Le métabolisme oxydatif joue un rôle important dans l’hématopoïèse normale et leucémique. L’homéostasie des espèces réactives de l’oxygène (ROS) est un élément crucial qui repose sur une balance finement régulée entre leur élimination et leur production. A ce niveau, des études ont montrés une différence entre cellules souches leucémique (CSL) présentant un faible niveau de ROS et cellules différenciées leucoblastiques présentant un plus fort niveau de ROS. Dans cette étude nous avons montré que les NADPH oxydases sont des producteurs majeurs de ROS des cellules de leucémies aiguës myéloïdes. Les cellules leucoblastiques, quelque soit le stade de différenciation présentent une activité NADPH oxydase constitutive qui contribue à leur niveau de ROS élevé et favorise leur prolifération en accélérant le cycle cellulaire. A l’inverse, les analyses réalisées sur des CSL grâce à des modèles murins de leucémies primaires induites par les oncogènes Hoxa9 et Meis1 suggèrent qu’il existerait une plus faible activité oxydase dans les cellules souches leucémiques. / Oxydative metabolism play a key role in normal and leukemic hematopoiesis. Reactive oxygen species (ROS) homeostasis is a crucial point which is the result of a finely regulated balance between elimination and production. Recent studies establishe a difference in ROS level between leukemic stem cells (LSC, ROSlow) and differentiated leucoblasts (higher level). In our study we have shown that NADPH oxidases are major ROS producers in acute myeloid leukemic cells. Leukoblasts, wathever their differentition stage, have a constitutive NADPH oxydase activity that contributes to the ROS level and promotes the proliferation by accelerating the cell cycle. Conversly, the analyses of LSCs performed by using murins primary leukemia induced by Hoxa9 and Meis1 oncogens suggest a potential lower NADPH oxidase activity in LSCs.

Dans le monde, 350 000 leucémies sont diagnostiquées chaque année. La rechute reste un problème majeur des leucémies aiguës myéloïdes (LAM) et le métabolisme oxydatif pourrait jouer un rôle essentiel dans la réponse au traitement. Un faible niveau des espèces réactives de l’oxygène (ROS) est associé à des propriétés des cellules souches leucémiques et la quiescence alors qu’un niveau plus élevé caractérise les leucoblastes proliférants. L’homéostasie des ROS repose sur un équilibre entre les systèmes oxydants et antioxydants. Les antioxydants sont bien documentés dans les LAM alors que les connaissances sur l’activité oxydante sont encore limitées. Dans ce travail nous avons choisi d’étudier les sept complexes NADPH oxydases (NOX) dans 25 lignées issues de LAM humaines et des LAM primaires. L’analyse des ARNm et des protéines montre des profils d’expression variables entre les lignées avec une expression plus forte des sous-unités du complexe NOX2 dans les lignées correspondant à des stades de différenciation myéloïde plus avancés. L’activité enzymatique des NOX est cependant équivalente entre les lignées. Deux inhibiteurs, DPI et VAS3947, ont été utilisés pour connaître la contribution des NOX à la production des ROS cellulaires. Alors qu’ils ont inhibé l’activité, ils ont aussi généré un stress oxydatif majeur conduisant à une diminution de la prolifération cellulaire et une forte apoptose, le DPI en augmentant les ROS mitochondriaux et VAS3047 les ROS cytoplasmiques. Afin de connaitre les sous-unités impliquées et de mieux comprendre les mécanismes, les sous-unités NOX2 et p22phox ont été inhibée par ARN interférence. Celle-ci n’ont pas affecté la prolifération mais ont montré des effets compensatoires. Nos data montrent qu’inhiber les NOX pourrait s’avérer une stratégie thérapeutique en augmentant le stress oxydatif dans les cellules leucémiques. / 350,000 leukaemia are diagnosed each year worldwide. In acute myeloid leukaemia (AML), relapse remains a major problem and the oxidative metabolism might play a crucial role in the therapeutic response. Low level of reactive