Les objectifs de cette thèse étaient d'apporter une percée conceptuelle pour une compréhension en profondeur des mécanismes moléculaires des protéines intrinsèquement désordonnées (IDPs) et de leurs rôles dans la physiologie cellulaire de Chlamydomonas reinhardtii. La combinaison d’approches expérimentale et bioinformatique m’a permis d’identifier 682 protéines thermorésistantes chez C. reinhardtii. Parmi celles-ci, 299 protéines sont systématiquement prédites comme potentielles IDP par quatre algorithmes de prédiction de désordre. Nos résultats indiquent que le pourcentage désordonné moyen de ces protéines prédites comme étant des IDPs est d'environ 20%, et la plupart d'entre elles (~70%) sont adressées à d'autres compartiments que la mitochondrie et le chloroplaste. Leur composition en acides aminés est biaisée par rapport à d'autres IDPs de la base de données de protéines désordonnées (DisProt). Ces IDPs potentielles jouent des fonctions moléculaires diverses, et 54% d'entre elles sont des cibles de phosphorylation.Notre travail a également augmenté l’état des connaissances sur l'adénylate kinase 3 (ADK3), une enzyme contenant une région intrinsèquement désordonnée (IDR). Cette enzyme a été isolée par notre approche globale pour caractériser les IDPs de l’algue verte. L’extension C-terminale désordonnée (CTE) de cette enzyme lui confère de nouvelles fonctions comme par exemple, la formation d’un complexe bi-enzymatique avec la glycéraldéhyde-3-phosphate déshydrogénase (GAPDH), la régulation (négative) de l'activité GAPDH avec le NADPH comme cofacteur, et le rôle de chaperon pour la GAPDH en la protégeant de la dénaturation par traitement thermique et de l’agrégation. / The objectives of this work were to bring a conceptual breakthrough for an in-depth understanding of the molecular mechanisms of intrinsically disordered proteins (IDPs) and their roles in the cellular physiology of Chlamydomonas reinhardtii. Using experimental approaches, 682 heat-resistant proteins were identified as putative IDPs. Among them, 299 proteins were consistently predicted as IDPs by all four disordered predictors. The mean percentage of disordered residues content of these IDPs is about 20%, and most of them (~70%) are addressed to other compartments than mitochondrion and chloroplast. These newly identified IDPs from C. reinhardtii have a biased amino acid composition as regard to other IDPs from the Database of protein disorder (DisProt). Furthermore, they play diverse molecular functions, and 54% of them are the targets for phosphorylation. Our work also revealed more knowledge of the IDR-containing protein adenylate kinase 3 (ADK3) that was extracted by heat-treatment. Its disordered C-terminal extension (CTE) brought new functions to this protein. For instance, via its CTE, ADK3 can form a bi-enzyme complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), down-regulates the NADPH-dependent GAPDH activity, and behaves as a chaperone for GAPDH against its aggregation and inactivation under heat-treatment.

La sélénocystéine est incorporée co-traductionnellement dans les sélénoprotéines en réponse à un codon UGA habituellement l'un des 3 codons stop. La protéine SBP2 joue un rôle majeur dans ce mécanisme de recodage en se liant à une structure en tige-boucle (SECIS) située dans la région 3'UTR de l'ARNm des sélénoprotéines. Nous avons isolé et caractérisé fonctionnellement SBP2 de Drosophila melanogaster. Par comparison avec SBP2 humaine, nous avons identifié un domaine de liaison à l'ARN additionnel essentiel à la liaison au SECIS et à la sous-unité ribosomique 60S et permettant une sélectivité structurale du SECIS. Des prédictions structurales et des analyses biophysiques ont établi que SBP2 était une protéine globalement désordonnée ou “Intrinsically Disordered Protein” qui ne se replie qu'en présence de partenaires. Enfin, nous avons établi que l'assemblage des mRNP de sélénoprotéines faisait appel à des facteurs communs et présentait de multiples similarités avec celui des sn/snoRNP.

