The aim of this thesis is to investigate molecular mechanism of reactive oxygen species production by flavin dehydrogenases mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) and succinate dehydrogenase (SDH). Together, they represent important source of reactive oxygen species in mammalian mitochondria, but the mechanism of electron leak is still poorly understood. Because mechanisms of reactive oxygen species production by other complexes of respiratory chain are better characterized, they can serve as case studies to get insight into mechanisms of reactive oxygen species by flavin dehydrogenases. Relevant knowledge is therefore summarized in the first part of the thesis. To study the production of reactive oxygen species by the isolated flavin dehydrogenases, we used brown adipose tissue mitochondria solubilized by digitonin as a model. Enzyme activity measurements, hydrogen peroxide production studies by Amplex UltraRed fluorescence and luminol luminescence revealed flavin as the most likely source of electron leak in SDH under in vivo conditions, while we propose coenzyme Q binding site as the site of reactive oxygen species production in the case of mGPDH. Distinct mechanism of this production by the two dehydrogenases is also apparent from induction of reactive oxygen species...

A Doença de Chagas, causada pelo parasito tripanossomatídeo Trypanosoma cruzi, é endêmica e se distribuí por toda América Latina. É uma das parasitoses mais negligenciadas pela indústria farmacêutica e os únicos fármacos disponíveis para seu tratamento foram introduzidos há décadas. Infelizmente, eles são ineficientes e apresentam sérios efeitos colaterais. Esse panorama mostra a necessidade do desenvolvimento de novos fármacos para a quimioterapia contra a doença de Chagas. As enzimas pertencentes a vias metabólicas essenciais para a sobrevivência do parasito tais como a via glicolítica e a de síntese de novo de nucleotídeos de pirimidinas, têm sido propostas como alvos interessantes no planejamento novos fármacos para o tratamento da doença de Chagas. Neste trabalho, as enzimas Gliceraldeído 3-fosfato desidrogenase (TcGAPDH) e a Diidroorotato desidrogenase (TcDHODH) de Trypanosoma cruzi foram estudadas como alvos para o planejamento de inibidores enzimáticos com propriedades físico-químicas e características estruturais similares à de compostos-líderes. Para isso, foram utilizados métodos e ferramentas de Quiminformática tanto baseadas nas estruturas dos ligantes (LBVS) quanto dos receptores (SBVS). Para a identificação e seleção de potenciais inibidores da enzima GAPDH, uma coleção virtual obtida do banco de dados ZINC, contendo aproximadamente 2,5 milhões de compostos, foi avaliada através de vários filtros de seleção com o objetivo de priorizar aqueles compostos mais interessantes do ponto de vista estrutural, de propriedades físico-químicas e farmacocinéticas. A aplicação desses filtros originou uma subcoleção de aproximadamente 450 mil estruturas que foram avaliadas segundo a complementaridade de interações com a estrutura da enzima através de métodos de docagem molecular. Com base nestes resultados, doze compostos que se mostraram promissores foram selecionados e adquiridos comercialmente para serem testadas in vitro contra a enzima TcGAPDH. Dos doze compostos testados, três exibiram afinidade (Ki) pela enzima em concentrações inferiores a 80 μM, Além disso, esses compostos também são caracterizados pelo baixo peso molecular (274 a 330 g mol-1) e no máximo 24 átomos diferentes do hidrogênio e, como consequência, apresentam eficiências do ligante entre 0,24 e 0,34 Kcal mol-1 átomo-1, o que os tornam ótimos candidatos à otimização molecular visando aumento da afinidade pelo alvo. Para a busca de inibidores da enzima TcDHODH, primeiramente foi realizada uma busca por cavidades na estrutura 3D do alvo para a identificação de regiões distintas do sítio catalítico e passíveis de serem exploradas no planejamento de ligantes. Através desta análise foi possível o estabelecimento de quatro novas regiões com forma, volume e localizações adequadas para acomodar pequenas moléculas capazes de modular a atividade da TcDHODH. Uma destas regiões, chamada S2, localizada sob a alça β4-αA e no canal de acesso dos substratos ao sítio ativo, foi escolhida para o planejamento baseado na estrutura do alvo. As estruturas de aproximadamente cem compostos derivados de pirimidinas, substituídos em posições estrategicamente definidas e selecionados através de buscas por subestruturas, foram docadas no sítio de interesse e nove compostos adquiridos e testados in vitro contra a enzima com o objetivo de validar as hipóteses estabelecidas inicialmente. Destes, cinco compostos mostraram potências (IC50) superiores à do produto de reação (inferior a 150 μM), Os resultados encontrados validaram as hipóteses geradas na primeira etapa e foram usados para direcionar a seleção de outras quinze novas moléculas através de um protocolo de docagem molecular com ajuste induzido. A avaliação in vitro desses compostos contra a enzima TcDHODH resultou na identificação de outros 11 compostos ativos, dos quais o mais potente exibiu afinidade pela enzima em concentração igual a 124 nM. Este composto possui eficiência do ligante igual a 0,56 Kcal mol-1 átomo-1 e pode ser considerado um fragmento molecular com excelentes características do ponto de vista do potencial para futuro desenvolvimento como agente terapêutico. Os resultados encontrados também evidenciaram a importância de determinadas características estruturais impressas nos inibidores da TcDHODH para a complementaridade com o novo sítio de interação identificado. Assim, novos compostos foram propostos para otimização molecular com o objetivo de melhorar afinidade e aumentar a diversidade de classes e, deste modo, ampliar o