Le transporteur de drogues membranaire MRP1 participe à la résistance des cellules cancéreuses à la chimiothérapie lorsqu’il est surexprimé. Cette surexpression peut être exploitée afin de provoquer l’apoptose sélective de ces cellules, MRP1 devenant leur talon d’Achille : c’est l’effet de sensibilité collatérale (SC). Ainsi, le vérapamil stimule l’efflux médié par MRP1 d’un tripeptide antioxydant indispensable aux cellules, le GSH ou glutathion réduit, et provoque la mort sélective des cellules surexprimant ce transporteur. La recherche d’autres agents de SC comme le vérapamil nous a menés à l’étude de composés flavonoïdiques pouvant induire un efflux rapide et massif de GSH. Parmi ces composés, nous avons identifié un puissant agent de SC des cellules résistantes surexprimant MRP1, le dimère de flavonoïde 4e, candidat très prometteur pour de futures études in vivo. Nous avons déterminé que la surexpression de MRP1 est effectivement responsable de la SC dans les cellules cancéreuses résistantes H69AR, et que l’efflux de GSH se doit d’être massif et prolongé pour induire l’apoptose des cellules. Nous avons montré que cet efflux perturbe l’homéostasie du glutathion et l’état redox des cellules, entraînant un stress oxydatif qui participe au déclenchement de la mort cellulaire. Enfin, nous nous sommes attachés à identifier d’éventuelles cibles secondaires des agents de SC dans les cellules surexprimant MRP1, via l’initiation de l’étude de leur transcriptome et métabolome. La compréhension du mécanisme d’action de ces agents de sensibilité collatérale vise, à terme, à l’éradication des cancers résistants surexprimant MRP1 / The membrane drug transporter MRP1 is involved in the resistance of cancer cells to chemotherapy, when overexpressed. This overexpression can be exploited in order to induce the selective apoptosis of these cells, so that MRP1 becomes their Achilles’ heel: this is called Collateral Sensitivity (CS). Thus verapamil stimulates the MRP1-mediated efflux of GSH (reduced form of glutathione) that is an antioxidant tripeptide essential for cells, and induces the selective death of MRP1- overexpressing cells. Seeking for other CS agents than verapamil led to the study of flavonoid compounds able to induce a massive and rapid efflux of GSH and to the identification of a powerful CS agent of resistant cells overexpressing MRP1, i.e. flavonoid dimer 4e, which is a very promising candidate for in vivo studies. We determined that overexpression of MRP1 is indeed responsible for CS in H69AR resistant cancer cells, and that GSH efflux must be massive and protracted in order to induce cell apoptosis. We showed that this efflux disturbs glutathione homeostasis and cell redox state, which leads to an oxidative stress that is involved in triggering cell death. At last, we sought to identify possible secondary targets of CS agents in MRP1-overexpressing cells, via the initiation of transcriptomic and metabolomic studies. Understanding the mechanism of action of these Collateral Sensitivity agents aims to the eradication of resistant cancers that overexpress MRP1

nÃo hà / Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / IntroduÃÃo: A Doxorubicina (DOX), antineoplÃsico antracÃclico, à largamente utilizada no tratamento dos mais diversos tipos de tumores sÃlidos e neoplasias hematolÃgicas. A Cardiotoxicidade provocada pelo uso de antibiÃticos antracÃclicos tem sido observada hà algumas dÃcadas como fator agravante e limitante do uso terapÃutico da DOX. Apesar do conhecimento de inÃmeros fatores que podem mediar a induÃÃo da cardiotoxicidade pela DOX, os mecanismos fisiopatolÃgicos continuam nÃo esclarecidos. Objetivo: Avaliar o efeito da amifostina e glutationa na cardiotoxicidade aguda induzida por doxorubicina e estudar a morfologia do tecido cardÃaco de camundongos tratados com doxorubicina por microscopia de forÃa atÃmica. Material e MÃtodos: Camundongos C57BL/6 fÃmeas (n=8) foram tratados com doxorubicina (25mg/Kg i.p.) ou salina (0,2mL i.p.) e sacrificados 96 horas apÃs tratamento. Outro grupo experimental foi tratado com amifostina (AMF 25, 50 e 100 mg/Kg s.c.), glutationa (GLT 125, 250 e 500mg/Kg s.c.) ou salina 30 mim antes da injeÃÃo de doxorubicina, no caso da glutationa a administraÃÃo foi diÃria atà o dia do sacrifÃcio. Os parÃmetros analisados foram: eletrocardiograma, Ãndices cardÃaco e esplÃnico, dosagem de grupos sulfidrilas nÃo protÃicos, dosagem de CK e CK-MB, parÃmetros histolÃgicos, dosagem por ELISA de TNF-&#61537;, IL-1&#61538;&#61484; expressÃo por imunohistoquÃmica de TNF-&#61537;, IL-1&#61538;, iNOS, apoptose e anÃlise por microscopia de forÃa atÃmica. Resultados: O tratamento com AMF nas doses de 50 e 100mg/Kg e GLT 250 e 500 mg/Kg foi capaz de aumentar a percentagem de sobrevivÃncia dos animais que foram submetidos a cardiotoxicidade aguda induzida por DOX (25 mg/Kg) quando comparados com o grupo injetado somente com DOX. A AMF e GLT tambÃm foram capazes de prevenir, em comparaÃÃo ao grupo DOX (p<0,05), as alteraÃÃes nos valores eletrocardiogrÃficos, (aumento do QRS e QTc e diminuiÃÃo da amplitude de R), as alteraÃÃes nos Ãndices cardÃacos e esplÃnicos, a elevaÃÃo dos nÃveis sÃricos das enzimas CK e CK-MB, a reduÃÃo dos nÃveis de grupos sulfidrilas nÃo protÃicos no tecido cardÃaco e as alteraÃÃes histolÃgicas (degeneraÃÃo hidrÃpica e vacuolizaÃÃo, focos de hialinizaÃÃo de fibras cardÃacas, picnose e necrose) induzidas pela DOX (25mg/Kg). A DOX induziu aumento da marcaÃÃo imunohistoquÃmica para cÃlulas apoptÃticas e expressÃo de iNOS e diminuiu a expressÃo de TNF-&#61537;. A AMF foi capaz de prevenir estas alteraÃÃes, sendo esta prevenÃÃo apenas discreta para a expressÃo de TNF-&#61537;. A microscopia de forÃa atÃmica revelou alteraÃÃes morfolÃgicas nÃo vistas pela microscopia Ãptica e mostrou ser uma ferramenta valiosa na avaliaÃÃo de efeitos de