A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine. / Post-translational modification of intracellular proteins through acetylation is recognized as an important regulatory mechanism of cellular energy homeostasis. Specific proteins called sirtuins deacetylate other mitochondrial proteins involved in glucose and lipid metabolism, activating them in metabolic processes. SIRT3 is a sirtuin of particular interest as it is found exclusively in mitochondria and has been shown to affect a variety of cellular metabolic processes. The activity of this enzyme is related to cellular insulin sensitivity. This study attempted to identify the relationship between insulin sensitivity and change in amount of SIRT3 following a bout of exercise in non-diabetic individuals. We find a moderate inverse correlation between insulin sensitivity and increase in SIRT3 abundance following exercise. This suggests that this protein may not be involved directly in cells’ ability to regulate energy homeostasis or that it may act through another mechanism not investigated in this study.

<p>The larva of the fruit fly Drosophila melanogaster is an excellent model to study the molecular control of innate cellular immune responses. Cellular responses take place, and can be studied, following infestation of the wasp Leptopilina boulardi. This response includes proliferation and activation (differentiation) of the blood cells (hemocytes). In a successful anti-parasitic response, an immune-induced lineage of hemocytes, the lamellocytes, forms a cellular capsule covering and killing the foreign intruder. I will in this thesis present data about the finding and characterization of a novel marker that is expressed specifically in the hemocytes, the Hemese gene. I furthermore describe the construction of a useful tool, the transgenic Hemese-Gal4 fly, which enables blood cell specific expression of any gene of interest. By using the Hemese-Gal4 fly in a directed screen, I have found that a surprisingly large number of genes, that in turn are members of seemingly diverse signaling pathways, are able to induce a cellular response. In many cases their expression is also associated with a blood cell tumor phenotype. Overexpression of certain genes, such as hopscotch (a Drosophila Jak homologue) and hemipterous (a c-jun kinase kinase) lead to the formation of lamellocytes. Other genes may control the cell number, such as Egfr and Ras, as their expression produced a massive in increase the numbers of hemocytes. A third group of genes, including, e.g. Alk, Rac1 and Pvr give a mixed response, promoting both hemocyte proliferation and activation. Surprisingly, the suppression of WNT signaling in hemocytes lead to hemocyte activation. In one case, with a UAS-Pvr dominant negative construct, we observe a reduction of the circulating blood cells in uninfested larva. The expression of DN-Pvr additionally contributes to reduce encapsulation rates in larvae subjected to Leptopilina infestation. In conclusion: the control of blood cells in larval hematopoiesis, and during parasitic wasp attacks, is complex and may involve multiple pathways. In a broader sense, the gene functions found in the directed screen may have implications also for understanding the molecular control of mammalian myeloid lineage blood cells.</p>

The larva of the fruit fly Drosophila melanogaster is an excellent model to study the molecular control of innate cellular immune responses. Cellular responses take place, and can be studied, following infestation of the wasp Leptopilina boulardi. This response includes proliferation and activation (differentiation) of the blood cells (hemocytes). In a successful anti-parasitic response, an immune-induced lineage of hemocytes, the lamellocytes, forms a cellular capsule covering and killing the foreign intruder. I will in this thesis present data about the finding and characterization of a novel marker that is expressed specifically in the hemocytes, the Hemese gene. I furthermore describe the construction of a useful tool, the transgenic Hemese-Gal4 fly, which enables blood cell specific expression of any gene of interest. By using the Hemese-Gal4 fly in a directed screen, I have found that a surprisingly large number of genes, that in turn are members of seemingly diverse signaling pathways, are able to induce a cellular response. In many cases their expression is also associated with a blood cell tumor phenotype. Overexpression of certain genes, such as hopscotch (a Drosophila Jak homologue) and hemipterous (a c-jun kinase kinase) lead to the formation of lamellocytes. Other genes may control the cell number, such as Egfr and Ras, as their expression produced a massive in increase the numbers of hemocytes. A third group of genes, including, e.g. Alk, Rac1 and Pvr give a mixed response, promoting both hemocyte proliferation and activation. Surprisingly, the suppression of WNT signaling in hemocytes lead to hemocyte activation. In one case, with a UAS-Pvr dominant negative construct, we observe a reduction of the circulating blood cells in uninfested larva. The expression of DN-Pvr additionally contributes to reduce encapsulation rates in larvae subjected to Leptopilina infestation. In conclusion: the control of blood cells in larval hematopoiesis, and during parasitic wasp attacks, is complex and may involve multiple pathways. In a broader sense, the gene functions found in the directed screen may have implications also for understanding the molecular control of mammalian myeloid lineage blood cells.

