En aquesta tesi s'apliquen eines bioinformàtiques a l'estudi de determinats sistemes biològics. En particular, l'estudi teòric de la influència de determinats aminoàcids sobre l'estructura i la dinàmica dels elements d'estructura secundària de les proteïnes s'aplica a la modelització per homologia dels receptors acoblats a proteïna G (GPCRs) i a l'estudi dels seus mecanismes d'activació.Se sap que determinats residus, com prolina, serina o treonina, provoquen distorsions locals en l'estructura de les hèlices a. L'anàlisi de bases de dades de seqüències de segments transmembrana mostra com certes combinacions d'aquests residus són més comunes que d'altres, i que algunes d'elles estan sobre-representades de manera significativa, mentre que d'altres estan clarament sots-representades. La restricció d'aquesta anàlisi de seqüències a la regió transmembrana dels GPCRs de la Classe A mostra com aquestes combinacions es troben en posicions específiques i, a més, es troben conservades en certes subfamílies de receptors.L'estructura i la dinàmica de les hèlices transmembrana que contenen aquestes combinacions de prolina i serina o treonina s'han estudiat mitjançant simulacions de dinàmica molecular en un entorn hidrofòbic explícit. Els resultats mostren com algunes d'aquestes combinacions indueixen distorsions importants en l'estructura de l'hèlix a, degut al seu efecte desestabilitzador de la xarxa de ponts d'hidrogen que dóna estabilitat a l'hèlix.Aquests resultats s'han aplicat a la construcció d'un model tridimensional del receptor de quimiocines CCR5 , utilitzant tècniques de modelització molecular per homologia. En aquest model es proposa que les hèlices transmembrana (TMH) 2 i 3 del receptor CCR5 són estructuralment diferents del patró de rodopsina. TMH2 està més doblegada degut a la presència d'un motiu Thr-X-Pro, que, a més, fa que aquesta hèlix es doblegui cap a TMH3. Així doncs, es proposa que, en aquest receptor, aquestes dues hèlices interaccionen. Aquesta interacció estaria mediada per la presència de residus hidrofòbics conservats i específics en les dues hèlices. Aquestes hipòtesis han estat posades a prova mitjançant experiments de mutagènesi dirigida, gràcies a la col·laboració amb l'Institut de Recherche Interdisciplinaire en Biologie Humaine et Nucléaire (IRIBHN), Université Libre de Bruxelles. Els resultats experimentals permeten establir la hipòtesi que la interfície TMH2-TMH3 participa en l'activació induïda per quimiocines del receptor CCR5.Com a conclusió, aquesta tesi pretén mostrar com, mitjançant la utilització d'eines bioinformàtiques, és possible traduir les seqüències primàries de proteïnes i les interaccions a nivell atòmic en estructures tridimensionals de proteïnes. A més, aquesta tesi mostra que, encara que l'estructura tridimensional de la rodopsina bovina és un patró útil per la modelització per homologia de GPCRs, s'han de tenir en compte de manera explícita les especificitats de seqüència de cada receptor per tal de construir models de receptors particulars. Aquestes especificitats de seqüència consisteixen en patrons de seqüència conservats en determinades famílies, que es tradueixen en divergències estructurals. Entre aquests patrons de seqüència, es proposa que els residus de serina i treonina, sols o combinats amb residus de prolina propers, poden modular la geometria de les TMHs, degut a la seva capacitat d'interferir amb la xarxa de ponts d'hidrogen que dóna estabilitat a les hèlices a.Finalment, es proposa que la influència dels motius de serina, treonina i prolina en l'estructura de les TMHs pot estar relacionada amb els processos d'activació dels GPCRs de la Classe A i, possiblement, d'altres proteïnes de membrana. En els GPCRs, aquests motius poden haver evolucionat per tal d'adaptar uns mecanismes d'activació conservats als lligands característics de cada família de receptors. / This thesis is framed in the study of particular biological systems through the use of bioinformatics. In particular, the theoretical study of the influence of certain amino acids on the structure and dynamics of the secondary structure elements of proteins has been applied to homology modelling of G protein-coupled receptors (GPCRs) and to the study of their mechanisms of activation.Certain residues, as proline, serine or threonine, are known to induce local distortions in the a-helical structure. Analysis of sequence databases of transmembrane segments evidence that certain combinations of these residues are more common than others, and that some of them are significantly over-represented, while others are clearly under-represented. The focusing this sequence analysis on the transmembrane region of Class A GPCRs illustrates that these combinations are located in some specific locations and conserved within certain subfamilies of receptors.The structure and dynamics of transmembrane a-helices containing these combinations of proline and serine or threonine have been studied using molecular dynamics simulations in an explicit hydrophobic environment. The results show how some of these combinations induce significant distortions in the a-helical structure, due to their effect on the hydrogen bond network that stabilizes the helix.These results have been applied to the building of a three-dimensional model of the chemokine CCR5 receptor, using homology modelling techniques. In this model, transmembrane helices (TMH) 2 and 3 of CCR5 are proposed to be different from the bovine rhodopsin template. TMH2 is more bent due to the presence of a Thr-X-Pro motif, which, in turn, induces this helix to lean towards TMH3. As a consequence, an interaction between these two helices is proposed for this particular receptor. This interaction would be mediated through the presence of specific and conserved hydrophobic and aromatic