Orientadores: Sérgio Tadeu Martins Marba, Maria Marluce dos Santos Vilela / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-19T19:35:04Z (GMT). No. of bitstreams: 1 Caldas_JamilPedrodeSiqueira_D.pdf: 1222861 bytes, checksum: f1f27f58131a25e1222f77de30548e3e (MD5) Previous issue date: 2011 / Resumo: O objetivo do estudo foi avaliar a associação entre marcadores de estresse oxidativo e de inflamação ao nascimento e o desenvolvimento de hemorragia peri e intraventricular (HPIV) em recém-nascidos pré-termo de muito baixo peso ao nascer (RNPTMBP). Estudo de coorte envolvendo todos os RNPTMBP nascidos no CAISM-UNICAMP entre maio de 2009 a outubro de 2010. Excluíram-se aqueles dos quais não se obteve a amostra de sangue, os com malformação grave do sistema nervoso central e os que morreram antes de seis horas de vida. A coleta de sangue de cordão umbilical foi realizada diretamente da placenta ainda implantada, tendo sido dosados a produção basal e estimulada de intermediários reativos de oxigênio (ROI) por leucócitos polimorfonucleares e o níveis séricos de glutationa reduzida (GR) e interleucina-6 (IL-6). A HPIV foi diagnosticada por ecografia transfontanelar seriada - 6, 12, 24 e 72 horas de vida e com 7 e 28 dias de vida; classificada segundo Papile et al. (1978). Dados maternos, do parto e do RN foram considerados como variáveis independentes de controle. A distribuição dos acometidos por HPIV foi dada por frequência. Os valores de ROI, GR e IL-6 foram expressos como mediana e percentis 25 e 75. As variáveis categóricas foram avaliadas por teste de qui-quadrado ou Fisher, e teste de Mann-Withney para comparação das medianas de ROI, GR e de IL-6 entre os RNs com e sem HPIV...Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital / Abstract: This study evaluated the relationship between four laboratorial tests of oxidative stress and inflammatory markers and the development of intraventricular and periventricular hemorrhage (IVH) in very low birth weight infants (VLBW) - less than < 1.500 g at birth. Cohort study accomplished in a neonatal intensive care at CAISM-UNICAMP, from May 2009 to October 2010. Infant with severe brain malformations, death before 6 hours of life and those whose umbilical blood samples weren't obtained were excluded. Basal and under stimulation production of reactive intermediaries of oxygen (BROI and SROI), reduced glutathione (RG) and interleukin-6 (IL-6) were evaluated from umbilical blood. IVH was diagnosed by serial transfontanellar brain ultrasonography and classified according to Papile et al (1978). It was made at 6,12, 24, 72 hours of life and 7 and 28 days of life. Maternal factors, labor and delivery characteristics, and neonatal parameters were considered control independent variables and they were expressed in frequency. BROI, SROI, RG and Il-6 levels were expressed in medians and 25 and 75 percentiles. Chi-square or Fisher tests were used to determine the association of categorical variables and IVH. Mann-Whitney test were used to compare IVH and non-IVH groups and laboratorial markers medians. Univariate analysis was performed to identify differences between the IVH and non-IVH groups...Note: The complete abstract is available with the full electronic document / Doutorado / Pediatria / Doutor em Saude da Criança e do Adolescente

Orientador: Wanda Pereira Almeida / Tese (doutorado) - Universiade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-20T10:19:37Z (GMT). No. of bitstreams: 1 Motta_LuizFrederico_D.pdf: 7487995 bytes, checksum: 147ff46d24e06394d18af036b2472d1e (MD5) Previous issue date: 2012 / Resumo: O objetivo do presente trabalho foi identificar os principais descritores dos derivados análogos de chalcona com intuito de correlacionar com a atividade anti-Candida albicans. A incidência de infecções sistêmicas por C. albicans vem crescendo bastante nos últimos anos, particularmente nos casos de HIV e também em função do aumento da resistência ao arsenal terapêutico existente. Realizamos um estudo QSAR-2D, obtendo um modelo multidimensional pelo método PLS. O modelo obtido possui quatro descritores: refratividade molar, potencial de ionização, comprimento molecular e Verloop B4(A). Com apenas 3 variáveis latentes (PCs), foi capaz de acumular 96,14% da informação original, elucidando 85% da variância total e predizendo 78% da atividade biológica. O modelo proposto possui bom grau de ajuste e significância estatística (R = 0,776 e SEC = 0,229). Os métodos LOO cross-validation, LNO cross-validation, Y-randomization e a validação externa indicaram que o modelo é significante, robusto e possui elevada previsibilidade interna e externa. Levando em consideração o modelo QSAR-2D, propusemos a síntese de