drogas. ConclusÃo: Nossos resultados sugerem o efeito citoprotetor da amifostina pelo aumento da atividade da glutationa peroxidase no tecido cardÃaco e que esta se mostra tÃo eficiente quanto a droga de referencia dexrazoxane. A utilizaÃÃo da microscopia atÃmica introduz uma ferramenta de anÃlise comparativa em escala nanomÃtrica, tornando possÃvel observar a destruiÃÃo membranar cardÃaco condizente com dano oxidativo. / IntroduÃÃo: A Doxorubicina (DOX), antineoplÃsico antracÃclico, à largamente utilizada no tratamento dos mais diversos tipos de tumores sÃlidos e neoplasias hematolÃgicas. A Cardiotoxicidade provocada pelo uso de antibiÃticos antracÃclicos tem sido observada hà algumas dÃcadas como fator agravante e limitante do uso terapÃutico da DOX. Apesar do conhecimento de inÃmeros fatores que podem mediar a induÃÃo da cardiotoxicidade pela DOX, os mecanismos fisiopatolÃgicos continuam nÃo esclarecidos. Objetivo: Avaliar o efeito da amifostina e glutationa na cardiotoxicidade aguda induzida por doxorubicina e estudar a morfologia do tecido cardÃaco de camundongos tratados com doxorubicina por microscopia de forÃa atÃmica. Material e MÃtodos: Camundongos C57BL/6 fÃmeas (n=8) foram tratados com doxorubicina (25mg/Kg i.p.) ou salina (0,2mL i.p.) e sacrificados 96 horas apÃs tratamento. Outro grupo experimental foi tratado com amifostina (AMF 25, 50 e 100 mg/Kg s.c.), glutationa (GLT 125, 250 e 500mg/Kg s.c.) ou salina 30 mim antes da injeÃÃo de doxorubicina, no caso da glutationa a administraÃÃo foi diÃria atà o dia do sacrifÃcio. Os parÃmetros analisados foram: eletrocardiograma, Ãndices cardÃaco e esplÃnico, dosagem de grupos sulfidrilas nÃo protÃicos, dosagem de CK e CK-MB, parÃmetros histolÃgicos, dosagem por ELISA de TNF-&#61537;, IL-1&#61538;&#61484; expressÃo por imunohistoquÃmica de TNF-&#61537;, IL-1&#61538;, iNOS, apoptose e anÃlise por microscopia de forÃa atÃmica. Resultados: O tratamento com AMF nas doses de 50 e 100mg/Kg e GLT 250 e 500 mg/Kg foi capaz de aumentar a percentagem de sobrevivÃncia dos animais que foram submetidos a cardiotoxicidade aguda induzida por DOX (25 mg/Kg) quando comparados com o grupo injetado somente com DOX. A AMF e GLT tambÃm foram capazes de prevenir, em comparaÃÃo ao grupo DOX (p<0,05), as alteraÃÃes nos valores eletrocardiogrÃficos, (aumento do QRS e QTc e diminuiÃÃo da amplitude de R), as alteraÃÃes nos Ãndices cardÃacos e esplÃnicos, a elevaÃÃo dos nÃveis sÃricos das enzimas CK e CK-MB, a reduÃÃo dos nÃveis de grupos sulfidrilas nÃo protÃicos no tecido cardÃaco e as alteraÃÃes histolÃgicas (degeneraÃÃo hidrÃpica e vacuolizaÃÃo, focos de hialinizaÃÃo de fibras cardÃacas, picnose e necrose) induzidas pela DOX (25mg/Kg). A DOX induziu aumento da marcaÃÃo imunohistoquÃmica para cÃlulas apoptÃticas e expressÃo de iNOS e diminuiu a expressÃo de TNF-&#61537;. A AMF foi capaz de prevenir estas alteraÃÃes, sendo esta prevenÃÃo apenas discreta para a expressÃo de TNF-&#61537;. A microscopia de forÃa atÃmica revelou alteraÃÃes morfolÃgicas nÃo vistas pela microscopia Ãptica e mostrou ser uma ferramenta valiosa na avaliaÃÃo de efeitos de drogas. ConclusÃo: Nossos resultados sugerem o efeito citoprotetor da amifostina pelo aumento da atividade da glutationa peroxidase no tecido cardÃaco e que esta se mostra tÃo eficiente quanto a droga de referencia dexrazoxane. A utilizaÃÃo da microscopia atÃmica introduz uma ferramenta de anÃlise comparativa em escala nanomÃtrica, tornando possÃvel observar a destruiÃÃo membranar cardÃaco condizente com dano oxidativo. / Introduction: Doxorubicin (DOX) is an antineoplasic anthracyclic agent used on the treatment of several solid tumors and hematological cancers. DOX-induced cardiotoxicity has been studied for decades as a limiting factor on the anticancer therapy with this drug. Despite the current knowledge concerning the mechanisms of DOX-induced cardotoxicity, its pathophysiology is still not clear. Purpose: To evaluate of the amifostine and glutathione citoprotective effect on the DOX-induced acute cardiotoxicity and to study the morphology of cardiac tissue through the use of atomic force microscopy as a tool. Materials and Methods: C57BL/6 female mice were treated with doxorubicin (25mg/Kg i.p.) or saline (0,2mL i.p.) and sacrificed 96 hours after treatment. In another experimental setting, mice were given amifostine (AMF 25, 50 e 100 mg/Kg s.c.), glutathione (GLT 125, 250 e 500mg/Kg s.c.) or vehicle, 30 mim before the administration of DOX, except glutathione that was injected daily. Analitical parameters included: electrocardiograms, cardiac and spleen indices, non protein suphidrils groups levels, CK and CK-MB cardiac enzymes levels, histological analysis, cardiac levels of (TNF-&#61537;, IL-1&#61538;, iNOS) determined by ELISA, immunohistochemistry for TNF-&#61537;, IL-1&#61538;, iNOS, apoptosis and atomic force microscopy tissue analysis. Results: AMF (100mg/Kg) and GLT (250 e 500 mg/Kg) treatments were able of improve the survival rate of animals in spite of the injection of DOX (25 mg/Kg) in comparison to DOX-treated only group (p<0.05). A AMF e GLT were also able to prevent electrocardiographic changes (rising of QRS e QTc and reduced R amplitude), changes in the cardiac and spleen indices, the augmentation of blood levels of CK e CK-MB, reduction of non proteic suphidrils groups levels, and histological changes induced by DOX (25mg/Kg). DOX induced the augmentation of the immunostaining for apoptotic cells and iNOS what was prevented by the administration of amifostine. The atomic force microscopy reveals morphological changes on the tissue organizational structure which is not possible to be observed through optical microscopy. Conclusion: Our results suggest that the amifostine citoprotective effect on DOX-induced acute cardiotoxicity is due the rising of glutathione peroxidase activity in the cardiac