Cette thèse se concentre sur la problématique suivante : comment assister un utilisateur novice pour l'aider à obtenir un dessin de son graphe qui soit adapté à ses besoins ? En effet, les méthodes de dessins actuelles, très nombreuses, nécessitent une grande expertise pour obtenir un dessin de bonne qualité. Or, par manque d'expertise, les utilisateurs novices ne peuvent pour l'instant pas produire des dessins d'une telle qualité à partir de leurs données. La solution proposée consiste à mettre en place un système interactif proposant à l'utilisateur différents dessins pour un même graphe afin qu'il obtienne un résultat qui réponde correctement à ses besoins. Ce système se base sur un algorithme de force modifié utilisé par un système d'algorithme génétique hautement modulable. L'objectif de la modification apportée à l'algorithme de dessin étant de pouvoir générer plusieurs dessins intéressants pour un même graphe. / The main objective of this thesis is to deal with assisting a novice user in drawinga graph which conforms to his/her needs. Currently, a lot of different methods for graph drawing exist, but they need an high level of expertise to be efficiently used. However, novice users don't have this kind of expertise, and thus they usually use the most common drawing methods. We design a solution to deal with this problem using an interactive system which generate several different drawings for a graph and then let the user choose which best conform to his/her constraints. This system is based on a modified force-directed algorithm controlled by a highly parameterisable genetic algorithm. The aim of the modification applied to the force-directed algorithm is to generate several different and interesting drawings of the same graph, by setting the parameters for each vertex (instead of global graph values).

L'application des techniques de séquençage à haut débit dans les dernières années a conduit à l'obtention de la séquence de génomes complets de plusieurs organismes. Le développement de nouveaux outils de génétique inverse était donc souhaitable afin de faire un usage optimal des données accumulées. Les nucléases hautement spécifiques représentent un outil unique pour induire des modifications ciblées du génome in vivo. L'induction d'une cassure double brin dans l'ADN est réparée soit par la voie de jonction d’extrémités nonhomologues soit par la voie très fidèle de la recombinaison homologue. Le ciblage d'un locus précis avec une nucléase spécifique stimule fortement la réparation de l'ADN, qui peut être utilisé pour induire des modifications ciblées dans le génome. Dans ma thèse, je vise à fournir une preuve de prinicipe pour l'utilisation des méganucléases et des transcription activator like effector nucléases (TALENs), deux classes communes de nucléases très spécifiques, comme nouveaux outils d'édition du génome chez le medaka, Oryzias latipes, et le poisson zèbre, Danio rerio. J'ai d’abord trouvé les conditions optimales d'utilisation de ces nucléases dans nos modèles de poissons. J'ai à cette occasion également développé une méthode très sensible et rapide pour la détection de modifications génomiques ciblées. J'ai ensuite induit des mutations au sein de trois gènes différents chez le poisson zèbre avec des TALENs. Les mutations dans le gène col6a1 ont conduit à la mise en évidence pour la première fois d’une technique de modification d’un site d’épissage d’un gène de poisson zèbre à l’aide d’une nucléase ciblée. Ce travail nous a permis d’établir une lignée de poisson avec une mutation dans le collagène VI alpha 1 qui est similaire à une mutation fréquemment trouvée chez les patients humains atteints de myopathie de Bethlem. De même, j’ai pu induire des mutations dans le gène nle1 du poisson zèbre qui vont permettre la mise en place de lignées de poissons mutants de ce gène. En outre, j'ai pu montrer qu’un nouveau type de nucléase, une TALEN Compact, était actif sur une cible chromosomique chez le poisson zèbre. En conclusion, les études que j'ai effectuées ont apporté la preuve de principe pour l'activité de TALENs et Compact TALENs ainsi que la première démonstration de modification de l'épissage à l’aide d’une TALEN chez le poisson zèbre et ont abouti à la mise en place d'une lignée de poisson dont le phénotype est proche d’un syndrome humain ouvrant la voie à la création de modèles pour d’autres mutations de ce type. Pour finir, je discute dans cette thèse des conditions permettant un usage le plus efficace des nucléases ciblées pour la génération de mutants et l’édition du génome. / The application of high throughput sequencing techniques in the recent years has led to obtaining the full genome sequences of many organisms. The development of novel tools for reverse genetics was thus desirable to make optimal use of the accumulated data. Site directed nucleases represent a unique platform to induce targeted genome modifications in vivo. Targeting a precise locus with a highly specific nuclease stimulates DNA repair, which can be harnessed for genome editing. Induction of a double strand break in DNA is repaired by either the error prone pathway of nonhomologous end joining or the high fidelity pathway of homologous recombination in the cell. Both mechanisms can be used to insert foreign DNA into the genome of the host. In my thesis, I aimed to provide proof of principle for the use of meganucleases and transcription activator like effector nucleases (TALENs), two common classes of site directed nucleases, as novel tools for genome editing in medaka, Oryzias latipes, and zebrafish, Danio rerio. During the first years of my thesis, I found the optimal conditions to use these nucleases in our fish models. I also developed a very sensitive and rapid method for detection of targeted genome modifications. I then induced mutations at three different endogenous loci in zebrafish with TALENs. The mutations in the col6a1 gene led to the first demonstration of splicing site modification in zebrafish using a TALE nuclease. This allowed the establishment of a fish line with a mutation in type VI collagen alpha 1 chain homologous to one mutation frequently found in human patients with Bethlem myopathy. Then I generated mutations in the nle1 gene which are heritable and from which establishment of mutant fish lines is in progress. In addition, by using the method for detection of targeted genome modifications I developed, I showed that a novel type of nuclease, a Compact TALEN, was active on a chromosomal target in zebrafish. In conclusion, the studies I performed provided proof of principle for the activity of TALENs and Compact TALENs as well as the first demonstration of TALEN-Mediated modification of splicing in zebrafish and resulted in the establishment of a fish line with mutated collagen VI. Induction of heritable mutations in the nle1 gene in zebrafish was also confirmed. Additionally, I proved that the choice of expression vector is crucial for the synthesis of active site directed nucleases for use in fish and established a novel efficient method for detection of targeted genomic mutations.