novos análogos de chalcona. Realizamos a síntese de 28 chalconas alvo derivadas de aldeídos aromáticos, empregando-se a condensação de Claisen-Schmidt, e avaliamos a atividade anti-Candida albicans. Os compostos foram caracterizados estruturalmente por métodos espectrométricos. Das 28 chalconas alvo, 18 são inéditas. Os rendimentos químicos variaram entre 53% e 98%. Com relação à avaliação da atividade antifúngica, foi realizado o teste de difusão em disco para todos os compostos, empregando o meio RPMI 1640 e seguiram-se os protocolos padrões publicados no documento M27-A2 (CLSI, 2002). As chalconas que apresentaram halo de inibição > 10 mm foram consideradas ativas, e a CIM e CFM foram determinadas pelo método da microdiluição em caldo. O estudo QSAR-2D corroborou o resultado experimental observado. A chalcona mais ativa apresentou CIM = 9 mg/mL, e no teste de citotoxicidade para células 3T3 não mostrou atividade, sugerindo toxicidade seletiva. A chalcona mais ativa apresentou um perfil Drug Likeness e Drug Score baixo em relação ao fluconazol, mas sobreviveu à Regra de Lipinski apresentando biodisponibilidade oral. O MEP, MDEHOMO e MDELUMO da chalcona mais ativa revelou que o orbital HOMO é evidenciado na carbonila, que o carbono C4 possui orbital LUMO e potencial eletrostático positivo, indicando que a chalcona possui centro eletrofílico em C4 sujeito à ocorrência de ataques nucleófilos. O MDEHOMO da glutationa reduzida revelou que o orbital HOMO está no átomo de enxofre do grupo sulfídrila do aminoácido cisteína. Em função da ocorrência do mecanismo de ressonância, as chalconas possuem uma estrutura química com centro eletrofílico no carbono C4, o que indica provável interação entre o orbital HOMO do átomo de enxofre da GSH e o orbital LUMO do carbono C4 da chalcona resultando em ligação covalente e na formação de conjugado glutationa-chalcona. A diminuição na concentração de glutationa reduzida no meio intracelular do fungo resulta em stress oxidativo celular e, portanto morte da Candida albicans / Abstract: The objective of this study is to identify the main descriptors of the derivatives of chalcone analogues with the aim to correlate with the activity anti-Candida albicans. The incidence of systemic infection by C. albicans has increased greatly in recent years, particularly in cases of HIV and also due to increased resistance to existing therapeutic arsenal. We performed a study QSAR-2D, obtained a multidimensional model by PLS method. The obtained model has four descriptors: molar refractivity, ionization potential, molecular length and Verloop B4 (A). With only three latent variables (PCs), was able to earn 96.14% of the original data, explaining 85% of the total variance and predicting 78% of biological activity. The proposed model has a good degree of fit and statistical significance (R = 0.776 and SEC = 0.229). The methods LOO cross-validation, LNO cross-validation, Y-randomization and external validation indicated that the model is significant, robust and has high internal and external predictability. Taking into account the model QSAR-2D, we proposed the synthesis of new analogues of chalcone. We performed the synthesis of 28 chalcones target derived from aromatic aldehydes, using the Claisen-Schmidt condensation, and evaluated the activity anti-Candida albicans. The compounds were characterized by spectrometric methods. Of the 28 chalcones target, 18 are new. The chemical yields ranged between 53% and 98%. Regarding the evaluation of antifungal activity, we performed the disk diffusion test for all compounds, using the RPMI 1640 and followed standard protocols published in document M27-A2 (CLSI, 2002). The chalcones that presented inhibition halo > 10mm were considered active, and the CIM and CFM were determined by broth microdilution. The study QSAR-2D corroborated with the observed experimental result. The most active chalcone showed MIC = 9 mg/mL, and the test of cytotoxicity to 3T3 cells had no activity, suggesting selective toxicity. The chalcone most active gave an overview Drug Likeness and Drug Score low in relation to fluconazole, but survived the Lipinsky Rule of presenting oral bioavailability. The MEP, MDEHOMO and MDELUMO of chalcone most active shown that the HOMO orbital is evidenced in the carbonyl, the carbon C4 has the LUMO orbital and a positive electrostatic potential, indicating that the chalcone has electrophilic center in C4 subject to the occurrence of nucleophilic attack. The MDEHOMO of reduced glutathione revealed that the HOMO orbital on sulfur atom of the sulfhydryl group of the amino acid cysteine. Because the occurrence of the resonance mechanism, the chemical structure of chalcones have a carbon C4 electrophilic, indicating a probable interaction between the HOMO orbital of the sulfur atom of GSH and the LUMO orbital of the carbon C4 of chalcone resulting covalent bond and the formation of glutathione-chalcone conjugated. The decrease in the concentration of reduced glutathione in the intracellular environment of the fungus results in cellular oxidative stress and therefore the death of Candida albicans / Doutorado / Físico-Química / Doutor em Ciências