oxygen species (ROS) is associated with properties of leukemic stem cells and quiescence whereas higher level promotes leukoblasts proliferation. ROS homeostasis relies on a tightly regulated balance between the oxidant and antioxidant systems. Although the antioxidant system is extensively studied in AML, the oxidant system remains poorly documented. In this work we aimed to study the seven NADPH oxidases (NOX) complexes in 25 AML human cell lines and primary samples. NOX transcriptional and protein profiles are variable with a higher expression of NOX2 in cell lines belonging to mature differentiation stages. An equivalent level of enzymatic activity was observed across all the cell lines. To reveal the contribution of NOX to global ROS production in the cells, two NOX inhibitors, DPI and VAS3947, were then used. Although both inhibitors efficiently blocked NOX activity they unexpectedly triggered strong oxidative stress leading to reduced cell proliferation and strong apoptosis, DPI by increasing mitochondrial ROS while VAS3947 by increasing cytoplasmic ROS production. To highlight which of the subunits were involved and to understand the mechanisms, NOX2 and p22phox subunits were inhibited using shRNA strategy. These did not affect cell proliferation but revealed a compensation effect. Our data suggest that NOX inhibition might be potential therapeutic strategy by increasing oxidative stress in leukemic cells.

Magister Scientiae - MSc (Biotechnology) / Soybean have been listed as a priority commodity crop in South Africa (SA) and provide a good source of protein to the population. Therefore, soybean has been earmarked as an important food security crop and strategies are currently being discussed at governmental level to increase and sustain soybean production. However, the SA landscape poses many challenges to the agricultural sector such as prolong drought periods, flooding, nutrient poor soils, saline soils and heavy metal contaminated soils. Heavy metal (HM) contamination is becoming a serious concern and is aggravated by historical mining in SA. Indeed, SA has established itself as the number one ranked mining country in the world and is frequently mining metals such as chromium, vanadium, gold, zirconium, platinum, and antimony. Prolong rainfall near mining areas leads to acid mine drainage which lowers the soil pH to approximately two. These highly acidic soils will solubilize the metals and cause the metals to leach into river systems as well as the water table leading to increase heavy metal contamination in nearby soil sites. This increase metal content negatively affects seed germination and overall plant development. Nonetheless, plants have evolved numerous internal mechanisms that help them to survive HM toxicity; by either avoiding or tolerating the stress. Two stress-activated pathways that help the plant tolerate stress have attracted much interest i.e. the glyoxalase system and reactive oxygen species (ROS) - antioxidant system as they detoxify methylglyoxal (MG) and ROS. / 2021-08-31

A prevalência da obesidade tem aumentado em todo mundo, afetando também mulheres em idade reprodutiva. Estudos têm demonstrado que o acúmulo excessivo de tecido adiposo resulta em prejuízos à reprodução feminina. O mecanismo pelo qual a obesidade diminui a fertilidade não está totalmente estabelecido. Atualmente, são propostas a condição inflamatória e a indução de estresse oxidativo como potenciais mecanismos de ação para as patologias relacionadas à obesidade. Adicionalmente, a angiotensina II (Ang II) é mais um fator que tem sido relacionado com alterações associadas à obesidade, e os efeitos deste peptídeo incluem ações pró-inflamatórias e pró-oxidativas. O presente estudo analizou o efeito da obesidade na ovulação e no metabolismo oxidativo ovariano, e a participação da Ang II como um possível modulador da formação de espécies reativas de oxigênio em ovários de ratas obesas e seus efeitos na ovulação. Foram utilizadas ratas