tissue. The citoprotective effect of amifostine is as efficient as the reference drug dexrazoxane. The use of atomic force microscopy as a new pharmacological tool for comparative analysis in nanometric scale allow us to observe DOX-induced membrane destruction what is suggestive of oxidative stress process. / Introduction: Doxorubicin (DOX) is an antineoplasic anthracyclic agent used on the treatment of several solid tumors and hematological cancers. DOX-induced cardiotoxicity has been studied for decades as a limiting factor on the anticancer therapy with this drug. Despite the current knowledge concerning the mechanisms of DOX-induced cardotoxicity, its pathophysiology is still not clear. Purpose: To evaluate of the amifostine and glutathione citoprotective effect on the DOX-induced acute cardiotoxicity and to study the morphology of cardiac tissue through the use of atomic force microscopy as a tool. Materials and Methods: C57BL/6 female mice were treated with doxorubicin (25mg/Kg i.p.) or saline (0,2mL i.p.) and sacrificed 96 hours after treatment. In another experimental setting, mice were given amifostine (AMF 25, 50 e 100 mg/Kg s.c.), glutathione (GLT 125, 250 e 500mg/Kg s.c.) or vehicle, 30 mim before the administration of DOX, except glutathione that was injected daily. Analitical parameters included: electrocardiograms, cardiac and spleen indices, non protein suphidrils groups levels, CK and CK-MB cardiac enzymes levels, histological analysis, cardiac levels of (TNF-&#61537;, IL-1&#61538;, iNOS) determined by ELISA, immunohistochemistry for TNF-&#61537;, IL-1&#61538;, iNOS, apoptosis and atomic force microscopy tissue analysis. Results: AMF (100mg/Kg) and GLT (250 e 500 mg/Kg) treatments were able of improve the survival rate of animals in spite of the injection of DOX (25 mg/Kg) in comparison to DOX-treated only group (p<0.05). A AMF e GLT were also able to prevent electrocardiographic changes (rising of QRS e QTc and reduced R amplitude), changes in the cardiac and spleen indices, the augmentation of blood levels of CK e CK-MB, reduction of non proteic suphidrils groups levels, and histological changes induced by DOX (25mg/Kg). DOX induced the augmentation of the immunostaining for apoptotic cells and iNOS what was prevented by the administration of amifostine. The atomic force microscopy reveals morphological changes on the tissue organizational structure which is not possible to be observed through optical microscopy. Conclusion: Our results suggest that the amifostine citoprotective effect on DOX-induced acute cardiotoxicity is due the rising of glutathione peroxidase activity in the cardiac tissue. The citoprotective effect of amifostine is as efficient as the reference drug dexrazoxane. The use of atomic force microscopy as a new pharmacological tool for comparative analysis in nanometric scale allow us to observe DOX-induced membrane destruction what is suggestive of oxidative stress process.

O presente experimento teve por objetivos comparar a adição de diferentes concentrações de glutationa reduzida (GSH) no diluidor para criopreservação do sêmen de cães, considerando-se as características morfofuncionais pós-descongelação; e verificar os índices de gestação e número de filhotes por ninhada obtidos a partir da inseminação artificial intra-uterina, por cateterização cervical endoscópica, com sêmen criopreservado contendo diferentes concentrações de glutationa. Na primeira fase do experimento, foram realizadas duas colheitas seminais de 11 reprodutores com intervalo mínimo de uma semana entre os procedimentos. Foram empregadas três concentrações diferentes de glutationa reduzida (0, 10 e 20 mM) ao meio de congelação tris-gema-citrato. O sêmen fresco, refrigerado, glicerolizado e descongelado foram avaliados por citometria de fluxo com uso de sondas específicas para determinar a integridade das membranas plasmática e acrossomal, potencial de membrana mitocondrial e fragmentação de DNA. Também foi realizada dosagem de substâncias reativas ao ácido tiobarbitúrico (TBARS) para avaliar os níveis de estresse oxidativo. Na segunda fase do experimento, foram inseminadas 6 fêmeas, alocadas em dois grupos: inseminação intra-uterina com sêmen congelado em diluidor contendo 10 mM (IA-GSH10, n=3) e sem a adição de glutationa (IA-GSH0, n=3). O acompanhamento do ciclo estral foi realizado por citologia vaginal, vaginoscopia, dosagem de progesterona e LH. Foram realizadas duas inseminações no 5o e 6o dias após pico de LH e a gestação diagnosticada aos 30 dias. A adição de 10 e 20 mM de GSH ao diluidor para criopreservação promoveu queda na motilidade espermática nas amostras descongeladas, sendo o maior prejuízo observado na concentração de 20 mM. Foi detectada maior porcentagem de acrossomos íntegros nos grupos tratados. A glicerolização e, principalmente, a exposição do espermatozóide ao nitrogênio líquido e posterior descongelação foram responsáveis pela diminuição da motilidade espermática, em consequência ao prejuízo da função mitocondrial promovida pela produção de radicais livres durante o processamento seminal. A gestação foi diagnosticada em duas fêmeas do grupo IA-GSH10 e duas do grupo IA-GSH0. Em conclusão, a adição de 10 e 20 mM de GSH ao diluidor para criopreservação seminal não promoveu os efeitos protetores esperados na espécie canina, sendo a concentração de 20 mM responsável por maiores prejuízos à amostra seminal. A adição de 10 mM de GSH ao diluidor para criopreservação seminal na espécie canina manteve o índice de fertilidade semelhante ao obtido com sêmen criopreservado sem a suplementação antioxidante. / The present experiment aimed to compare the addition of different concentrations of reduced glutathione (GSH) on frozen-thawed canine semen, considering the morphofunctional characteristics; and to verify the pregnancy rate and number of puppies per litter