residues in both helices. These hypothesis have been tested through site-directed mutagenesis experiments, thanks to a collaboration with the Institut de Recherche Interdisciplinaire en Biologie Humaine et Nucléaire (IRIBHN), Université Libre de Bruxelles. The experimental results let us to hypothesize that the TMH2-TMH3 interface is involved in the chemokine-induced activation of the CCR5 receptor.As a conclusion, this thesis aims to show how through the use of bioinformatics tools, primary sequences of proteins and interactions at an atomic level can be translated to three-dimensional protein structures. In addition, this thesis illustrates that, even though the three-dimensional structure of bovine rhodopsin is a very useful template for homology modelling of GPCRs, the sequence specificities of each receptor have to be explicitly taken into account in order to build models. These sequence specificities consist in sequence patterns conserved within certain families, which are translated into structural divergences. Among these sequence patterns, we hypothesize that serine and threonine, alone or combined with nearby proline residues, can modulate the geometry of TMHs, due to its capability to interfere with the hydrogen bond network that stabilize a-helices.Finally, we propose that the influence of serine, threonine and proline motifs in the structure of TMHs may be related to processes of activation in the Class A of GPCRs, and, possibly, other membrane proteins as well. In GPCRs, these motifs may have evolved in order to adapt a conserved mechanism of activation of the G protein to the cognate ligands of each receptor family.

Carbohydrates had historically been associated to two biological functions: energy storage and structural support. However, in the last decades, new complex structures of oligosaccharides have been found to play vital roles in many biological processes, such as signal transduction, immune response, cell differentiation and cancer development, among others. Advances in the functional understanding of carbohydrate-protein interactions represented a breakthrough in the field of glycobiology and glycochemistry, opening a new branch of potential therapeutic targets (carbohydrate acting enzymes), glycomimetic drugs and biomarkers. The bottleneck in the field of glycochemistry is the synthesis of complex saccharides; hence many efforts have been devoted to the development of novel enzymatic strategies for carbohydrate synthesis. Glycoside transferases (GT) and glycoside hydrolases (GH) are the enzymes that catalyze the formation and the cleavage of the glycosidic linkage respectively. They are used in complex oligosaccharides synthesis, and recently they have been engineered to produce enzymes with particular substrate specificities or even activities. In spite of these advances, the understanding of the molecular mechanisms of enzymatic carbohydrate synthesis and degradation is far from complete. Structural studies have shown that the puckering of the sugar ring at the cleavage point must change during catalysis. Knowing the conformational catalytic itinerary has an impact in the design of GHs inhibitors. However, these itineraries are not known for all families of GHs. On the other hand, the saccharide puckering is not an issue in GTs, but the reaction mechanism is not known. In fact, the glycosidic bond formation in GTs remains one of the most intriguing and unanswered questions in the field of glycobiology. The coming of age of powerful theoretical methods such as quantum mechanics / molecular mechanics (QM/MM) and ab initio molecular dynamics (AIMD) has enabled the elucidation of complex reactive processes in proteins and enzymes. In particular, the modeling of the Michaelis complex and the reaction mechanisms of GHs highlighted the interplay between electronic and structural changes that preactivate the substrate for catalysis. Some of these changes can already be anticipated by analyzing the conformational energy landscape of the substrate. Part of the research of this Thesis complements previous studies of our group by analyzing the factors that govern substrate distortion in GHs. In this respect, it extends the use of conformational free energy landscapes of simple sugars to predict the conformation of the substrate in Michalis complexes. Additionally, the molecular mechanism of retaining glycoside transferases is elucidated. This Thesis is organized as follows: Chapter I contains an introduction of the enzymes studied (GHs and GTs) and presents the main objectives of this work. The theoretical methods used are detailed in Chapter II. Chapters III to V are focused on enzyme-substrate interactions affecting the conformation of the substrate in GHs. Concretely; in Chapter III we test how mutation of the acid/base catalytic residue, the use of a substrate-like thio-analogue inhibitor or fluorometric aglycons affects the distortion of the substrate. In Chapter IV we study the influence of the enzyme-substrate interactions through the 2-OH, in particular the effect of the commonly used 2-deoxy-2-fluoro substitution. The conformational itinerary of this inhibitor during catalysis is modeled in Chapter V. In Chapter VI, the conformational flexibility of β-D-mannopyranose and α-L-fucopyranose molecules is investigated. The topologies of their corresponding conformational free energy landscapes are related with the observed crystallographic structures of β-mannosidases and α-fucosidases, and the predictive potential of such calculations is discussed. Chapter VII focuses on trehalose 6-phosphate synthase (a family 20 retaining GT that belongs to fold type B). The mechanism of glycosidic bond formation in this enzyme is elucidated. Finally, in Chapter VI, the main conclusions of this work are summarized.