submetidas a uma dieta hipercalórica composta por alimentos palatáveis, conhecida como dieta de cafeteria, a partir do desmame até a idade adulta, por um período de 17 semanas. Os animais foram divididos em grupos: CTL (controle), CTL LOS (controle + losartan), CAF (cafeteria) e CAF LOS (cafeteria + losartan). Avaliamos o consumo de alimentos e líquidos, o peso corporal, o peso da gordura abdominal e retroperitonial, a concentração de insulina plasmática, o número de oócitos, as atividades das enzimas antioxidantes (superóxido dismutase e catalase), concentração de peróxido de hidrogênio (H2O2) e parâmetros de dano oxidativo (lipoperoxidação e oxidação de proteínas). As fêmeas obesas do grupo CAF apresentaram maior consumo de energia e reduzido consumo de água e ração padrão, hiperinsulinemia, aumento das gorduras abdominal e retroperitonial, e aumento de peso corporal. A obesidade não reduziu significativamente a ovulação, mas aumentou a atividade das enzimas antioxidantes e a concentração de H2O2 no ovário. A administração do losartan, em ratas alimentadas com a dieta de cafeteria, reduziu a ingestão energética total, a concentração plasmática de insulina e o ganho de gordura abdominal, mas não evitou o desenvolvimento da obesidade. O losartan inibiu a atividade da enzima antioxidante superóxido dismutase no ovário das ratas obesas do grupo CAF, porém não alterou a atividade da enzima antioxidante catalase. Os resultados deste estudo demonstram que a obesidade resulta em alterações no metabolismo e sugerem a participação da Ang II na formação de espécies reativas de oxigênio no ovário. / The prevalence of obesity has increased worldwide, also affecting women of reproductive age. Studies have shown that excessive accumulation of adipose tissue results in impaired female reproduction. The mechanism by which obesity reduces fertility is not fully established. Currently, proposals are the inflammatory condition and the induction of oxidative stress as a potential mechanism of action for diseases related to obesity. Additionally, angiotensin II (Ang II) is another factor that has been related to changes associated with obesity, and the effects of this peptide include pro-inflammatory and pro-oxidative actions. The present study considers the effect of obesity on ovulation and ovarian oxidative metabolism, and the participation of Ang II as a possible modulator of the formation of reactive oxygen species in ovaries of obese rats and their effects on ovulation. Female rats fed a diet consisting of high calorie foods palatable, known as cafeteria diet from weaning to adulthood, for a period of 17 weeks. The animals were divided into groups: Control (CTL) CTL LOS (control + losartan), CAF (cafeteria) and CAF LOS (losartan + cafeteria). We evaluate the consumption of food and fluids, body weight, abdominal and retroperitoneal fat weight, the plasma insulin concentration, the number of oocytes, the activities of antioxidant enzymes (superoxide dismutase and catalase), concentration of hydrogen peroxide (H2O2 ) and parameters of oxidative damage (lipid peroxidation and protein oxidation). The females of the CAF group had higher energy consumption and reduced consumption of water and standard chow, hyperinsulinemia, increased abdominal and retroperitoneal fat, and weight gain. Obesity not significantly reduced ovulation, but increased the activity of antioxidant enzymes and the concentration of H2O2 in the ovary. The administration of losartan in rats fed the cafeteria diet, reduced total energy intake, plasma insulin concentration and abdominal fat gain, but did not prevent the development of obesity. Losartan inhibited the activity of the antioxidant enzyme superoxide dismutase in the ovary of rats CAF group, but did not alter the activity of the catalase antioxidant enzyme. The results of this study demonstrate that the cafeteria diet results in changes in metabolism and suggests the involvement of Ang II in the formation of ROS in the ovary.