obtained from intrauterine insemination by endoscopic catheterization of the cervix, with frozen-thawed semen containing different concentrations of glutathione. During the first phase of the experiment, two seminal samples collected weekly from 11 mature dogs were used. Three different concentrations of reduced glutathione (0, 10 and 20 mM) were supplemented in a tris-citrate egg yolk extender. The fresh, chilled, glycerolized and post-thawed semen were assessed by flow cytometry using specific probes to determine plasma membrane and acrosomal integrity, mitochondrial membrane potential and DNA fragmentation. Thiobarbituric acid reactive substances (TBARS) assay was also performed to assess the occurrence of oxidative stress. In the second phase of the experiment, six canine females were allocated into two groups: intrauterine insemination with frozen-thawed semen containing 10 mM GSH (IA-GSH10, n = 3) and without the addition of glutathione (IA-GSH0, n = 3). Bitches were monitored by vaginal cytology, vaginoscopy, progesterone and LH assays. Two inseminations were performed on days 5 and 6 post LH peak. We verified that the addition of 10 and 20 mM of GSH to semen extender promoted a decrease in post-thaw sperm motility, as well as a higher damage degree at the 20 mM concentration. We also detected a higher percentage of acrossomal integrity in treated groups. At glycerolization and moreover, the sperm exposure to liquid nitrogen and further thawing, were responsible for the decrease in sperm motility, due to the loss of mitochondrial function through the free radicals production. Gestation was diagnosed in two female from IA-GSH10 group and two bitches of the IA-GSH0 group. In conclusion, we did not observe the expected protective effects of the addition of 10 and 20 mM GSH to semen extender. Furthermore, the concentration of 20 mM was responsible for the more extreme damage to semen sample. The supplementation of 10 mM GSH to semen extender for cryopreservation maintained the fertility rates similar to the ones observed for cryopreservation without antioxidant supplementation in dogs.

Com o aumento de atividades humanas nocivas ao meio ambiente e principalmente ao meio aquático, torna-se importante a elucidação dos mecanismos de defesa que envolvem os organismos expostos. As algas têm particular importância por serem a base da cadeia alimentar do ecossistema marinho. Ao acumular as substâncias tóxicas presentes no ambiente e servirem de alimento para outras espécies, elas podem provocar biomagnificação do agente tóxico ao longo da cadeia. A escolha da microalga Lingulodinium polyedrum deve-se à sua ampla distribuição em âmbito nacional e mundial e por ser um organismo modelo para estudos de toxicologia envolvendo metais. Este trabalho tem como objetivos padronizar as condições do meio de cultivo de forma a proporcionar o crescimento ideal da espécie, traçar a curva de crescimento e monitorar os aspectos biológicos que sofrem alterações frente a metais, tais como: taxa fotossintética, atividade da enzima antioxidante superóxido dismutase, balanço entre glutationa reduzida e oxidada, bioacumulação intracelular dos metais a que foram expostas e identificação de substâncias quelantes sintetizadas pelas algas, conhecidas como fitoquelatinas. Para alcançar estes objetivos foram utilizadas técnicas analíticas tais como espectrofotometria, espectrometria de massas e espectrometria de emissão atômica com plasma acoplado indutivamente. Dentre os resultados obtidos estão diminuição da quantidade de glutationa reduzida e oxidada quando algas são expostas a metais, diminuição da quantidade de cádmio presente no meio, aumento da atividade de superóxido dismutase e síntese de fitoquelatinas. Com os resultados obtidos podemos concluir que as fitoquelatinas podem ser usadas como biomarcadores de exposição ao cádmio e o organismo, por ser capaz de diminuir a quantidade de metais disponíveis no ambiente, tem potencial para biorremediar ambientes poluídos. / Given the increase of environmentally harmful human activities, in particular the ones injurious to the aquatic environment, it is important to elucidate the defense mechanisms utilized by organisms exposed to damaging agents. Those species can later be suggested to be used as pollution bioindicators or bioremediatiors. Algae are of particular importance because they are the basis of the marine ecosystem food chain. Given that these organisms can accumulate toxic substances from the environment and they serve as food for other species, it will cause biomagnification of the toxic agent in the chain. The choice of the microalgae Lingulodinium polyedrum was due to its wide national and global distribution and the fact that it is a model organism for toxicology studies involving metals. This work aims to standardize the medium conditions in order to provide the ideal growth of the species; plot the growth curve; and monitor the biological aspects that change in the presence of metals, such as: photosynthetic rate, antioxidant enzyme superoxide dismutase activity, balance between oxidized and reduced glutathione, intracellular accumulation of metals and identification of chelating substances synthesized by the alga, known as phytochelatins. To achieve these objectives there were used analytical techniques such as spectrophotometry, mass spectrometry and inductively coupled plasma atomic emission spectroscopy. Among the obtained results there are the decrease in the amount of reduced and oxidized glutathione when algae are exposed to the metal; reduction in the quantity of cadmium in the medium; and increase in superoxide dismutase activity and phytochelatin synthesis. Based on the results it can be concluded that phytochelatins can be used as biomarkers of exposure to cadmium and the organism have potential to bioremediate polluted environments.