El cáncer de mama es una enfermedad con elevada incidencia, prevalencia y mortalidad que puede estar influida por factores nutricionales, entre los que destacan los lípidos de la dieta. A su vez, en cáncer se han descrito alteraciones del epigenoma. Los patrones epigenéticos son susceptibles de ser modificadas por factores ambientales. Así, el objetivo del presente trabajo ha sido definir cambios epigenéticos implicados en el desarrollo del cáncer de mama, así como determinar si el estilo de vida, y especialmente los hábitos dietéticos en relación al consumo de lípidos, influyen en este tipo de neoplasia a través de la alteración de patrones epigenéticos. Dicho objetivo se ha desarrollado en dos líneas de investigación, una en cáncer de mama humano y otra en cáncer de mama experimental. El estudio en humanos se ha realizado en una población de mujeres sanas y de enfermas de cáncer de mama, caracterizadas a nivel clínico y de estilo de vida, a partir de muestras de sangre periférica de las sanas y de sangre periférica, glándula mamaria y tumor de las enfermas. En dichas muestras se determinó la metilación del ADN a nivel global y de un panel de 12 genes implicados en los “hallmarks” del cáncer (BRCA1, p16, RARβ2, ESR1, PGR, RASSF1A, NES1, TWIST1, MASPINA, CDH1, CXCL12 y HLA-A). Por su parte, el estudio experimental se ha desarrollado en el modelo de cáncer de mama inducido con DMBA en la rata Sprague-Dawley, a partir de muestras de glándula mamaria y tumor de animales alimentados con diferentes dietas hiperlipídicas (rica en aceite de maíz o rica en aceite de oliva virgen extra). Por lo que respecta al efecto del cáncer sobre los patrones epigenéticos, los resultados obtenidos mostraron que la influencia que ejerce la propia enfermedad sobre el epigenoma alteraría mecanismos epigenéticos comunes, en términos generales, entre el cáncer de mama humano y el experimental. Estas alteraciones se caracterizarían, entre otras, por el incremento de la metilación gen-específica asociado a un aumento de la actividad ADN metiltransferasa, la disminución de la expresión de genes supresores tumorales (RASSF1A y TIMP3) y la alteración de modificaciones postraduccionales de las histonas (H3K4me2, H3K27me3, H4K20me3 y H4K16ac). Estos cambios, además, podrían estar influidos por factores dietéticos y de estilo de vida, como el consumo de alcohol, la realización de actividad física, la ingesta calórica total así como de proteínas y vitaminas B2, B6 y B12 y, especialmente, de lípidos (tanto en cantidad total como en función del tipo). Asimismo, la influencia de estos y otros factores de riesgo relacionados con la reproducción sobre el desarrollo de cáncer de mama, tales como la edad en la primera gestación y de aparición de la menopausia, podría ser, entre otros mecanismos, a través de la alteración de los patrones epigenéticos. El interés de hallar que determinados factores de riesgo, incluido el estilo de vida, alteran dichos patrones se basa en que los mecanismos epigenéticos, a diferencia de los genéticos, son modificables. Así, los resultados del presente trabajo permiten formular opiniones científicas sobre la importancia que tienen los hábitos dietéticos y el estilo de vida en relación a la salud o al riesgo de enfermedad. En este sentido, se podrían definir factores de riesgo y/o protectores a los que está sometida la población en base a sus hábitos alimenticios en relación al consumo de grasas. Por todo ello, este trabajo en su conjunto se enmarcaría en el campo de la prevención primaria y secundaria del cáncer de mama. / Breast cancer is a disease with high incidence, prevalence and mortality, that may be influenced by nutritional factors, and especially dietary lipids. Furthermore, disruption of epigenome is a major change occurring in all types of cancers. Epigenetic patterns are reversible and may be modified by environmental factors. Thus, the aim of this study was to define epigenetic changes involved in breast cancer and determine if lifestyle, particularly dietary habits in relation to fat intake, influence this type of neoplasia through the alteration of epigenetic patterns. This objective has been developed in two lines of research, one in human breast cancer and the other in an experimental model of breast cancer. The human study was conducted in a population of healthy volunteers and breast cancer patients, who were characterized clinically and in which we evaluated their lifestyle. We obtained blood from healthy volunteers and blood, mammary gland and tumor from breast cancer patients. In such samples we determined global DNA methylation and gene-specific methylation of a panel of 12 genes with an important role on the key hallmarks of cancer (BRCA1, p16, RARβ2, ESR1, PGR, RASSF1A, NES1, TWIST1, MASPINA, CDH1, CXCL12 and HLA-A). On the other hand, the experimental study was developed in the rat dimethylbenz(a)anthracene (DMBA)-induced breast cancer model, using samples from mammary gland and tumor from animals fed different high fat diets (rich in corn oil or in extra virgin olive oil). In relation to the effect of cancer on the epigenetic patterns, the results showed that the influence of the disease on the epigenome alter common epigenetic mechanisms, in general, in human and experimental breast cancer. These alterations would be characterized, among others, by the increase of gene-specific DNA methylation associated with an increase in DNA methyltransferase activity, the decrease of tumor-suppressor genes expression and the alteration of post-translational histone modifications (H3K4me2, H3K27me3, H4K20me3 and H4K16ac). These changes could be influenced by dietary and lifestyle factors, such