Esta dissertação apresenta o desenvolvimento, a validação e a aplicação de algoritmos para inferência de conectividade neural em registros de EEG contendo potenciais evento-relacionados (ERP). Os sinais foram caracterizados via modelos auto-regressivos multivariados (MVAR) e empregou-se a coerência parcial direcionada (PDC) no estudo das relações de causalidade entre eles. Certas características dos ERPs, como sua transitoriedade intrínseca e as múltiplas repetições em experimentos, levaram ao desenvolvimento de novos algoritmos, como a estimação de modelos conjuntos a partir de vários segmentos de sinal e um procedimento em janela deslizante capaz de descrever a evolução temporal da estatística dos sinais de interesse. Ademais, mostrou-se a possibilidade de estender os resultados da análise assintótica da estatística da PDC ao caso multi-trecho, tornando possível o estudo de sua significância estatística sem recorrer a procedimentos de reamostragem. Os algoritmos foram validados em exemplos com neural mass models, modelos não-lineares capazes de gerar sinais com características muito semelhantes a sinais de EEG reais, e aplicados a uma base de dados pública contendo resultados de experimentos com ratos. / This dissertation presents the development, validation, and application of algorithms for inferring neural connectivity in EEG signals containing event-related potentials (ERP). The time series were described via multivariate auto-regressive models (MVAR) and partial directed coherence (PDC) was used to study causal relations between them. Certain features of the ERPs, such as their transitory behavior and the existence of multiple trials in an experiment, lead to the development of a new algorithm capable of estimating a joint model from multiple segments and a sliding-window procedure for describing the nonstationarity behavior of the signals of interest. Furthermore, the possibility of extending the asymptotic results for PDC\'s statistics to the multi-trial case was demonstrated, allowing, therefore, the study of its statistical significance without recurring to resampling methods. The algorithms were validated in examples with neural mass models, non-linear models capable of generating signals with features very similar to real EEG recordings, and then applied to a publicly available dataset of experiments in rats.