L’ANS recombinante de Vitis vinifera (VvANS) a été exprimée chez E. coli, et purifiée par chromatographie d’affinité sur colonne Nickel. La production et purification de l'holoenzyme chargée en fer a été mise en point, afin d’éviter les réactions d'oxydation non-enzymatique incontrôlée en présence de sel de Fe(II). Un complexe VvANS-Fe(II) stable est formé en présence d'α- cétoglutarate et d'ascorbate, complexe qui est catalytiquement actif à PO2 ambiante en l'absence de sel de Fe(II). La transformation de la (+)-catéchine par VvANS a été étudiée avec ou sans ascorbate, en utilisant le complexe VvANSFe( II) ou l’holoenzyme co-incubée en présence de sulfate ferreux, afin d’étudier le rôle de l’ascorbate. Aucune activité enzymatique n’a été observée en l'absence d’ascorbate, ce qui indique qu'il s'agit d'un cofacteur indispensable de VvANS. Un adduit covalent ascorbate-cyanidine est produit in vitro, mais seulement en l'absence d'un autre réducteur nucléophile majeur, le glutathion GSH. Les deux stéréoisomères de la leucocyanidine (flavan-diols 3,4-cis et 3,4-trans), substrats potentiels de VvANS, mais non commerciaux, ont été synthétisés par réduction de la dihydroquercétine par NaBH4, puis identifiés par RMN du proton. L'analyse des deux stéréoisomères par spectrométrie de masse en tandem (MS/MS) montre que leurs voies de fragmentation MS/MS sont distinctes et peuvent être utilisées pour les distinguer lors de leur production. Les deux stéréoisomères sont stables en milieu aqueux congelé à -20°C. Douze flavonoïdes de quatre familles distinctes (flavanones, dihydroflavonols, flavan-3-ols et flavan-3,4-diols) ont été testés comme substrats potentiels. Tous les produits enzymatiques ont été purifiés par HPLC en phase inverse, puis identifiés par MS/MS, avec les résultats suivants: 1) Seuls les dihydroflavonols de configuration (2R,3R) sont acceptés comme substrats par VvANS dont l'activité diminue avec le nombre de groupements hydroxyles du cycle B. 2) Seuls les flavan-3-ols ou flavan-3,4-diols de configuration (2R,3S) ayant un catéchol ou trois OH phénoliques vicinaux sur le cycle B sont acceptés comme substrats. 3) La naringénine n'est pas substrat de VvANS, sans doute en raison de l'absence de groupement hydroxyle en C3. […] Le glutathion GSH est un puissant nucléophile, réducteur et piégeur de radicaux libres, qui est abondant dans la baie de raisin. Nous avons donc étudié son effet sur l'activité de VvANS avec tous les substrats identifiés. GSH n’a pas d'effet sur la transformation des dihydroflavonols et des flavan-3,4-diols, mais il modifie considérablement le mode de transformation de la (+)-catéchine et de la (+)-gallocatéchine. En présence de (+)-catéchine et de GSH, on observe deux produits majeurs, la cyanidine et un adduit thioéther cyanidine-glutathion, et le rendement de production est beaucoup plus élevé qu'en l'absence de GSH. De plus, l’adduit covalent ascorbate-cyanidine et le dimère issu de la (+)-catéchine obtenus lors de la réaction réalisée en l'absence de GSH ont disparu. Nos données suggèrent que l'adduit covalent cyanidine-glutathion est un thioéther en C4 qui fait l'objet d'un équilibre de tautomérisation céto-énolique en C3, et se décompose en cyanidine et GSH. En présence de (+)-gallocatéchine, un adduit thioéther similaire delphinidine-glutathion est aussi observé. Pour tester l'éventuelle spécificité de GSH, trois autres mercaptans (thiomalate, cystéine et cystéamine) ont été testés et aucun adduit similaire n’a été observé, ce qui suggère que GSH est un ligand spécifique, et pourrait être un coenzyme de VvANS. Nos résultats suggèrent que les anthocyanidines pourraient être produites in vivo à partir d'un substrat flavan-3-ol (catéchine ou gallocatéchine) via un intermédiaire thioéther de glutathion, alors que le stéréoisomère naturel (3,4-cis) de la leucocyanidine n'est pas transformé en cyanidine. / Recombinant anthocyanidin synthase from Vitis vinifera (VvANS) has been expressed in E. coli, and purified by nickel affinity chromatography. The production and purification of the iron-loaded enzyme has been developed in order to avoid uncontrolled nonenzymatic oxidation reactions in the presence of Fe(II) salt. A stable VvANS-Fe(II) complex is formed in the presence of 2-oxoglutarate and ascorbate, and this complex is catalytically active at ambient PO2 in the absence of Fe(II) salt. The transformation of (+)-catechin by VvANS has been studied with and without ascorbate, by using either the VvANSFe( II) complex or the holoenzyme co-incubated with ferrous sulfate, to investigate the role of ascorbate. No enzyme activity has been observed in the absence of ascorbate, which means that it is an essential enzyme cofactor. A covalent adduct ascorbate-cyanidin is produced in vitro, but only in the absence of glutathione (GSH), another major nucleophilic and reducing agent. The two stereoisomers of leucocyanidin (3,4-cis et 3,4-trans flavan-diols) which were expected to behave as substrates of VvANS, are not commercial and were synthesized by reduction of dihydroquercetin by NaBH4, and characterized by proton NMR. The analysis of the two stereoisomers by means of tandem mass spectrometry (MS/MS) shows that their fragmentation pathways are distinct and may be used to distinguish them during their production. The two stereoisomers are stable in frozen aqueous medium at -20°C. Twelve flavonoids of four distinct families (flavanones, dihydroflavonols, flavan-3-ols et flavan-3,4-diols) were tested as potential substrates of VvANS. All enzymatic products were purified by means of reverse-phase HPLC and characterized by MS/MS, with the following results: 1) Only dihydroflavonols of (2R,3R) configuration are accepted as substrates by VvANS, the activity decreasing with the number of hydroxyl groups of ring B. 2) Only flavan-3-ols or flavan-3,4-diols of (2R,3S) configuration having either a catechol or three vicinal phenolic OH on ring B are accepted as substrates. 3) Naringenin is not substrate of VvANS, most likely because a C3 hydroxyl group is missing. […] Glutathione GSH is a powerful nucleophilic and reducing agent as well as a free radical scavenger, which is abundant in grape berries. We therefore studied its effect on VvANS activity with all identified substrates. GSH has no effect on the transformation of dihydroflavonols and flavan-3,4-diols, but it considerably modifies the transformation pattern of (+)- catechin and (+)-gallocatechin. In the presence of (+)-catechin and GSH, we observe two major products, cyanidin and a cyanidin-glutathione thioether, with production yields which are much higher than in the absence of GSH. Moreover, the ascorbate-cyanidin covalent adduct and the (+)-catechin dimer that had been obtained in the absence of GSH have disappeared. Our data suggest that the cyanidin-glutathione adduct is a C4-thioether which is in equilibrium between the two keto-enolic tautomeric forms at C3, and decomposes into cyanidin and GSH. In the presence of (+)-gallocatechin, a similar delphinidin-glutathione thioether adduct is also observed. In order to test the possible specificity of GSH as a cofactor, three other mercaptans (thiomalate, cysteine and cysteamine) were tested, and no similar product was observed, which suggests that GSH is a specific ligand, and might be a coenzyme of VvANS. Our results suggest that anthocyanidins could be produced in vivo from a flavan-3-ol substrate (catechin or gallocatechin) via a glutathione thioether intermediate, whereas the natural 3,4-cis stereoisomer of leucocyanidin is not transformed into cyanidin by VvANS.

