In this thesis, I present a model of multidomain evolution with associated algorithms and software for phylogenetic analysis of multidomain families, as well as applications of this novel methodology to case-studies and the human genome. Phylogenetic analysis is of central importance to understanding the origins and evolution of life on earth. In biomedical research, molecular phylogenetics has proved an essential tool for practical applications. Current molecular phylogenetic methods are not equipped, however, to model many of the unique characteristics of multidomain families. Genes that encode this large and important class of proteins are characterized by a mosaic of sequence fragments that encode structural or functional modules, called domains. Multidomain families evolve via domain shuffling, a process that includes insertion, internal duplication, and deletion of domains. This versatile evolutionary mechanism played a transformative role in major evolutionary transitions, including the emergence of multicellular animals and the vertebrate immune system. Multidomain families are ill-suited to current methods for phylogeny reconstruction due to their mosaic composition. Different regions of the same protein may have different evolutionary histories. Moreover, a protein may contain domains that also occur in otherwise unrelated proteins. These attributes pose substantial obstacles for phylogenetic methods that require a multiple sequence alignment as input. In addition, current methods do not incorporate a model of domain shuffling and hence, cannot infer the events that occurred in the history of the family. I address this problem by treating a multidomain family as a set of co-evolving domains, each with its own history. If the family is evolving by vertical descent from a conserved set of ancestral domains, then all constituent domains will have the same phylogenetic history. Disagreement between domain tree topologies is evidence that the family evolved through processes other than speciation and gene duplication. My algorithms exploit this information to reconstruct the history of domain shuffling in the family, as well as the timing of these events and the ancestral domain composition. I have implemented these algorithms in software that outputs the most parsimonious history of events for each domain family. The software also reconstructs a composite family history, including duplications, insertions and losses of all constituent domains and ancestral domain composition. My approach is capable of more detailed and accurate reconstructions than the widely used domain architecture model, which ignores sequence variation between domain instances. In contrast, my approach is based on an explicit model of events and captures sequence variation between domain instances. I demonstrate the utility of this method through case studies of notch-related proteins, protein tyrosine kinases, and membrane-associated guanylate kinases. I further present a largescale analysis of domain shuffling processes through comparison of all pairs of domain families that co-occur in a protein in the human genome. These analyses suggest that (1) a remarkably greater amount of domain shuffling may have occurred than previously thought and (2) that it is not uncommon for the same domain architecture to arise more than once through independent events. This stands in contrast to earlier reports that convergent evolution of domain architecture is rare and suggests that incorporating sequence variation in evolutionary analyses of multidomain families is a crucial requirement for accurate inference.

No description available.

A crescente demanda de tráfego em redes de acesso pressiona a melhor utilização das redes backbone, que são utilizadas para transporte de grandes taxas de dados em diversos domínios (Sistemas Autônomos, SAs). Com o aumento destas redes, aumenta-se a complexidade de topologia das interligações entre domínios. Desta maneira, roteamento de tráfego e pontos de interconexão de SAs (nós de borda) são questões importantes para o desempenho destas redes, que são operadas por diversos provedores que podem utilizar protocolos de comunicação distintos. Neste sentido, o roteamento interdomínio apresenta desafios como a publicação ou não de informações de parâmetros de rede de SAs e como tratar esta questão de maneira globalizada, com novos protocolos e suas especificações. Em termos de pontos de interconexão de SAs, a especificação dos locais onde enlaces inter-redes são conectados aos domínios são importantes para seu desempenho, já que são responsáveis por toda troca de tráfego entre redes distintas. O trabalho considera redes ópticas opacas e translúcidas em cenário multidomínio com bandas multigranulares. Neste cenário é estudado um algoritmo de roteamento multidomínio. No trabalho também é feito um planejamento, especificando em quais nós serão conectados enlaces interdomínio. A principal contribuição deste trabalho é o estudo de planejamento de enlaces interdomínio, com a proposta de um método para escolha de nós de borda (sistematização), com objetivo de diminuir a probabilidade de bloqueio interdomínio. A sistematização é baseada em estudos de resultados de algoritmo genético desenvolvido para o mesmo propósito e sua utilização diminui em até 42% o bloqueio interdomínio. Um algoritmo de alocação de banda também foi desenvolvido para redes multidomínio, que considera parâmetros da camada de rede e óptica para o cálculo de peso de enlaces para encontrar caminhos ópticos entre nós fonte e destino. Os resultados mostram diminuição de até 35% no bloqueio interdomínio com a modificação feita em algoritmo proposto na literatura. / The huge demand for traffic in last mile networks