Os programas de computador têm apoiado o estudo de sistemas biomédicos em que um volume considerável de dados são empregados. Na biomecânica, a análise das influências entre as articulações pode melhorar o conhecimento das lesões relacionadas à corrida associadas ao uso excessivo durante a atividade de corrida. Compreender os padrões de interação entre diferentes articulações anatômicas, durante o movimento, pode contribuir para o aprimoramento de programas de treinamento, reabilitação e prevenção a lesões. Neste trabalho, um software personalizado foi desenvolvido para implementar a Coerência Parcial Direcionada (PDC), uma abordagem no domínio da freqüência da Causalidade de Granger (GC), adequado às especificidades da fisioterapia. Com entradas independentes e padronizadas, modularização e parametrização, as rotinas investigaram a direção de interação entre diferentes canais, registrando e salvando arquivos intermediários. Separados nos três planos anatômicos, sagital, frontal e transverso, foram utilizados dados cinemáticos para analisar as interações entre tornozelo, joelho, quadril, pelve e tronco durante a corrida. Três modificações de técnica de corrida foram abordadas: com aterrissagem iniciada com o antepé, com aumento de 10% na taxa de passo e com aumento de flexão de tronco, além da habitual. As análises foram realizadas para o ciclo completo (apoio e balanço) e com separação da fase de apoio, e revelaram que essas duas estratégias de processamento são complementares. Comparando as influências proximal e distal, os procedimentos sugeriram uma predominância das interações proximal a distal, mostrando uma origem central de movimentos. Dessa forma, destaca-se a relevância em controlar e fortalecer tronco e quadril para a minimização de lesões. Considerando os resultados e a oportunidade de configuração, o software pode ser empregado para estudar outras articulações e aplicações, bem como evoluir para um sistema automatizado de apoio à decisão. / Computer programs have supported the study of biomedical systems in which a considerable amount of data is employed. In biomechanics, analysis of influences between joints can improve the knowledge of the Running-Related-Injuries (RRI) associated to overuse during running activity. Understanding the patterns of interaction among anatomical joints during movement can contribute to the improvement of training, rehabilitation and injury prevention programs. In this work, a customized software was developed to implement Partial Directed Coherence (PDC), an approach in the frequency domain of Granger Causality (GC), adapted to the physical therapy specificities. With independent and standardized inputs, modularization and parameterization, the routines investigated the interaction direction between different channels, logging and saving intermediate files. Separated in the three anatomical planes, sagittal, frontal and transverse, kinematic data were employed to analyze the interactions between ankle, knee, hip, pelvis and trunk during running. Three running technique modifications were addressed: forefoot strike landing pattern, increasing 10% of the step rate and increasing trunk flexion, in addition to usual running. The analyzes were performed for the complete cycle (stance and swing) and with separation of the stance phase, and revealed that these two processing strategies are complementary. Comparing proximal and distal influences, procedures suggested a predominance of proximal to distal interactions, showing a central origin of movements. In this way, the importance of controlling and strengthening trunk and hip to minimize injuries is highlighted. Considering the results and the processing configuration opportunity, the software can be employed to study other joints and applications, as well as evolve to an automated decision support system.

Les interactions protéine-protéine (IPPs), complexes et dynamiques, sont le cœur des réseaux protéiques cellulaires. Au niveau des synapses excitatrices, la densité post-synaptique (PSD) est un exemple typique de réseau protéique dont la structure et la composition à l’échelle nanoscopique détermine la fonction cellulaire. Ainsi, la régulation dynamique de la composition de la PSD et des mouvements des récepteurs au glutamate dans ou hors de la PSD constitue la base des théories moléculaires actuelles sur l’apprentissage et la mémoire. Dans ce contexte, durant ma thèse, j’ai étudié une classe d’IPPs faisant intervenir les domaines PDZ. En effet, durant ces dernières années, de nombreuses études ont démontré l’implication de ces interactions impliquant les domaines PDZ de la famille de PSD95 dans le ciblage synaptique et l’ancrage des récepteurs au glutamate. Cependant, en partie dû au manque d’outils adaptés, les mécanismes moléculaires sous-jacents qui contrôlent de façon dynamique leur rétention à la synapse restent mal compris. Dans le but d’étudier ces interactions impliquant des domaines PDZ, j’ai développé plusieurs stratégies de sélection par phage display basées sur l’utilisation du dixième domaine de type III de la fibronectine humaine (10Fn3) dans le but de cibler les motifs d’interaction aux domaines PDZ des récepteurs (Stargazin pour les rAMPA et GluN2A pour les rNMDA) ou les domaines PDZ eux-mêmes. En utilisant une approche multidisciplinaire, mes objectifs principaux ont été de concevoir de petits anticorps synthétiques qui nous permettront de rompre ou de stabiliser spécifiquement ces complexes protéiques, ainsi que d’observer les interactions endogènes. / Complex and dynamic protein-protein interactions are the core of protein-based networks in cells. At excitatory synapses, the postsynaptic density (PSD) is a typical example of protein-based network whose nanoscale structure and composition determines the cellular function. For instance, the dynamic regulation of PSD composition and glutamate receptors movements into or out of the PSD are the base of current molecular theories of learning and memory. In this context, during my PhD, I focused on a class of protein-protein interactions mediated by PDZ domains. Indeed, over the last decade, numerous studies have shown the critical implication of PDZ domain-mediated interactions from the PSD95 scaffolding protein family in the synaptic targeting and anchoring of glutamate receptors. However, in part due to the lack of adapted tools, the molecular mechanisms that dynamically govern their respective synaptic retention remain poorly understood. In order to investigate these PDZ domain-mediated interactions, I developed several selection strategies by phage-display based on the fibronectin type III (FN3) scaffold in order to either target the PDZ domain-binding motifs of the receptors complexes (e.g., stargazin for AMPARs and GluN2A for NMDARs) or the PDZ domains themselves. Using a multidisciplinary approach, my main objectives were to engineer small synthetic antibodies that will allow us to acutely and specifically disrupt or stabilize these protein complexes, as well as monitor endogenous interactions.