espectro de perfis farmacocinéticos para posteriores ensaios celulares e in vivo, Através da realização deste trabalho foi possível validar as estratégias adotadas na utilização dos métodos computacionais e também as hipóteses construídas a partir da aplicação dos mesmos. A taxa de acerto (TA) alcançada foi superior a 30% no planejamento de inibidores para ambos os alvos, ou seja, muito superiores às encontradas em experimentos de ensaio em massa. Deste modo, este estudo contribuiu com a proposição de novos esqueletos moleculares que podem ser usados como compostos-líderes no desenvolvimento de novos agentes tripanocidas focando nas enzimas TcGAPDH e TcDHODH como alvos. / Chagas\' disease, an endemic illness widely distributed throughout Latin America, is caused by the protozoa parasite Trypanosoma cruzi. It is one of the tropical diseases that are among the most neglected by the pharmaceutical industry, for which available treatments were launched more than 30 years ago. In addition, these drugs are ineffective and cause severe side effects to patients. This panorama shows the need for the development of new and more effective chemotherapeutic agents for the treatment of the disease. Enzymes belonging to metabolic pathways that are essential for the parasite survival such as the glycolysis and pyrimidine nucleotide biosynthesis have been proposed as attractive targets for the design of new drugs for the treatment of Chagas disease. In this work, the enzymes Gyceraldehyde-3-phosphate dehydrogenase (TcGAPDH) and the Dihydroorotate dehydrogenase (TcDHODH) from Trypanosoma cruzi were studied as targets for the design of inhibitors with physicochemical properties and structural characteristics similar to lead-compounds. Methods in Cheminformatics within the Ligand- and Structure-based Virtual Screening (LBVS and SBVS, respectively) approaches were thoroughly employed as tools to identify new hits. For the selection and identification of potential inhibitors of the GAPDH enzyme, a compound database containing nearly 2.5 million of small molecules retrievable from ZINC was evaluated through several molecular filters aiming at prioritizing those compounds more interesting from the point of view of their structures, physicochemical and predicted ADME/Tox properties. The application of Filter originated a subcollection of approximately 450 thousand structures that were then scored according to their complementary interactions with the 3D structure of the enzyme through molecular docking. Based on docking results, twelve compounds that showed to be promising ligands were selected and commercially acquired for in vitro assays against the TcGAPDH. Of the twelve compounds evaluated in vitro, three exhibited affinity constants (Ki) at concentrations lower than 80 μM. Furthermore, the selected compounds are also characterized by the low molecular weight (274 to 330 g mol-1) and a maximum of non-hydrogen atom count of 24, as a result, they have Ligand Efficiencies between 0,24 and 0,34 Kcal mol-1 átomo-1, what grant them great potential as candidates for molecular optimization and potency improvement. For the search of TcDHODH inhibitors, cavities in the 3D structure of the target for the identification of areas apart from catalytic site but likely to be explored in the design of ligands were selected a priori. This resulted in four new regions with appropriate shape, volume and locations to accommodate small molecules capable of modulating the activity of TcDHODH. One of the areas, called S2 site, is located under the α4 - βA loop and in the access channel of the substrate to the active site and was chosen to be explored in the SBDD studies. Approximately one hundred of pyrimidine derivatives containing strategically defined posítions for molecular substitution were retrieved from commercially available compounds database through substructure searching and docked into the previously defined site. Based on the docking results nine compounds were selected, purchased and assayed in vitro against the enzyme with the objective of validating the hypothesis. Of these, five compounds showed potencies (IC50) better than that exhibited by the product of the reaction (values lower than 150 μM). Thus, the results found validated the hypotheses generated in the first stage of the designing and they were used to drive the selection of other fifteen new molecules through induced fit molecular docking protocol. The in vitro evaluation of those compounds against the TcDHODH enzyme resulted in the identification of other eleven ligands, of which the most potent exhibited affinity for enzyme at the concentration of 124 nM. This molecule has a Ligand Efficiency of 0.56 Kcal mol-1 atom-1 and can be considered a fragment-like compound with excellent characteristics from the point of view of its potential for future development as therapeutic agent. The results found also evidenced the importance of certain structural characteristics in the inhibitor of TcDHODH for the complementarily with the new identified site of interaction. Thus, new compounds were proposed for potency and class diversity improvement. By doing so we hope to enlarge ADME profile spectrum for further cellular and in vivo assays. Through the success of this work, it was possible to validate the strategies adopted in the use of computational methods and also the hypotheses built from the application of that. The success rate (TA) obtained was higher than 30% in the design of ligands for both studied targets, which is much better than that usually found along High Throughput Screening assays. Thus, this study contributed with the proposítion of new molecular scaffolds that can be used as lead compounds in the development of new tripanocidal agents having as targets the enzymes TcGAPDH or TcDHODH.