Moduler l’agrégation de protéines amyloïdes est thérapeutiquement pertinent (p. ex. la polyneuropathie amyloïde familiale traitée avec le Tafamidis). Cependant, pour de nombreuses amyloïdoses, il n’existe pas encore de modulateur d'agrégation efficace pour thérapie. Il a été récemment montré que combiner des composés qui modulent l’agrégation d’amyloïdes peut résulter en des effets synergiques. Une stratégie de criblage combinatoire, pour identifier des cocktails synergiques de composés, pourrait donc conduire à une percée thérapeutique pour de nombreuses amyloïdoses. Cependant, les technologies à haut débit existantes ne sont pas adaptées pour le criblage combinatoire.J’ai développé SynAggreg – une technologie in vitro à haut débit très sensible, précise, reproductible, peu coûteuse et flexible - qui permet d'identifier à la fois des inhibiteurs et des accélérateurs d'agrégation, de caractériser leur mécanisme d'action sur la cinétique d'agrégation et de les classer par leur efficacité. SynAggreg est également la première technologie adaptée au criblage combinatoire et pour l’étude d’effets synergiques de manière fiable. Enfin, cette nouvelle technologie peut être facilement adaptée à plusieurs amyloïdoses en remplaçant la partie amyloïde de la protéine de fusion par des techniques de biologie moléculaire. Ainsi, SynAggreg apparaît comme une boîte à outils pour la recherche fondamentale et appliquée et possède un fort potentiel de valorisation. / Modulating amyloid proteins aggregation is therapeutically relevant (e.g. the familial amyloid polyneuropathy treated with Tafamidis). However, for many amyloidoses, there is yet no efficient aggregation modulator for therapy. It was recently shown that combining compounds that modulate the aggregation of amyloids can result in synergistic effects. A combinatorial screening strategy to identify synergistic cocktails of compounds could thus lead to a therapeutic break through for many amyloidoses. However, existing high-throughput technologies are not adapted for combinatorial screening.I developed SynAggreg - a very sensitive, accurate, reproducible, cost effective, flexible and high-throughput in vitro technology - which allows identifying both aggregation inhibitors and accelerators, characterizing their mechanism of action on aggregation kinetics and ranking them by their efficiency. SynAggreg is also the first technology suitable for combinatorial screening and for studying reliably synergistic effects of combinations of compounds. Finally, this new technology can be easily adapted to several amyloidoses by replacing the amyloid part of the fusion protein with molecular biology techniques. Thus, SynAggreg appears as a toolbox for fundamental and applied research, and has a high potential for valorization.

Dans ses documents officiels, le Magistère de l’Église catholique qualifie souvent la contraception artificielle intraconjugale d’acte intrinsèquement mauvais. Le recours à cette notion semble considérer la nature biologique comme l’index impératif d’une norme morale sans autre considération. De quoi dépend alors la moralité d’un acte ? Une technique, prise en elle-même, comporte-t-elle une valeur morale définie ? Pour répondre à ces questions, nous avons retracé l’évolution de cette notion à travers une relecture de ses enjeux théologiques en les fondant sur la circulation de plusieurs pôles de réflexion (Écriture, Tradition, raison, sciences, vécu). Pour dissiper les ambiguïtés liées à cette notion, nous avons estimé qu’il serait préférable d’employer l’expression d’acte injustifiable plutôt que celle d’acte intrinsèquement mauvais. La notion d’acte injustifiable a l’avantage de faire droit à l’herméneutique et à l’exercice de la raison pratique dans le discernement éthique. Ainsi, on peut faire droit à la pluralité et articuler au mieux la temporalité et la complexité systémique dans le jugement moral des actes humains / In its official documents, the Magisterium of the Catholic Church often refers to intra-marital artificial contraception as an intrinsically evil act. The use of this concept seems to consider the biological nature as an imperative index of a moral standard without other considerations. If artificial contraception is wrong in itself, then what place do we give to conscience and freedom of couples in birth control? Is there an irreducible opposition between the natural law and contraceptive techniques? Does a technique, if taken by itself, hold a defined moral value? To answer these questions, we have traced back the evolution of this concept through a rereading of its anthropological-theological issues based on the circulation of several poles of thought (Scripture, Tradition and Reason, Humanities, Experience, etc.). To dispel any equivocities and ambiguities related to this concept, we believe it is better to use the term unjustifiable act rather than that of an intrinsically evil act. The concept of unjustifiable act has the advantage of granting hermeneutics and the use of practical reason in ethical discernment. In this way, we can honor otherness (recognition of particularities and subjectivities, while searching for the universal), grant the plurality of actions, norms and systems of legitimation of standards and allow regulation to better enable the regulation of historicity, temporality and systemic complexity in moral judgment of human actions.

