Hedgehog (Hh) morphogens play fundamental roles in development whilst dysregulation of Hh signalling leads to disease. Multiple receptors are involved in the modulation of Hh morphogens at the cell surface. Among these, the interactions of Hh ligands with glycosaminoglycan (GAG) (for example heparan or chondroitin sulphate) chains of proteoglycans in the extracellular matrix play a key role in shaping morphogen gradients and fulfil important functions in signal transduction. Several high resolution crystal structures of Sonic Hh (Shh)-GAG complexes have been determined. The interaction determinants, confirmed by binding studies and mutagenesis reveal a novel Hh site for GAG interactions, which appears to be common to all Hh proteins. This novel site is supported by a wealth of published functional data, and resides in a hot spot region previously found to be crucial for Hh receptor binding. Crystal packing analysis combined with analytical ultracentrifugation on Hh-GAG complexes suggest a potential mechanism for GAG-dependent multimerisation. A key step in the Hh pathway is the transduction of the Hh signal into the receiving cell. The Hh signal transducer, Smoothened, is a key target drug target in the pathway with several modulators in clinical trials, despite an absence of structural data. Smoothened is required to activate all levels of Hh signalling. Recent evidence points to the conserved N-terminal ectodomain (ECD) in regulating Smo activity, from vertebrates to invertebrates. Despite the central importance of the ECD, its precise function remains elusive. A crystal structure of the ECD at 2.2 Å resolution is reported here. Structural analysis and biophysical experiments are discussed with reference to the potential function of this intriguing domain.

Le couplage oxydant du méthane (OCM) est une réaction complexe de catalyse hétérogène, permettant la conversion directe du méthane en éthylène, pour un coût énergétique moindre par rapport aux procédés industriels indirects actuels. L’OCM nécessite une température supérieure à 700°C, à pression atmosphérique. Il y a donc compétition avec l’oxydation totale. Dans les nombreuses études rapportées dans la littérature, la limite de 25 % de rendement en C2 (éthane + éthylène) n’a pas été franchie. Les mécanismes proposés ne sont pas applicables à tous les catalyseurs actifs ou valables pour un large domaine de conditions opératoires. Une nouvelle manière d’aborder cette réaction est de prendre en compte la plus large diversité possible des paramètres intervenant dans ce procédé, de la formulation aux réacteurs en vue d'optimiser les performances. La présente étude a permis d’extraire des descripteurs pertinents du processus de l’OCM à partir de données expérimentales et d’établir certaines corrélations entre descripteurs et performances. Des catalyseurs LaSrCaO ont été sélectionnés après tests à haut débit en réacteur parallèle à lit fixe et un modèle micro-cinétique de l’OCM dans ce réacteur a été validé grâce aux données obtenues. D’autres expériences ont été menées avec succès en réacteur à membrane dense pour améliorer la productivité en éthylène. Le rôle joué par la composition de surface des catalyseurs a été identifié et une analyse critique de la méthode générale mise en œuvre conclut ce travail / The oxidative coupling of methane (OCM) is a complex heterogeneous catalytic reaction allowing the direct conversion of methane to ethylene, at a lower energetic cost than the current industrial processes. OCM requires a temperature higher than 700°C at atmospheric pressure. Hence, there is competition with total oxidation. In the numerous studies reported in literature, the limit of 25% C2 (ethane + ethylene) yield could not be overtaken. Proposed mechanisms are not relevant for all active materials or on all operating condition ranges. A new way to approach the reaction would be to take into account the wider possible panel of parameters involved in this process, from formulation to reactors targeting at process optimisation. The present study permitted to extract relevant descriptors of OCM process from experimental data and establish relationships between descriptors and performances. LaSrCaO catalysts were selected and tested in a parallel fixed-bed reactor and the data obtained were used to validate a micro-kinetic model in this reactor. Experiments were also performed successfully in a dense membrane reactor to improve ethylene productivity. The role played catalyst surface composition was also identified and a critical analysis of the global method implemented concludes this work