push the better utilization of backbone networks, which are used to transport large data rates in several domains (Autonomous Systems, ASs). With this growth, the topology complexity of interdomain links increases. Then, traffic routing and interconnection points of ASs (border nodes) are relevant questions for the performance of these networks, which are managed by several providers that can use distinct communications protocols. Thus, the interdomain routing presents challenges such as the decision on publishing or not the network´s parameters from ASs and how to deal with this issue in a global way, with new protocols and its specifications. For interconnection points between ASs, the points where interdomain links are connected are important for their performances, since they are responsible for all traffic exchange between distinct networks. This work considers opaque and translucent optical networks in a multidomain scenario with multigranular data rates. In this scenario a multidomain routing algorithm is studied and a network planning is developed, specifying the nodes where interdomain links are connected. The main contribution of this work is the planning of interdomain links, with the proposal of a method for border nodes selection (systematization), with the objective of decreasing the interdomain blocking probability. The systematization is based on the results from a genetic algorithm developed for the same purpose and its utilization decrease up to 42% of the interdomain blocking. A bandwidth allocation algorithm was also created for multidomain scenarios, that considers parameters from network and optical layer for the link weight calculation in order to find optimal paths. The results show a decreasing of up to 35% for interdomain blocking with a contribution based on literature\'s work.

A crescente demanda de tráfego em redes de acesso pressiona a melhor utilização das redes backbone, que são utilizadas para transporte de grandes taxas de dados em diversos domínios (Sistemas Autônomos, SAs). Com o aumento destas redes, aumenta-se a complexidade de topologia das interligações entre domínios. Desta maneira, roteamento de tráfego e pontos de interconexão de SAs (nós de borda) são questões importantes para o desempenho destas redes, que são operadas por diversos provedores que podem utilizar protocolos de comunicação distintos. Neste sentido, o roteamento interdomínio apresenta desafios como a publicação ou não de informações de parâmetros de rede de SAs e como tratar esta questão de maneira globalizada, com novos protocolos e suas especificações. Em termos de pontos de interconexão de SAs, a especificação dos locais onde enlaces inter-redes são conectados aos domínios são importantes para seu desempenho, já que são responsáveis por toda troca de tráfego entre redes distintas. O trabalho considera redes ópticas opacas e translúcidas em cenário multidomínio com bandas multigranulares. Neste cenário é estudado um algoritmo de roteamento multidomínio. No trabalho também é feito um planejamento, especificando em quais nós serão conectados enlaces interdomínio. A principal contribuição deste trabalho é o estudo de planejamento de enlaces interdomínio, com a proposta de um método para escolha de nós de borda (sistematização), com objetivo de diminuir a probabilidade de bloqueio interdomínio. A sistematização é baseada em estudos de resultados de algoritmo genético desenvolvido para o mesmo propósito e sua utilização diminui em até 42% o bloqueio interdomínio. Um algoritmo de alocação de banda também foi desenvolvido para redes multidomínio, que considera parâmetros da camada de rede e óptica para o cálculo de peso de enlaces para encontrar caminhos ópticos entre nós fonte e destino. Os resultados mostram diminuição de até 35% no bloqueio interdomínio com a modificação feita em algoritmo proposto na literatura. / The huge demand for traffic in last mile networks push the better utilization of backbone networks, which are used to transport large data rates in several domains (Autonomous Systems, ASs). With this growth, the topology complexity of interdomain links increases. Then, traffic routing and interconnection points of ASs (border nodes) are relevant questions for the performance of these networks, which are managed by several providers that can use distinct communications protocols. Thus, the interdomain routing presents challenges such as the decision on publishing or not the network´s parameters from ASs and how to deal with this issue in a global way, with new protocols and its specifications. For interconnection points between ASs, the points where interdomain links are connected are important for their performances, since they are responsible for all traffic exchange between distinct networks. This work considers opaque and translucent optical networks in a multidomain scenario with multigranular data rates. In this scenario a multidomain routing algorithm is studied and a network planning is developed, specifying the nodes where interdomain links are connected. The main contribution of this work is the planning of interdomain links, with the proposal of a method for border nodes selection (systematization), with the objective of decreasing the interdomain blocking probability. The systematization is based on the results from a genetic algorithm developed for the same purpose and its utilization decrease up to 42% of the interdomain blocking. A bandwidth allocation algorithm was also created for multidomain scenarios, that considers parameters from network and optical layer for the link weight calculation in order to find optimal paths. The results show a decreasing of up to 35% for interdomain blocking with a contribution based on literature\'s work.

