Germ line mutations of the gene encoding the tricarboxylic acid (TCA) cycle enzyme fumarate hydratase (FH) cause a hereditary cancer syndrome known as hereditary leiomyomatosis and renal cell cancer (HLRCC). HLRCC-associated tumors harbor biallelic FH inactivation that results in the accumulation of the TCA cycle metabolite fumarate. Although it is known that fumarate accumulation can alter cellular signaling, if and how fumarate confers a growth advantage remain unclear. Here we show that fumarate accumulation confers a chronic proliferative signal by disrupting cellular iron signaling. Specifically, fumarate covalently modifies cysteine residues on iron regulatory protein 2 (IRP2), rendering it unable to repress ferritin mRNA translation. Simultaneously, fumarate increases ferritin gene transcription by activating the NRF2 (nuclear factor [erythroid-derived 2]-like 2) transcription factor. In turn, increased ferritin protein levels promote the expression of the promitotic transcription factor FOXM1 (Forkhead box protein M1). Consistently, clinical HLRCC tissues showed increased expression levels of both FOXM1 and its proliferation-associated target genes. This finding demonstrates how FH inactivation can endow cells with a growth advantage.

PURPOSE: The Nrf2/Keap1/ARE pathway is a major regulator of cytoprotective responses to oxidants. Gluatredoxin-1 (Glrx-1), a small thiol transferase removes glutathione (GSH) adducts from proteins and participates in redox signaling. Glrx-/- mice exhibit increased protein GSH adducts (PSSG) and non-alcoholic fatty liver disease (NAFLD). Unexpectedly, our Glrx-/- mice showed increased hepatic glutathione (GSH) levels. The Nrf2/Keap1/ARE pathway, as an important regulator of glutathione synthesis, could be regulated by Glrx-1 activity. METHODS: To determine the role of Nrf2 in vivo, we treated Glrx-/- mice with high fat high sucrose (HFHS) diet to induce metabolic and oxidative stress. Livers were harvested at 10 months of age after 8 months on HFHS diet. Gene expression of Nrf2 and its down-signaling targets were determined using RT-qPCR and protein expression was accessed via WB. To determine the role of Nrf2 in Glrx-deficiency in vitro, Glrx siRNA was transfected in HEK293A and HepG2 cells and exposed to high palmitate high glucose (HPHG) to mimic metabolic stress and hydrogen peroxide to mimic oxidative stress. RESULTS: Glrx-/- deficiency increased Nrf2 activity and gene expression, and decreased Keap1 activity and gene expression. Glrx silencing in liver promoted Nrf2 activity and translocation to the nucleus, and downstream targets of Nrf2 were upregulated. CONCLUSION: Our findings indicate that the Nrf2/Keap1/ARE pathway is regulated by Glrx in vitro and in vivo.

Indiana University-Purdue University Indianapolis (IUPUI) / Type 1 and Type 2 diabetes (T1D/T2D) share a common etiology that involves an increase in oxidative stress that leads to dysfunction and subsequent β cell death. Lipoxygenases are enzymes that catalyze the oxygenation of polyunsaturated fatty acids to form lipid metabolites involved in a variety of biological functions including cellular oxidative stress response. On the other hand, Interleukin 6 (IL-6) signaling has been demonstrated to be protective in islets. In this study, we explored the effect of lipoxygenase enzymes 12-Lipoxygenase, 12/15 Lipoxygenase and IL-6 on β cell function and survival in mice using both STZ and high-fat diet (HFD) models of diabetes. Alox12-/- mice showed greater impairment in glucose tolerance following STZ and HFD compared to wild-type mice (WT), whereas Alox15-/- were protected against dysglycemia. These findings were accompanied by evidence of islet oxidative stress in Alox12-/- mice and reduced oxidative stress in Alox15-/- mice, consistent with alterations in the expression of antioxidant response enzymes in islets from these mice. Additionally, islets from Alox12-/- mice showed a compensatory increase in Alox15 gene expression and treatment of these mice with the 12/15-lipoxygenase inhibitor ML-351 rescued the dysglycemic phenotype. IL-6 was able to significantly attenuate the generation of reactive oxygen species by proinflammatory cytokines in human pancreatic islets. Furthermore, we find that IL-6 regulates the master antioxidant response protein NRF2. Collectively these results show that loss of Alox12 activates a compensatory increase in Alox15 that sensitizes β cells to oxidative stress and signaling by IL-6 is required for maximal antioxidant response under conditions of increased ROS formation, such as obesity.