Made available in DSpace on 2017-07-20T12:40:20Z (GMT). No. of bitstreams: 1 Agnes Thiane Machado.pdf: 7176772 bytes, checksum: 01a4049f5c4aed0935803a0cf3a6468d (MD5) Previous issue date: 2017-03-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Study of protein three-dimensional structures allow us to investigate the relations between amino acid sequence, structure and function, what is important chiefly for proteins from pathogenic organisms or ones that belong to the same genus of these, such that they can be used as a structural model. In this context, this work aims at the structural characterization of the enzymes alanine racemase long isoform from Trypanosoma cruzi and glyceraldehyde-3-phosphate dehydrogenase from Naegleria gruberi. The long isoform of alanine racemase catalyzes the conversion between L and D-alanine which, in turn, is part of one of the metabolic pathways in Trypanosoma cruzi, the etiologic agent of Chagas disease. The heterologous expression of this enzyme in Escherichia coli BL21 (DE3) GroEL was analyzed by SDS-PAGE, which revealed that the protein is in higher proportion in the insoluble fraction, thus it was necessary to establish a recovery protocol followed by an in vitro refolding. Data from enzymatic assays and circular dichroism revealed the success of the recovery/refolding protocol, which may in the future contribute to the search for specific inhibitors. Glyceraldehyde-3-phosphate dehydrogenase from Naegleria gruberi catalyzes the sixth step of the organism’s glycolytic pathway. NgGAPDH enzyme was expressed in E. coli (DE3) using the pET-15b vector, and then purified by tree chromatographic steps, two of nickel affinity and one of size exclusion. The enzymatic characterization was investigated with the enzyme without the his-tag; NgGAPDH presented higher activity at pH 8.0, 25 °C and 10 mM of arsenate, and positive cooperativity for substrates G3P and NAD+. His-tag depleted NgGAPDH crystals appeared in 3 days after drop settings, the best crystal diffracted to 1.94 A resolution and belongs to space group P21 with cell parameters a = 83.74 A, b = 94.55 A, c = 90.93 A, = 99.96 °. The final refined structure presents R = 0.1652 and Rfree = 0.2029. The catalytic domain formed by residues 134 to 313 is highly conserved, as expected, with the exception of Asn145, present only in NgGAPDH, while the other GAPDHs present either Ser or Thr on the corresponding position. Molecular dynamics analysis revealed that Asn145 has correlated motion with residues Ala123, Thr125 and Pro126 that belong to what was called "bonded loop". It should be emphasized that this is the first GAPDH from the phylum Percolozoa that has its three dimensional structure determined and kinetic parameters established, such that we expect to have contributed to the understanding of the evolution of this class of proteins. / O estudo da estrutura tridimensional de proteínas nos permite investigar as relações entre sequência de aminoácidos, estrutura e função, o que é importante principalmente para proteínas de organismos patogênicos ou mesmo pertencente ao gênero destes, que podem ser utilizadas como modelo estrutural. Neste contexto, o presente trabalho visa caracterizar estruturalmente as enzimas alanina racemase isoforma longa de Trypanosoma cruzi e gliceraldeído-3-fosfato desidrogenase de Naegleria gruberi. A alanina racemase isoforma longa catalisa a conversão entre L e D-alanina em uma das vias metabólicas do T. cruzi, que é o agente etiológico da doença de Chagas. A análise por SDS-PAGE de amostras da expressão heteróloga dessa enzima em Escherichia coli BL21(DE3) GroEL revelou que a proteína está em maior proporção na fração insolúvel, por isso, foi necessário estabelecer um protocolo de recuperação seguido de um reenovelamento in vitro. Dados de ensaios enzimáticos e dicroísmo circular revelaram o sucesso do protocolo de recuperação/reenovelamento, o que poderá no futuro contribuir para a busca de inibidores específicos. A gliceraldeído-3-fosfato desidrogenase de Naegleria gruberi catalisa a sexta etapa da via glicolítica do organismo. A enzima NgGAPDH foi expressa em E. coli (DE3) usando-se o vetor pET-15b e então purificada em três passos de cromatografia, dois por afinidade a níquel e um por exclusão por tamanho. A caracterização enzimática foi realizada com a enzima sem a ―his-tag‖; a NgGAPDH apresentou maior atividade em pH 8,0, 25 °C e 10 mM de arsenato, e cooperatividade positiva frente aos substratos G3P e NAD+. Cristais de NgGAPDH sem a ―his-tag‖ apareceram em 3 dias após montagem das gotas e o melhor difratou a 1,94 A de resolução, pertencendo ao grupo espacial P21 com parâmetros de cela a = 83,74 Å, b = 94,55 A, c = 90,93 A e = 99,96 °. A estrutura final refinada apresenta R = 0,1652 e Rfree = 0,2029. O domínio catalítico formado pelos resíduos 134 a 313 é altamente conservado, como esperado, com exceção da Asn145, presente somente em NgGAPDH, enquanto que as demais GAPDHs apresentam Ser ou Thr na posição correspondente. Análises por dinâmica molecular revelaram que a Asn145 tem correlação de movimento com os resíduos Ala123, Thr125 e Pro126, pertencentes ao que se chamou de ―bonded loop‖. Ressalte-se que esta é a primeira GAPDH do filo Percolozoa que tem sua estrutura tridimensional determinada e parâmetros cinéticos estabelecidos, tal que se espera contribuir para o entendimento da evolução dessa classe de proteínas.