Domínio proteico é uma sequência de aminoácidos evolutivamente conservada e funcionalmente independente. Um dos aspectos mais importantes do estudo de uma proteína que contem múltiplos domínios é o entendimento da comunicação, entre os diferentes domínios, e seu papel biológico. Essa comunicação em maior parte é feita pela interação direta entre domínios. A interação poderia ser tratada como uma clássica interação proteína-proteína. Entretanto, proteínas multidomínio possuem restrições determinadas por suas regiões conectoras. Os conectores interdomínio impõem restrições e limitam espaço conformacional dos domínios. Apresentamos aqui o MAD, uma rotina capaz de obter modelos tridimensionais de alta resolução para proteínas, contendo qualquer número de domínios, a partir de sua sequencia primária. Os domínios conservados são identificados utilizando a base de domínios conservados (CDD) e seus limites são utilizados para definir as regiões conectoras. É criado um ensamble de possíveis dobramentos dos conectores e sua distribuição de distâncias C/N-terminais são utilizadas como restrição espacial na busca pela interação entre os domínios.Os modelos dos domínios são obtidos por uma modelagem comparativa. Foi implementada uma heurística, capaz de lidar com a natureza combinatorial dos múltiplos domínios e com a necessidade imposta pela limitação computacional de realizar o docking dos domínios em forma de pares. Todas combinações de domínios são submetidas as rotinas de docking. Aplica-se filtro de distância e energético, excluindo as conformações que apresentam distância C/N-terminal entre domínios maior do que o valor máximo observado no ensamble de conectores e seleciona as conformações energeticamente mais favoráveis. As conformações são submetidas a uma rotina de agrupamento hierárquico baseada em sua similaridade estrutural. Para a segunda fase as conformações selecionadas são pareadas com seu domínio complementar e ressubmetidas a rotina de docking até que todas as fases tenham sido completadas. Foi criado um conjunto de testes a partir do Protein Data Bank contendo 54 proteínas multidomínio para que a rotina de docking do MAD fosse comparada com outros softwares utilizados pela comunidade cientifica, mostrou-se superior ou equivalente aos métodos testados. A capacidade de utilizar dados experimentais foi demostrada através da proposição de um modelo da forma ativa da enzima tirosina fosfatase 2, nunca observado experimentalmente. A rotina de docking foi expandida paralelamente em uma aplicação standalone e utilizada na resolução de diversos problemas biológicos. Concluímos que a inovação metodológica proposta pelo MAD é de grande valia para a modelagem molecular e tem potencial de gerar uma nova perspectiva a respeito da interação de proteína multidomínio, visto que é possível analisar essas proteínas em sua plenitude e não como domínios separados. / Protein domain is an evolutionary conserved and functionally independent amino acid sequence. One of the most important aspects of the study of a protein that contains multiple domains is the understanding of communication between the different areas, and their biological role. This communication is made mostly by direct interaction between domains. The interaction could be treated as a classical protein-protein interaction. However, multidomain proteins have certain restrictions for its connector regions. The intra connectors impose restrictions and limit conformational space of the domains. We present the MAD, a routine able to get three-dimensional models of high-resolution protein, containing any number of domains, from its primary sequence. The conserved domains are identified using the basic conserved domains database (CDD) and its boundaries are used to define the connector regions. This creates a ensemble of possible folding of the connectors and distribution of distances C/N-terminals are used as spatial restriction in the search for interaction between domains.Os models of the domains are obtained by comparative modelling. A heuristic able to handle the combinatorial nature of the multiple areas and the need imposed by the computer to perform the limitation of the docking areas as pairs was implemented. All combinations of domains are referred to the docking routines. Distance and energy filters are applied, excluding conformations that have C/N-terminal domains distances larger than the maximum value observed in the connectors ensemble and selects the most favourable energy conformations. Conformations are subjected to hierarchical clustering routine based on their structural similarity. For the second phase, the selected conformations are paired with its complementary domain and resubmitted to the docking routine until all phases have been completed. A test set has been created from the Protein Data Bank containing 54 multidomain proteins so that the docking routine of MAD could be compared with other software used by the scientific community, it has been shown to be superior or equivalent to the tested methods. The ability to use experimental data was demonstrated by proposing a model of the active form of tyrosine phosphatase enzyme 2, never observed experimentally. The docking routine was expanded in a standalone application and used in solving various biological problems. We conclude that the methodological innovation proposed by the MAD is very useful for molecular modelling and has the potential to generate a new perspective on multidomain protein interaction as you can analyse these proteins in its entirety and not as separate domains.