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18125号 / 医博第3845号 / 新制||医||1001(附属図書館) / 30983 / 京都大学大学院医学研究科医学専攻 / (主査)教授 横出 正之, 教授 柳田 素子, 教授 岩井 一宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Introdução: a epilepsia é uma doença neurológica, cuja prevalência é de 0,5 a 1% na população, sendo que 20 a 30% dos pacientes apresentam crises refratárias ao tratamento medicamentoso. A dieta cetogênica (DC) composta por alto teor de gorduras, baixo teor de carboidratos e quantidade proteica adequada tem emergido como um tratamento adjuvante e eficaz no controle de crises. Apesar de sua eficácia ser bem descrita na literatura, em humanos pouco se sabe sobre influência da DC em marcadores oxidativos e na modulação de fatores de transcrição como o NF-kB e Nrf2. Além disso, não se sabe sobre o impacto que diferentes tipos de ácidos graxos dietéticos ofertados na DC poderiam ocasionar nesses marcadores. Objetivo: avaliar o impacto da modificação do perfil de ácidos graxos dietéticos ofertados na DC SAFA sobre o perfil lipídico, oxidativo e ativação de fatores de transcrição NF-kB e Nrf2 em crianças e adolescentes com epilepsia refratária submetidos ao tratamento com dieta cetogênica. Metodologia: tratase de um ensaio clínico com seguimento de 6 meses, no qual o grupo DC SAFA recebeu uma dieta rica em colesterol e gordura saturada, e o grupo DC NSAFA recebeu uma dieta com redução de pelo menos 20% de gorduras saturadas; aumento em > 50% da oferta de ácido graxo monoinsaturados e ácidos graxos poli-insaturados quando comparados ao grupo DC SAFA. Foram avaliadas características socioeconômicas e clínicas e marcadores: antropométricos; bioquímicos de adesão a dieta; de consumo alimentar; de estresse oxidativo; e de capacidade antioxidante em ambos os grupos. Esses parâmetros foram avaliados em três momentos: antes do início da DC (T0), três meses (T1) e seis meses (T2) após o início do tratamento. As análises estatísticas foram realizadas através do software SPSS. Resultados: Foram incluídas 26 crianças e adolescentes no grupo DC clássica e 26 no grupo DC modificada. A DC NSAFA conferiu alterações menos intensas no perfil lipídico com menor percentual de aumento de colesterol total, LDL-C, APO B, razão LDL-C por HDL-C, razão HDL-C por APO A, razão LDL-C por APO B, colesterol não HDL e ácidos graxos não esterificados em comparação com a DC SAFA. Em ambas as dietas houve aumento significativo da concentração de LDLox. Foi observado o aumento significativo do NFkB na DC SAFA, sem diferenças significativas no percentual de mudança dessa variável entre as intervenções, além disso na DC SAFA houve redução de Nrf2 e o mesmo se comportou de forma distinta entre as intervenções, sendo que na DC SAFA houve redução significativa desse marcador. Conclusão: a DC SAFA demonstrou apresentar pior perfil de resposta lipídica, inflamatória e oxidante em comparação a DC NSAFA, a adoção da DC NSAFA para o tratamento da epilepsia refratária poderia minimizar os efeitos negativos observados na DC SAFA e atenuar o principal efeito adverso observado que são as dislipidemias. / Introduction: epilepsy is a neurological disease, its prevalence is estimated about 0,5 to 1 % in the population and 20 to 30% are refractory to the drug treatment. The ketogenic diet (KD) composed by high content of fat, low quantity of carbohydrate and adequate content of protein it is an adjuvant treatment with high efficacy in seizure control. Although it is well known about the efficacy of the diet, in humans there are few studies about the influence of the