as alcohol consumption, physical activity, calorie intake, protein and vitamins B2, B6 and B12 consumption, and especially lipid intake (including total amount and type of lipid). In addition, the influence on the development of breast cancer of these and other risk factors related to reproduction, such as age at first pregnancy and onset of menopause, could be done, among other mechanisms, through the alteration of epigenetic patterns. Finding that certain risk factors, including lifestyle, alter these patterns is of interest since epigenetic mechanisms, unlike genetic, are modifiable. Thus, the results of this study allow to formulate scientific opinions about the importance of dietary habits and lifestyle in relation to health or disease risk. In this sense, they could be defined risk and/or protective factors to which the population is exposed based on their dietary habits in relation to fat intake. Therefore, this work as a whole would be framed in the field of primary and secondary prevention of breast cancer.

Orientador: Elso Drigo Filho / Banca: Álvaro de Souza Dutra / Banca: Gerald Weber / Banca: Carla Goldman / Banca: Jorge Chaine / Resumo: Este trabalho apresenta um estudo termodinâmico de modelos de redes unidimensionais, usados para simular o DNA. Primeiramente, o modelo de Peyrard-Bishop (PB) com o potencial "Corcunda" on site foi utilizado para analisar a termodinâmica da molécula. Com o método do operador integral de transferência foram obtidas as propriedades termodinâmicas do sistema e as soluções da equação tipo - Schrödinger que emerge desse formalismo foram determinadas pelo método variacional. Com os parâmetros do potencialnormalmente utilizados na literaturao valor obtido para a temperatura de desnaturação foi extremamente alto. Por isso, são sugeridos diferentes parâmetros para descrever a termodinâmica da molécula de DNA com esse modelo. Além disso, também é estudada uma das extensões do modelo original de PB, na qual a interação de empilhamento puramente harmônica é modificada por um potencial não harmônico. São apresentados os passos e as aproximações necessárias para determinar a equação tipo - Schrödinger com massa dependente da posição que descreve as propriedades termodinâmicas desse modelo. As soluções dessa equação são determinadas a partir de uma adaptação do método variacionalpara o estudo desse problema. Para adquirir confiança na aplicação do método na solução da equação de Schrödinger com massa dependente da posição, alguns exemplos da literatura foram resolvidos.Por fim,a termodinâmica da extensão do modelo de PB foi determinada com a aplicação desse método semi - analítico. Os resultados mostraram que é possível utilizar o método variacional para solucionar esse tipo de problema e quea transição de fase da molécula ocorre em uma temperatura compatível com a apresentada na literatura / Abstract: This work shows a thermodynamical study of one-dimensional lattice models, used to simulate the DNA. The Peyrard-Bishop (PB) model with the "Hump" potential on site was used to analyze the molecule thermodynamics. The thermodynamical properties of the system were obtained with the transfer integral operator method and the solutions of the type-Schrödinger equation that emerges from the formalism were determined with the variational method. With the potential parameters normally used in the literature the obtained value to the denaturation temperature was extremely high. Because of this, it is suggested different parameters to describe the thermodynamics of the DNA molecule. Besides, it is also studied an extension of the original PB model, in which the purely harmonic stacking interaction is modified to a non-harmonic potential. The steps and necessary approximations to determine the type-Schrödinger equation with position-dependent mass that describes the thermodynamical properties of this model are shown. The solutions of this equation are determined from an adaptation of the variational method to the study of this kind of problem. To gain confidence in the application of this method in the solution of the Schrödinger equation with position-dependent mass, some examples of the literature were solved. Finally, the thermodynamic of the extension of the PB model was determined with the application of this semi-analytical method. The results showed that it is possible to use the variational method to solve this kind of problem and that the phase transition of the molecule occurs in a temperature in agreement with the one present in the literature / Doutor

Na última década, o estudo de espécies ativas de oxigênio e nitrogênio e o seus papeis em um grande número de patologias revelou que substâncias antioxidantes são capazes de prevenir os efeitos do estresse oxidativo. A reação quimiluminescente de luminol e peróxido de hidrogênio, na presença de hemina como catalisador, tem sido utilizada como método de avaliação de atividade antioxidante, uma vez que a emissão pode ser suprimida por este tipo de substância. Foi realizado um estudo cinético para estabelecer as condições experimentais do ensaio. As concentrações dos reagentes foram variadas em diferentes condições experimentais na ausência e na presença de antioxidantes, e os resultados obtidos levaram ao melhor entendimento acerca do sistema. Um novo método para o tratamento de dados foi também utilizado, permitindo a correlação entre o efeito antioxidante e o número de fótons suprimidos. Vários antioxidantes conhecidos (trolox, ácido ascórbico e ácido úrico) foram utilizados para estabelecer a metodologia. A atividade antioxidante foi calculada a partir da correlação entre a área de supressão obtida e a concentração