Cette étude s'intègre dans le cadre d'un développement accru de nouvelles techniques de contrôle non destructif des matériaux magnétiques basées sur les phénomènes de couplage magnéto-mécanique. L'objectif est de promouvoir des méthodes originales de mesure des propriétésmagnétiques permettant d'évaluer quantitativement l'état thermo-métallurgico-mécanique d'un matériau par une simple identification inverse.Nous proposons dans ce document un modèle magnéto-mécanique couplé dit modèle multidomaine compatible avec la procédure de contrôle magnétique. Ce modèle analytique permet de simuler le comportement magnétique et magnétostrictif d'un matériau magnétique soumis à un chargement mécanique unidirectionnel. Il a montré une bonne adaptabilité à des états de contraintes et structures magnétiques variées. Le modèle multidomaine a pu être validé dans le cas d'un chargement élastique uniaxial et multixial par un simple recours à une contrainte équivalente magnéto-mécanique. Il a pu également intégrer les éléments nécessaires à la modélisation de l'influence de la plasticité sur l'état magnétique. La plasticité est introduite via un état de contrainteinterne caractérisant une structure hétérogène biphasée. Des corrélations intéressantes entre variables d'écrouissage macroscopiques et propriétés magnétiques ont été élaborées et l'approche a été validée sur un acier dual phases.Nous nous sommes enfin intéressées à a mise en place d'un protocole expérimental novateur assurant un suivi continuel du comportement piézomagnétique du matériau au cours d'un essai de fatigue. Cette technique permet d'estimer la limite d'endurance des matériaux magnétiques. / This study is within a recent research largely motivated by the possibility of development of new non-destructive techniques based on the magneto-mechanical coupling. Thus, the issue is to propose original magnetic methods allowing a quantitative evaluation of the thermo-metallurgicomechnical state of ferromagnetic materials by a simple inverse identification.We propose in this document a coupled magneto-mechanical modeling called multidomain modeling suitable for the non-destructive process. This model is able to simulate magnetic and magnetostrictive behaviors of materials submitted to an uniaxial mechanical loading. It exhibits an adaptability to various mechanical states and magnetic structures. Multidomain modeling provides good results in the case of elastic loading either uniaxial or multiaxial by the use of an equivalent stress. It can also integrate the key elements for modeling the effect of plasticity on the magneticbehavior. Plasticity is introduced through internal stress characterizing heterogenous biphasic structure. Interesting correlations between macroscopic hardening parameters and magnetic properties are shown and the plasticity approach is confirmed by experiments carried out on a dual phase steel.Finally, we propose an experimental protocol allowing in situ continuous investigation of the piezomagnetic behavior during fatigue test. This experimental technique permits the estimation of fatigue limit of ferromagnetic materials.