Over the past 30 years, transcription factor nuclear factor kappa B (NF-κB) has been extensively characterized in organisms ranging from flies to humans, where it is known to play key roles in developmental and immune-related processes. More recently, DNA sequencing approaches have identified homologs of NF-κB and many upstream signaling components in basal phyla, including Cnidaria (sea anemones, corals, hydras, and jellyfish), Porifera (sponges), and single-celled protists, including Capsaspora owczarzaki and some choanoflagellates. However, little is known about the activity and regulation of NF-κB proteins in these basal organisms. In this dissertation, the structure, activity, and biology of NF-κB in three basal phyla is examined and the extent of conservation with more derived organisms as well as phylum-specific properties are investigated. In the coral Orbicella faveolata (Of) a simplified but nearly complete Toll-like receptor (TLR)-to-NF-κB pathway exists, but basal to cnidarians, there are fewer upstream signaling molecules present. For example, in the poriferan Amphimedon queenslandica (Aq) and the protist Capsaspora owczarzaki (Co), singular NF-κBs and some upstream signaling proteins are encoded in their genomes, but no canonical TLRs exist. In contrast, the expanded family of choanoflagellates, including the choanoflagellate Acanthoeca spectabilis (As), contains TLR-like and up to three NF-κB-like homologs, although their domain structures differ from NF-κB pathway members of higher organisms. Of-NF-κB, Aq-NF-κB, and Co-NF-κB all resemble the mammalian NF-κB protein p100 in that they contain an N-terminal DNA-binding domain, a C-terminal Ankyrin (ANK) repeat domain, and similar DNA binding-site profiles. C-terminal truncation results in translocation of these basal NF-κBs to the nucleus and increases their DNA-binding and transcriptional activation activities. Nevertheless, unlike mammalian NF-κB p100, the C-terminal sequences of Aq-NF-κB do not inhibit its DNA-binding activity. The three As-NF-κB-like proteins all consist of primarily the N-terminal conserved Rel Homology domain sequences of NF-κB, but lack C-terminal ANK repeats. All three As-NF-κB proteins constitutively enter the nucleus of human and Co cells, but differ from one another in DNA-binding and transcriptional activation activities. Furthermore, all three As-NF-κB proteins can form heterodimers, indicating that NF-κB diversified into multi-subunit families at least two times during evolution. Expression of IKKs induce proteasome-dependent C-terminal processing of Of-NF-κB and Aq-NF-κB in human cells, and processing requires C-terminal serines. In contrast, C-terminal processing of Co-NF-κB is not induced by co-expression of IKK in human cells and no IKK homolog exists in the Co genome, suggesting that IKK-mediated processing of NF-κB is a mechanism that evolved solely in animals. Treatment of Of and sponge tissue with lipopolysaccharide (LPS), a ligand for mammalian innate immunity, results in gene expression changes consistent with NF-κB pathway mobilization in Of and increases both DNA-binding activity and processing of sponge NF-κB. Furthermore, sponge tissue contains constitutive NF-κB site DNA-binding activity, as well as nuclear and processed NF-κB. Moreover, exogenously expressed Co-NF-κB localizes to the nucleus in Co cells. Together, these data suggest that the mechanism as well as level of activation of NF-κB in basal organisms is different from what is observed in higher organisms. Additionally, NF-κB mRNA and DNA-binding levels differ across three life stages of Capsaspora, suggesting distinct roles for NF-κB in these life stages. RNA-seq and GO analyses identify possible gene targets and biological functions of Co-NF-κB. Overall, these data represent the first functional characterization of NF-κB signaling proteins in an endangered coral, in any organisms basal to cnidarians (i.e., an evolutionary important sponge), and outside the Kingdom Animalia (protists). These findings suggest that these seemingly simple organisms contain conserved innate immune-like pathways that may be regulated by NF-κB and provide information about the evolution and diversification of this biologically important transcription factor.

