A participação das espécies reativas de oxigênio (ERO) no mecanismo de ação dos antifúngicos azólicos, bem como a relação entre resistência aos antifúngicos e resposta ao estresse oxidativo, têm sido sugeridos. Entretanto, os dados ainda são inconclusivos e diferem entre os micro-organismos. Neste estudo estão apresentados os resultados da geração de ERO pelo fluconazol em isolados de C. glabrata sensíveis e resistentes a esse antifúngico e a resposta antioxidante da levedura. Nesses isolados, tratados e não tratados com fluconazol em concentração subinibitória, de acordo com sua concentração inibitória mínima (CIM), até fase de crescimento estacionário, foi avaliado se o fluconazol geraria ERO. Subsequentemente, foram analisadas as defesas antioxidantes glutationa peroxidase (GPx), superóxido dismutase (SOD), glutationa-S-transferase (GST), consumo de peróxido de hidrogênio e glutationa total, bem como possível dano oxidativo causado pelo fluconazol em lipídeos, proteínas e ácidos nucleicos e os níveis de nitritos e nitratos. Os resultados mostram que nos isolados de C. glabrata sensíveis e resistentes ao fluconazol, na presença do antifúngico, houve um aumento da geração ERO e maior atividade enzimática da GPx e SOD comparada a dos isolados não tratados com fluconazol, não havendo diferença estatística entre isolados sensíveis e resistentes nesses três parâmetros citados. Em relação à enzima GST, os isolados sensíveis mostraram maior atividade enzimática comparada aos resistentes, e quando as células sensíveis foram tratadas com fluconazol, a atividade da GST diminuiu. O fluconazol não induziu dano oxidativo em proteínas e em lipídeos, entretanto foi observado dano oxidativo ao DNA. Diante disso, sugere-se que o fluconazol gera ERO como parte do seu mecanismo antifúngico em C. glabrata em fase de crescimento estacionário, induzindo dano oxidativo no DNA. Como resposta, observa-se aumento na atividade enzimática da SOD e da GPx na levedura. O entendimento da resposta antioxidante de leveduras patogênicas tem importante interesse clínico, uma vez que o desenvolvimento racional de novas drogas antifúngicas requer conhecimento do metabolismo fúngico. / The participation of reactive oxygen species (ROS) in azoles antifungal mechanism of action has been suggested, as well as the relation between antifungal resistance and oxidative stress response. However, data are still inconclusive and differ between microorganisms. This study presents the results of ROS generation by fluconazole in fluconazole-susceptible and resistant C. glabrata strains and their antioxidant response. It was evaluated whether fluconazole generates ROS in those isolates treated and untreated with fluconazole at sub-inhibitory concentration according to their minimal inhibitory concentration (MIC). This treatment was conducted until stationary growth phase was reached. Subsequently, the antioxidant defenses glutathione peroxidase (GPx), superoxide dismutase (SOD), glutathione-S-transferase (GST), consumption of hydrogen peroxide and total glutathione, the possible oxidative damage in lipids, proteins and nucleic acids and the levels of nitrites and nitrates were analyzed. Results showed increased ROS generation in fluconazole-susceptible and resistant C. glabrata strains treated with fluconazole, and also higher GPx and SOD enzymatic activity, compared to untreated cells. No statistical difference of those three parameters was observed between susceptible and resistant strains. In relation to GST, susceptible strains demonstrated higher activity compared to the resistant ones, and when susceptible cells were treated with fluconazole the GST activity decreased compared to untreated. Fluconazole did not induce oxidative damage in proteins and in lipids, however oxidative DNA damage was observed. Therefore, it is suggested that fluconazole generates ROS as part of its antifungal mechanism in C. glabrata at stationary growth phase, inducing oxidative DNA damage. In response, there was increase in the enzymatic activity of SOD and GPx in yeast. The understanding of the pathogenic yeast antioxidant response has important clinical interest, since the rational development of new antifungal drugs requires knowledge about the fungal metabolism.