Domínio proteico é uma sequência de aminoácidos evolutivamente conservada e funcionalmente independente. Um dos aspectos mais importantes do estudo de uma proteína que contem múltiplos domínios é o entendimento da comunicação, entre os diferentes domínios, e seu papel biológico. Essa comunicação em maior parte é feita pela interação direta entre domínios. A interação poderia ser tratada como uma clássica interação proteína-proteína. Entretanto, proteínas multidomínio possuem restrições determinadas por suas regiões conectoras. Os conectores interdomínio impõem restrições e limitam espaço conformacional dos domínios. Apresentamos aqui o MAD, uma rotina capaz de obter modelos tridimensionais de alta resolução para proteínas, contendo qualquer número de domínios, a partir de sua sequencia primária. Os domínios conservados são identificados utilizando a base de domínios conservados (CDD) e seus limites são utilizados para definir as regiões conectoras. É criado um ensamble de possíveis dobramentos dos conectores e sua distribuição de distâncias C/N-terminais são utilizadas como restrição espacial na busca pela interação entre os domínios.Os modelos dos domínios são obtidos por uma modelagem comparativa. Foi implementada uma heurística, capaz de lidar com a natureza combinatorial dos múltiplos domínios e com a necessidade imposta pela limitação computacional de realizar o docking dos domínios em forma de pares. Todas combinações de domínios são submetidas as rotinas de docking. Aplica-se filtro de distância e energético, excluindo as conformações que apresentam distância C/N-terminal entre domínios maior do que o valor máximo observado no ensamble de conectores e seleciona as conformações energeticamente mais favoráveis. As conformações são submetidas a uma rotina de agrupamento hierárquico baseada em sua similaridade estrutural. Para a segunda fase as conformações selecionadas são pareadas com seu domínio complementar e ressubmetidas a rotina de docking até que todas as fases tenham sido completadas. Foi criado um conjunto de testes a partir do Protein Data Bank contendo 54 proteínas multidomínio para que a rotina de docking do MAD fosse comparada com outros softwares utilizados pela comunidade cientifica, mostrou-se superior ou equivalente aos métodos testados. A capacidade de utilizar dados experimentais foi demostrada através da proposição de um modelo da forma ativa da enzima tirosina fosfatase 2, nunca observado experimentalmente. A rotina de docking foi expandida paralelamente em uma aplicação standalone e utilizada na resolução de diversos problemas biológicos. Concluímos que a inovação metodológica proposta pelo MAD é de grande valia para a modelagem molecular e tem potencial de gerar uma nova perspectiva a respeito da interação de proteína multidomínio, visto que é possível analisar essas proteínas em sua plenitude e não como domínios separados. / Protein domain is an evolutionary conserved and functionally independent amino acid sequence. One of the most important aspects of the study of a protein that contains multiple domains is the understanding of communication between the different areas, and their biological role. This communication is made mostly by direct interaction between domains. The interaction could be treated as a classical protein-protein interaction. However, multidomain proteins have certain restrictions for its connector regions. The intra connectors impose restrictions and limit conformational space of the domains. We present the MAD, a routine able to get three-dimensional models of high-resolution protein, containing any number of domains, from its primary sequence. The conserved domains are identified using the basic conserved domains database (CDD) and its boundaries are used to define the connector regions. This creates a ensemble of possible folding of the connectors and distribution of distances C/N-terminals are used as spatial restriction in the search for interaction between domains.Os models of the domains are obtained by comparative modelling. A heuristic able to handle the combinatorial nature of the multiple areas and the need imposed by the computer to perform the limitation of the docking areas as pairs was implemented. All combinations of domains are referred to the docking routines. Distance and energy filters are applied, excluding conformations that have C/N-terminal domains distances larger than the maximum value observed in the connectors ensemble and selects the most favourable energy conformations. Conformations are subjected to hierarchical clustering routine based on their structural similarity. For the second phase, the selected conformations are paired with its complementary domain and resubmitted to the docking routine until all phases have been completed. A test set has been created from the Protein Data Bank containing 54 multidomain proteins so that the docking routine of MAD could be compared with other software used by the scientific community, it has been shown to be superior or equivalent to the tested methods. The ability to use experimental data was demonstrated by proposing a model of the active form of tyrosine phosphatase enzyme 2, never observed experimentally. The docking routine was expanded in a standalone application and used in solving various biological problems. We conclude that the methodological innovation proposed by the MAD is very useful for molecular modelling and has the potential to generate a new perspective on multidomain protein interaction as you can analyse these proteins in its entirety and not as separate domains.