La Chimie Verte s’engage entre autres à mettre au point des procédés plus respectueux de l’environnement et à émanciper de la pétrochimie les filières industrielles de fabrication de produits. Dans ce contexte, les enzymes représentent des alternatives de choix pour réaliser des réactions de synthèse de molécules écoresponsable à partir de la biomasse végétale. - L’endoglycocéramidase II de Rhodoccoccus sp. M-777, une glycoside-hydrolase, a été la cible d’un travail d'ingénierie du site actif afin de réorienter son activité hydrolytique vers la synthèse de polyglucosides d’alkyles, de potentiels biosurfactants. Une transglycosylase permettant d’atteindre des rendements de production de plus de 70% a été obtenue. La modélisation de la mutation permet de proposer des pistes sur les raisons de cette inversion du ratio hydrolyse/transglycosylation.- Une stratégie d'évolution dirigée a été appliquée à la féruloyle-estérase A d’Aspergillus niger pour la rendre plus résistante aux chocs thermiques et à la présence de solvants, deux propriétés requises pour utiliser cette enzyme pour des réactions de transfert dans des conditions thermodynamiquement favorables. Un catalogue d’enzymes améliorées, pour les deux propriétés, a été obtenu. L'accumulation de ces connaissances permettra de pouvoir plus efficacement rationaliser le design de biocatalyseur pour la synthèse de molécules, en accord avec les attentes de la chimie verte. / Green chemistry promotes the development of more environmentally friendly processes and the ending of polluting petrochemical industries by promoting the use of renewable resources. In this context, enzymes represent interesting alternatives catalysts for chemical transformations. Notably, they constitute tools of choice for synthesis of organic molecules from plant biomass.- Endoglycoceramidase II from Rhodococcus sp. M-777, a retaining glycoside hydrolase, was subjected to active-site remodelling in order to reorient its activity towards the synthesis of alkyl-polyglucosides, molecules with potential biosurfactant properties. Thus, an efficient transglycosylase able to reach production yield of more than 70% of alkyl-cellobiosides was obtained. A modelling study help to identify the determinants of this complete reversion of the transfer / hydrolysis ratio.- A directed evolution strategy was applied to Aspergillus niger feruloyl-esterase A, in order to make it more resistant to heat shocks and to the presence of solvents, two prerequisites to use this enzyme for transfer reactions under thermodynamically favourable conditions. This led to the establishment of a catalog of optimized enzymes for their thermostability, their solvent resistance, or both properties.These results will pave the way towards a more efficient way to rationally design biocatalysts meeting the expectations of green chemistry.