This thesis describes the rational design of a novel enzyme, a thiolester hydrolase, derived from human glutathione transferase (GST) A1-1 by the introduction of a single histidine residue. The first section of the thesis describes the design and the determination of the reaction mechanism. The design was based on the crystal structure of human GST A1-1 complexed with S-benzylglutathione. The resulting enzyme, A216H, catalyzed the hydrolysis of the non-natural substrate GSB, a thiolester of glutathione and benzoic acid. The reaction followed saturation kinetics with a kcat of 0.00078 min-1 and KM of 5 μM. The rate constant ratio, (kcat/KM)/kuncat, was found to be more than 107 M-1. The introduction of a single His residue in position 216 opened up a novel reaction pathway in human GST A1-1 and is a nice example of catalytic promiscuity. The substrate requirements were investigated and A216H was found to be selective since only two out of 18 GS-thiolesters tested were substrates for A216H. The reaction mechanism of the A216H-catalyzed hydrolysis of GSB was determined and found to proceed via an acyl intermediate at Y9. The hydrolysis was catalyzed by H216 that acts as a general base and the deacylation was found to be the rate-determining step. The Y9-intermediate could be selectively trapped by oxygen nucleophiles and primary alcohols, in particular 1-propanol and trifluoroethanol, were the most efficient. In addition, saturation kinetics was obtained in the acyl transfer reaction with 1-propanol indicating the presence of a second binding site in A216H. The second section of this thesis describes the site-specific covalent modification of human GST A1-1. The addition of GSB to the wild-type protein results in a site-specific benzoylation of only one tyrosine residue, Y9, out of ten present in the protein (one out of totally 51 nucleophiles). The reaction was tested with five GST classes (Alpha, Mu, Pi, Theta and Omega) and found to be specific for the Alpha class isoenzymes. The covalent modification reaction was further refined to target a single lysine residue, K216, providing a more stable linkage in the form of an amide bond. The reaction was found to be versatile and approximately 50% of the GS-thiolesters tested acylated K216, including a fluorophore. / <p>On the day of the public defence the status of article II was: Submitted and article IV was: In press.</p>

Le bois représente une des ressources en polymères les plus abondantes de l’écosystème terrestre. Les champignons dégradant la matière lignocellulosique jouent un rôle important dans le cycle du carbone. Ils présentent un fort intérêt au niveau biotechnologique en particulier pour la production d’enzymes. Parmi les champignons saprophytes, ceux de la classe des Agaricomycota sont particulièrement intéressants puisqu’ils possèdent la capacité de dégrader les différents composés du bois : cellulose, hémicelloloses et lignine. De plus, ces champignons ont développé un système de détoxication impliquant des enzymes telles que les glutathion transférases (GST). Celles-ci sont impliquées dans la dégradation de composés potentiellement toxiques générés lors de la dégradation du bois mais également la dégradation de xénobiotiques. L’étude des systèmes extracellulaires et intracellulaires de Trametes versicolor impliqués dans les processus de décomposition du bois, décrite dans ce manuscrit, avait pour objectif d’identifier les facteurs moléculaires impliqués dans l’adaptation des champignons à leur environnement. Les approches pluridiciplinaires mises en œuvre lors de cette thèse ont permis d’identifier une variabilité phénotypique intraspécifique chez une dizaine de souches de T. versicolor, cette variabilité semblant être liée à la nature de l’essence ligneuse d’origine de ces souches. De plus, les travaux réalisés sur les GSTs apparteant aux classes oméga et GHR ont contribué à améliorer nos connaissances sur l’implication de cette famille multigénique dans l’adaptation des champignons xylophages à leur mode de vie / Wood is one of the most abundant polymer resources of the Earth’s ecosystem. Wood decaying fungi play an important role in the carbon cycle. They have a strong interest in biotechnology level in particular for the production