La concurrence industrielle pousse les entreprises à développer de nouveaux produits innovants. En outre, les industriels sont soumis à de plus en plus de contraintes issues des différents acteurs du processus de conception (fabricants, utilisateurs) ou liées aux évolutions de leur environnement (réglementations, technologies). Il leur est donc nécessaire d’adapter leurs activités de conception et plus particulièrement les étapes d’évaluation pour que les produits développés correspondent aux exigences multiples de toutes les parties prenantes. Ces contraintes se traduisent par l’intégration de nouveaux domaines ou l’évolution de domaines existants dans le processus de conception.Dans ce contexte, l’objectif de ces travaux de thèse est de formaliser et d’apporter des méthodes permettant l’évaluation multidomaine dans le processus de conception. Ce manuscrit s’intéresse donc à la problématique suivante : « Comment les phases d’évaluation dans un processus de conception multidomaine peuvent-elles permettre d’accroître les performances du produit à partir des représentations intermédiaires ? ». Pour cela nous proposons une méthodologie permettant d’améliorer le processus d’évaluation en créant un outil d’évaluation multidomaine et en formalisant les étapes du processus d’évaluation pour apporter des méthodes d’amélioration adaptées aux domaines concernés par le produit à concevoir. Des expérimentations sont menées afin de valider leurs apports dans le processus de conception en s’intéressant notamment à trois domaines : l’innovation, la prise en compte des impacts environnementaux et la fabrication additive. Ces expérimentations ont permis de valider la méthodologie de création de l’outil d’évaluation et l’apport des étapes d’amélioration. / Industrial competition is driving companies to develop new innovative products. In addition, industrial companies are increasingly subject to constraints from the various actors in the design process (manufacturers, users) or linked to changes in their environment (regulations, technologies). It is therefore necessary for them to adapt their design activities and more particularly the evaluation steps so that the products developed correspond to the multiple requirements of all stakeholders. These constraints result in the integration of new domains or the evolution of existing domains into the design process.In this context, the objective of this thesis work is to formalize and provide methods for multidomain evaluation in the design process. This manuscript therefore addresses the following question: "How can evaluation phases in a multidomain design process help to increase the performance of intermediate representations? ». To this end, we propose a methodology to improve the evaluation process by creating a multidomain evaluation tool and formalizing the steps of the evaluation process to provide improvement methods adapted to the areas concerned by the product to be designed. Experiments are being carried out to validate their contribution to the design process, focusing in particular on three areas: innovation, taking into account environmental impacts and additive manufacturing. These experiments made it possible to validate the methodology for creating an evaluation tool and the contribution of the improvement steps.

La création de nouvelles fonctions basées sur la reconnaissance protéique et sur l'assemblage de domaines est un enjeu majeur en biotechnologie et est un moyen de comprendre les relations structures/fonctions des protéines engagées dans des processus d'interactions. Aujourd’hui, des bibliothèques de protéines artificielles obtenues par ingénierie peuvent être sources de protéines aux propriétés de reconnaissance analogues à celles des dérivés d’anticorps.L’équipe Modélisation et Ingénierie des Protéines a ainsi construit une banque de protéines à motifs structuraux répétés appelées « alphaReps ». Les alphaReps présentent des propriétés remarquables en termes de production et de stabilité. Contrairement à la plupart des anticorps et dérivés d’anticorps, elles peuvent même s’exprimer sous forme fonctionnelle dans le cytoplasme de cellules eucaryotes. De tels objets peuvent donc maintenant être utilisés comme des briques