Les spermatozoïdes acquièrent leur pouvoir fécondant et leur mobilité lors de leur transit le long de l’épididyme. Paradoxalement, cette maturation épididymaire nécessite la présence d’espèces oxygénées réactives (EOR) pour condenser la chromatine spermatique afin de mieux protéger l’ADN de ces mêmes molécules. Nous avons étudié la façon dont l’épididyme parvient à assurer l’équilibre entre un déficit et un excès d’EOR en caractérisant le phénotype épididymaire de souris dont la production de deux enzymes antioxydantes glutathion peroxydases a été invalidée, GPx5 et snGPx4. L’épididyme de ces souris génère une réponse antioxydante élevée et augmente l’activité de pontage disulfure sur les gamètes en modulant l’expression génique d’enzymes antioxydantes (Trx, Prx, GST, SOD3, catalase) et de protéines disulfide isomérases (Pdia). Bien que ce sursaut d’activité soit efficace pour protéger les membranes du tissu et des spermatozoïdes, la chromatine des spermatozoïdes présente un défaut de condensation, laissant l’ADN spermatique vulnérable face aux EOR. Les gamètes présentent alors des dommages oxydants dans le noyau qui s’aggravent avec la diminution de l’activité antioxydante lors du vieillissement. Des approches immunologiques et biochimiques ont montré que les dommages oxydants se produisent préférentiellement sur l’ADN spermatique situé à la périphérie du noyau, qui est enrichi en nucléosomes persistants et qui est associé à la matrice nucléaire. Afin de déterminer s’il est possible de diminuer ces atteintes oxydantes sur les gamètes, nous avons étudié les effets d’une supplémentation orale antioxydante sur des souris sauvages et sur l’un de nos modèles murins mutant. / The spermatozoa acquire their fertilizing ability and their motility through the epididymis. Paradoxically, this epididymal maturation needs reactive oxygen species (ROS) to condense the sperm chromatin permitting to protect the DNA against these molecules. We studied how the epididymis ensure the equilibrium between deficit and excess of ROS by characterizing the epididymal phenotype of mice lacking two antioxidant activities of the glutathione peroxidase family, GPx5 & snGPx4. The epididymis of these mice produce a strong antioxidant response and also increase the disulfide bridging activity by adjusting the gene expression of antioxidant enzymes (Trx, Prx, GST, SOD3, catalase) and disulfide isomerase proteins (Pdia). This protection is efficient for tissue and sperm membranes but not for sperm chromatin which is susceptible to decondensation. The sperm cells show oxidative damage in the nucleus, which worsen with the decrease of the antioxidant activity upon aging. Immunological and biochemical approaches indicated that oxidative damage occurred only on the sperm DNA at the periphery of the nucleus, which is enriched with persisting nucleosomes and associated to the nuclear matrix. Finally, to determine if these oxidative damages on the spermatozoa can be lowered, we studied the effects of an oral antioxidant supplement on wild type and gpx5-/- mice (Chabory et al., 2009).

No sistema cardiovascular, os hormônios da tireoide exercem uma importante ação, influenciando a captação de cálcio, o inotropismo e o cronotropismo cardíaco e a resistência vascular periférica. No entanto, uma exacerbação destas ações, causada pelo aumento da secreção dos hormônios da tireoide, gera uma quebra desta homeostase e o desenvolvimento de hipertireoidismo. O hipertireoidismo leva ao aumento do consumo de oxigênio, gerando uma situação de estresse oxidativo. A exposição crônica ao estresse oxidativo leva à ativação de fatores de transcrição e citocinas, causando hipertrofia de cardiomiócitos e progressão para insuficiência cardíaca com inflamação e apoptose. Logo, o objetivo deste trabalho foi avaliar o papel dos hormônios da tireoide sobre a ativação de vias inflamatórias e apoptóticas mediada pelo estresse oxidativo. Neste estudo, nós avaliamos parâmetros de estresse oxidativo e algumas citocinas envolvidas com as vias de sinalização inflamatória e apoptótica. Para isso, utilizamos 60 ratos wistar, divididos em 2 grupos: Controle e Tratado (T4), com um n de 30 animais por grupo. O grupo T4 foi submetido à indução de hipertireoidismo através da adição de L-tiroxina (T4 – 12mg/L) na água de beber por 28 dias. O grupo controle não recebeu tratamento