diet in oxidative biomarkers and its modulation in transcription factors such as NF-kB and Nrf2. Moreover it is not known about the impact of different types of dietetic fatty acids offers in KD could be influence in oxidative biomarkers and transcription factors. Objective: to evaluate and compare the impact of two different ketogenic diet, one composed by high content of saturated fatty acids (KD SAFA) and another one composed by high content of monounsaturated fatty acids and polyunsaturated fatty acids (KD NSAFA), on lipid and oxidative profile and activations of NF-kB and Nrf2 transcriptions factors in children and adolescents with refractory epilepsy on dietetic treatment with KD. Methods: clinical study, with 6 months of follow up, the patients was divided in two groups: one received a KD rich in saturated fatty acids and cholesterol (KD SAFA) and other received a KD with 20% less content of saturated fatty acids, increase in 50% of monounsaturated and polyunsaturated fatty acids comparing to KD SAFA (KD NSAFA). It was evaluated socioeconomic and clinical characteristics, anthropometric measure, food intake and lipids, oxidative and inflammatory biomarkers. The patients was evaluated in three different moments: before start the diet (T0), three months after start the KD (T1) and six months after start the KD (T2). The statistical analysis was performed using the software SPSS. Results: It was included 26 children and adolescents in KD SAFA and 26 in KD NSAFA. The participants treated with KD NSAFA had less modifications on classical lipid profile with less increase of: total cholesterol, LDL-C, APO B, LDL-C/HDL-C, HDL-C/APO A, LDL-C/APO B, non HDL cholesterol and non esterified fatty acids comparing to the participants treated with KD SAFA. In both diets there was a significant increase in oxidized LDL. It was observed increase of NFkB in the group treated with KD SAFA, without differences on the percentage of change of this biomarker between the interventions, in addition on the KD SAFA there was decrease in Nrf2 and the percentage of change it was different between the interventions with more reduction of this biomarker on KD SAFA. Conclusion: the KD SAFA showed worst profile in lipid, oxidative and inflammatory parameters comparing to KD NSAFA, suggesting that the use of KD NSAFA could be attenuate one of the main negative effect that are dyslipidemias.

Introdução: a epilepsia é uma doença neurológica, cuja prevalência é de 0,5 a 1% na população, sendo que 20 a 30% dos pacientes apresentam crises refratárias ao tratamento medicamentoso. A dieta cetogênica (DC) composta por alto teor de gorduras, baixo teor de carboidratos e quantidade proteica adequada tem emergido como um tratamento adjuvante e eficaz no controle de crises. Apesar de sua eficácia ser bem descrita na literatura, em humanos pouco se sabe sobre influência da DC em marcadores oxidativos e na modulação de fatores de transcrição como o NF-kB e Nrf2. Além disso, não se sabe sobre o impacto que diferentes tipos de ácidos graxos dietéticos ofertados na DC poderiam ocasionar nesses marcadores. Objetivo: avaliar o impacto da modificação do perfil de ácidos graxos dietéticos ofertados na DC SAFA sobre o perfil lipídico, oxidativo e ativação de fatores de transcrição NF-kB e Nrf2 em crianças e adolescentes com epilepsia refratária submetidos ao tratamento com dieta cetogênica. Metodologia: tratase de um ensaio clínico com seguimento de 6 meses, no qual o grupo DC SAFA recebeu uma dieta rica em colesterol e gordura saturada, e o grupo DC NSAFA recebeu uma dieta com redução de pelo menos 20% de gorduras saturadas; aumento em > 50% da oferta de ácido graxo monoinsaturados e ácidos graxos poli-insaturados quando comparados ao grupo DC SAFA. Foram avaliadas características socioeconômicas e clínicas e marcadores: antropométricos; bioquímicos de adesão a dieta; de consumo alimentar; de estresse oxidativo; e de capacidade antioxidante em ambos os grupos. Esses parâmetros foram avaliados em três momentos: antes do início da DC (T0), três meses (T1) e seis