élémentaires en vue d’une ingénierie modulaire. Ainsi la construction de nouvelles fonctions de reconnaissance optimisées tant au niveau de la spécificité que de l’affinité sera possible en réarrangeant et/ou dupliquant ces briques élémentaires.Un premier volet de ce projet de thèse a consisté à construire puis étudier les propriétés biophysiques de protéines bidomaines basées sur les alphaReps afin de mieux comprendre les comportements adoptés par de telles constructions. Outre l’aspect fondamental de cette question, cette étude donnera « les règles » pour moduler de façon contrôlée les interactions entre ces protéines. Les résultats montrent qu'il est possible de créer de nouvelles fonctions par simple ajout d'un linker entre deux alphaReps : avidité, coopérativité, changement de conformation.Dans un second temps, l’objectif a été de développer, à partir des protéines bidomaines précédemment étudiées, de nouveaux biosenseurs basés sur le FRET (Förster Resonance Energy Transfer) pouvant être utilisés in vivo et in vitro. Cette deuxième partie présente deux biosenseurs avec des limites de détection de l'ordre du nanomolaire. Les alphaReps utilisées dans ces constructions pouvant être changées en fonction de la cible souhaitée, il s'agit ici d'une preuve de concept pouvant être généralisée à n'importe quelle cible.Enfin la dernière partie de cette thèse s'est portée sur la conception et l'étude de nouveaux biosenseurs génétiquement codables. Ces biosenseurs présentent notamment l’avantage d’être utilisables immédiatement après production et ne nécessitent donc plus d’étape de couplage chimique. Les résultats obtenus montrent que la création de tels biosenseurs est possible mais qu’une optimisation reste encore nécessaire pour améliorer leur spécificité, leur stabilité et leur capacité de détection. / The creation of new protein functions based on recognition and molecular assembly is not only a major goal in biotechnology but is also a means to understand the relation structure/function of proteins involved in interaction processes. Today, libraries of artificial proteins obtained by engineering can be a source of proteins with recognition properties similar to the properties of antibodies.The team Protein Engineering and Modeling has thus created a library of proteins with structural repeats called the “alphaReps”. The alphaReps present remarkable properties in terms of production and stability. Unlike most of the antibodies and their derivatives, they can even be expressed and functional in the cytoplasm of eukaryotic cells. Such objects can therefore be used as building bricks in modular engineering. The construction of new optimized recognition functions both in specificity and in affinity can then be possible by rearranging or duplicating these elementary bricks.The first part of this thesis project consisted in the construction and study of the biophysical properties of bidomain proteins based on alphaRep in order to have a better understanding of the behaviour of such constructions. Beside the fundamental aspect of this question, this study will give the “rules” to modulate the interactions between these proteins in a controlled way. The results show that it is possible to create new functions such as avidity, cooperativity, conformational change, simply by adding a linker between two alphaReps.In a second step, the goal was to develop, with the bidomain proteins previously studied, new biosensors based on the FRET (Förster Resonance Energy Transfer) which can be used in vivo and in vitro. This second part presents two biosensors with limits of detection in the nanomolar range. Since the alphaReps used in these constructions can be changed depending on the chosen target, it is a proof of concept which can be adapted to any desired target.Finally, the third part of this thesis focused on the development of genetically codable biosensors. These biosensors have the particular advantage of being usable directly after production and therefore no longer require a chemical coupling step. The results show that the development of such biosensors is worth considering but an optimization is still required in order to improve their specificity, their stability and their detection capacity.