A Doença de Chagas, causada pelo parasito tripanossomatídeo Trypanosoma cruzi, é endêmica e se distribuí por toda América Latina. É uma das parasitoses mais negligenciadas pela indústria farmacêutica e os únicos fármacos disponíveis para seu tratamento foram introduzidos há décadas. Infelizmente, eles são ineficientes e apresentam sérios efeitos colaterais. Esse panorama mostra a necessidade do desenvolvimento de novos fármacos para a quimioterapia contra a doença de Chagas. As enzimas pertencentes a vias metabólicas essenciais para a sobrevivência do parasito tais como a via glicolítica e a de síntese de novo de nucleotídeos de pirimidinas, têm sido propostas como alvos interessantes no planejamento novos fármacos para o tratamento da doença de Chagas. Neste trabalho, as enzimas Gliceraldeído 3-fosfato desidrogenase (TcGAPDH) e a Diidroorotato desidrogenase (TcDHODH) de Trypanosoma cruzi foram estudadas como alvos para o planejamento de inibidores enzimáticos com propriedades físico-químicas e características estruturais similares à de compostos-líderes. Para isso, foram utilizados métodos e ferramentas de Quiminformática tanto baseadas nas estruturas dos ligantes (LBVS) quanto dos receptores (SBVS). Para a identificação e seleção de potenciais inibidores da enzima GAPDH, uma coleção virtual obtida do banco de dados ZINC, contendo aproximadamente 2,5 milhões de compostos, foi avaliada através de vários filtros de seleção com o objetivo de priorizar aqueles compostos mais interessantes do ponto de vista estrutural, de propriedades físico-químicas e farmacocinéticas. A aplicação desses filtros originou uma subcoleção de aproximadamente 450 mil estruturas que foram avaliadas segundo a complementaridade de interações com a estrutura da enzima através de métodos de docagem molecular. Com base nestes resultados, doze compostos que se mostraram promissores foram selecionados e adquiridos comercialmente para serem testadas in vitro contra a enzima TcGAPDH. Dos doze compostos testados, três exibiram afinidade (Ki) pela enzima em concentrações inferiores a 80 μM, Além disso, esses compostos também são caracterizados pelo baixo peso molecular (274 a 330 g mol-1) e no máximo 24 átomos diferentes do hidrogênio e, como consequência, apresentam eficiências do ligante entre 0,24 e 0,34 Kcal mol-1 átomo-1, o que os tornam ótimos candidatos à otimização molecular visando aumento da afinidade pelo alvo. Para a busca de inibidores da enzima TcDHODH, primeiramente foi realizada uma busca por cavidades na estrutura 3D do alvo para a identificação de regiões distintas do sítio catalítico e passíveis de serem exploradas no planejamento de ligantes. Através desta análise foi possível o estabelecimento de quatro novas regiões com forma, volume e localizações adequadas para acomodar pequenas moléculas capazes de modular a atividade da TcDHODH. Uma destas regiões, chamada S2, localizada sob a alça β4-αA e no canal de acesso dos substratos ao sítio ativo, foi escolhida para o planejamento baseado na estrutura do alvo. As estruturas de aproximadamente cem compostos derivados de pirimidinas, substituídos em posições estrategicamente definidas e selecionados através de buscas por subestruturas, foram docadas no sítio de interesse e nove compostos adquiridos e testados in vitro contra a enzima com o objetivo de validar as hipóteses estabelecidas inicialmente. Destes, cinco compostos mostraram potências (IC50) superiores à do produto de reação (inferior a 150 μM), Os resultados encontrados validaram as hipóteses geradas na primeira etapa e foram usados para direcionar a seleção de outras quinze novas moléculas através de um protocolo de docagem molecular com ajuste induzido. A avaliação in vitro desses compostos contra a enzima TcDHODH resultou na identificação de outros 11 compostos ativos, dos quais o mais potente exibiu afinidade pela enzima em concentração igual a 124 nM. Este composto possui eficiência do ligante igual a 0,56 Kcal mol-1 átomo-1 e pode ser considerado um fragmento molecular com excelentes características do ponto de vista do potencial para futuro desenvolvimento como agente terapêutico. Os resultados encontrados também evidenciaram a importância de determinadas características estruturais impressas nos inibidores da TcDHODH para a complementaridade com o novo sítio de interação identificado. Assim, novos compostos foram propostos para otimização molecular com o objetivo de melhorar afinidade e aumentar a diversidade de classes e, deste modo, ampliar o espectro de perfis farmacocinéticos para posteriores ensaios celulares e in vivo, Através da realização deste trabalho foi possível validar as estratégias adotadas na utilização dos métodos computacionais e também as hipóteses construídas a partir da aplicação dos mesmos. A taxa de acerto (TA) alcançada foi superior a 30% no planejamento de inibidores para ambos os alvos, ou seja, muito superiores às encontradas em experimentos de ensaio em massa. Deste modo, este estudo contribuiu com a proposição de novos esqueletos moleculares que podem ser usados como compostos-líderes no desenvolvimento de novos agentes tripanocidas focando nas enzimas TcGAPDH