CYBA SNPs and inflammation scores or fibrosis stages in the overall population. The presence of the CA + AA genotypes of the NOX4 SNP was nominally associated with a lower alaninoamine transferase (ALT) concentration in the male population (CA + AA = 72.23 ± 6.34U/L versus CC = 100.22 ± 9.85; mean ± SEM; P = 0.05). The TT genotype of the CYBA SNP was also nominally associated with a lower ALT concentration in the male population (TT = 84.01 ± 6.77U/L versus TA + AA = 109.67 ± 18.37U/L; mean ± SEM; P = 0.047). The minor A-allele of the NOX4 SNP was inversely associated with the frequency of metabolic syndrome (MS) in the male population (odds ratio (OR): 0.15; 95% confidence interval (CI): 0.03 to 0.79; P = 0.025). CONCLUSIONS: The results suggest that the evaluated NOX4 and CYBA SNPs are not direct genetic determinants of fibrosis in HCV patients, but nevertheless NOX4 rs3017887 SNP could indirectly influence fibrosis susceptibility due to its inverse association with MS in male patients