Influenza virus causes severe respiratory infections in birds and mammals and it is responsible for up to half a million deaths of human beings worldwide each year. Two molecular targets in influenza viral life cycle, neuraminidase and M2 proton channel are exploited in treatment. However, the recent emergence of new pandemic type along with increasing resistance against approved drugs has urged the need for a new drug target discovery and potential search of its inhibitor. Recently, an interesting protein-protein interaction between two subunits PA and PB1 of influenza A viral polymerase has been identified by X-ray crystallography as a new promising drug target. The fact that relatively few residues drive the binding and that the binding interface is highly conserved presents an intriguing possibility to identify antiviral lead compounds effective against all subtypes of influenza A virus. In our laboratory, we expressed and purified two fusion tag constructs of the recombinant C-terminal domain of polymerase acidic subunit (CPA) from the pandemic isolate A/California/07/2009 H1N1. First, GST-CPA fusion protein was used for kinetic evaluation of PA-PB1 interaction by surface plasmon resonance. Moreover, this construct was used in the development of high-throughput screening method for search of...

Les études d'interaction protéine-ligand aident à mieux comprendre la structure et la fonction des protéines. Dans la première partie de la thèse, la cyt bd oxydase a été étudiée. La protéine d'E. coli a été immobilisée avec succès sur des électrodes modifiées par des nanoparticules d'or. Ainsi, un biocapteur électrochimique a été créé, permettant de tester certains inhibiteurs potentiels de cyt bd provenant d’E. coli. Le cyt bd issue de G. thermodenitrificans thermophile a également été étudié. En faisant appel aux spectroscopies IR et Raman ainsi que l’électrochimie, il a été démontré que la protéine est distincte du cyt bd d’E. coli. Une influence mutuelle du pH et de la température sur la catalyse a été aussi démontrée. La deuxième partie de la thèse portait sur la protéine mitochondriale mitoNEET. L'influence du pH et de divers ligands (pioglitazone, resvératrol, ions phosphates) a été examinée. / Protein-ligand interaction studies help to better understand the structure and function of proteins. In the first part of the thesis cyt bd oxidase was studied. The protein from E. coli was successfully immobilised at gold nanoparticles modified electrodes. Thus, an electrochemical biosensor was created allowing testing some potential inhibitors of cyt bd from E. coli. The cyt bd from thermophilic G. thermodenitrificans was also studied. By means of IR, Raman spectroscopy and electrochemistry the protein was shown to be distinct from cyt bd from E. coli. A mutual influence of pH and temperature was demonstrated on the electrochemical and catalytical properties. The second part of the thesis focused on mitochondrial mitoNEET protein. The influence of the pH and various ligands was studied.

Unnatural amino acids (uAA) expand the structural and functional possibilities of proteins. Numerous previous studies have demonstrated uAA as a powerful tool for protein engineering, but challenges also remain. Three notable such challenges include: (1) the fitness of uAA-incorporated proteins are difficult to predict and time-consuming to screen with conventional methods, (2) uAA incorporation in difficult-to-express proteins (e.g. membrane proteins such as G-protein coupled receptors) remain challenging, and (3) the incorporation of multiple types of uAA are still limited. In response, we pose cell-free protein synthesis (CFPS), a rapid and versatile in vitro expression system, as a platform to explore solutions to these challenges. The "cell-free" nature of CFPS enables it to accelerate protein expression and tolerate extensive modifications to its translational environment. In this work, these advantages were utilized to address the aforementioned challenges by: (1) rapidly expressing and screening uAA-containing proteins, (2) incorporating uAA in functional G-protein coupled receptor in the presence of membrane-mimicking lipid additives, and (3) engineer the translational environment extensively towards multiple uAA incorporation.