of enzymes. Among the saprophytic fungi, those of the class of agaricomycota are particularly studied since they possess the ability to degrade varous compounds from wood : cellulose, hemicelluloses dand lignin. In addition, these fungi have developed a detoxification system involving enzymes such as glutathione transferases (GST). These latter are involved in degradation of wood but also in the degradation of xenobiotics. In this manuscript, the study of extracellular and intracellular system from Trametes versicolor, involved in wood decay process is described, the main goal being to identify the molecular factors involved in adaptation of the to their environment. Multidisciplinary approaches used in this PhD led to identification of an intraspecific phenotypic variability among ten strains of T. versicolor, this variability appearing to be related to the tree species where these strains have been isolated. Moreover, the work done on GSTs belonging to GHR and omega classes have improved our knowledge of the involvement of this gene family in adaptating the wood decayers to thrit lifestyle

Free radicals have been implicated in the progression of various diseases, such as cancers and cardiomyopathies. When the body is overburdened with free radicals and endogenous antioxidants become depleted, oxidative stress ensues with resultant damage to biomolecules. During oxidative stress high levels of reactive oxygen species are generated, cellular viability decreases, and apoptosis and lipid peroxidation are induced. Supplementation with exogenous supplements rich in antioxidants, such as herbal remedies containing polyphenols, could result in increased protection against oxidative stress. The aim of the study was to assess the effect of Burkea africana and Syzygium cordatum in a cellular oxidative stress model for the potential development of an antioxidant supplement. Crude aqueous and methanolic extracts were prepared by solvent maceration, while a polyphenolic-rich extract was created through liquid-liquid extraction. Polyphenolic content and antioxidant activity was assessed in cell-free systems. Polyphenolic content was determined through the Folin-Ciocalteau and aluminium trichloride methods, while antioxidant activity was assessed by the Trolox Equivalence Antioxidant Capacity and 1,1-diphenyl-2-picrylhydrazyl radical assays. Identification of phytochemical classes was done through thin layer chromatography and biochemical reactions. Inherent cytotoxicity of samples was determined in four cell cultures (3T3-L1 pre-adipocytes, C2C12 myoblasts, normal human dermal fibroblasts and U937 macrophage-like cells) using the neutral red uptake assay. The effect on oxidative stress was assessed in 2,2`-azobis-(2-methylpropionamidine) dihydrochloride-exposed U937 macrophage-like cells with regards to reactive oxygen species generation, cytotoxicity, apoptosis, lipid peroxidation and GSH depletion. Both B. africana and S. cordatum showed enrichment of polyphenols from the aqueous extract, to methanolic extract, to polyphenolic-rich extract. Antioxidant activity showed the same trend, which correlated well with the increased concentration of polyphenols, such as catechin, gallic acid and myricetin. Samples indicated toxicity in the 3T3-L1 and C2C12 cell lines, though no toxicity was noted in the U937 cell line and normal human dermal fibroblast cultures. Free radical-induced generation of reactive oxygen species, cytotoxicity, lipid peroxidation and apoptosis was successfully reduced by crude extracts of B. africana and the polyphenolic-rich extracts of both plants between concentrations of 10 and 20 ìg/ml. The crude extracts of S. cordatum were mostly ineffective in reducing these parameters, even though cell viability was increased. B. africana pre-treatment decreased reduced glutathione concentrations significantly in a dose-dependent manner, while the methanolic and polyphenolic-rich extract of S. cordatum increased concentrations moderately. Polyphenolic-rich extracts of B. africana and S. cordatum had the most potent decrease in oxidative stress-related parameters in the present study, which could be attributed to the polyphenolic content and antioxidant activity. Limited cytotoxicity was apparent in two of the four cell lines tested; further isolation and purification needs to be carried out to assess the bioactive constituents which do not elicit a toxic response. Further investigation through the use of quantitative structure–activity relationship modeling could give more insight on conformational and chemical changes that need to be brought about to modify the bioactive phytochemicals for reduced cytotoxicity, but increased antioxidant activity. Copyright / Dissertation (MSc)--University of Pretoria, 2013. / Pharmacology / unrestricted