de antioxidante, usando trolox como composto de referência. Empregando esta metodologia foi possível determinar a atividade antioxidante dos extratos e do produto majoritário isolado de Photomorphe umbellata, o 4-nerolidicatecol. / In the last decade the study of active oxygen and nitrogen species and their role in a large number of chronicle diseases, including cancer, heart disease and even aging itself, revealed that natural and synthetic antioxidants are able to prevent the effects caused by oxidative stress. Several methods can be used to evaluate the total antioxidant activity in body fluids, complex mixtures and isolated substances. Simple trapping assays can quantify the total antioxidant content in a sample, which is expressed as TRAP (total radical-trapping potential) or TEAC (trolox equivalent antioxidant capacity) and these indexes are well accepted due to its high sensitivity and operational facilities. The determination of the antioxidant potential of plant extracts and isolated natural products may constitute a simple tool to evaluate the potential biological activity of plant constituents. The chemiluminescence reaction of luminol and hydrogen peroxide in the presence of hemin as catalyst has been used as the method to evaluate the antioxidant activity, since the chemiluminescence emission can be suppressed by antioxidants, and a linear relationship between antioxidant concentration and the observed induction time is obtained. A kinetic study was performed to establish the ideal experimental conditions for the assay. The reactant concentrations were varied in the absence and the presence of antioxidants, and the results lead to a better understanding of the system. A new method for data treatment is also used, which allows the correlation of the antioxidant effect to the number of photons suppressed. Several well-known antioxidants (trolox, ascorbic and uric acid) were used to establish the methodology. TRAP values were calculated from the correlations between the number of photons suppressed and the antioxidant concentration, using trolox as reference compound. Using this methodology we were able to determine the antioxidant activity of Photomorphe umbellata extracts, and of its isolated major compound, 4-nerolidylcatechol.

Na última década, o estudo de espécies ativas de oxigênio e nitrogênio e o seus papeis em um grande número de patologias revelou que substâncias antioxidantes são capazes de prevenir os efeitos do estresse oxidativo. A reação quimiluminescente de luminol e peróxido de hidrogênio, na presença de hemina como catalisador, tem sido utilizada como método de avaliação de atividade antioxidante, uma vez que a emissão pode ser suprimida por este tipo de substância. Foi realizado um estudo cinético para estabelecer as condições experimentais do ensaio. As concentrações dos reagentes foram variadas em diferentes condições experimentais na ausência e na presença de antioxidantes, e os resultados obtidos levaram ao melhor entendimento acerca do sistema. Um novo método para o tratamento de dados foi também utilizado, permitindo a correlação entre o efeito antioxidante e o número de fótons suprimidos. Vários antioxidantes conhecidos (trolox, ácido ascórbico e ácido úrico) foram utilizados para estabelecer a metodologia. A atividade antioxidante foi calculada a partir da correlação entre a área de supressão obtida e a concentração de antioxidante, usando trolox como composto de referência. Empregando esta metodologia foi possível determinar a atividade antioxidante dos extratos e do produto majoritário isolado de Photomorphe umbellata, o 4-nerolidicatecol. / In the last decade the study of active oxygen and nitrogen species and their role in a large number of chronicle diseases, including cancer, heart disease and even aging itself, revealed that natural and synthetic antioxidants are able to prevent the effects caused by oxidative stress. Several methods can be used to evaluate the total antioxidant activity in body fluids, complex mixtures and isolated substances. Simple trapping assays can quantify the total antioxidant content in a sample, which is expressed as TRAP (total radical-trapping potential) or TEAC (trolox equivalent antioxidant capacity) and these indexes are well accepted due to its high sensitivity and operational facilities. The determination of the antioxidant potential of plant extracts and isolated natural products may constitute a simple tool to evaluate the potential biological activity of plant constituents. The chemiluminescence reaction of luminol and hydrogen peroxide in the presence of hemin as catalyst has been used as the method to evaluate the antioxidant activity, since the chemiluminescence emission can be suppressed by antioxidants, and a linear relationship between antioxidant concentration and the observed induction time is obtained. A kinetic study was performed to establish the ideal experimental conditions for the assay. The reactant concentrations were varied in the absence and the presence of antioxidants, and the results lead to a better understanding of the system. A new method for data treatment is also used, which allows the correlation of the antioxidant effect to the number of photons suppressed. Several well-known antioxidants (trolox, ascorbic and uric acid) were used to establish the methodology. TRAP values were calculated from the correlations between the number of photons suppressed and the antioxidant concentration, using trolox as reference compound. Using this methodology we were able to determine the antioxidant activity of Photomorphe umbellata extracts, and of its isolated major compound, 4-nerolidylcatechol.