e TcDHODH como alvos. / Chagas\' disease, an endemic illness widely distributed throughout Latin America, is caused by the protozoa parasite Trypanosoma cruzi. It is one of the tropical diseases that are among the most neglected by the pharmaceutical industry, for which available treatments were launched more than 30 years ago. In addition, these drugs are ineffective and cause severe side effects to patients. This panorama shows the need for the development of new and more effective chemotherapeutic agents for the treatment of the disease. Enzymes belonging to metabolic pathways that are essential for the parasite survival such as the glycolysis and pyrimidine nucleotide biosynthesis have been proposed as attractive targets for the design of new drugs for the treatment of Chagas disease. In this work, the enzymes Gyceraldehyde-3-phosphate dehydrogenase (TcGAPDH) and the Dihydroorotate dehydrogenase (TcDHODH) from Trypanosoma cruzi were studied as targets for the design of inhibitors with physicochemical properties and structural characteristics similar to lead-compounds. Methods in Cheminformatics within the Ligand- and Structure-based Virtual Screening (LBVS and SBVS, respectively) approaches were thoroughly employed as tools to identify new hits. For the selection and identification of potential inhibitors of the GAPDH enzyme, a compound database containing nearly 2.5 million of small molecules retrievable from ZINC was evaluated through several molecular filters aiming at prioritizing those compounds more interesting from the point of view of their structures, physicochemical and predicted ADME/Tox properties. The application of Filter originated a subcollection of approximately 450 thousand structures that were then scored according to their complementary interactions with the 3D structure of the enzyme through molecular docking. Based on docking results, twelve compounds that showed to be promising ligands were selected and commercially acquired for in vitro assays against the TcGAPDH. Of the twelve compounds evaluated in vitro, three exhibited affinity constants (Ki) at concentrations lower than 80 μM. Furthermore, the selected compounds are also characterized by the low molecular weight (274 to 330 g mol-1) and a maximum of non-hydrogen atom count of 24, as a result, they have Ligand Efficiencies between 0,24 and 0,34 Kcal mol-1 átomo-1, what grant them great potential as candidates for molecular optimization and potency improvement. For the search of TcDHODH inhibitors, cavities in the 3D structure of the target for the identification of areas apart from catalytic site but likely to be explored in the design of ligands were selected a priori. This resulted in four new regions with appropriate shape, volume and locations to accommodate small molecules capable of modulating the activity of TcDHODH. One of the areas, called S2 site, is located under the α4 - βA loop and in the access channel of the substrate to the active site and was chosen to be explored in the SBDD studies. Approximately one hundred of pyrimidine derivatives containing strategically defined posítions for molecular substitution were retrieved from commercially available compounds database through substructure searching and docked into the previously defined site. Based on the docking results nine compounds were selected, purchased and assayed in vitro against the enzyme with the objective of validating the hypothesis. Of these, five compounds showed potencies (IC50) better than that exhibited by the product of the reaction (values lower than 150 μM). Thus, the results found validated the hypotheses generated in the first stage of the designing and they were used to drive the selection of other fifteen new molecules through induced fit molecular docking protocol. The in vitro evaluation of those compounds against the TcDHODH enzyme resulted in the identification of other eleven ligands, of which the most potent exhibited affinity for enzyme at the concentration of 124 nM. This molecule has a Ligand Efficiency of 0.56 Kcal mol-1 atom-1 and can be considered a fragment-like compound with excellent characteristics from the point of view of its potential for future development as therapeutic agent. The results found also evidenced the importance of certain structural characteristics in the inhibitor of TcDHODH for the complementarily with the new identified site of interaction. Thus, new compounds were proposed for potency and class diversity improvement. By doing so we hope to enlarge ADME profile spectrum for further cellular and in vivo assays. Through the success of this work, it was possible to validate the strategies adopted in the use of computational methods and also the hypotheses built from the application of that. The success rate (TA) obtained was higher than 30% in the design of ligands for both studied targets, which is much better than that usually found along High Throughput Screening assays. Thus, this study contributed with the proposítion of new molecular scaffolds that can be used as lead compounds in the development of new tripanocidal agents having as targets the enzymes TcGAPDH or TcDHODH.