Proteins are the fundamental macromolecules within a cell that carry out most of the biological functions. The computational study of protein structure and its functions, using machine learning and data analytics, is elemental in advancing the life-science research due to the fast-growing biological data and the extensive complexities involved in their analyses towards discovering meaningful insights. Mapping of protein’s primary sequence is not only limited to its structure, we extend that to its disordered component known as Intrinsically Disordered Proteins or Regions in proteins (IDPs/IDRs), and hence the involved dynamics, which help us explain complex interaction within a cell that is otherwise obscured. The objective of this dissertation is to develop machine learning based effective tools to predict disordered protein, its properties and dynamics, and interaction paradigm by systematically mining and analyzing large-scale biological data. In this dissertation, we propose a robust framework to predict disordered proteins given only sequence information, using an optimized SVM with RBF kernel. Through appropriate reasoning, we highlight the structure-like behavior of IDPs in disease-associated complexes. Further, we develop a fast and effective predictor of Accessible Surface Area (ASA) of protein residues, a useful structural property that defines protein’s exposure to partners, using regularized regression with 3rd-degree polynomial kernel function and genetic algorithm. As a key outcome of this research, we then introduce a novel method to extract position specific energy (PSEE) of protein residues by modeling the pairwise thermodynamic interactions and hydrophobic effect. PSEE is found to be an effective feature in identifying the enthalpy-gain of the folded state of a protein and otherwise the neutral state of the unstructured proteins. Moreover, we study the peptide-protein transient interactions that involve the induced folding of short peptides through disorder-to-order conformational changes to bind to an appropriate partner. A suite of predictors is developed to identify the residue-patterns of Peptide-Recognition Domains from protein sequence that can recognize and bind to the peptide-motifs and phospho-peptides with post-translational-modifications (PTMs) of amino acid, responsible for critical human diseases, using the stacked generalization ensemble technique. The involved biologically relevant case-studies demonstrate possibilities of discovering new knowledge using the developed tools.

Seleção de características é um tópico muito importante em aplicações de reconhecimento de padrões, especialmente em bioinformática, cujos problemas são geralmente tratados sobre um conjunto de dados envolvendo muitas variáveis e poucas observações. Este trabalho analisa aspectos de seleção de características no problema de identificação de redes de regulação gênica a partir de sinais de expressão gênica. Particularmente, propusemos um modelo de redes gênicas probabilísticas (PGN) que devolve uma rede construída a partir da aplicação recorrente de algoritmos de seleção de características orientados por uma função critério baseada em entropia condicional. Tal critério embute a estimação do erro por penalização de amostras raramente observadas. Resultados desse modelo aplicado a dados sintéticos e a conjuntos de dados de microarray de Plasmodium falciparum, um agente causador da malária, demonstram a validade dessa técnica, tendo sido capaz não apenas de reproduzir conhecimentos já produzidos anteriormente, como também de produzir novos resultados. Outro aspecto investigado nesta tese é o fenômeno da predição intrinsecamente multivariada (IMP), ou seja, o fato de um conjunto de características ser um ótimo caracterizador dos objetos em questão, mas qualquer de seus subconjuntos propriamente contidos não conseguirem representá-los de forma satisfatória. Neste trabalho, as condições para o surgimento desse fenômeno foram obtidas de forma analítica para conjuntos de 2 e 3 características em relação a uma variável alvo. No contexto de redes de regulação gênica, foram obtidas evidências de que genes alvo de conjuntos IMP possuem um enorme potencial para exercerem funções vitais em sistemas biológicos. O fenômeno conhecido como canalização é particularmente importante nesse contexto. Em dados de microarray de melanoma, constatamos que o gene DUSP1, conhecido por exercer função canalizadora, foi aquele que obteve o maior número de conjuntos de genes IMP, sendo que todos eles possuem lógicas de predição canalizadoras. Além disso, simulações computacionais para construção de redes com 3 ou mais genes mostram que o tamanho do território de um gene alvo pode ter um impacto positivo em seu teor de IMP com relação a seus preditores. Esta pode ser uma evidência que confirma a hipótese de que genes alvo de conjuntos IMP possuem a tendência de controlar diversas vias metabólicas cruciais para a manutenção das funções vitais de um organismo. / Feature selection is a crucial topic in pattern recognition applications, especially in bioinformatics, where problems usually involve data with a large number of variables and small number of observations. The present work addresses feature selection aspects in the problem of gene regulatory network identification from expression profiles. Particularly, we