O estudo da evolução da concentração de elementos de uma reação química, conhecida como Cinética Química, é de extrema importância para a compreensão das complexas interações em sistemas biológicos. Uma maneira de descrever a cinética de uma reação química é utilizando um sistema de equações diferenciais ordinárias (EDOs). Uma vez que para resolver um sistema de equações diferenciais ordinárias pode ser uma tarefa difícil (ou mesmo inviável), métodos numéricos são utilizados para realizar simulações, ou seja, para obter concentrações aproximadas das espécies químicas envolvidas durante um determinado período de tempo. No entanto, quanto maior for o sistema simulado de EDOs, mais os métodos numéricos estão sujeitos a erros. Além disso, o aumento do tamanho do sistema muitas vezes resulta em simulações que são mais exigentes do ponto de vista computacional. Assim, o objetivo deste projeto de mestrado é o desenvolvimento de regras para simplificar os sistemas de equações diferenciais ordinárias que modelam a cinética de reações químicas e, portanto, a obtenção de um algoritmo para executar simulações numéricas de um modo mais rápido e menos propenso a erros. Mais do que diminuir o erro e o tempo de execução, esta simplificação possibilita o biólogo escolher a solução mais factível do ponto de vista de medida. Isso porque, a identificação dos sistemas (i.e., inferência dos parâmetros) requer que a concentração de todas as espécies químicas seja conhecida, ao menos em um certo intervalo de tempo. Contudo, em muitos casos, não é possível medir a concentração de todas as espécies químicas consideradas. Esta simplificação gera sistemas equivalentes ao original, mas que dispensa a utilização de certas concentrações de espécies químicas. Um sistema de equações diferenciais ordinárias pode ser simplificado considerando as relações de conservação de massa, que são equações algébricas. Além disso, no caso de reações enzimáticas, o sistema de equações diferenciais ordinárias pode ser simplificado pelo pressuposto de que a concentração do complexo enzima-substrato mantém-se constante, o que permite a utilização da equação de Michaelis-Menten. De todas as combinações possíveis das equações algébricas com as equações diferenciais, uma família de sistemas simplificados de EDOs foi construída, permitindo a escolha do sistema mais simples. Esta escolha segue um critério guloso que favorece a minimização do número de equações diferenciais e do número total de termos. As regras em desenvolvimento de simplificação dos sistemas de equações diferenciais ordinárias foram utilizados para projetar um algoritmo, que foi implementado usando a linguagem de programação Python. O algoritmo concebido foi testado utilizando instâncias artificiais. / The study of the evolution of the concentration of species in a chemical reaction, known as Chemical Kinetics, is of paramount importance for the understanding of complex interactions in biological systems. One way to describe the kinetics of a chemical reaction is using a system of ordinary differential equations (ODEs). Once to solve a system of ODEs can be a difficult (or even unfeasible) task, numerical methods are employed to carry out simulations, that is, to obtain approximated concentrations of the involved chemical species for a certain time frame. However, the larger is the simulated system of ODEs, the more numerical methods are subject to error. Moreover, the increase of the system size often results in simulations that are more demanding from the computational point of view. Thus, the objective is the development of rules to simplify systems of ODEs that models the kinetics of chemical reactions, hence obtaining an algorithm to execute numerical simulations in a faster way and less prone to error. More than decrease error and run time, this simplification allows the biologist to choose the most feasible solution from the point of view of measurement. This is because the identification of systems (i.e., inferring parameters) requires that the concentration of all chemical species is known, at least in a certain time interval. However, in many cases it is not possible to measure the concentration of all chemical species considered. This simplification creates systems equivalent to the original, but that does not require the use of certain concentrations of chemical species. A system of ODEs can be simplified considering the relations of mass conservation, which are algebraic equations. Furthermore, in the case of enzymatic reactions, the system of ODEs can be simplified under the assumption that the concentration of enzyme-substrate complex remains constant, which allows us to use the Michaelis-Menten equation. From all possible combinations of the algebraic equations with differential equations, a family of simplified systems of ODEs will be built, allowing the choice of a simplest system. This choice will follow a greedy criterion which favors the minimization of number of differential equations and the total number of terms. The rules under development to simplify systems of ODEs will be used to design an algorithm, which will be implemented using Python programming language. The designed algorithm will be tested using synthetic data.