Le diabète est une maladie chronique de l’homéostasie du glucose caractérisée par une hyperglycémie non contrôlée qui est le résultat d’une défaillance de la sécrétion d’insuline en combinaison ou non avec une altération de l’action de l’insuline. La surnutrition et le manque d’activité physique chez des individus qui ont des prédispositions génétiques donnent lieu à la résistance à l’insuline. Pendant cette période dite de compensation où la concentration d’acides gras plasmatiques est élevée, l’hyperinsulinémie compense pleinement pour la résistance à l’insuline des tissus cibles et la glycémie est normale. Le métabolisme du glucose par la cellule pancréatique bêta entraîne la sécrétion d’insuline. Selon le modèle classique de la sécrétion d’insuline induite par le glucose, l’augmentation du ratio ATP/ADP résultant de la glycolyse et de l’oxydation du glucose, induit la fermeture des canaux KATP-dépendant modifiant ainsi le potentiel membranaire suivi d’un influx de Ca2+. Cet influx de Ca2+ permet l’exocytose des granules de sécrétion contenant l’insuline. Plusieurs nutriments comme les acides gras sont capables de potentialiser la sécrétion d’insuline. Cependant, le modèle classique ne permet pas d’expliquer cette potentialisation de la sécrétion d’insuline par les acides gras. Pour expliquer l’effet potentialisateur des acides gras, notre laboratoire a proposé un modèle complémentaire où le malonyl-CoA dérivé du métabolisme anaplérotique du glucose inhibe la carnitine palmitoyltransférase-1, l’enzyme qui constitue l’étape limitante de l’oxydation des acides gras favorisant ainsi leur estérification et donc la formation de dérivés lipidiques signalétiques. Le modèle anaplérotique/lipidique de la sécrétion d'insuline induite par le glucose prédit que le malonyl-CoA dérivé du métabolisme du glucose inhibe la bêta-oxydation des acides gras et augmente la disponibilité des acyl-CoA ou des acides gras non-estérifiés. Les molécules lipidiques agissant comme facteurs de couplage du métabolisme des acides gras à l'exocytose d'insuline sont encore inconnus. Des travaux réalisés par notre laboratoire ont démontré qu’en augmentant la répartition des acides gras vers la bêta-oxydation, la sécrétion d’insuline induite par le glucose était réduite suggérant qu’un des dérivés de l’estérification des acides gras est important pour la potentialisation sur la sécrétion d’insuline. En effet, à des concentrations élevées de glucose, les acides gras peuvent être estérifiés d’abord en acide lysophosphatidique (LPA), en acide phosphatidique (PA) et en diacylglycérol (DAG) et subséquemment en triglycérides (TG). La présente étude a établi l’importance relative du processus d’estérification des acides gras dans la production de facteurs potentialisant la sécrétion d’insuline. Nous avions émis l’hypothèse que des molécules dérivées des processus d’estérification des acides gras (ex : l’acide lysophosphatidique (LPA) et le diacylglycerol (DAG)) agissent comme signaux métaboliques et sont responsables de la modulation de la sécrétion d’insuline en présence d’acides gras. Afin de vérifier celle-ci, nous avons modifié le niveau d’expression des enzymes clés contrôlant le processus d’estérification par des approches de biologie moléculaire afin de changer la répartition des acides gras dans la cellule bêta. L’expression des différents isoformes de la glycérol-3-phosphate acyltransférase (GPAT), qui catalyse la première étape d’estérification des acides gras a été augmenté et inhibé. Les effets de la modulation de l’expression des isoenzymes de GPAT sur les processus d’estérifications, sur la bêta-oxydation et sur la sécrétion d’insuline induite par le glucose ont été étudiés. Les différentes approches que nous avons utilisées ont changé les niveaux de DAG et de TG sans toutefois altérer la sécrétion d’insuline induite par le glucose. Ainsi, les résultats de cette étude n’ont pas associé de rôle pour l’estérification de novo des acides gras dans leur potentialisation de la sécrétion d’insuline. Cependant, l’estérification des acides gras fait partie intégrante d’un cycle de TG/acides gras avec sa contrepartie lipolytique. D’ailleurs, des études parallèles à la mienne menées par des collègues du laboratoire ont démontré un rôle pour la lipolyse et un cycle TG/acides gras dans la potentialisation de la sécrétion d’insuline par les acides gras. Parallèlement à nos études des mécanismes de la sécrétion d’insuline impliquant les acides gras, notre laboratoire s’intéresse aussi aux effets négatifs des acides gras sur la cellule bêta. La glucolipotoxicité, résultant d’une exposition chronique aux acides gras saturés en présence d’une concentration élevée de glucose, est d’un intérêt particulier vu la prépondérance de l’obésité. L’isoforme microsomal de GPAT a aussi utilisé comme outil moléculaire dans le contexte de la glucolipotoxicité afin d’étudier le rôle de la synthèse de novo de lipides complexes dans le contexte de décompensation où la fonction des cellules bêta diminue. La surexpression de l’isoforme