Orientador: Fernando Alves de Melo / Banca: Júlio César Borges / Banca: Luis Octávio Regasini / Resumo: A proteína adaptadora Grb2 é uma importante reguladora da proteína quinase FGFR2 antes de estímulos extracelulares e é conhecida por formar complexos protéicos responsáveis por ativar a via de sinalização da MAPK, relacionada com a proliferação celular. Quando Grb2 é fosforilada ela se dissocia de FGFR2. Está por sua vez, adquire a capacidade de recrutar proteínas parceiras do citosol para iniciar vias de sinalização importantes para realização do metabolismo celular. A desfosforilação de Grb2 pela fosfatase Shp2 refaz o complexo FGFR2-Grb2 retomando controle sobre FGFR2. Desta maneira, devido a esta versatilidade em executar funções variadas dentro da célula, Grb2 torna-se um alvo importante para testar sua interação com moléculas conhecidas por apresentar propriedades farmacológicas. Sendo assim, as moléculas Morina (2',3,4',5,7-pentahidroxiflavona) e Rutina (Quercetina-3-O-α-L-Rhamnopiranosil-(1→6)-β-D-Glucopiranosídeo), foram escolhidas devido a suas propriedades anti-tumorais conhecidas na literatura e pela ausência de estudos em nível molecular que relacionem as propriedades destas moléculas com as proteínas que atuam nesta via de sinalização. Por conseguinte, utilizou-se espectroscopia de fluorescência em estado estacionário e ressonância magnética nuclear, com o objetivo de caracterizar a interação entre Morina e Rutina com Grb2. Através da determinação do perfil termodinâmico de interação destas moléculas com Grb2, obtidos por fluorescência, pudemos inferir um... / Abstract: Grb2 adaptor protein is an important regulator of FGFR2 before extracellular stimuli and is known to form complexes that activate MAPKinase pathway. When phosphorylated Grb2 dissociates from FGFR2 that gets able to recruit protein partners from the cytosol in order to start important signaling pathways inside cells. Dephosphorylating of Grb2 by Shp2 recue the FGFR2-Grb2 complex resulting in a control upon FGFR2. Because Grb2 shows to be versatile to performing functions inside the cell other than adaptor protein, it becomes an important target to test the interaction with molecules known to have anti-tumor properties. Therefore, the molecules Morin (2',3,4',5,7-pentahydroxyflavone) and Rutin (quercetin-3-O-α-L-Ramnopiranosil-(1→6)-β-D-glucopyranoside), were chosen because to its anti-inflammatory and anti-tumor properties known in the literature and because the absence of studies at the molecular level that brings up information about the properties of these molecules to specific protein targets. Thus, we have used static fluorescence spectroscopy and nuclear magnetic resonance in order to characterize the interaction between those molecules and Grb2. The thermodynamic profile got from fluorescence assays allow us to infer that the interaction between the molecules with Grb2 is entropically driven, compatible with hydrophobic interactions for both molecules where the equilibrium constants can be found between 104 and 105 M-1. Furthermore, nuclear magnetic resonance has ... / Mestre

No presente trabalho é explorada em detalhe a implementação de um modelo de neurônio de tempo discreto proposto por Nikolai Rulkov, tanto em neurônios isolados como em redes de neurônios acoplados. Primeiramente, o neurônio individual é analisado com a construção do espaço de fases e com a identificação dos estados assintóticos do sistema no espaço de parâmetros através do cálculo do maior expoente de Lyapunov. Em seguida, duas células idênticas são conectadas via sinapse elétrica simétrica e homogênea. Fixando um dos parâmetros de controle do mapa, relações entre bacias de atração e diferenças de fase entre trens de pulsos são estudadas, assim como medidas de variância e covariância do sistema. Finalmente, é criada uma rede quadrada composta por neurônios idênticos, com conexões elétricas homogêneas e simétricas entre primeiros vizinhos, e é investigada a estabilidade dos diferentes regimes de sincronização que emergem macroscopicamente. Com o auxílio dos parâmetros de ordem variância e covariância e da distribuição de fases, também é construído um diagrama resumindo as configurações em que ocorrem cada regime e suas respectivas transições. / In the present work the implementation of a map-based neuron model proposed by Nikolai Rulkov is studied, both in isolated neurons and in coupled neurons networks. Initially, the