La préparation physique du joueur de rugby requière le développement simultané des qualités de force et d’endurance (Duthie et coll., 2003) et nécessite alors la combinaison d’efforts antagonistes pouvant induire un « conflit physiologique » au sein de l’organisme. Hickson (1980) fut le premier chercheur à mettre en évidence que la combinaison des qualités de force et d’endurance, au sein d’une même programmation, semble interférer sur le développement des qualités neuromusculaires. Cette interférence semblerait intervenir préférentiellement sur la production de force à vitesse rapide, la puissance et l’explosivité et concernerait essentiellement les groupes musculaires mobilisés au cours des deux formes d’entraînements. Plusieurs hypothèses, telles que la fatigue et les adaptations physiologiques contradictoires, ont été mises en avant pour expliquer ce phénomène. A l’inverse, l’entraînement de musculation ne semble pas présenter d’effets négatifs sur les adaptations oxydatives. Plusieurs variables, liées à la programmation de l’entraînement, influenceraient l’interférence sur le développement de la force. L’objectif général de ce travail de recherche sera alors d’étudier les effets de différentes configurations d’entraînement permettant de limiter l’intervention du phénomène interférentiel. Dans une première étude, nous avons mis en évidence une fatigue aigue induite par différents types d’entraînements d’endurance de haute intensité pouvant aller jusqu’à 24h. Ces résultats mettaient alors en avant la pertinence de placer les séances qualitatives de force avant celles d’endurance au sein d’une programmation combinée. Dans une deuxième étude, nous avons proposé de vérifier l’effet du temps de récupération entre les séances de force et d’endurance sur les adaptations neuromusculaires et oxydatives. Il s’avérerait alors qu’une durée de récupération de 24h soit plus efficace sur les gains de force et de VO2pic qu’une durée intermédiaire de 6h. Enfin, dans une troisième étude, nous nous sommes centrés sur les effets du type d’entraînement aérobie de haute intensité sur les adaptations physiologiques. L’entraînement de répétition de sprints longs perturberait davantage les gains de force à l’issue d’une période combinée caractérisée pourtant par 24h de récupération entre les séances. Ce type d’entraînement induirait en revanche des gains plus importants de VO2pic et de la performance moyenne au cours d’un test de sprints répétés qu’un entraînement intermittent court. / Physical training of rugby players requires simultaneous development of strength and endurance qualities (Duthie et al., 2003) and therefore the combination of antagonistic exercises inducing a "physiological conflict". Hickson (1980) was the first to demonstrate that the combination of strength and endurance qualities within a same program seems to reduce the development of neuromuscular qualities. This interference would impair preferentially maximal strength production at fast velocity, maximal power and explosivity and would concern mainly muscle groups solicited during the two forms of training (strength and aerobic). Several hypotheses, such as fatigue and conflicting physiological adaptations have been put forward to explain the interference. Conversely, strength training does not appear to have negative effects on oxidative adaptations. Many variables related to schedule of training, would influence the impairment of strength quality development. The main aim of this research was to measure the effects of different configurations of training in order to limit the interference on neuromuscular adaptations. In a first study, we emphasized an acute fatigue up to 24 hours induced by different types of high-intensity interval training. These results showed the relevance to program strength before endurance sessions during a concurrent training program. Then, in a second study, we proposed to measure the effect of recovery delay between strength and aerobic sessions on neuromuscular and oxidative adaptations. We observed a 24h recovery period was more effective than lower duration of 6h on strength gains and VO2peak. Last, in a third study, we focused on the effects of the type of high-intensity interval training. Sprint interval training would more impair strength gains after a concurrent training period, despite 24h recovery delay between sessions. However, this type of training would induce greater gains of VO2peak and consequently of repeated sprint ability than short intermittent training.