A doença inflamatória intestinal (DII) e a esclerose sistêmica (ES) são doenças inflamatórias crônicas de difícil diagnóstico e tratamento. A etiologia da DII e da ES ainda não é completamente compreendida, mas sabe-se que fatores genéticos, imunológicos e ambientais estão envolvidos na sua patogênese. A DII possui dois principais subtipos clínicos: a doença de Crohn (DC) e a retocolite ulcerativa (RCU), caracterizados pela inflamação do intestino delgado e/ou cólon. Evidências sugerem que o aumento do estresse oxidativo desempenha um papel importante na fisiopatologia da DII. A ES é uma doença inflamatória autoimune rara, caracterizada pela fibrose progressiva da pele e de órgãos internos. A hipótese de que o aumento do dano oxidativo pode iniciar o dano vascular e desencadear os eventos patológicos observados na ES vem sendo investigada. Genes e enzimas envolvidos na metabolização (Fase I) e detoxificação (Fase II) de xenobióticos são utilizados como marcadores de susceptibilidade para o desenvolvimento de doenças que possuem fatores ambientais como fatores de risco. Em uma reação de Fase I, as enzimas do Citocromo P450 (CYP) inserem um átomo de oxigênio em um substrato deixando-o eletrofílico e reativo, criando um sítio para posterior conjugação pelas enzimas de Fase II. As enzimas Glutationa S-tranferases (GST) de Fase II catalisam a conjugação da glutationa com uma grande variedade de compostos eletrofílicos, detoxificando substâncias endógenas e exógenas. A atividade catalítica aumentada das enzimas CYP, bem como a falha na detoxificação de metabólitos pelas GST pode contribuir para o aumento do estresse oxidativo. O objetivo deste estudo foi investigar o papel de polimorfismos nos genes que codificam enzimas de metabolização (CYP1A*2C e CYP2E1*5B) e detoxificação (GSTT1 nulo, GSTM1 nulo e GSTP1 Ile105Val) na susceptibilidade a estas doenças. O grupo de pacientes com DII era constituído por 235 indivíduos e o grupo controle por 241 indivíduos, todos eurodescendentes. Na ES, 122 pacientes (99 eurodescendentes e 23 afrodescendentes) e 329 controles (241 eurodescendentes e 87 afrodescendentes) foram analisados. Os polimorfismos CYP foram genotipados por PCR-RFLP, enquanto que os polimorfismos em GSTT1 e GSTM1 foram genotipados por PCR multiplex e PCR-RFLP para GSTP1. As frequências alélicas e genotípicas foram comparadas entre pacientes e controles usando o teste de Qui-Quadrado. A respeito dos resultados das análises em DII, as frequências alélicas e genotípicas dos polimorfismos CYP1A1*2C, CYP2E1*5B e GSTP1 Ile105Val, bem como as frequências genotípicas do polimorfismo de presença/ausência de GSTM1, foram similares nos três grupos de pacientes (DII, DC e RCU) quando comparados ao grupo controle (P>0,05). Observouse uma frequência significativamente aumentada do genótipo nulo de GSTT1 no grupo de pacientes com DII quando comparado ao grupo controle [0,28 vs 0,18; χ² com Yates P=0,02; OR=1,71 (IC 95% 1,09 –2,71)]. Quando separamos o grupo de pacientes em DC ou RCU, esta frequência permaneceu significativamente aumentada somente no grupo de pacientes com RCU comparado ao grupo controle [0,29 vs 0,18; χ² com Yates P=0,035; OR=1,84 (IC 95% 1,03 –3,24)]. Com relação aos resultados das análises na ES, uma frequência significativamente aumentada do genótipo *1A/*1A (P=0,03; 0,74 vs. 0,61) e do alelo *1A (P=0,013; 0,86 vs 0,78; OR=0,57, IC 95% 0,36–0,90) do polimorfismo CYP1A1*2C foi observada entre os indivíduos controles eurodescendentes. Em contrapartida, a frequência do alelo *2C estava significativamente aumentada entre os pacientes de mesma etnia (P=0,013; 0,22 vs 0,14; OR=1,75, IC 95% 1,11–2,74). Com relação às frequências alélicas e genotípicas dos polimorfismos CYP2E1*5B e GSTP1 Ile105Val, e as frequências genotípicas do polimorfismo de presença/ausência de GSTM1, nenhuma diferença significativa foi observada quando os grupos de pacientes de ambas as etnias foram comparados aos grupos controle (P>0,05). Uma frequência significativamente aumentada do genótipo nulo de GSTT1 [0,29 vs 0,18; χ² com Yates P=0,035; OR=1,85 (IC 95% 1,03–3,29)], bem como uma alta frequência da dupla deleção de GSTT1/GSTM1 [0,19 vs 0,08; χ² com Yates P=0,007; OR=2,62 (IC 95% 1,25 –5,46)], foi observada no grupo de pacientes comparado aos controles (eurodescendentes). Estas associações não se repetiram entre indivíduos afrodescendentes. Concluindo, nossos resultados sugerem que o genótipo nulo de GSTT1 está associado à susceptibilidade a DII e pode influenciar na definição do curso da doença para a RCU. Além disso, o genótipo nulo de GSTT1 sozinho ou em combinação com o genótipo