meses (T2) após o início do tratamento. As análises estatísticas foram realizadas através do software SPSS. Resultados: Foram incluídas 26 crianças e adolescentes no grupo DC clássica e 26 no grupo DC modificada. A DC NSAFA conferiu alterações menos intensas no perfil lipídico com menor percentual de aumento de colesterol total, LDL-C, APO B, razão LDL-C por HDL-C, razão HDL-C por APO A, razão LDL-C por APO B, colesterol não HDL e ácidos graxos não esterificados em comparação com a DC SAFA. Em ambas as dietas houve aumento significativo da concentração de LDLox. Foi observado o aumento significativo do NFkB na DC SAFA, sem diferenças significativas no percentual de mudança dessa variável entre as intervenções, além disso na DC SAFA houve redução de Nrf2 e o mesmo se comportou de forma distinta entre as intervenções, sendo que na DC SAFA houve redução significativa desse marcador. Conclusão: a DC SAFA demonstrou apresentar pior perfil de resposta lipídica, inflamatória e oxidante em comparação a DC NSAFA, a adoção da DC NSAFA para o tratamento da epilepsia refratária poderia minimizar os efeitos negativos observados na DC SAFA e atenuar o principal efeito adverso observado que são as dislipidemias. / Introduction: epilepsy is a neurological disease, its prevalence is estimated about 0,5 to 1 % in the population and 20 to 30% are refractory to the drug treatment. The ketogenic diet (KD) composed by high content of fat, low quantity of carbohydrate and adequate content of protein it is an adjuvant treatment with high efficacy in seizure control. Although it is well known about the efficacy of the diet, in humans there are few studies about the influence of the diet in oxidative biomarkers and its modulation in transcription factors such as NF-kB and Nrf2. Moreover it is not known about the impact of different types of dietetic fatty acids offers in KD could be influence in oxidative biomarkers and transcription factors. Objective: to evaluate and compare the impact of two different ketogenic diet, one composed by high content of saturated fatty acids (KD SAFA) and another one composed by high content of monounsaturated fatty acids and polyunsaturated fatty acids (KD NSAFA), on lipid and oxidative profile and activations of NF-kB and Nrf2 transcriptions factors in children and adolescents with refractory epilepsy on dietetic treatment with KD. Methods: clinical study, with 6 months of follow up, the patients was divided in two groups: one received a KD rich in saturated fatty acids and cholesterol (KD SAFA) and other received a KD with 20% less content of saturated fatty acids, increase in 50% of monounsaturated and polyunsaturated fatty acids comparing to KD SAFA (KD NSAFA). It was evaluated socioeconomic and clinical characteristics, anthropometric measure, food intake and lipids, oxidative and inflammatory biomarkers. The patients was evaluated in three different moments: before start the diet (T0), three months after start the KD (T1) and six months after start the KD (T2). The statistical analysis was performed using the software SPSS. Results: It was included 26 children and adolescents in KD SAFA and 26 in KD NSAFA. The participants treated with KD NSAFA had less modifications on classical lipid profile with less increase of: total cholesterol, LDL-C, APO B, LDL-C/HDL-C, HDL-C/APO A, LDL-C/APO B, non HDL cholesterol and non esterified fatty acids comparing to the participants treated with KD SAFA. In both diets there was a significant increase in oxidized LDL. It was observed increase of NFkB in the group treated with KD SAFA, without differences on the percentage of change of this biomarker between the interventions, in addition on the KD SAFA there was decrease in Nrf2 and the percentage of change it was different between the interventions with more reduction of this biomarker on KD SAFA. Conclusion: the KD SAFA showed worst profile in lipid, oxidative and inflammatory parameters comparing to KD NSAFA, suggesting that the use of KD NSAFA could be attenuate one of the main negative effect that are dyslipidemias.