microsomal de la GPAT, menant à l’augmentation de l’estérification des acides gras et à une diminution de la bêta-oxydation, nous permet de conclure que cette modification métabolique est instrumentale dans la glucolipotoxicité. / Diabetes is a chronic disease of glucose homeostasis characterized by hyperglycemia and the result of a failure of insulin secretion in combination or not with impaired insulin action. Overnutrition and lack of physical activity in individuals who have acquired or inherited genetic predispositions lead to insulin resistance. During the period of compensation where the concentration of plasma fatty acids is high, hyperinsulinemia fully compensates for the insulin resistance of target tissues and blood sugar is normal. Glucose promotes insulin secretion through its metabolism by the pancreatic β cell. According to the classical model of glucose-induced insulin secretion, the increase in the ATP/ADP ratio resulting from glycolysis and glucose oxidation induces the closure of KATP channels thus changing membrane potential followed by an influx of Ca2+. This influx of Ca2+ allows the exocytosis of secretory granules containing insulin. Several nutrients like fatty acids are capable of potentiating insulin secretion. However, the classical model does not explain the potentiation of insulin secretion by fatty acids. To explain the potentiating effect of fatty acids, our laboratory has proposed a complementary model in which malonyl-CoA derived from glucose anaplerotic metabolism inhibits carnitine palmitoyltransferase 1, the enzyme catalyzing the limiting step of fatty acid oxidation, thereby promoting their esterification and thus the formation signaling derivatives. The anaplerotic model of insulin secretion predicts that malonyl-CoA derived from glucose metabolism inhibits β-oxidation of fatty acids and increases the availability of acyl-CoA or non esterified fatty acids. Thus, lipid molecules can act as coupling factors for insulin exocytosis. Fatty acid-derived signalling molecules that are active remain to be identified. Work performed by our laboratory has shown that increasing the partition of fatty acids toward β-oxidation reduced glucose-induced insulin secretion, suggesting that derivatives of fatty acid esterification are important for the potentiation of insulin secretion. Indeed, at high concentrations of glucose, fatty acids are esterified into lysophosphatidic acid (LPA), phosphatidic acid (PA) and diacylglycerol (DAG) and subsequently in triglycerides (TG). The present study established the relative importance fatty acid esterification in the production of factors potentiating insulin secretion. We hypothesized that molecules derived from the process of esterification of fatty acid (eg lysophosphatidic acid (LPA) and diacylglycerol (DAG)) act as metabolic signals and are responsible for the modulation of the secretion of insulin in the presence of fatty acids. Thus, the level of expression of key enzymes controlling the process of esterification has been altered by molecular biology approaches to increase distribution of fatty acids toward esterification in the β cell. The expression of various isoforms of glycerol-3-phosphate acyltransferase (GPAT), which catalyzes the first step of esterification of fatty acids was increased and inhibited. The effects of GPAT isoenzyme modulation on the esterification process, on β-oxidation and on glucose-induced insulin secretion were investigated. The various approaches we used have changed the levels of DAG and TG without altering insulin secretion induced by glucose in the presence or absence of fatty acids. Thus, the results of this study do not suggest a role for de novo synthesis of glycerolipid intermidiates via esterification of fatty acids in the potentiation of insulin secretion. However, the esterification of fatty acids is an integral part of a TG/fatty acid cycle with its counterpart lipolysis. Moreover, parallel studies conducted by colleagues of the laboratory have demonstrated a role for lipolysis and a cycle TG/fatty acid in the potentiation of insulin secretion by fatty acids. In parallel with our studies of the mechanisms of insulin secretion involving fatty acids, our laboratory is also interested in the negative effects of fatty acids on the β cell. The glucolipotoxicity resulting from chronic exposure to saturated fatty acids in the presence of high glucose concentrations is of particular interest in the context of obesity rates. The microsomal isoform of GPAT was also used as a molecular tool under glucolipotoxicity conditions to study the role of de novo synthesis of complex lipids in the context of decompensation when β-cell function decreases. Increased esterification of fatty acids by the overexpression of microsomal isoform of GPAT has increased the toxic effects of fatty acids in the context of glucolipotoxicity. Thus, our results allow us to conclude that the distribution of lipids toward esterification and a decrease in β-oxidation is instrumental in glucolipotoxicity.