com L-tiroxina. Houve desenvolvimento de hipertireoidismo e indução de hipertrofia cardíaca no grupo T4. Verificamos também o aumento de H2O2 no coração e redução de -SH em eritrócitos no grupo T4. Houve aumento de LDH no grupo T4, indicando dano tecidual. Por fim, houve redução de PGC1-α, além de uma redução do p53 e de Bcl2 e aumento da razão Bax/Bcl2 no grupo T4. Os resultados apontam para a ocorrência de estresse oxidativo, o que com potencial dano induziu uma redução de PGC1-α e de p53, que podem estar relacionados à ativação de proteínas apoptóticas, como observado pelo aumento da razão Bax/Bcl2 no grupo tratado. / In the cardiovascular system, the thyroid hormones play an important action, influencing the uptake of calcium, the cardiac inotropy and chronotropy and peripheral vascular resistance. However, an exacerbation of these actions, caused by increased secretion of thyroid hormones, generates a breach of this homeostasis and could lead to development of hyperthyroidism. Hyperthyroidism leads to increased oxygen consumption, generating oxidative stress. Chronic exposure to oxidative stress leads to the activation of transcription factors and cytokines, causing cardiomyocyte hypertrophy and progression to heart failure with inflammation and apoptosis. Therefore, the aim of this study was to evaluate the role of thyroid hormones on the activation of inflammatory pathways and apoptotic mediated by oxidative stress. In this study, we evaluated some oxidative stress parameters and cytokines involved in inflammatory and apoptosis signaling pathways. For this, we used 60 Wistar rats, divided into 2 groups: control and treated (T4), with an n of 30 animals per group. The group T4 was subjected to hyperthyroidism induction by the addition of L-thyroxine (T4 - 12mg / L) in their drinking water for 28 days. The control group received no treatment with L-thyroxine. There was development of hyperthyroidism and induction of cardiac hypertrophy in the T4 group. We noticed the increase of H2O2 in the heart and reduced -SH in erythrocytes in the T4 group. There was LDH increase in the T4 group, indicating tissue damage. Finally, a reduction of PGC1-α, as well as a reduction of p53 and Bax/Bcl2 ratio increase in the T4 group. The results point to the occurrence of oxidative stress, which with potential damage induced a PGC1-α and p53 reduction, wich can be related to the activation of apoptotic proteins, as observed by increased Bax / Bcl2 ratio in the treated group.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Evidências crescentes têm demonstrado que o exercício prejudica a estrutura da membrana eritrocitária, como consequência do aumento do estresse físico e químico. O óxido nítrico (NO) derivado dos eritrócitos afeta a fluidez da membrana e a sua biodisponibilidade depende do equilíbrio entre a sua síntese e eliminação por espécies reativas de oxigênio. Nós investigamos se o exercício realizado em diferentes intensidades afetaria biodisponibilidade do NO eritrocitário e se levaria a um quadro de estresse oxidativo. Dez homens (26 4 anos, VO2pico 44,1 4,3 mL.kg-1.min-1) realizaram um teste cardiopulmonar máximo em esteira e um teste de exercício submáximo a 70% VO2pico durante 30 min. O sangue foi coletado em repouso e imediatamente após os exercícios para isolamento dos eritrócitos. O exercício máximo aumentou a contagem de eritrócitos, hemoglobina e hematócrito, sem levar a qualquer alteração na massa corporal que pudesse sugerir hemoconcentração devido a uma redução do volume plasmático. Observou-se uma diminuição do influxo de L-arginina depois do teste submáximo, mas não no teste máximo. No entanto, a atividade da óxido nítrico sintase, ou seja, a produção de NO, foi aumentada após o teste máximo. Os níveis de GMPc não se alteraram após ambos os teste de exercício. Em relação aos biomarcadores de estresse oxidativo, o exercício submáximo reduziu a oxidação proteica e aumentou a atividade da catalase e a expressão da glutationa peroxidase, enquanto que o exercício máximo levou a uma maior peroxidação lipídica e diminuição da atividade da SOD. Nem a atividade glutationa peroxidase ou a expressão NADPH oxidase foram afetadas pelo exercício. Estes resultados sugerem que o exercício induziu alterações no estresse oxidativo de eritrócitos, que parecem estar mais associadas com a intensidade do que