Les auxines sont des composants clés essentiels pour le contrôle du développement des racines et la réponse aux contraintes environnementales en raison de leur rôle central dans la division, l’élongation et la différenciation cellulaires. L’auxine endogène, acide indole-3-butyrique (IBA), bien que moins abondante et moins connue que l'acide indole-3-acétique (IAA), joue un rôle important dans le développement racinaire, en particulier lors de la formation des racines latérales et de l'élongation des poils absorbants. Il est généralement admis que les fonctions de l’IBA dépendent entièrement de la conversion peroxysomale de l’IBA en IAA. Bien que nos connaissances concernant les enzymes impliquées dans cette conversion soient très avancées, nous en savons peu sur les mode de régulation de ces fonctions. Au cours de ma thèse, j'ai démontré que les fonctions de l’IBA lors de l'induction des racines latérales et de l'élongation des poils absorbants dépendent du glutathion, un petit tripeptide rédox qui constitue l'une des molécules les plus importantes impliquées dans les réponses des plantes au stress oxydatif. De plus, j'ai démontré que le lien entre le glutathion et l'auxine IBA est essentiel pour les réponses à l’auxine dans la zone de transition de la racine primaire. Ce contrôle des fonctions de l’IBA par le glutathion pourrait être déterminant dans des conditions de stress abiotiques telles que la carence en phosphore.Une des fonctions du glutathion étant de réduire des réductases de fonctions thiols, les glutaredoxines (GRX), nous avons recherché si certaines GRX sont impliquées dans le développement racinaire. Nous avons constaté que ROXY19 et GRXS17 sont essentiels à la croissance des racines primaires et que ces deux GRX sont également impliqués, mais dans des rôles différents, lors du développement des racines latérales. Un crible suppresseur des phénotypes racinaires du mutant grxs17 avait été mis en place dans le laboratoire. J'ai utilisé des approches bio-informatiques pour isoler les mutations causales après reséquençage du génome de candidats capables de restaurer la croissance des racines primaires et / ou le développement normal des primordia des racines latérales. Malheureusement, cette approche ne nous a pas encore permis d’isoler de nouveaux acteurs. Cependant, elle jette les bases d’un futur grand progrès dans la compréhension de la manière dont GRXS17 contrôle le système racinaire.En conclusion, les résultats de ma thèse soulignent l’importance du glutathion et des glutarédoxines dans le contrôle de la plasticité du système racinaire et lors de conditions de stress abiotiques, notamment via la modulation de la voie auxinique IBA. / Auxins are critical key components for the control of root development and response to environmental constraints by its pivotal roles in cell division, elongation, and cell differentiation. The endogenous auxin Indole-3-butyric acid (IBA), although less abundant than the better-known Indole-3-Acetic Acid (IAA), plays important roles during root development especially during the formation of lateral roots and root hairs elongation. It is generally accepted that IBA functions are fully dependent on peroxisomal IBA-to-IAA conversion. While there is a great advance in our knowledge regarding the enzymes involved in the peroxisomal conversion of IBA-to-IAA, little is known about the mechanisms that modulate its functions. During my thesis, I demonstrated that the IBA functions during the induction of lateral roots and root hairs elongation are dependent on glutathione, which is a small redox tripeptide that constitutes one of the most important molecules involved in plant responses to oxidative stresses. Moreover, I demonstrated that the link between glutathione and the auxin IBA is critical for the auxin distribution that takes place in the transition zone of the primary root. The relevance of the control of IBA functions by glutathione might be determinant during abiotic stress conditions such as phosphorus deprivation.Since glutathione is a reducer of thiol reductases glutaredoxins (GRXs), we investigate if some GRXs are involved in root development. We found that ROXY19 and GRXS17 are critical for the primary root growth, and both GRX proteins play different roles during the formation of lateral roots. Based on this root phenotype, a suppressor screen in grxs17 background had been set up in the lab. I developed bioinformatic pipelines to isolate causal mutations from genome resequencing of candidates that are able to restore the primary root growth and/or the normal development of lateral roots primordia. Unfortunately, this approach did not yet allow us to isolate new actors, however it builds foundations for future big advance in understanding how glutaredoxins control the root system.In conclusion, the results showed in my Ph.D. thesis highlight the importance of glutathione and glutaredoxins in the control of the root system plasticity and during abiotic stress conditions, particularly via the modulation of the auxinic IBA pathway.