Philosophiae Doctor - PhD / Electroanalytical and optical properties of nanoscale materials are very important for biosensing applications as well as for understanding the unique one-dimensional carrier transport mechanism. One-dimensional semiconductor nanomaterials such as semiconductor quantum dots are extremely attractive for designing high-density protein arrays. Because of their high surfaceto-volume ratio, electro-catalytic activity as well as good biocompatibility and novel electron transport properties make them highly attractive materials for ultra-sensitive detection of biological macromolecules via bio-electronic or bio-optic devices. A genosensor or gene based biosensor is an analytical device that employs immobilized deoxyribonucleic acid (DNA) probes as the recognition element and measures specific binding processes such as the formation of deoxyribonucleic acid-deoxyribonucleic acid (DNA-DNA), deoxyribonucleic acid- ribonucleic acid (DNA-RNA) hybrids, or the interactions between proteins or ligand molecules with DNA at the sensor surface.In this thesis, I present four binary and two ternary-electrochemically and optically modulated selenide and telluride quantum dots, all synthesised at room temperature in aqueous media. Cationic gallium (Ga3+) synthesized in form of hydrated gallium perchlorate salt[Ga(ClO4)3.6H2O] from the reaction of hot perchloric acid and gallium metal was used to tailor the optical and electrochemical properties of the selenide and telluride quantum dots. The synthesized cationic gallium also allowed successful synthesis of novel water soluble and biocompatible capped gallium selenide nanocrystals and gallium telluride quantum dots. Cyclic voltammetric studies inferred that presence of gallium in a ZnSe-3MPA quantum dot lattice improved its conductivity and significantly increased the electron transfer rate in ZnTe-3MPA.Utraviolet-visible (UV-vis) studies showed that incorporation of gallium into a ZnSe-3MPA lattice resulted in a blue shift in the absorption edge of ZnSe-3MPA from 350 nm to 325 nm accompanied by decrease in particle size. An amphiphilic bifunctional molecule, 3-Mercaptopropionic acid (3-MPA) was used as a capping agent for all quantum dots. It was found that 3-MPA fully solubilised the quantum dots, made them stable, biocompatible, non agglomerated and improved their electron transfer kinetics when immobilized on gold electrodes.Retention of the capping agent on the quantum dot surface was confirmed by Fourier transform infrared spectroscopy (FTIR) which gave scissor type bending vibrations of C-H groups in the region 1365 cm-1 to 1475 cm-1, stretching vibrations of C=O at 1640 cm-1, symmetric and asymmetric vibrations of the C-H in the region 2850 cm-1 to 3000 cm-1 as well as stretching vibrations of –O-H group at 3435 cm-1. The particle size and level of non-agglomeration of the quantum dots was studied by high resolution transmission electron microscopy (HRTEM). The optical properties of the quantum dots were studied using UV-vis and fluorescence spectroscopic techniques.Quantum dot/nanocrystal modified gold electrodes were prepared by immersing thoroughly cleaned electrodes in the quantum dot/nanocrystal solution, in dark conditions for specific periods of time. The electrochemical properties of the modified electrodes were characterized by cyclic voltammetry (CV), square wave voltammetry (SWV), electrochemical impedance and spectroscopy (EIS). Six sensing platforms were then prepared using quantum dot/nanocrystal, one of which was used for detection of dopamine while the rest were used for detection of a DNA sequence related to 5-enolpyruvylshikimate-3-phosphate synthase, a common vector gene in glyphosate resistant transgenic plants.The first sensing platform, consisting of ZnSe-3MPA modified gold electrode (Au|ZnSe-3MPA) gave rise to a novel method of detecting dopamine in presence of excess uric acid and ascorbic acid. Using a potential window of 0 to 400 mV, the ZnSe-3MPA masked the potential for oxidation of uric and ascorbic acids, allowing detection of dopamine with a detection limit of 2.43 x 10-10 M (for SWV) and 5.65 x 10-10 M (for steady state amperometry), all in presence of excess uric acid (>6500 higher) and ascorbic acid (>16,000 times higher). The detection limit obtained in this sensor was much lower than the concentration of dopamine in human blood(1.31 x 10-9 M), a property that makes this sensor a potential device for detection of levels of dopamine in human blood.The other sensing platforms were prepared by bioconjugation of amine-terminated 20 base oligonucleotide probe DNA (NH2-5′-CCC ACC GGT CCT TCA TGT TC-3′) onto quantum dot modified electrodes with the aid of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The prepared DNA electrodes were electrostatically hybridized with different sequences which included 5′-GAA CAT GAA GGA CCG GTG GG-3′ (complementary target), 5′-CATAGTTGCAGCTGCCACTG-3′ (non complementary target) and 5′-GATCATGAAGCACCGGAGGG-3′ (3-base mismatched target).The hybridization events were monitored using differential pulse voltammetry (DPV) and SWV by monitoring the guanine oxidation signal or using EIS by monitoring changes in the charge transfer resistance. The quantum dot genosensors were characterized by low detection limits (in the nanomolar range), long linear range (40 - 150 nM) and were able to discriminate among complementary, non-complementary and 3-base mismatched target sequences.

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Biotic stress in the form of viral infection, as well as abiotic salt stress, cause leaves injuries, stomata closure and decreased rate of photosynthesis. These factors lead to the limitation of plant growth and to reduced amount of coenzyme NADPH. However NADPH is an important coenzyme for many metabolic pathways such as synthesis of fatty acids, amino acids and secondary metabolites involved in stress responses. NADPH is also a coenzyme for key enzymes of antioxidant system and for many regulatory enzymes. NADP-dependent enzymes are alternative source of NADPH in plants under stress conditions. In this work, activities of four NADP-dependent enzymes: Glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), NADP-isocitrate dehydrogenase (NADP-ICDH, EC 1.1.1.42), NADP-malic enzyme (decarboxylating) (NADP-ME, EC 1.1.1.40) and Shikimate dehydrogenase (SDH, EC 1.1.1.25) were studied. Activities of all these enzymes but SDH increased in leaves of tobacco plants (Nicotiana tabacum L.) infected by PVYNTN , The most sensitive enzymes to viral infection were NADP-ICDH and NADP-ME, whose activity was increased in comparison with control plants 3-fold and 2,4-fold, respectively. Changes in activity of studied enzymes were also determined in plants exposed to viral infection in combination with heat-shock...