nulo de GSTM1 é um fator genético de susceptibilidade para a ES, enquanto que o genótipo *1A/*1A ou a presença do alelo *1A do polimorfismo CYP1A1*2C pode exercer um papel protetor contra o desenvolvimento da ES em indivíduos eurodescendentes. / Inflammatory bowel disease (IBD) and systemic sclerosis (SSc) are chronic inflammatory diseases of difficult diagnosis and treatment. The etiology of IBD and SSc is not completely understood but it is known that genetic, immunologic and environmental factors are involved in its pathogenesis. Crohn’s disease (CD) and ulcerative colitis (UC) are the two major subtypes of IBD, characterized by inflammation of the small intestine and/or colon. Evidences suggest that the increase of oxidative stress plays an important role in the pathophysiology of IBD. SSc is a rare autoimmune inflammatory disease of the connective tissue characterized by progressive fibrosis of the skin and internal organs. The hypothesis that the increase of oxidative stress can initiate vascular damage and triggers the pathological events in SSc has been investigated. Genes and enzymes involved in metabolism (Phase I) and detoxification (Phase II) of xenobiotics are used as markers of susceptibility to the development of diseases that have environmental factors as risk factors. In a Phase I reactions, the Cytochrome P450 (CYP) enzymes insert an oxygen atom in a substrate that making it more electrophilic and reactive, and creating a site for subsequent conjugation by Phase II enzymes. Phase II Glutathione S-transferases (GSTs) enzymes catalyze the conjugation of glutathione with a variety of electrophilic compounds, detoxifying endogenous and exogenous substances. A higher catalytic activity of CYP enzymes, as well as the failure in detoxifying of metabolites by GST enzymes may to contribute for the increase of oxidative stress. The aim of this study was investigated the role of polymorphisms in genes coding Phase I enzymes (CYP1A*2C and CYP2E1*5B) and Phase II (GSTT1 null, GSTM1 null and GSTP1 Ile105Val) in susceptibility to these diseases. IBD group was constituted by 235 patients and the control group by 241 individuals, all European-derived. In SSc group, 122 patients (99 European-derived and 23 African-derived) and 329 controls (241 European-derived and 87 African-derived) were analyzed. The CYP polymorphisms were genotyped by PCR-RFLP, whereas polymorphisms in GSTM1 and GSTT1 were genotyped by multiplex PCR and PCRRFLP for GSTP1. Allelic and genotypic frequencies were compared between patients and controls using the Chi-square test. Concerning IBD, allelic and genotypic frequencies of CYP1A1*2C, CYP2E1*5B and GSTP1 Ile105Val polymorphisms, as well as genotypic frequencies of GSTM1 presence/absence polymorphism were similar in all groups patients (IBD, CD, and UC) and controls (P>0.05). We observed a significantly increased frequency of GSTT1 null genotype in IBD group as compared to controls [0.28 vs. 0.18, χ ² with Yates P=0.02, OR=1.71 (95% CI 1.09 – 2.71)]. When patients were classified in CD or UC group, this frequency remained significantly increased only among UC patients [0.29 vs. 0.18, χ ² with Yates P=0,035, OR=1.84 (95% CI 1.03 – 3.24)] as compared to controls. Regarding results in SSc, a frequency significantly increased of *1A/*1A genotype (P=0.03; 0.74 vs. 0.61) and *1A allele (P=0.013; 0.86 vs 0.78; OR=0.57, 95% CI 0.36–0.90) from CYP1A1*2C polymorphism was observed among European-derived controls. On the other hand, the frequency of *2C allele was significantly increased among patients of same ethnic group (P=0.013; 0.22 vs 0.14; OR=1.75, 95% CI 1.11–2.74). The allelic and genotypic frequencies of CYP2E1*5B and GSTP1 Ile105Val polymorphisms, as well as genotypic frequencies of GSTM1 presence/absence polymorphism were similar between SSc patients and controls of both ethnic groups (P>0.05). We observed a significantly increased frequency of GSTT1 null genotype [0.29 vs. 0.18, χ ² with Yates P=0.035, OR=1.85 (95% CI 1.03–3.29)], as well as an increased frequency of GSTT1/GSTM1 double-null in SSc patients as compared to controls [0.19 vs. 0.08; χ ² with Yates P=0.007, OR=2.62 (95% CI 1.25 – 5.46)]. These associations were exclusive to European-derived individuals. In conclusion, our results suggest that the GSTT1 null genotype is associated with susceptibility to IBD and may influence in defining the course of the disease for RCU. Furthermore, the GSTT1 null genotype alone or combined with GSTM1 null genotype is a susceptibility genetic factor to SSc, while the *1A/*1A genotype or the presence of *1A allele from CYP1A1*2C polymorphism may plays a protector role in SSc development in Brazilian Europeanderived individuals.