O estudo da evolução da concentração de elementos de uma reação química, conhecida como Cinética Química, é de extrema importância para a compreensão das complexas interações em sistemas biológicos. Uma maneira de descrever a cinética de uma reação química é utilizando um sistema de equações diferenciais ordinárias (EDOs). Uma vez que para resolver um sistema de equações diferenciais ordinárias pode ser uma tarefa difícil (ou mesmo inviável), métodos numéricos são utilizados para realizar simulações, ou seja, para obter concentrações aproximadas das espécies químicas envolvidas durante um determinado período de tempo. No entanto, quanto maior for o sistema simulado de EDOs, mais os métodos numéricos estão sujeitos a erros. Além disso, o aumento do tamanho do sistema muitas vezes resulta em simulações que são mais exigentes do ponto de vista computacional. Assim, o objetivo deste projeto de mestrado é o desenvolvimento de regras para simplificar os sistemas de equações diferenciais ordinárias que modelam a cinética de reações químicas e, portanto, a obtenção de um algoritmo para executar simulações numéricas de um modo mais rápido e menos propenso a erros. Mais do que diminuir o erro e o tempo de execução, esta simplificação possibilita o biólogo escolher a solução mais factível do ponto de vista de medida. Isso porque, a identificação dos sistemas (i.e., inferência dos parâmetros) requer que a concentração de todas as espécies químicas seja conhecida, ao menos em um certo intervalo de tempo. Contudo, em muitos casos, não é possível medir a concentração de todas as espécies químicas consideradas. Esta simplificação gera sistemas equivalentes ao original, mas que dispensa a utilização de certas concentrações de espécies químicas. Um sistema de equações diferenciais ordinárias pode ser simplificado considerando as relações de conservação de massa, que são equações algébricas. Além disso, no caso de reações enzimáticas, o sistema de equações diferenciais ordinárias pode ser simplificado pelo pressuposto de que a concentração do complexo enzima-substrato mantém-se constante, o que permite a utilização da equação de Michaelis-Menten. De todas as combinações possíveis das equações algébricas com as equações diferenciais, uma família de sistemas simplificados de EDOs foi construída, permitindo a escolha do sistema mais simples. Esta escolha segue um critério guloso que favorece a minimização do número de equações diferenciais e do número total de termos. As regras em desenvolvimento de simplificação dos sistemas de equações diferenciais ordinárias foram utilizados para projetar um algoritmo, que foi implementado usando a linguagem de programação Python. O algoritmo concebido foi testado utilizando instâncias artificiais. / The study of the evolution of the concentration of species in a chemical reaction, known as Chemical Kinetics, is of paramount importance for the understanding of complex interactions in biological systems. One way to describe the kinetics of a chemical reaction is using a system of ordinary differential equations (ODEs). Once to solve a system of ODEs can be a difficult (or even unfeasible) task, numerical methods are employed to carry out simulations, that is, to obtain approximated concentrations of the involved chemical species for a certain time frame. However, the larger is the simulated system of ODEs, the more numerical methods are subject to error. Moreover, the increase of the system size often results in simulations that are more demanding from the computational point of view. Thus, the objective is the development of rules to simplify systems of ODEs that models the kinetics of chemical reactions, hence obtaining an algorithm to execute numerical simulations in a faster way and less prone to error. More than decrease error and run time, this simplification allows the biologist to choose the most feasible solution from the point of view of measurement. This is because the identification of systems (i.e., inferring parameters) requires that the concentration of all chemical species is known, at least in a certain time interval. However, in many cases it is not possible to measure the concentration of all chemical species considered. This simplification creates systems equivalent to the original, but that does not require the use of certain concentrations of chemical species. A system of ODEs can be simplified considering the relations of mass conservation, which are algebraic equations. Furthermore, in the case of enzymatic reactions, the system of ODEs can be simplified under the assumption that the concentration of enzyme-substrate complex remains constant, which allows us to use the Michaelis-Menten equation. From all possible combinations of the algebraic equations with differential equations, a family of simplified systems of ODEs will be built, allowing the choice of a simplest system. This choice will follow a greedy criterion which favors the minimization of number of differential equations and the total number of terms. The rules under development to simplify systems of ODEs will be used to design an algorithm, which will be implemented using Python programming language. The designed algorithm will be tested using synthetic data.