Orientador: Iris Concepción Linares de Torriani / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Fisica Gleb Wataghin / Made available in DSpace on 2018-08-15T22:30:16Z (GMT). No. of bitstreams: 1 Silva_JulioCesarda_D.pdf: 7791031 bytes, checksum: 5711654f743b7d6fb045861e9239ad8c (MD5) Previous issue date: 2010 / Resumo: As técnicas de caracterização estrutural de macromoléculas tradicionais se baseiam no fato de uma macromolécula possuir uma conformação compacta e estruturada. Partes flexíveis ou regiões desordenadas têm sido sempre consideradas como grandes obstáculos para técnicas como a cristalografia de raios-X e a ressonância magnética nuclear (RMN). A necessidade de entender a atividade funcional de proteínas nativamente desenoveladas e de proteínas flexíveis com múltiplos domínios tem adquirido grande importância recentemente, mesmo porque essas proteínas desafiam o paradigma de que uma proteína precisa de uma estrutura bem definida para ser funcional. É bem nesse ponto que a técnica de espalhamento de raios-X a baixos ângulos (SAXS) surge oferecendo ferramentas únicas para realizar estudos de macromoléculas flexíveis ou parcialmente desestruturadas, com aplicações muito bem sucedidas em polímeros, matéria mole e macromoléculas em solução. Neste trabalho de tese decidimos enfrentar o desafio de caracterizar proteínas que não possuem uma estrutura bem definida. A teoria do espalhamento mereceu especial cuidado para se adequar tanto aos métodos experimentais da técnica quanto aos tratamentos matemáticos em cálculos usados para estudar esse tipo de proteínas. Apresentamos aqui o estudo de duas proteínas pertencentes à classe das proteínas nativamente desenoveladas: (1) a proteína FEZ1, que é necessária para o crescimento de axônios; (2) a proteína Ki-1/57, que é encontrada em diversas células com câncer principalmente em tumores do sistema linfático. Estudamos também algumas proteínas com múltiplos domínios conectados por regiões flexíveis e que são: (1) duas chaperonas da classe das HSP40 (proteínas Sis1 e Ydj1) juntamente com construções onde alguns domínios dessas proteínas foram cortados; (2) a proteína ribonucléica heterogênea hnRNP-Q que está envolvida em importantes funções do RNA. Experiências de SAXS foram realizadas, fornecendo parâmetros dimensionais e informações de forma dessas proteínas em solução. Modelos de baixa resolução das possíveis conformações foram calculados a partir das curvas de SAXS usando métodos de modelagem ab initio combinados com modelagem de corpos rígidos. Os resultados forneceram informações importantes para elucidar as funções biológicas dessas proteínas. É importante ressaltar que, para realizar os estudos com proteínas em solução, é necessário contar com uma instrumentação adequada e devidamente montada para a aplicação da técnica de SAXS. Para isso, durante o período de desenvolvimento deste doutorado houve um grande investimento na montagem, teste e caracterização de instrumentos, junto à equipe de profissionais do Laboratório Nacional de Luz Síncrotron (LNLS), completando o comissionamento da estação experimental SAXS2 do LNLS / Abstract: The traditional techniques for structural characterization of macromolecules are based on a compact and structured conformation of the macromolecule. Flexible or disordered regions have usually been regarded as a great hindrance to techniques like X-ray protein crystallography and nuclear magnetic resonance (NMR). The need to study functional activity of natively unfolded proteins and flexible multidomain proteins came to the light rather recently, defying the classical structure¿function paradigm where a protein must have a well-defined 3-D structure to be functional. In this type of situation, the small-angle X-ray scattering (SAXS) technique appears as a unique tool to deal with this problem. Indeed, the application of SAXS methods to the characterization of soft matter (e.g. polymers) and macromolecules in solution has already succeeded during the last years. In this work we decided to face the challenge of characterizing proteins that do not have a well defined structure. The SAXS experimental technique as well as the mathematical methods and calculations needed special attention in order to be correctly applied to study the specific problem of unstructured proteins in solution. Thus, it was possible to find evidence of the structural details of these proteins and obtain a low resolution 3-D average structure. Here we present the study of two proteins that belong to the group of natively unfolded proteins: (1) The FEZ1 protein, which is necessary for axon growth, and (2) the proteins indentified as Ki-1/57, which is found in diverse cancer cells mainly in lymphatic systems tumors. We also studied some flexible multidomain proteins: (1) two chaperones from the groups of HSP40 (the proteínas Sis1 e Ydj1), and two mutant constructions where some domains were deleted; (2) the heterogeneous ribonucleoprotein hnRNP-Q which is related to an array of important functions of RNA. Several SAXS experiments were performed providing overall parameters and important shape information about those proteins in solution. Low resolution models for the possible conformations of these proteins were restored from the SAXS curves using ab initio modeling methods combined with rigid body modeling. The SAXS results provided a unique structural background for the biologists to deal with the function of these proteins. SAXS experiments with proteins in solution demand the use of a specific instrumentation properly developed for those studies. So, it is important to mention that, throughout the duration of this doctorate, specific instrumentation development and testing was done together with the technical staff of the Brazilian Synchrotron Light Laboratory (LNLS, Campinas, SP, Brazil), collaborating with the commissioning of the new SAXS2 workstation, completed in 2008 / Doutorado / Física / Doutor em Ciências