proposed a probabilistic genetic network model (PGN) that recovers a network constructed from the recurrent application of feature selection algorithms guided by a conditional entropy based criterion function. Such criterion embeds error estimation by penalization of rarely observed patterns. Results from this model applied to synthetic and real data sets obtained from Plasmodium falciparum microarrays, a malaria agent, demonstrate the validity of this technique. This method was able to not only reproduce previously produced knowledge, but also to produce other potentially relevant results. The intrinsically multivariate prediction (IMP) phenomenon has been also investigated. This phenomenon is related to the fact of a feature set being a nice predictor of the objects in study, but all of its properly contained subsets cannot predict such objects satisfactorily. In this work, the conditions for the rising of this phenomenon were analitically obtained for sets of 2 and 3 features regarding a target variable. In the gene regulatory networks context, evidences have been achieved in which target genes of IMP sets possess a great potential to execute vital functions in biological systems. The phenomenon known as canalization is particularly important in this context. In melanoma microarray data, we verified that DUSP1 gene, known by having canalization function, was the one which composed the largest number of IMP gene sets. It was also verified that all these sets have canalizing predictive logics. Moreover, computational simulations for generation of networks with 3 or more genes show that the territory size of a target gene can contribute positively to its IMP score with regard to its predictors. This could be an evidence that confirms the hypothesis stating that target genes of IMP sets are inclined to control several metabolic pathways essential to the maintenance of the vital functions of an organism.

Les oreillons sont une maladie très contagieuse causée par le virus ourlien. La méthode préventive (le vaccin) contre ce virus a été déjà mise au point. Par contre, les épidémies récentes restent incontrôlables. Il est donc très important de comprendre le mécanisme moléculaire de son cycle de vie afin d’élaborer le traitement effectif et spécifique. Ce virus appartient à la famille des Paramyxoviridae. Son génome, l’ARN non segmenté monocaténaire de polarité négative, est protégé par la nucléoprotéine (N) en formant des structures filamenteuses nucléocapsides. N joue un rôle essentiel dans la synthèse du génome viral. En effet, cette protéine avec la polymérase et son cofacteur phosphoprotéine (P) constitue la machinerie de transcription-réplication du virus. La N et la P sont composées des régions pliées et dépliées. Malgré que la morphologie du virus ourlien est conservée parmi les autres membres de la famille, il existe quelques différences. Il a été démontré que la P est un oligomère antiparallel avec les deux extrémités d’un côté qui interagissent avec la partie structurale de N (Ncore). Tandis que la fonction de la région désordonnée (Ntail) est compliquée à identifier pour le moment. En comparant avec les autres paramyxovirus connus, Ntail n’interagit pas avec le domaine C-terminal de la P. Le rôle des régions déstructurées de P n’a pas été défini. Dans ce projet, nous dévoilons les mécanismes des interactions entre diverses régions de N et P et nous expliquons comment les domaines intrinsèquement désordonnés de N et P sont impliqués dans la régulation de la machine complexe de réplication virale. Nous avons utilisé la résonance magnétique nucléaire qui est la méthode la plus puissante afin de déterminer la structure, la dynamique et les partenaires d’interaction dont la fonction des protéines dépliées virales. / Mumps is a highly contagious disease caused by the mumps virus. The prevention treatment (vaccine) against it is already in the routine use. However, recent outbreaks still remain uncontrollable. Therefore, it is important to understand the molecular mechanism of the mumps virus life cycle. This virus belongs to the family of Paramyxoviridae. Its genome, negative strand non-segmented RNA is protected by the nucleoprotein (N) by forming filamentous structures called nucleocapsids. N plays an important role in viral genome synthesis. Together with the polymerase and its cofactor phosphoprotein (P) they constitute the transcription-replication machinery. Both N and P contain folded and unfolded regions. Despite mumps virus common morphology with other paramyxovirus, there are some differences. It has been proposed that P is an antiparallel oligomer with two extremities on the one side being in interaction with the structural part of N (Ncore). The function of the disordered domain (Ntail) remains unclear, as it does not seem to bind to the C-terminal part of P, as is the case for other paramyxoviruses. The role of the disordered domains of P is also not known. In this project we revealed mechanisms of interaction between different regions of N and P and we explain how disordered regions of N and P are implicated in the regulation of the complex machinery of viral replication. We used the nuclear magnetic resonance which is the most powerful method to determine structure, dynamics and potential interaction partners, and therefore, function of disordered viral proteins.