Les voies métaboliques les plus importantes dans le contrôle du stress oxydant chez la plante restent à élucider. Celles liées au glutathion jouent un rôle important. Cependant, les réactions responsables de l'oxydation du glutathion (du GSH en GSSG) n'ont pas encore été clairement identifiées. L’analyse des données biochimiques, transcriptomiques et génétiques soulèvent des questions pour mieux comprendre comment la régulation redox liée au stress pourrait influer sur la signalisation hormonale chez les plantes. Par une approche de génétique inverse utilisant, notamment, le mutant photorespiratoire conditionnel cat2, nous avons étudié la réponse et l'importance fonctionnelle de trois voies potentielles, médiées par les glutathion S-transférases, les peroxirédoxines dépendant de la glutarédoxine et les déhydroascorbate réductases (DHARs) chez Arabidopsis. Ainsi, l'interaction entre les DHARs semble être nécessaire pour coupler les pools d'ascorbate et de glutathion lors d’un stress oxydant. En complément à l'approche expérimentale, une modélisation mécaniste a permis d'étudier la production de H2O2 et son métabolisme, en lien avec l'activité catalase et la voie ascorbate-glutathion. Le modèle révèle que la catalase et l'ascorbate peroxydase prennent en charge de concert le traitement de H2O2, y compris dans les conditions optimales de croissance. Nos simulations suggèrent que la disponibilité en NADPH peut déterminer l'oxydation du glutathion via la monodéshydroascorbate réductase. Nos résultats expérimentaux et le modèle cinétique valident que la sensibilité du statut du glutathion au stress oxydant constitue un senseur approprié des augmentations du H2O2. / The most important metabolic pathways in the control of oxidative stress remain to be elucidated in plants. Those linked to glutathione play an important role. However, the reactions responsible for its oxidation have not been clearly identified. Here, analysis based on available biochemical, transcriptomic and genetic data emphasized likely important questions to be elucidated for a full understanding of how stress-related redox regulation might impinge on phytohormone-related signaling pathways. Using a reverse genetics approach and the photorespiratory conditional cat2 mutant, we studied the response and functional importance of three potential routes for glutathione oxidation pathways mediated by glutathione S-transferases, glutaredoxin dependent peroxiredoxins, and dehydroascorbate reductases (DHAR) in Arabidopsis during oxidative stress. Hence, interplay between different DHARs appears to be necessary to couple ascorbate and glutathione pools and to allow glutathione-related signaling during enhanced H2O2 metabolism. In addition to experimental work, modelling is another way to investigate H2O2 production and its metabolism related to catalase activity and ascorbate glutathione pathway. This approach led to major conclusions, that catalase and ascorbate peroxidase can share the load in H2O2 processing even in optimal growth conditions. Furthermore, simulations propose that NADPH availability may determine glutathione oxidation through its influence on monodehydroascorbate reduction. Taken together, experimental results and our kinetic model strengthen that the sensitivity of glutathione status to oxidative stress acts as a suitable sensor of increased H2O2.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22080号 / 農博第2372号 / 新制||農||1072(附属図書館) / 学位論文||R1||N5234(農学部図書室) / 京都大学大学院農学研究科農学専攻 / (主査)教授 冨永 達, 教授 奥本 裕, 教授 白岩 立彦 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Parkinson's disease (PD) is the second most common chronic neurodegenerative disease characterized by dopamine decrease in the substantia nigra. Currently, there is no promising cure for PD and this has resulted in extensive research into alternative medicines. The aim of this study was to investigate the effect of methanol and ethyl acetate extracts of Lannea schweinfurthii (Engl. Engl) (Anacardiaceae), Zanthoxylum capense (Thunb. Harv) (Rutaceae), Scadoxus puniceus ((L.) Friis & Nordal) (Amaryllidaceae) and Crinum bulbispermum (Burm. f.) Milne-Redh. & Schweick) (Amaryllidaceae) on rotenone-induced toxicity in SH-SY5Y neuroblastoma cells. The latter which mimics PD symptoms in vitro. Cytotoxicity of the plant extracts was assessed using sulforhodamine B (SRB) assay. Intracellular reactive oxygen species (ROS) were measured fluorometrically with the use of the fluorescent dye 2‟,7‟-dichlorodihydrofluorescein diacetate (H2DCF-DA). Intracellular glutathione content was measured fluorometrically after staining with monochlorobimane (MCB). Fluorescent dye 5,5‟ ,6,6‟ -tetrachloro-1,1‟ ,3,3‟ -tetraethylbenzimidazolcarbocyanine iodide (JC-1) was used to assess the mitochondrial membrane potential (MMP) status of cells. Apoptosis was assessed by determining caspase-3 activity through detection of 7-amino-4-methylcoumarin (AMC) which is a product of caspace-3 substrate, acetyl-Asp-Glu-Val-Asp 7-amino-4-methylcoumarin (Ac-DEVD-AMC), cleaved by the caspase-3 enzyme. Rotenone was used as an in vitro model to induce PD-like symptoms. Cytotoxicity studies for methanol extract of Zanthoxylum capense revealed the highest IC50 value of 121.3 μg/mL, indicating low toxicity. The ethyl acetate extract of Crinum bulbispermum was observed to have no effect on the normal proliferation of the SH-SY5Y cells and produced an IC50 value >100 μg/mL. The calculated IC50 value obtained from rotenone cytotoxicity studies was 112 iv nM. Zanthoxylum capense and Scadoxus puniceus plant extracts were observed to be neuroprotective against rotenone-induced toxicity. A decrease in intracellular glutathione content as well as MMP was also observed in cells exposed to rotenone alone (50 nM). There was no intracellular ROS generation observed in cells exposed to rotenone alone (50 nM) after 24 h and 72 h. However, apoptotic cell death was observed in cells treated with rotenone (50 nM). Intracellular ROS production was observed to be elevated by methanol and ethyl acetate extracts of C. bulbispermum. Methanol extracts of Z. capense was observed to increase intracellular glutathione content. MMP was increased effectively following treatment with ethyl acetate extract of C. bulbispermum. Moreover, both methanol and ethyl acetate plant extracts were found to decrease caspase-3 activity significantly (p<0.05), in cells exposed to 50 nM rotenone. Z. capense methanol extract reduced caspase-3 activity the most effectively. Treatment with plant extracts was protective and decreased cell death. Furthermore, L. schweinfurthii, Z. capense, S. puniceus and C. bulbispermum, demonstrated strong antioxidant and anti-apoptotic effects against rotenone-toxicity, making them potential agents in developing therapies for treating PD. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Pharmacology / unrestricted

Les canaux anioniques ont toujours été sous-estimés par rapport aux canaux cationiques principalement en raison des difficultés à caractériser leurs identités moléculaires et par l'absence d'inhibiteurs sélectifs puissants. C’est une classe comportant une grande hétérogénéité et une importante complexité comprenant entre autres le CFTR activé par l’AMP cyclique, les anoctamines activées par le calcium intracellulaire et les canaux VRAC activés lors d’un gonflement cellulaire. L’identité moléculaire d’une protéine essentielle aux courants ioniques déclenchés par le gonflement cellulaire a été découverte en 2014, permettant de pouvoir enfin investiguer le fonctionnement et le rôle de ces canaux. Dans une première partie, nous avons démontré l’absence de spécificité des inhibiteurs des principaux canaux chlorures. Cette étude met en exergue le besoin de développer de nouveaux inhibiteurs permettant de discriminer les différents canaux chlorures, mais aussi la nécessité de coupler l’approche pharmacologique à une approche génétique. Dans une deuxième partie, nous avons mis en évidence la perméabilité des canaux anioniques sensibles au gonflement cellulaire au glutathion, principal antioxydant cellulaire. Nos résultats suggèrent donc que les canaux VRAC pourraient être impliqués dans la régulation du stress oxydatif. Dans la troisième partie de ma thèse, nous avons montré que le facteur de croissance TGFβ1 stimule l’ouverture des canaux anioniques sensibles au gonflement cellulaire. Cette activation s’accompagne d’une perte de glutathion et d’une accumulation intracellulaire d’espèces réactives de l’oxygène. Cette dernière est à l’origine d’une transition épithélio-mésenchymateuse des cellules rénales. L’inhibition pharmacologique ainsi que la répression génétique des canaux VRAC réduit significativement les effets du TGFβ1. Enfin dans la dernière partie, nous avons caractérisé le rôle des canaux anioniques sensible au gonflement cellulaire dans la sécrétion de facteurs pro-inflammatoires par les macrophages stimulés par un choc hypotonique. En collaboration, nous avons montré que le blocage de la régulation du volume cellulaire, sous le contrôle de ces canaux, entraine une inhibition de l’activation de l’inflammasome et donc de la sécrétion des facteurs pro-inflammatoires. Bien que les mécanismes de signalisation cellulaire restent à déterminer, ces travaux mettent en lumière l’importance des canaux anioniques sensibles au gonflement cellulaire dans la transition épithéliomésenchymateuse et l’inflammasome. Il apparait que ces canaux pourraient être des cibles thérapeutiques prometteuses dans un certain nombre de pathologies et qu’il est primordial de poursuivre les investigations dans ce domaine. / Anion channels have always been underestimated compared to cationic channels mainly because of the difficulty in characterizing their molecular identities and the absence of potent selective inhibitors. It is a class with great heterogeneity and complexity, including CFTR activated by cAMP, anoctamines activated by intracellulaire calcium and VRAC channels activated by cell swelling. The molecular identity of an essential protein to the ionic currents triggered by cell swelling was discovered in 2014, allowing the scientist community to investigate the role of these channels. In a first part, we highlighted the lack of specificity of the inhibitors of different chloride channels. This study shed lights on the need to develop new inhibitors to discriminate the different chloride channels, but also the need to couple the pharmacological approach to a genetic approach. In a second part, we have demonstrated the glutathione permeability of Volume Regulated Anion Channels. Our results therefore suggest that VRAC channels might be involved in the regulation of oxidative stress as glutathione is the main antioxidant within the cell. In the third part of my thesis, we have shown that TGFβ1 growth factor stimulates the opening of Volume Regulated Anion Channels which is correlated with both a loss of glutathione and an intracellular accumulation of reactive oxygen species. The latter is at the origin of an epitheliomesenchymal transition of renal cells. The pharmacological inhibition as well as the genetic repression of the VRAC channels significantly reduces the effects of TGFβ1. Finally, in the last part, we characterized the role of Volume Regulated Anion Channels in the secretion of pro-inflammatory factors by macrophages stimulated by hypotonic shock. In collaboration, we have shown that blocking the regulation of cell volume, under the control of these channels, leads to an inhibition of the inflammasome and therefore of the secretion of pro-inflammatory factors. Although cell signaling mechanisms remain to be determined, this work highlights the importance of VRAC in the epithelial-mesenchymal transition and inflammasome. It appears that these channels could be promising therapeutic targets in a certain number of pathologies and that it is essential to continue the investigations in this field.