A participação das espécies reativas de oxigênio (ERO) no mecanismo de ação dos antifúngicos azólicos, bem como a relação entre resistência aos antifúngicos e resposta ao estresse oxidativo, têm sido sugeridos. Entretanto, os dados ainda são inconclusivos e diferem entre os micro-organismos. Neste estudo estão apresentados os resultados da geração de ERO pelo fluconazol em isolados de C. glabrata sensíveis e resistentes a esse antifúngico e a resposta antioxidante da levedura. Nesses isolados, tratados e não tratados com fluconazol em concentração subinibitória, de acordo com sua concentração inibitória mínima (CIM), até fase de crescimento estacionário, foi avaliado se o fluconazol geraria ERO. Subsequentemente, foram analisadas as defesas antioxidantes glutationa peroxidase (GPx), superóxido dismutase (SOD), glutationa-S-transferase (GST), consumo de peróxido de hidrogênio e glutationa total, bem como possível dano oxidativo causado pelo fluconazol em lipídeos, proteínas e ácidos nucleicos e os níveis de nitritos e nitratos. Os resultados mostram que nos isolados de C. glabrata sensíveis e resistentes ao fluconazol, na presença do antifúngico, houve um aumento da geração ERO e maior atividade enzimática da GPx e SOD comparada a dos isolados não tratados com fluconazol, não havendo diferença estatística entre isolados sensíveis e resistentes nesses três parâmetros citados. Em relação à enzima GST, os isolados sensíveis mostraram maior atividade enzimática comparada aos resistentes, e quando as células sensíveis foram tratadas com fluconazol, a atividade da GST diminuiu. O fluconazol não induziu dano oxidativo em proteínas e em lipídeos, entretanto foi observado dano oxidativo ao DNA. Diante disso, sugere-se que o fluconazol gera ERO como parte do seu mecanismo antifúngico em C. glabrata em fase de crescimento estacionário, induzindo dano oxidativo no DNA. Como resposta, observa-se aumento na atividade enzimática da SOD e da GPx na levedura. O entendimento da resposta antioxidante de leveduras patogênicas tem importante interesse clínico, uma vez que o desenvolvimento racional de novas drogas antifúngicas requer conhecimento do metabolismo fúngico. / The participation of reactive oxygen species (ROS) in azoles antifungal mechanism of action has been suggested, as well as the relation between antifungal resistance and oxidative stress response. However, data are still inconclusive and differ between microorganisms. This study presents the results of ROS generation by fluconazole in fluconazole-susceptible and resistant C. glabrata strains and their antioxidant response. It was evaluated whether fluconazole generates ROS in those isolates treated and untreated with fluconazole at sub-inhibitory concentration according to their minimal inhibitory concentration (MIC). This treatment was conducted until stationary growth phase was reached. Subsequently, the antioxidant defenses glutathione peroxidase (GPx), superoxide dismutase (SOD), glutathione-S-transferase (GST), consumption of hydrogen peroxide and total glutathione, the possible oxidative damage in lipids, proteins and nucleic acids and the levels of nitrites and nitrates were analyzed. Results showed increased ROS generation in fluconazole-susceptible and resistant C. glabrata strains treated with fluconazole, and also higher GPx and SOD enzymatic activity, compared to untreated cells. No statistical difference of those three parameters was observed between susceptible and resistant strains. In relation to GST, susceptible strains demonstrated higher activity compared to the resistant ones, and when susceptible cells were treated with fluconazole the GST activity decreased compared to untreated. Fluconazole did not induce oxidative damage in proteins and in lipids, however oxidative DNA damage was observed. Therefore, it is suggested that fluconazole generates ROS as part of its antifungal mechanism in C. glabrata at stationary growth phase, inducing oxidative DNA damage. In response, there was increase in the enzymatic activity of SOD and GPx in yeast. The understanding of the pathogenic yeast antioxidant response has important clinical interest, since the rational development of new antifungal drugs requires knowledge about the fungal metabolism.

A participação das espécies reativas de oxigênio (ERO) no mecanismo de ação dos antifúngicos azólicos, bem como a relação entre resistência aos antifúngicos e resposta ao estresse oxidativo, têm sido sugeridos. Entretanto, os dados ainda são inconclusivos e diferem entre os micro-organismos. Neste estudo estão apresentados os resultados da geração de ERO pelo fluconazol em isolados de C. glabrata sensíveis e resistentes a esse antifúngico e a resposta antioxidante da levedura. Nesses isolados, tratados e não tratados com fluconazol em concentração subinibitória, de acordo com sua concentração inibitória mínima (CIM), até fase de crescimento estacionário, foi avaliado se o fluconazol geraria ERO. Subsequentemente, foram analisadas as defesas antioxidantes glutationa peroxidase (GPx), superóxido dismutase (SOD), glutationa-S-transferase (GST), consumo de peróxido de hidrogênio e glutationa total, bem como possível dano oxidativo causado pelo fluconazol em lipídeos, proteínas e ácidos nucleicos e os níveis de nitritos e nitratos. Os resultados mostram que nos isolados de C. glabrata sensíveis e resistentes ao fluconazol, na presença do antifúngico, houve um aumento da geração ERO e maior atividade enzimática da GPx e SOD comparada a dos isolados não tratados com fluconazol, não havendo diferença estatística entre isolados sensíveis e resistentes nesses três parâmetros citados. Em relação à enzima GST, os isolados sensíveis mostraram maior atividade enzimática comparada aos resistentes, e quando as células sensíveis foram tratadas com fluconazol, a atividade da GST diminuiu. O fluconazol não induziu dano oxidativo em proteínas e em lipídeos, entretanto foi observado dano oxidativo ao DNA. Diante disso, sugere-se que o fluconazol gera ERO como parte do seu mecanismo antifúngico em C. glabrata em fase de crescimento estacionário, induzindo dano