a duração do execicio. Além disso, nas intensidades recomendadas para a promoção da saúde, o exercício mostrou ser protetor, aumentando a atividade e a expressão de enzimas antioxidantes importantes e reduzindo os danos oxidativos. / Growing evidence has shown that exercise impairs erythrocyte membrane structure as a consequence of increased physical and chemical stress. Erythrocyte-derived nitric oxide (NO) affects membrane fluidity, and its bioavailiability depends on the balance between its synthesis and scavenging by reactive oxygen species. Here, we investigated whether aerobic exercise performed at different intensities would affect erythrocyte NO bioavailability and oxidative stress. Ten men (26 4 years old, VO2peak 44.1 4.3 mL.kg-1.min-1) performed a treadmill maximal cardiopulmonary exercise test, and a submaximal exercise at 70% VO2peak during 30 min. Blood was collected at rest and immediately after exercises for erythrocytes isolation. Maximal exercise increased erythrocytes count, haemoglobin and haematocrit levels, without any change in body mass that could suggest haemoconcentration due to a plasma volume reduction. It was observed a decrease in L-arginine influx after moderate, but not maximal exercise. Yet, nitric oxide synthase activity, and thus, NO production, was increased after maximal exercise. Cyclic GMP levels did not change after both exercise bouts. In relation to biomarkers of oxidative stress, moderate exercise reduced protein oxidation, and increased catalase activity and glutathione peroxidise expression; whereas maximal exercise led to greater lipid peroxidation, and diminished SOD activity. Neither glutathione peroxidase activity nor NADPH expression were affected by exercise. These findings suggest that exercise induced changes in erythrocyte oxidative stress are more associated with intensity than duration. Furthermore, at intensities recommended for health promotion, exercise was shown to be protective, increasing the activity and expression of important antioxidant enzymes and reducing oxidative damage.

Dans ce travail, nous avons étudié la dégradation de la sulfaclozine, un antibiotique, dans des solutions aqueuses par photocatalyse (TiO2 en suspensions) ainsi que par oxydation par les ions persulfate. L'utilisation d'inhibiteurs spécifiques (KI et alcools) nous a permis de comprendre l'intervention de chacune des espèces réactives (électrons, trous, radicaux •OH) dans la dégradation de la sulfaclozine. En outre, l'identifications des produits de dégradation par LC-MS/MS et le suivi de leur cinétique d'apparition et de disparition avec et sans alcool nous a permis de proposer un mécanisme de dégradation photocatalytique faisant intervenir les trous, les radicaux •OH, les électrons, et les radicaux O2•–. Nous avons également évalués plusieurs méthodes d'activation du persulfate (UV, irradiation solaire, UV/TiO2 et Fe(II)) afin de générer des radicaux SO4•– pour dégrader la sulfaclozine. Nous avons montré qu'à pH 7, le système présentant la plus grande efficacité quelque soit la concentration de persulfate, était le système UV/TiO2/K2S2O8. L'utilisation des inhibiteurs spécifiques des radicaux •OH et SO4•– a permis de constater que le pH a un effet important sur le rôle de chacun de ces radicaux dans la dégradation de la sulfaclozine. Les constantes de vitesse de la réaction de la sulfaclozine avec les radicaux •OH et SO4•– ont été déterminées et des valeurs proches ont été trouvées (?109 M-1s-1). Nous avons également étudié l'effet des principaux ions constituants de l'eau sur la dégradation de la sulfaclozine dans les trois systèmes suivants: UV/TiO2, UV/TiO2/K2S2O8 et UV/K2S2O8. Cette étude a montré que les bicarbonates et les phosphates accélèrent la dégradation photocatalytique alors qu'aucun effet n'a été observé dans le système UV/K2S2O8. En ce qui concerne les ions chlorures et nitrates nous avons montré qu'ils augmentaient l'adsorption de la sulfaclozine à la surface de TiO2 mais n'accéléraient pas significativement la réaction de dégradation / In this work, we studied the degradation of the antibiotic sulfaclozine in aqueous solutions by photocatalysis (on TiO2 suspensions) as well as by persulfate ions. The use of specific inhibitors (KI and alcohols) allowed us to understand the intervention of each of the reactive species (electrons, holes, radicals •OH) in the degradation of sulfaclozine. In addition, the identification of the by-products by LC-MS / MS and the monitoring of their appearance and disappearance kinetics, allowed us to propose a photocatalytic degradation mechanism involving TiO2 holes, •OH radicals, electrons, and O2•– radicals. We also evaluated several methods for persulfate activation (UV, sunlight, UV / TiO2 and Fe (II)) to generate SO4•–. We have shown that at pH 7, the system having the highest efficiency, regardless of persulfate concentration, was the UV/TiO2/K2S2O8 system. The use of specific inhibitors of •OH and SO4•– radicals showed that pH has a significant effect on the role of each of these radicals in the sulfaclozine degradation. Moreover, the reaction rate constants of sulfaclozine with •OH radicals and with SO4•– radicals were determined and close values were found (?109 M-1s-1). We also studied the effect of the main water constituents on the degradation of sulfaclozine in the following three systems: UV/TiO2, UV/TiO2/K2S2O8 and UV/K2S2O8. This study showed that bicarbonate and phosphate accelerated the photocatalytic degradation of sulfaclozine while no effect was observed in the UV/K2S2O8 system. Regarding chloride and nitrate ions, we obtained an enhancement in sulfaclozine adsorption on the surface of TiO2 but no significant enhancement of the degradation rate was observed

La migration cellulaire dirigée est un processus crucial lors du développement embryonnaire, de la cicatrisation, de la réponse immunitaire mais aussi lors de la formation de métastases. La réussite de ces processus nécessite que les cellules perçoivent un signal asymétrique, l'interprète et s'oriente pour migrer de façon dirigée. In vivo, la migration est dirigée par de nombreux signaux du microenvironnement cellulaire. L'hypoxie, ou diminution du niveau d'oxygène tissulaire, est une caractéristique importante de l'environnement cellulaire dans l'embryon et dans les tumeurs solides. Du fait de la limitation de la diffusion de l'oxygène, l'hypoxie génère in vivo des gradients d'oxygène. Nous avons développé une méthode originale dans laquelle des cellules épithéliales génèrent elles-mêmes gradient d'oxygène in vitro. Et de façon très intéressante, ces cellules sont capables de migrer de façon directionnelle vers des concentrations en oxygène plus élevées. Cette capacité d'aérotaxie est indépendante de la respiration mitochondriale et des acteurs de réponse à l'hypoxie. Les dérivés réactifs de l'oxygène (ROS) seraient les médiateurs de la réponse migratoire au gradient. La production asymétrique de ROS entre l'avant et l'arrière des cellules serait à l'origine de l'activation différentielle du récepteur EGFR et de la persistance des cellules vers des concentrations plus importantes en oxygène. Cette capacité chimio-attractante de l'oxygène, connue chez les bactéries, mais non décrite pour des cellules eucaryotes, pourrait jouer un rôle majeur lors du développement embryonnaire et dans la dissémination métastatique / Cell migration is a crucial process during embryonic development, wound healing, immune system but also metastasis. Success of these processes relies on the capacities of cells to sense an asymmetric signal, interpret it and orient themselves to migrate in a directed manner. In vivo, migration is guided by several signals from the cellular microenvironment. Hypoxia, or decrease in the level of tissue oxygen, is an important feature of the cellular environment in the embryo and in solid tumors. Owing to the limitation of oxygen diffusion, hypoxia often generates oxygen gradients in vivo. We have developed an original method in which epithelial cells themselves generate oxygen gradient in vitro. And interestingly, these cells are able to migrate directionally to higher oxygen concentrations. This aerotaxis ability is independent of mitochondrial respiration and hypoxia response pathway. The reactive oxygen species (ROS) would mediate the migratory response to the gradient. The asymmetric production of ROS between the front and the back of the cells would be at the origin of the differential activation of the EGFR receptor and the persistence of cells towards higher oxygen concentrations. This chemoattractant capacity of oxygen, known in bacteria, but not described for eukaryotic cells, could play a major role in embryonic development and in metastatic dissemination