individual neuron is analyzed through the construction of the phase space and the identification of asymptotic states of the system on the parameters space using the larger Lyapunov exponent calculation. Then, two identical cells are connected trough symmetric and homogeneous electrical synapses. Fixing one of the control parameters of the map, relations between basins of attraction and phase difference between spike bursts are studied, as well as measures of the system variance and covariance. Finally, a square network composed by identical neurons is created, the connections are among the first neighbors, are homogenous and symmetric. The stability of the different synchronization regimes that emerge macroscopically is investigated. With the aid of the order parameters variance and covariance and of the phase distributions, a diagram is also constructed, summarizing the configurations set in which each regime occurs and their respective transitions.

Made available in DSpace on 2014-06-11T19:30:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-06-18Bitstream added on 2014-06-13T19:40:25Z : No. of bitstreams: 1 ribeiro_nf_dr_sjrp_parcial.pdf: 71860 bytes, checksum: a27e6229408d27223218d0c5e3a87d2e (MD5) Bitstreams deleted on 2015-06-03T11:42:37Z: ribeiro_nf_dr_sjrp_parcial.pdf,. Added 1 bitstream(s) on 2015-06-03T11:44:04Z : No. of bitstreams: 1 000716639_20150718.pdf: 62536 bytes, checksum: 09cec3d191a740604459d84f516c0442 (MD5) Bitstreams deleted on 2015-08-03T12:21:11Z: 000716639_20150718.pdf,. Added 1 bitstream(s) on 2015-08-03T12:22:23Z : No. of bitstreams: 1 000716639.pdf: 403805 bytes, checksum: 597085aba1fcb00175bd67f7336a0df5 (MD5) / Este trabalho apresenta um estudo termodinâmico de modelos de redes unidimensionais, usados para simular o DNA. Primeiramente, o modelo de Peyrard-Bishop (PB) com o potencial “Corcunda” on site foi utilizado para analisar a termodinâmica da molécula. Com o método do operador integral de transferência foram obtidas as propriedades termodinâmicas do sistema e as soluções da equação tipo – Schrödinger que emerge desse formalismo foram determinadas pelo método variacional. Com os parâmetros do potencialnormalmente utilizados na literaturao valor obtido para a temperatura de desnaturação foi extremamente alto. Por isso, são sugeridos diferentes parâmetros para descrever a termodinâmica da molécula de DNA com esse modelo. Além disso, também é estudada uma das extensões do modelo original de PB, na qual a interação de empilhamento puramente harmônica é modificada por um potencial não harmônico. São apresentados os passos e as aproximações necessárias para determinar a equação tipo – Schrödinger com massa dependente da posição que descreve as propriedades termodinâmicas desse modelo. As soluções dessa equação são determinadas a partir de uma adaptação do método variacionalpara o estudo desse problema. Para adquirir confiança na aplicação do método na solução da equação de Schrödinger com massa dependente da posição, alguns exemplos da literatura foram resolvidos.Por fim,a termodinâmica da extensão do modelo de PB foi determinada com a aplicação desse método semi - analítico. Os resultados mostraram que é possível utilizar o método variacional para solucionar esse tipo de problema e quea transição de fase da molécula ocorre em uma temperatura compatível com a apresentada na literatura / This work shows a thermodynamical study of one-dimensional lattice models, used to simulate the DNA. The Peyrard-Bishop (PB) model with the “Hump” potential on site was used to analyze the molecule thermodynamics. The thermodynamical properties of the system were obtained with the transfer integral operator method and the solutions of the type-Schrödinger equation that emerges from the formalism were determined with the variational method. With the potential parameters normally used in the literature the obtained value to the denaturation temperature was extremely high. Because of this, it is suggested different parameters to describe the thermodynamics of the DNA molecule. Besides, it is also studied an extension of the original PB model, in which the purely harmonic stacking interaction is modified to a non-harmonic potential. The steps and necessary approximations to determine the type-Schrödinger equation with position-dependent mass that describes the thermodynamical properties of this model are shown. The solutions of this equation are determined from an adaptation of the variational method to the study of this kind of problem. To gain confidence in the application of this method in the solution of the Schrödinger equation with position-dependent mass, some examples of the literature were solved. Finally, the thermodynamic of the extension of the PB model was determined with the application of this semi-analytical method. The results showed that it is possible to use the variational method to solve this kind of problem and that the phase transition of the molecule occurs in a temperature in agreement with the one present in the literature