ErbB2/HER2 est un récepteur tyrosine kinase de la famille d'EGFR (ErbB1) surexprimé dans plus de 20% des cancers du sein et associé à une forme particulièrement agressive de la maladie. Les récepteurs ErbBs sont actifs seulement sous forme de dimères, permettant la phosphorylation de leur queue C-terminale par leur domaine tyrosine kinase. La phosphorylation entraine l'interaction avec des protéines adaptatrices et l'activation de voies de signalisation, Ras/MAPK et PI3K/Akt principalement. Ces voies contrôlent la prolifération, la motilité cellulaire et la résistance à l'apoptose. Contrairement à ErbB1/3/4, ErbB2 dimérise en l'absence de ligand. Comprendre les autres mécanismes de régulation de la phosphorylation de ses tyrosines et de ses interactions est donc particulièrement intéressant.ErbB2 a fait l'objet de nombreuses études structurales et fonctionnelles. Elles ont permis la mise au point de traitements ciblés efficaces mais sujets à l'apparition de résistance, dont l'anticorps Trastuzumab, ciblant sa partie extracellulaire. La queue C-terminale d'ErbB2 (CtErbB2) a été très souvent ignorée dans ces études. Cette partie étant intrinsèquement désordonnée, il a fallu attendre ces dernières années pour que les concepts et les outils permettant de l'étudier émergent.Dans cette thèse, j'ai d'abord effectué la caractérisation structurale et dynamique de CtErbB2. J'ai montré que bien qu'étant dépourvue de toute structure stable, cette région riche en prolines possède plusieurs structures secondaires transitoires et un contact longue-distance participant très probablement à la régulation de ses interactions intra- et inter-moléculaires. Dans une deuxième partie je me suis intéressée à la caractérisation de la protéine adaptatrice Grb2, partenaire essentiel de ErbB2 pour l'activation de la voie des MAP kinases. L'organisation en solution des domaines de cette protéine modulaire dans sa forme libre était jusque là inconnue. J'ai ensuite étudié l'interaction entre Grb2 et CtErbB2, et montré que CtErbB2 interagit non seulement avec le domaine SH2 de Grb2 (par l'intermédiaire d'une phosphotyrosine), mais aussi avec son domaine SH3 N-terminal (grâce à un motif polyproline). Enfin, j'ai mis en place plusieurs stratégies de phosphorylation des tyrosines de CtErbB2, dans le but d'étudier plus largement l'effet des phosphorylations sur l'ensemble de cette région. / ErbB2/HER2 is a receptor tyrosine kinase of the EGFR (ErbB1) family overexpressed in 20% of breast cancers and associated to a particularly aggressive form of the disease. ErbB receptors are only active upon dimerization that enables phosphorylation of their C-terminal tail by their tyrosine kinase domain. Phosphorylation then triggers interaction with adaptor proteins and activation of signaling pathways, mainly Ras/MAPK and Akt/PI3K. Those pathways control cell proliferation, motility and resistance to apoptosis. Contrary to ErbB1/3/4, ErbB2 can dimerize without any ligand. Understanding other mechanisms of regulation of its tyrosine phosphorylation and of its interactions is thus particularly interesting.ErbB2 structure and function have been extensively studied. This has led to the development of several FDA-approved targeted drugs, that are effective but to which resistance occurs, amongst which the Trastuzumab antibody that targets ErbB2 extracellular domain. The C-terminal tail of ErbB2 (CtErbB2) has been widely ignored in these studies. Since it is intrinsically disordered, the concepts and tools to study it have only emerged in the last few years.In the present work, I have performed the structural and dynamic study of CtErbB2. I showed that despite its lack of any stable structure, this proline-rich region exhibits several transient secondary structures and a long-range contact