A scientific and cultural revolution occurred with the sequencing of the human genome. The information provided by this accomplishment has provided tools for researchers to test new ideas in silico and on the bench. In redox biology many of the genes, transcripts, proteins, and redox active species have been well characterized. However, the vast majority have not been quantitated in an absolute manner. This is a necessary step to provide the tools for mathematical modeling and systems biology approaches for predicting changes in the cellular redox environment and the biochemical and biological consequences. Here we demonstrate techniques for the absolute quantitation of human catalase, glutathione peroxidase, peroxiredoxin, thioredoxin, and superoxide dismutase within cells. These techniques can be parsed into two groups: detection of activity and detection of total amount of species. Methods for the absolute quantitation of active catalase, peroxiredoxins, and superoxide dismutase have been developed by utilizing specific characteristics of each enzyme. Catalase generates oxygen in the presence of hydrogen peroxide that can easily be detected with a Clark electrode (oxygen monitor); the data are fit to a single-exponential to determine the observed pseudo-first-order rate constant. From this the effective number of fully active catalase enzymes in the sample can be determined. Peroxiredoxin in the disulfide state can be reduced by thioredoxin; thioredoxin from E. coli loses fluorescence upon oxidation. The loss of fluorescence over time is mathematically fit to a single-exponential to determine the observed pseudo first-order rate constant from which the number of active enzymes can be determined. Using an inhibition assay to detect superoxide dismutase activity along with the rate constants at which superoxide reacts with the dismutase and the competing superoxide-reacting-indicator-molecule, the concentration of active superoxide dismutase can be determined. To detect the total amount of protein of an enzyme in a biological sample, an immunoassay was first implemented. This method utilized Bio-Plex® beads from Bio-Rad; however, it was problematic because the antibodies applied did not perform satisfactorily not allowing sufficient signal-to-noise to be deployed. Quantitative mass spectrometry was then implemented to detect total catalase, glutathione peroxidase 1, peroxiredoxin 2, and thioredoxin 1 in human red blood cells. With the absolute concentration of these enzymes and proteins along with data for oxygen consumption rates and peroxisomal hydrogen peroxide concentration for several cell lines, we hypothesize that a reasonable model of hydrogen peroxide and superoxide flux can be constructed. Quantitative data such as these provide the foundation for the new redox biology of the 21st century. Presented here is a roadmap for the obligatory first steps to dissect quantitatively the cellular and tissue metabolic pathways and redox networks that are the basis of all of biology.

Hematopoietic stem cells (HSCs) that function to maintain the hematopoietic compartment through self-renewal and differentiation capacities, as well as their downstream progeny, are susceptible to transformation resulting in the generation of the leukemic stem cell (LSC). Chief in the factors that control HSC regulation and protection of the HSC compartment is the cellular redox environment. Deregulation of the Hematopoietic Stem/Progenitor Cell (HSPC) redox environment results in loss of HSPC function and exhaustion. The characteristic developments of HSPC exhaustion via exposure to redox stress closely mirror phenotypic traits of hematopoietic malignancies, presenting the HSPC/LSC redox environment as a potential therapeutic target. While myelosuppression and HSPC exhaustion are detrimental side effects of classical chemotherapies, new approaches that differentially modify the HSPC/LSC redox environment may demonstrate LSC cytotoxicity while offering protection of normal HSPC function via differential activation of internal signaling pathways. Precisely how the redox environment and downstream signaling events are affected by these treatments remains unclear; thus highlighting the need for robust methods that evaluate the HSPC/LSC redox state. Because the glutathione (GSH), glutathione disulfide (GSSG) redox couple heavily contributes to the management of HSPC function and redox environment, characterizing the GSH/GSSG redox potential at the HSPC level would provide great insight for therapeutic opportunities. However, accurate measurement the GSH/GSSG redox potential within HSPCs/LSCs has been difficult due to their inherently low numbers. Here, we describe the development and validation of a sensitive method used for the direct and simultaneous quantitation of both oxidized and reduced GSH via LC-MS/MS. We use these methodologies to establish a difference in GSH-GSSG half-cell reduction potentials between normal and malignant HSPCs and examine the therapeutic effect of a redox active MnSOD mimetic, Mn(III) mesotetrakis (N-n-butoxyethylpyridinium-2yl) porphyrin, MnTnBuOE-2-PyP5+ (MnP), within these populations in vitro as well as within a human xenograft model in vivo. MnP demonstrates significant cytotoxic effects in several malignant models, while inducing an opposite cytoprotective effect in normal HSPC populations. The GSH/GSSG redox balance, specifically managed by glutathione reductase activity, is identified as a determining factor of MnP efficacy in various malignant populations. Treatment of the human myelodysplastic cell line (MDSL) offers mechanistic insights into MnP efficacy through hydrogen peroxide mediated activation of activator protein 1 (AP-1) signaling. We identify the redox dependent activation of JunB, a known regulator of normal myeloid lineage HSC proliferation, as a transcriptional mechanistic mediator of MnP treatment induced AP-1 signaling resulting in malignant cytotoxicity. The development of this novel method allowing for the identification of targetable differences between normal and malignant cell populations has provided insight to the underpinnings of potential redox based therapies. Additionally, the finding that MnP can target varying cellular redox states and exert selective cytotoxicity in malignant over normal populations by re-gaining lost control of AP-1 signaling demonstrates the potential for development of safe therapeutics within a variety of clinical applications.