La famille des NADPH oxydases est constituée de complexes multi protéiques, dont un des composants est la sous unité catalytique NOX. Il s'agit de protéine transmembranaire qui assure le transport des électrons à travers la membrane, à partir d'un donneur d'électrons, le NADPH, à un accepteur final, l'oxygène moléculaire. Il en résulte la production de superoxydes, précurseur d'espèces réactives de l'oxygène. Les NOX sont impliquées dans divers processus physiologiques et pathologiques qui les ont placés au rang des cibles thérapeutiques à forte valeur ajoutée.Les NOX sont reportées comme des protéines membranaires propres aux eucaryotes supérieures. Ainsi toutes les données fonctionnelles disponibles pour cette famille d'enzyme, ont été le fruit des études structure-fonction et de données putatives indirectes. D'où l'idée et l'intérêt d'identifier chez les procaryotes des candidats homologues aux Nox eucaryotes et susceptibles d'être de bons modèles pour des études structurales. En se servant d'outils bio-informatiques, nous avons définis des signatures de séquences propres aux NOX et avons identifié chez les procaryotes des centaines de séquences homologues. SpNox de chez Streptococcus Pneumoniae, a été sélectionnée et surexprimée chez E. Coli. SpNox a été purifiée et a fait l'objet d'une caractérisation fonctionnelle et biochimique approfondie. Au vue des résultats obtenus, SpNox se rapproche de part ses propriétés structurales et mechanistiques des NOX eucaryotes. Ainsi elle se présente comme le premier modèle procaryote de protéine NOX. Les premiers cristaux de cette famille de protéines ont été obtenus et son rôle in vivo reste à exploiter.En parallèle à l'approche procaryote, nous avons mené une étude structure-fonction sur la protéine NOX2 des neutrophiles PLB-985. Deux arginines conservées chez toutes les NOX eucaryotes ont été sélectionnées et leur rôle a été étudié par mutagenèse dirigée. Apres évaluation des propriétés enzymatiques des mutants NOX2, nous avons pu identifier l'arginine 513 comme étant impliquée dans la spécificité de NOX2 vis a vis du NADPH. Ces résultats nous ont permis de proposer une nouvelle orientation du NADPH dans son site d'ancrage à la protéine NOX2. / The NADPH oxidase complex was the first identified example of a system that generates reactive oxygen species in a dedicated manner. NOX are proteins involved in the transmembrane transfer of electrons to the molecular oxygen, resulting in the production of superoxides. In addition to ROS related damages, deregulation of Nox-dependant ROS production induces pathological consequences. Accordingly, the Nox family became a potential drug target, making the understanding of their function at molecular basis crucial.In the literature, it has always been reported that Nox proteins exist only in eukaryotes. Since eukaryotic membrane proteins have proven to be difficult to study, all the data available on Nox enzymes are obtained from putative assignments or structure-function studies.In our project, to overcome the difficulty of working on eukaryotic membrane proteins, we used an original approach based on bioinformatics tools. Through using specific filters and a novel program, we were able to identify hundreds of prokaryotic candidates. Among them, we selected SpNox, as a prokaryotic model from Streptococcus Pneumoniae. We have developed its expression in E. Coli as well as a multistep purification scheme. We also conducted an extensive enzymatic and mechanistic characterization of the purified enzyme. Our data support a strong structural and functional homology with known NOX enzymes. Finally, crystallization trials are performed leading to first crystals ever obtained for this family of protein. The understanding of Nox's physiological function in bacteria remains to investigate.In parallel to the prokaryotic approach, a structure-function study was conducted on the human model NOX2 in the PLB-985 neutrophils. Conserved arginines among eukaryotic Nox sequences were selected. Site directed mutagenesis followed by activity tests, lead us to identify a crucial role for arginine 513. It is implicated in the specificity towards NADPH as an electron donor for NOX2. With these data, we were able to suggest a new orientation of the NADPH, notably the phosphate moiety, in the binding site.