A capacidade das células para responder corretamente a sinais externos e perceber mudanças no seu microambiente é a base do desenvolvimento, reparação de tecidos e de imunidade, bem como a homeostase do tecido normal. Transdução de sinal é o principal meio pelo qual as células respondem a sinais externos de seu ambiente e coordenam alterações celulares complexas. O estudo das vias de sinalização molecular permite-nos tentar compreender o funcionamento dessas transduções de sinais e, consequentemente, as respostas celulares a estímulos externos. Uma abordagem adequada para tais estudos é o uso de modelos matemáticos para simular a cinética das reações químicas que descrevem uma dada via de sinalização, o que nos permite gerar predições testáveis de processos celulares. Construir modelos cinéticos preditivos de vias de sinalização molecular através de dados de alto rendimento produzidos utilizando técnicas ômicas (i.e., genômica, transcriptômica, (fosfo-)proteômica) constitui um dos atuais desafios enfrentados pelos pesquisadores na área de Biologia Molecular. Recentemente, para lidar com este desafio, o arcabouço de e-Science SigNetSim foi introduzido pelo Grupo de Biologia Computacional e de Bioinformática do Instituto Butantan. Esse arcabouço permite fazer a descrição de vias de sinalização molecular através da descrição da estrutura de um modelo através de um conjunto de reações químicas, que por sua vez é mapeado para um sistema de Equações Diferencias Ordinárias (EDOs), numericamente simuladas e avaliadas. Todavia, modificações na estrutura das vias precisam ser feitas manualmente, o qual restringe severamente o número de estruturas da via que precisam ser testadas, especialmente no caso de modelos grandes. Portanto, diante desse panorama, este trabalho propõe o desenvolvimento de um método para modificar vias de sinalização molecular. Esse método se baseia no uso de bancos de dados de interatomas para fornecer um conjunto de espécies químicas candidatas para serem incluídas na via de sinalização. Um componente integrado ao arcabouço SigNetSim capaz de testar diferentes hipóteses de modificação de vias foi desenvolvido neste projeto utilizando a metodologia de heurística incremental. Para avaliar a eficiência do componente implementado, utilizamos como estudo de caso um modelo de vias sinalização de MAPKs e PI3K/Akt para realizar testes experimentais e analisar os resultados obtidos. / The ability of cells to respond correctly external signals and to perceive changes in their microenvironment is the basis for development, tissue repair and immunity as well as normal tissue homeostasis. Signal transduction is the primary means by which cells respond to external signals from their environment and coordinate complex cellular changes. The study of molecular signaling pathways allows us to understand the operation of each process of cellular signal transduction. The use of mathematical models to simulate the kinetics of chemical reactions that describe a given signaling pathway, allow us to generate testable predictions of the cell processos. To Build Kinetic predictive models to molecular signaling pathways through massive data omics produced using modern techniques, Genomics, transcriptomics, (Phospho) proteomics, is one of the current challenges faced by researchers in the field of molecular biology. Recently, the \\textit SigNetSim e-Science was introduced by the Biological Computacional and Bioinformatical Group from the Butantan Institute to face this challenge. This \\textit makes the description of molecular signaling pathways through a set of chemical reactions, which are mapped into a system of ordinary differential equations, this system will be numerically simulated and evaluated . However, changes in the structure of the pathways need to be updated manually presented in this work, which severely restricts the number of track structures that need to be tested, especially for the large models. Therefore, given this background, we present the method to modify the molecular signaling pathways. This method relies on the use of interactome database to provide a set of chemical species candidates to be included in the signaling pathway. An component integrated to SigNetSim framework able to test different hypotheses of pathways modification was developed in this project using the incremental heuristic methodology. To evaluate the implemented component, we used the MAPKs and PI3K/Akt pathways model as case study, in order to perform experimental tests and to analyze the obtained results.