Le monoxyde d’azote (NO) est une molécule gazeuse synthétisée par les NO Synthases à partir de L-arginine. NO est une puissante molécule de signalisation dans de nombreux processus physiologiques comme la vasodilatation et la neurotransmission. Il module l’activité de multiples protéines (ex : guanylate cyclase soluble et ribonucléotide réductase) grâce à la nitrosylation de groupements thiol ou de métaux de transition. En tant que radical libre, NO peut réagir avec de nombreuses espèces comme l’oxygène moléculaire, et ainsi former des dérivés réactifs. Grâce à ces propriétés, NO est un acteur majeur de l’immunité innée et de l’inflammation. Les phagocytes produisent de grandes quantités de NO en réponse à des stimuli proinflammatoires, via l’activité NO Synthase inductible (iNOS). En raison des effets délétères des dérivés de NO, l’activité iNOS doit être finement régulée. Le suppresseur de tumeur p53 est capable de réprimer l’expression du gène Nos2 après avoir été lui-même activé en réponse à une accumulation de NO. La protéine p73 est un homologue de p53 encodé par un gène qui génère à la fois des isoformes actives (TAp73) et des isoformes qui sont dépourvues du domaine de transactivation N-terminal et exercent un effet dominant négatif (ΔNp73). Cette étude se focalise sur le rôle des isoformes TAp73 dans la régulation de l’expression de la iNOS. Nous démontrons que les isoformes TAp73 régulent négativement l’expression de la iNOS aux niveaux transcriptionnel et post-traductionnel en potentialisant l’effet répresseur du TGF-β, ce qui résulte en une forte surexpression de la iNOS dans les cellules TAp73-/-. Ces résultats confortent le rôle de la famille p53 comme un réseau essentiel de protéines régulatrices des fonctions du TGF-β. / Nitric oxide (NO) is a gaseous molecule synthesized from L-arginine by Nitric Oxide Synthases. NO acts as a potent signaling molecule in various physiological processes like vasorelaxation and neurotransmission. It modulates the activity of many proteins (e.g. soluble guanylate cyclase and ribonucleotide reductase) through nitrosylation of thiol moieties or transition metal ions. As a free radical, NO can also react with a number of cellular species, notably molecular oxygen, to form reactive oxygen species and reactive nitrogen species. Thanks to these properties, NO appears as a major component of innate immune response and inflammation. Phagocytes produce large amounts of NO in response to proinflammatory through inducible Nitric Oxide Synthase (iNOS) activity. Because of the harmful effects of NO derivatives on cellular components, iNOS activity needs to be tightly regulated. The p53 tumor suppressor has been shown to repress Nos2 after being activated by NO itself. The p73 protein is an homologous encoded by the TP73 gene that generate transcriptionally active TAp73 isoforms and ΔNp73 isoforms that lack the transactivation domain and exert a dominant negative effect. This study focuses on the role of TAp73 isoforms in regulation of iNOS expression. We demonstrate that TAp73 isoforms potentiate the repressive effect of TGF-β on iNOS expression at transcriptional and post-traductional levels, resulting in a substantial iNOS overexpression in TAp73-/- cells. These results emphasize the emerging role of p53 family as a master regulator of TGF-β functions.