oxidativo no DNA. Como resposta, observa-se aumento na atividade enzimática da SOD e da GPx na levedura. O entendimento da resposta antioxidante de leveduras patogênicas tem importante interesse clínico, uma vez que o desenvolvimento racional de novas drogas antifúngicas requer conhecimento do metabolismo fúngico. / The participation of reactive oxygen species (ROS) in azoles antifungal mechanism of action has been suggested, as well as the relation between antifungal resistance and oxidative stress response. However, data are still inconclusive and differ between microorganisms. This study presents the results of ROS generation by fluconazole in fluconazole-susceptible and resistant C. glabrata strains and their antioxidant response. It was evaluated whether fluconazole generates ROS in those isolates treated and untreated with fluconazole at sub-inhibitory concentration according to their minimal inhibitory concentration (MIC). This treatment was conducted until stationary growth phase was reached. Subsequently, the antioxidant defenses glutathione peroxidase (GPx), superoxide dismutase (SOD), glutathione-S-transferase (GST), consumption of hydrogen peroxide and total glutathione, the possible oxidative damage in lipids, proteins and nucleic acids and the levels of nitrites and nitrates were analyzed. Results showed increased ROS generation in fluconazole-susceptible and resistant C. glabrata strains treated with fluconazole, and also higher GPx and SOD enzymatic activity, compared to untreated cells. No statistical difference of those three parameters was observed between susceptible and resistant strains. In relation to GST, susceptible strains demonstrated higher activity compared to the resistant ones, and when susceptible cells were treated with fluconazole the GST activity decreased compared to untreated. Fluconazole did not induce oxidative damage in proteins and in lipids, however oxidative DNA damage was observed. Therefore, it is suggested that fluconazole generates ROS as part of its antifungal mechanism in C. glabrata at stationary growth phase, inducing oxidative DNA damage. In response, there was increase in the enzymatic activity of SOD and GPx in yeast. The understanding of the pathogenic yeast antioxidant response has important clinical interest, since the rational development of new antifungal drugs requires knowledge about the fungal metabolism.

Le cancer du sein est le cancer le plus fréquent chez les femmes. Malgré les progrès thérapeutiques, les mécanismes de résistance restent la cause de la morbidité et de la mortalité. L'acide lipoïque (LA) est un cofacteur essentiel du métabolisme oxydatif via sa fonction dans les complexes de pyruvate déshydrogénase et d'α-céto déshydrogénase. Il a été démontré des effets anticancéreux, mais ses mécanismes d'action ne sont pas entièrement compris. Mon projet de thèse vise à évaluer l’effet inhibiteur de LA sur la prolifération de diverses lignées cellulaires du cancer du sein et d’étudier ses mécanismes. Nos résultats ont montré que LA inhibe la prolifération cellulaire en inhibant les voies de signalisation prolifératives PI3K/Akt et MAPK/ERK. Nos résultats ont contribué à une meilleure compréhension des mécanismes d’action conduisant à cet effet anti-prolifératif de LA. En effet, nous avons mis en évidence la réduction de l’expression de la proprotéine convertase, furine, responsable de la maturation d’IGF-1R en réponse à LA aboutissant in fine à l’inhibition de la maturation de ce récepteur. En outre, nous avons démontré que l’effet pro-oxydant de LA aboutit à la réduction de l’expression du facteur de transcription, CREB, qui est impliqué dans l’induction de l’expression de la furine. En conclusion, nous avons élucidé, pour la première fois, le mécanisme d’action suivant : LA induit rapidement et brutalement des ROS par la phosphorylation oxydative qui vont inhiber l’expression du facteur de transcription CREB. Cette inhibition bloque alors l’activation transcriptionnelle de la furine, qui est cruciale pour permettre le passage de la pro-forme d’IGF-1R, qui est immature et non fonctionnelle, à la forme mature, IGF-1R qui est alors recrutée à la membrane plasmique / Breast cancer is the most common cancer in women. Despite therapeutic advances, the mechanisms of resistance remain the underlying cause of morbidity and mortality. Lipoic acid (LA) is and an essential cofactor of oxidative metabolism via its function in pyruvate dehydrogenase and α-keto dehydrogenase complexes. Its potential therapeutic effects have been well documented in the treatment of pathologies associated with oxidative stress, such as diabetes, atherosclerosis, liver diseases and neurodegenerative diseases. In addition, it has been demonstrated its anticancer effects in various cancers, but its mechanisms of action are not fully understood. My PhD project aims to evaluate the inhibitory effect of LA on the proliferation of various breast cancer cell lines and to study the mechanisms of action likely to be involved in this process. Our results showed that LA inhibits cell proliferation by inhibiting PI3K/Akt and MAPK/ERK proliferative signaling. Our results contributed to a better understanding of the mechanisms of action leading to this anti-proliferative effect of LA. Indeed, we have demonstrated the reduction of the expression of the proprotein convertase, furin, responsible for the maturation of IGF-1R in response to LA resulting the inhibition of the maturation of this receptor. In addition, we have demonstrated that the pro-oxidative effect of LA reduces the the expression of the transcription factor, CREB, which is involved in the induction of furin expression. In conclusion, we demonstrated for the first the following mechanism of action: LA rapidly induces ROS by oxidative phosphorylation that inhibit the expression of the CREB transcription factor. This inhibition then blocks the transcriptional activation of furin, an enzyme that is crucial to allow the passage of the IGF-1R pro-form, which is immature and non-functional, to the mature form, IGF-1R, which is then recruited to the plasma membrane