Les modèles climatiques indiquent qu’il est très probable que les végétaux soient de plus en plus exposés à deux facteurs de stress environnementaux : l’ozone troposphérique (O3) et le déficit hydrique du sol, tous deux pouvant provoquer un stress oxydant pour le végétal. Dans des conditions naturelles, ces deux facteurs peuvent être concomitants ou se succéder. L’impact de l’O3 et de la sécheresse nécessite donc une attention particulière. Afin de déterminer les réponses de défense mises en place par les arbres, deux génotypes de Populus nigra x deltoides (Carpaccio et Robusta) ont été exposés aux contraintes séparées ou à leur combinaison en conditions contrôlées dans des chambres de culture. Pour explorer les effets des stress et l’interaction entre les deux contraintes, nous avons ciblé les deux premiers niveaux de défense des plantes que sont le contrôle de l’ouverture/fermeture des stomates et les processus de détoxication cellulaire. Nos résultats montrent que Carpaccio et Robusta sont tous deux relativement tolérants à une sécheresse modérée grâce à un contrôle efficient des stomates. Face à l’O3, cependant, les deux génotypes adoptent des stratégies de réponse différentes : un évitement important pour Carpaccio et une maximisation de l’assimilation au détriment des feuilles pour Robusta. Cela se traduit par une différence de fermeture des stomates. Les deux génotypes ne font alors pas face au même flux d’O3 entrant dans les feuilles, ce qui impacte la détoxication cellulaire, dans laquelle le glutathion semble jouer un rôle majeur. En lien avec les modifications de capacité antioxydante, l’activité des enzymes du cycle ascorbate-glutathion (MDHAR, DHAR et GR) et/ou l’expression des gènes codant pour ces protéines sont modifiées. En combinaison de stress, le déficit hydrique protège le végétal du stress oxydant induit par l’O3 en amplifiant la fermeture des stomates. En revanche, la croissance de l’arbre est impactée par l’effet additif des deux contraintes. De plus, l’induction de voies de régulation hormonales différentes par les deux contraintes pourrait modifier le « cross-talk » complexe régulant la réponse au stress combiné. Enfin, dans le cas d’une succession de stress, l’exposition à l’O3 avant un épisode de sécheresse impacte faiblement la réponse de l’arbre. Cependant, un ralentissement de la fermeture des stomates induit par l’O3 est observé malgré l’arrêt de la fumigation. Il est donc nécessaire de prendre en compte le ralentissement et la fermeture des stomates induit par l’O3 et le déficit hydrique dans les modèles de conductance stomatique utilisés pour calculer l’indicateur du flux d’O3 entrant, le PODy (Phytotoxic Ozone Dose above a threshold of y nmol O3 m-2.s-1). / Climate models indicate that it is very likely that plants will be more and more exposed to two environmental stressors: ground-level ozone (O3) and soil water deficit, both causing oxidative stress to the plant. Under natural conditions, these two factors can be concomitant or successive. Therefore, the impact of O3 and drought requires special attention. In order to determine the defensive responses adopted by trees, two genotypes of Populus nigra x deltoides (Carpaccio and Robusta) were exposed to separate or combined stresses under controlled conditions in growing chambers. To explore the effects of stresses and their interaction, we targeted the plant’s first two levels of defence: i) the control of stomatal opening and closing, ii) the cellular detoxification processes. Our results show that both Carpaccio and Robusta are relatively tolerant to moderate drought thanks to an efficient stomatal control. However, different response strategies were adopted by the two genotypes to cope with O3. For Carpaccio, the strategy is avoidance, and for Robusta, the strategy is maximization of net CO2 assimilation at the expense of leaves. This results in a difference in the stomatal closure. The two genotypes do not face the same flow of O3 entering the leaves. This impacts cellular detoxification in which glutathione seems to play a major role. Also, the activity of ascorbate-glutathione cycle enzymes (MDHAR, DHAR and GR) and/or the expression of genes encoding these proteins are modified. Under combined stresses, the water deficit protects the plant from the O3-induced oxidative stress by amplifying the stomatal closure. Nevertheless, the tree growth is impacted by the additive effect of the two stresses. Furthermore, the induction of different hormonal regulatory pathways by the two stressors could modify the complex "cross-talk" regulating the response to combined stress. Finally, in the case of a succession of stresses, exposure to O3 prior to a drought episode has a weak impact on the tree's response. However, O3 induced a stomatal sluggishness in closure despite the cessation of fumigation. It is therefore necessary to take into account stomatal closure and sluggishness induced by O3 and water deficit in the stomatal conductance models used to calculate the indicator of O3 flux inside the leaves, PODy (Phytotoxic Ozone Dose above a threshold of y nmol O3 m-2.s-1).

Tumours of the central nervous system (CNS) are the second most common cancers diagnosed in children, yet the cause of this disease remains largely unknown. This thesis examines whether polymorphisms in folate-metabolising and glutathione S transferase (GST) genes influence the risk and disease outcome of childhood CNS tumours. 204 children aged ≤18 years diagnosed with a CNS tumour at the Sydney Children??s Hospital between 1989 and 2004 were included in the study. DNA samples were isolated from archival frozen and formalin-fixed paraffin-embedded tumour tissue. Polymorphisms in GST and folate pathway genes were examined using real-time PCR. Genotype distributions in children with CNS tumours were compared to those observed in a control panel of cord blood samples from 363 healthy newborns. Children carrying at least one variant allele for each of MTHFR 677 C>T, MTHFR 1298 A>C, MTR 2756 A>G, MTRR 66 A>G, and RFC 80 G>A were found to have a 2.8-fold greater risk of developing a CNS tumour than non-carriers (OR=2.80; 95%CI: 1.08-7.56, P=0.022), an association which was even more apparent in those children with an embryonal tumour (OR=4.54; 95%CI: 1.13-15.85, P=0.016). Results also showed that children with the GSTP1 105 Val/Val genotype were three times more likely to develop a CNS tumour of embryonal cell origin than children with the GSTP1 105 Ile/Ile or Ile/Val genotypes (OR=3.02; 95%CI: 1.34-6.46, P=0.005). No such association was observed for CNS tumours of glial cell origin. The GSTM1, GSTT1, and GSTP1 Ala114Val polymorphisms did not appear to be associated with the development of a childhood CNS tumour. In addition, children with the MTHFR 677 TT or RFC 80 AA genotypes were found to have a higher risk of death within 5 years of diagnosis compared to children with one or more MTHFR 677 C or RFC 80 G alleles, respectively (HR=5.52, 95%CI: 1.00-30.37, P=0.049 and HR=5.69, 95%CI: 1.38-23.51, P=0.016, respectively), after adjusting for other prognostic factors such as sex, age at diagnosis, period of diagnosis, and tumour grade. Conversely, children with the MTR 2756 AG or GG genotypes, or MTRR 66 AG or GG genotypes, were more likely to survive compared to those with the MTR 2756 AA or MTRR 66 AA genotypes, respectively (HR=0.21, 95%CI: 0.05-0.93, P=0.040 and HR=0.11, 95%CI: 0.02-0.53, P=0.006). Results presented in this thesis indicate that polymorphisms in folate-metabolising and GST genes may play a role in the aetiology and survival of childhood CNS tumours, and that this may vary depending on the histological sub-type of tumour.