La dermatite allergique de contact (DAC) est une réaction inflammatoire aiguë, médiée par les cellules dendritiques (DCs) survenant suite à l’exposition répétée de la peau avec une molécule allergisante. La prévalence estimée des cas de DAC aux substances parfumantes est de 1,7 % à 4,1 % dans la population générale. Les molécules allergisantes sont des molécules appelées haptènes, qui vont se conjuguer avec des protéines de l’épiderme ou du derme. C’est le cas du cinnamaldéhyde (CinA), une molécule retrouvée dans la cannelle. Le linalol et le limonène sont des terpènes présents dans la lavande et l’orange, qui vont s’autoxyder au contact de l’air pour former des allergènes puissants, tels que les hydroperoxydes allyliques. Le premier objectif de cette thèse a été d’étudier le mécanisme d’action de ces terpènes et leurs hydroperoxydes allyliques respectifs sur la lignée cellulaire THP-1, qui sert de substitut aux cellules dendritiques. Le rôle du facteur de transcription Nrf2, majeur dans la lutte contre le stress oxydant, a également été investigué.Les consommateurs de produits cosmétiques sont exposés à de faibles concentrations de molécules allergisantes, mais plusieurs fois par jour ou par semaine. Nous avons souhaité étudier l’exposition répétée à de faibles doses d’haptène sur la peau.Les kératinocytes jouent également un rôle dans la DAC : ce sont les premières cellules qui vont rencontrer la molécule allergisante dans la peau. La deuxième partie de ce travail a été d’étudier l’impact d’une exposition répétée de CinA à de faible concentration sur ces KCs et plus particulièrement sur la différenciation de l’épiderme, en utilisant un modèle organotypique de peau en 3D. / Allergic contact dermatitis (ACD) represents a severe health problem. It is a dendritic cells (DCs) mediated skin disease caused by repeated exposure to an allergenic compound. ACD cases of fragrances in general population is estimated from 1.7 % to 4.1%. Contact sensitizers are compounds termed haptens and they will form a conjugate with epidermis and dermis proteins. Example is cinnamaldehyde (CinA), a molecule found in cinnamon. Linalool and limonene are terpenes found in lavender and oranges. In contact with the air, they will autoxidize to form highly allergenic compounds: allylic hydroperoxides. The first aim of this thesis was to study the mechanism of action of those terpenes and their respective allylic hydroperoxides on THP-1 cell-line, described as a surrogate of DCs. The transcription factor Nrf2 is playing a major role in oxidative stress and was also investigated.Consumers of cosmetic products are exposed to low quantities of allergenic compounds, but several times a day or a week. We wanted to study repeated exposure of low concentration of haptens on the skin.KCs also play a key role in ACD: they are the first cells that will encounter the allergenic compound in the skin. The second aim of this thesis was to study the impact of repeated exposure of low concentrations of CinA on those KCs and more particularly on the epidermis differenciation, using a 3D organotypic culture of skin.

Chaque année, la grippe provoque des centaines de milliers de décès dans le monde. Dans le cas d’infections sévères, il a été démontré que la génération excessive de molécules inflammatoires telles que les cytokines et les chimiokines, la sécrétion d’espèces réactives dérivées de l'oxygène ainsi que l’afflux massif de cellules immunitaires innées et adaptatives dans les voies respiratoires contribuent à la génération de dommages pulmonaires aigus et contribuent à l'immunopathologie reliée à l’infection. Tenant compte de ce fait, le défi actuel dans le traitement de la grippe est de contrôler la réponse inflammatoire tout en inhibant la réplication virale afin de permettre à l'organisme de se défendre contre les infections sévères à l'influenza. Des études récentes ont montré que l’activation du récepteur nucléaire PPARγ par ses ligands, tel que la 15d-PGJ[indice inférieur 2], diminuait l’inflammation pulmonaire et améliorait la survie des souris infectées avec des doses létales du virus influenza. Mis à part ses effets sur PPARγ, le ligand 15d-PGJ[indice inférieur 2] est aussi connu pour activer le facteur nucléaire antioxydant Nrf2. Il a été montré que Nrf2 réduit la réplication du virus influenza. Cependant, son mode d'action dans cette fonction nécessite une clarification. De manière intéressante, une étude a montré que Nrf2 réduit l’inflammation pulmonaire en régulant l’expression de PPARγ et ceci dans un modèle murin du syndrome de détresse respiratoire aigu. Les résultats de ces études précédentes mènent à l’hypothèse que les voies de PPARγ et Nrf2 interagissent fonctionnellement et qu'elles sont impliquées dans la réduction de l’inflammation induite lors d'infections sévères causées par l'influenza. L’objectif général de cette étude est donc de mieux comprendre les mécanismes protecteurs de PPARγ et Nrf2 dans la régulation de l’inflammation et la réplication virale suite à une infection par le virus influenza. Nos résultats ont démontré premièrement que le fait de cibler les deux voies moléculaires PPARγ et Nrf2, permet une inhibition significative de l’inflammation et de la morbidité liée à l’infection. Dans un deuxième temps, nos résultats dévoilent le mécanisme antiviral de Nrf2 et démontrent que l’activation de cette voie réduit la réplication du virus influenza d’une façon dépendante de l’expression de l’antiprotéase SLPI.