that might participate in the regulation of its intra- and inter-molecular interactions. Then, I characterized the adaptor protein Grb2, which is a partner of ErbB2 that is essential for the activation of the MAPK pathway. The solution organization of the domains of this modular protein in its apo-form was unknown so far. I also studied the interaction between Grb2 and CtErbB2, showing that in addition to the known SH2-phosphotyrosine interaction, a polyproline motif of CtErbB2 binds to the N-terminal SH3 domain of Grb2. Finally, I implemented several strategies to phosphorylate CtErbB2 tyrosines, to study more extensively the effect of phosphorylation on the whole tail.

Indiana University-Purdue University Indianapolis (IUPUI) / Intrinsically disordered proteins (IDPs) are flexible proteins without defined 3D structures. Studies show that IDPs are abundant in nature and actively involved in numerous biological processes. Two crucial subjects in the study of IDPs lie in analyzing IDPs’ functions and identifying them. We thus carried out three projects to better understand IDPs. In the 1st project, we propose a method that separates IDPs into different function groups. We used the approach of CH-CDF plot, which is based the combined use of two predictors and subclassifies proteins into 4 groups: structured, mixed, disordered, and rare. Studies show different structural biases for each group. The mixed class has more order-promoting residues and more ordered regions than the disordered class. In addition, the disordered class is highly active in mitosis-related processes among others. Meanwhile, the mixed class is highly associated with signaling pathways, where having both ordered and disordered regions could possibly be important. The 2nd project is about identifying if an unknown protein is entirely disordered. One of the earliest predictors for this purpose, the charge-hydropathy plot (C-H plot), exploited the charge and hydropathy features of the protein. Not only is this algorithm simple yet powerful, its input parameters, charge and hydropathy, are informative and readily interpretable. We found that using different hydropathy scales significantly affects the prediction accuracy. Therefore, we sought to identify a new hydropathy scale that optimizes the prediction. This new scale achieves an accuracy of 91%, a significant improvement over the original 79%. In our 3rd project, we developed a per-residue C-H IDP predictor, in which three hydropathy scales are optimized individually. This is to account for the amino acid composition differences in three regions of a protein sequence (N, C terminus and internal). We then combined them into a single per-residue predictor that achieves an accuracy of 74% for per-residue predictions for proteins containing long IDP regions.

The corrosion protection of intrinsically conducting polymers (ICP) for mild steel is studied with polymethylthiophene films (PMT). Homogeneous and very adherent PMT films are formed on mild steel if the surface is pretreated with 2(3-thienyl)ethylphosphono acid used as adhesion promoter (AP). The corrosion protection properties of such films are investigated with and without topcoats in some corrosive media. Possible protection mechanisms of ICP could be discussed. / Korrosionsschutz von Baustahl durch intrinsisch leitfähige Polymere (ILP) mit dem Polymethylthiophen-Film (PMT) wurde untersucht. Homogene, kompakte und haftfeste PMT-Filme auf Baustahlsubstrat nach einer speziellen Vorbehandlung mit 2(3-Thienyl)-ethylphosphonosäure als Haftvermittler können elektrochemisch abgeschieden werden. Die weiteren Untersuchungen betreffen die Wirkung von PMT ohne und mit Topcoat für den Korrosionsschutz auf Baustahl. Mögliche Korrosionsschutzmechanismen von ILP werden diskutiert.