No presente trabalho estudou-se os processos de interação entre uma série de porfirinas catiônicas e a melanina. Utilizou-se as porfirinas (Zn-(tetra(4-N-Metilpiridil)porfina): (\'ZN\'-(tetra(4-\'N\'-Metilpiridil)porfina): \'ZN\'-\'T\'\'M\'\'PY\'\'P\', (\'ZN\'-(tetra(4-\'N\'-Benzilpiridil)porfina): \'ZN\'-\'T\'\'BZ\'\'PY\'\'P\', as respectivas porfirinas de base livre (\'T\'\'M\'\'PY\'\'P\' e \'T\'\'BZ\'\'PY\'\'P\') e a melanina sintética obtida a partir da auto-oxidação da dihidroxifenilalanina (L-DOPA). O trabalho baseou-se em três técnicas espectroscópicas complementares: espectroscopia eletrônica (absorção na região do ultravioleta-visível), fluorescência e espalhamento Raman ressonante. Demonstrou-se a formação de complexos estáveis como resultado da interação porfirina-melanina. A partir dos dados de espectroscopia Raman fez-se uma atribuição de bandas vibracionais para as porfirinas, comparando-se com os dados disponíveis na literatura para porfirinas semelhantes. Discutiu-se as possíveis mudanças de estruturas provocadas pela melanina na porfirina, baseadas nas diferenças entre os espectros Raman das porfirinas puras e dos complexos porfirina-melanina. Sugeriu-se uma nova interpretação para os espectros eletrônicos das porfirinas baseada no modelo de Gouterman (modelo de quatro orbitais). A partir dessa interpretação do espectro eletrônico analisou-se a perturbação causada pela melanina nos orbitais moleculares da porfirina fazendo=se uma discussão dos espectros de emissão fluorescente das mesmas. Estimou-se a taxa de transferência de energia da porfirina para melanina através da supressão da fluorescência da porfirina, usando-se medidas de fluorescência com resolução temporal. Demonstrou-se que a fluorescência do complexo porfirina-melanina tem um tempo de vida menor que 5 ps. Utilizou-se as medidas de supressão de fluorescência em regime estacionário para determinar as constantes de dissociação do complexo porfirina-melanina e os possíveis sítios de ligação da melanina. Os resultados evidenciam a importante propriedade do polímero de melanina em atuar como eficiente meio para a dissipação não radiativa de estados eletrônicos excitados. / In this work we studied the interaction between some cationic porphyrins and melanin. The porphyrins used were (\'ZN\'-(tetra(4-\'N\'-Metilpiridil)porfina): \'ZN\'-\'T\'\'M\'\'PY\'\'P\', (\'ZN\'-(tetra(4-\'N\'-Benzilpiridil)porfina): \'ZN\'-\'T\'\'BZ\'\'PY\'\'P\', and their respective free bases porphyrins (\'T\'\'M\'\'PY\'\'P\' e \'T\'\'BZ\'\'PY\'\'P\'). Synthetic melanin was obtained from the auto-oxidation of the 3,4-dihydroxyphenylalanine (L-DOPA). From resonance Raman spectra an attribution is made to the vibrational bands of the porphirins, analyzing the effects of the substituents. The possible changes in the porphyrin structure due to the interaction with melanin are discussed based on the modifications of the resonance Raman spectra of the porphyrins in the presence of melanin. From optical absorption and fluorescence measurements it is suggested a new interpretation to the eletronic spectra of the porphyrins based on the Goutermans model. The perturbation due to the melanin in the molecular orbitals of the porphyrins is also analysed. An estimation of the energy transfer rate to the melanin by time resolved fluorescence measurements of the porphyrin-melanin solution is made. It is demonstrated that the fluorescence lifetime of the porphyrin-melanin complexes are lesser than 5ps. The dissociation constants of the porphyrin-melanin complexes and possible sites of binding of the melanin are determined by fluorescence quenching of the porphyrins as observed by steady state measurements. The results show clearly the important role of the melanin to act as an efficient way to the non-radiative dissipation of the excited electronic states of the porphyrins.