Nicotine has multiple behavioral effects as a result of its action in the central nervous system. Nicotine strengthens the behaviors that lead to nicotine administration (primary reinforcement), and this effect of nicotine depends on mesotelencephalic systems of the brain that are critical to goal directed behavior, reward, and reinforcement. Nicotine also serves as a ‘reinforcement enhancer’ – drug administration enhances behaviors that lead to other drug and nondrug reinforcers. Although the reinforcement enhancing effects of nicotine may promote tobacco use in the face of associated negative health outcomes, the neuroanatomical systems that mediate this effect of nicotine have never been described. The ventral tegmental area (VTA) is a nucleus that serves as a convergence point in the mesotelencephalic system, plays a substantial role in reinforcement by both drug and nondrug rewards and is rich in both presynaptic and postsynaptic nicotinic acetylcholine receptors (nAChRs). Therefore, these experiments were designed to determine the role of the VTA and nAChR subtypes in the reinforcement enhancing effect of nicotine. Transiently inhibiting the VTA with a gamma amino butyric acid (GABA) agonist cocktail (baclofen and muscimol) reduced both primary reinforcement by a visual stimulus and the reinforcement enhancing effect of nicotine, without producing nonspecific suppression of activity. Intra-VTA infusions of a high concentration of mecamylamine a nonselective nAChR antagonist, or methylycaconitine, an α7 nAChR antagonist, did not reduce the reinforcement enhancing effect of nicotine. Intra-VTA infusions of a low concentration of mecamylamine and dihydro-beta-erythroidine (DHβE), a selective antagonist of nAChRs containing the *β2 subunit, attenuated, but did not abolish, the reinforcement enhancing effect of nicotine. In follow-up tests replacing systemic nicotine injections with intra-VTA infusions (70mM, 105mM) resulted in complete substitution of the reinforcement enhancing effects – increases in operant responding were comparable to giving injections of systemic nicotine. These results suggest that *β2-subunit containing nAChRs in the VTA play a role in the reinforcement enhancing effect of nicotine. However, when nicotine is administered systemically these reinforcement enhancing effects may depend on the action of nicotine at nAChRs in multiple brain nuclei.

Various solutions have been proposed to “fix” performance management (PM) over the last several decades. Pulakos, Mueller Hanson, Arad, and Moye (2015) have presented a holistic approach to improving PM in organizations. Although this approach addresses several key elements related to the social context of PM, namely the buy-in of organizational stakeholders, timely and regular feedback, and future-directed feedback, we believe that several robust findings from the PM research literature could further improve this process. Are Pulakos et al. looking at the forest but missing the trees? In the following commentary, we offer several reasons that performance judgments and perhaps even informal ratings are still operating and occurring in the proposed holistic system. Therefore, advancements in other areas of PM research may offer additional ways to fix PM.

Glucosyltransferases (GTs) are the important group of enzymes which facilitates the incorporation of UDPactivated glucose to a corresponding acceptor molecule through glucosylation. Glucosylation is a common alteration reaction in plant metabolism and is regularly associated with the production of secondary metabolites. Glucosylation serves a number of roles within metabolism including: stabilizing structures, affecting solubility, transport, and regulating the bioavailability of the compounds for other metabolic processes. GTs involved in secondary metabolism share a conserved 44 amino acid residue motif (60–80% identity) known as the plant secondary product glucosyltransferase (PSPG) box, which has been demonstrated to include the UDP-sugar binding moiety. Among the secondary metabolites, flavonoid glycosides affect taste characteristics in citrus making the associated glucosyltransferases particularly interesting targets for biotechnology applications in these species. Custom design of enzymes requires understanding of structure/function of the protein. The present study focuses on creating mutant Flavonol- 3-O- Glucosyltransferases proteins using site-directed mutational analysis and testing the effect of each mutation on substrate specificity and kinetic properties of the enzyme.

Citrus paradisi 3-O-glucosyltransferase (Cp3GT, Genbank Protein ID: ACS15351) and Citrus sinensis 3-O-glucosyltransferase (Cs3GT, Genbank Protein ID: AAS00612.2) share 95% amino acid sequence identity. Cp3GT was previously established as a flavonol-specific 3-O-glucosyltransferase by direct enzymatic analysis. Cs3GT is annotated as a flavonoid-3-O-glucosyltransferase and predicted to use anthocyanidins as substrates based on gene expression analysis correlated with the accumulation of anthocyanins in C. sinensis cv. Tarocco, a blood orange variety. Mutant enzymes in which amino acids found in Cs3GT were substituted for position equivalent residues in Cp3GT were generated, heterologously expressed in yeast, and characterized for substrate specificity. Structure–function relationships were investigated for wild type and mutant glucosyltransferases by homology modelling using a crystallized Vitis viniferaanthocyanidin/flavonol 3-O-GT (PDB: 2C9Z) as template and subsequent substrate docking. All enzymes showed similar patterns for optimal temperature, pH, and UDP/metal ion inhibition with differences observed in kinetic parameters. Although changes in the activity of the mutant proteins as compared to wild type were observed, cyanidin was never efficiently accepted as a substrate.