Biochemical reactions make up most of the activity in a cell. There is inherent stochasticity in the kinetic behavior of biochemical reactions which in turn governs the fate of various cellular processes. In this work, the precision of a method for dimensionality reduction for stochastic modeling of biochemical reactions is evaluated. Further, a method of stochastic simulation of reaction kinetics is implemented in case of a specific biochemical network involved in maintenance of long-term potentiation (LTP), the basic substrate for learning and memory formation. The dimensionality reduction method diverges significantly from a full stochastic model in prediction the variance of the fluctuations. The application of the stochastic simulation method to LTP modeling was used to find qualitative dependence of stochastic fluctuations on reaction volume and model parameters. / text

A capacidade das células para responder corretamente a sinais externos e perceber mudanças no seu microambiente é a base do desenvolvimento, reparação de tecidos e de imunidade, bem como a homeostase do tecido normal. Transdução de sinal é o principal meio pelo qual as células respondem a sinais externos de seu ambiente e coordenam alterações celulares complexas. O estudo das vias de sinalização molecular permite-nos tentar compreender o funcionamento dessas transduções de sinais e, consequentemente, as respostas celulares a estímulos externos. Uma abordagem adequada para tais estudos é o uso de modelos matemáticos para simular a cinética das reações químicas que descrevem uma dada via de sinalização, o que nos permite gerar predições testáveis de processos celulares. Construir modelos cinéticos preditivos de vias de sinalização molecular através de dados de alto rendimento produzidos utilizando técnicas ômicas (i.e., genômica, transcriptômica, (fosfo-)proteômica) constitui um dos atuais desafios enfrentados pelos pesquisadores na área de Biologia Molecular. Recentemente, para lidar com este desafio, o arcabouço de e-Science SigNetSim foi introduzido pelo Grupo de Biologia Computacional e de Bioinformática do Instituto Butantan. Esse arcabouço permite fazer a descrição de vias de sinalização molecular através da descrição da estrutura de um modelo através de um conjunto de reações químicas, que por sua vez é mapeado para um sistema de Equações Diferencias Ordinárias (EDOs), numericamente simuladas e avaliadas. Todavia, modificações na estrutura das vias precisam ser feitas manualmente, o qual restringe severamente o número de estruturas da via que precisam ser testadas, especialmente no caso de modelos grandes. Portanto, diante desse panorama, este trabalho propõe o desenvolvimento de um método para modificar vias de sinalização molecular. Esse método se baseia no uso de bancos de dados de interatomas para fornecer um conjunto de espécies químicas candidatas para serem incluídas na via de sinalização. Um componente integrado ao arcabouço SigNetSim capaz de testar diferentes hipóteses de modificação de vias foi desenvolvido neste projeto utilizando a metodologia de heurística incremental. Para avaliar a eficiência do componente implementado, utilizamos como estudo de caso um modelo de vias sinalização de MAPKs e PI3K/Akt para realizar testes experimentais e analisar os resultados obtidos. / The ability of cells to respond correctly external signals and to perceive changes in their microenvironment is the basis for development, tissue repair and immunity as well as normal tissue homeostasis. Signal transduction is the primary means by which cells respond to external signals from their environment and coordinate complex cellular changes. The study of molecular signaling pathways allows us to understand the operation of each process of cellular signal transduction. The use of mathematical models to simulate the kinetics of chemical reactions that describe a given signaling pathway, allow us to generate testable predictions of the cell processos. To Build Kinetic predictive models to molecular signaling pathways through massive data omics produced using modern techniques, Genomics, transcriptomics, (Phospho) proteomics, is one of the current challenges faced by researchers in the field of molecular biology. Recently, the \\textit SigNetSim e-Science was introduced by the Biological Computacional and Bioinformatical Group from the Butantan Institute to face this challenge. This \\textit makes the description of molecular signaling pathways through a set of chemical reactions, which are mapped into a system of ordinary differential equations, this system will be numerically simulated and evaluated . However, changes in the structure of the pathways need to be updated manually presented in this work, which severely restricts the number of track structures that need to be tested, especially for the large models. Therefore, given this background, we present the method to modify the molecular signaling pathways. This method relies on the use of interactome database to provide a set of chemical species candidates to be included in the signaling pathway. An component integrated to SigNetSim framework able to test different hypotheses of pathways modification was developed in this project using the incremental heuristic methodology. To evaluate the implemented component, we used the MAPKs and PI3K/Akt pathways model as case study, in order to perform experimental tests and to analyze the obtained results.