La caractérisation de l'activité enzymatique des (phospho)lipases requiert des tests enzymatiques spécifiques, continus, utilisant des substrats lipidiques et adaptés au criblage à haut débit des activités et/ou des inhibiteurs de (phospho)lipases. Afin de développer de tels tests, la synthèse de glycérophosphatidylcholine (PC) estérifiée en position sn-1 et/ou sn-2 par l'acide alpha-éléostéarique (acide 9Z, 11E, 13E, octadécatriénoïque) a été effectuée. La triple insaturation conjuguée présente au sein de cet acide gras constitue un chromophore intrinsèque qui confère une forte absorption dans le domaine de l'ultra-violet à cet acide gras et aux lipides le contenant. Les PC contenant l'acide alpha-éléostéarique ont été adsorbées par « coating » au fond des puits d'une microplaque de titration. L'hydrolyse du substrat lipidique par une phospholipase A1 (PLA1) ou phospholipase A2 (PLA2), injectée dans le milieu réactionnel, est suivie en continu par l'augmentation de l'absorbance à 272 nm, due à la transition de l'acide alpha-éléostéarique de la phase adsorbée à la phase aqueuse. Des PC hétérogènes ont été synthétisées à partir de rac-glycidol pour effectuer un marquage sélectif de la PC par l'acide alpha-éléostéarique sur la position sn-1 (EOPC) ou sn-2 (OEPC). Pour empêcher la migration de la chaîne acyle, un lien éther non hydrolysable par les PLA1 ou PLA2 a été introduit sur l'autre position sn de la PC avec une chaîne alkyl (C18). Ces PC chimiquement définies ont permis d'élaborer une méthode de dosage en continu de l'activité enzymatique et discriminant les activités PLA1 ou PLA2, ce qui représente un caractère innovant par rapport à toutes les méthodes existantes / The characterization of the catalytic activity of (phospho)lipases requires specific assays, that are continuous, sensitive, use lipidic substrates and could be applied to high throughput screening. In order to perform these tests, several tailor-made alpha-eleostearic (9Z, 11E, 13E-octadecatrienoic acid) containing glycerophosphatidylcholines (PC) have been synthetized with the alpha-eleostearic acid at the position sn-1 and/or sn-2. The conjugated triene present in this fatty acid constitutes an intrinsic chromophore and, consequently, confers strong UV absorption properties of the fatty acid and the lipids harboring it. PC substrates were coated onto a microplate well and the phospholipase A1 (PLA1) or phospholipase A2 (PLA2) activity was measured continuously by the increase in absorbance, at 272 nm, due to the transition of alpha-eleostearic acid from the adsorbed to the soluble state. Moreover, two structured analogues of PC labeled at the sn-1 (EOPC) or sn-2 (OEPC) position with the alpha-eleostearic acid have been synthetized from rac-glycidol. A non-absorbing and non-hydrolysable by PLA1 and PLA2 O-ether alkyl(C18) was introduced at the other sn position to prevent intramolecular acyl chain migration during the synthesis and the lipolysis. These structured PC were coated onto a microplate and used in a continuous assay, to discriminate, with excellent accuracy, between PLA1 or PLA2 activities. The development of a sensitive enzymatic method using coated substrates analogues to natural lipid is a relevant improvement from current assays for measuring continuously (phosphor)lipases activities and/or their inhibitors due to the alpha-eleostearic acid UV spectroscopic properties

Motivation: Although various high-throughput technologies provide a lot of valuable information, each of them is giving an insight into different aspects of cellular activity and each has its own limitations. Thus, a complete and systematic understanding of the cellular machinery can be achieved only by a combined analysis of results coming from different approaches. However, methods and tools for integration and analysis of heterogenous biological data still have to be developed. Results: This work presents systemic analysis of basic cellular processes, i.e. cell viability and cell cycle, as well as embryonic stem cell pluripotency and differentiation. These phenomena were studied using several high-throughput technologies, whose combined results were analysed with existing and novel clustering and hit selection algorithms. This thesis also introduces two novel data management and data analysis tools. The first, called DSViewer, is a database application designed for integrating and querying results coming from various genome-wide experiments. The second, named PhenoFam, is an application performing gene set enrichment analysis by employing structural and functional information on families of protein domains as annotation terms. Both programs are accessible through a web interface. Conclusions: Eventually, investigations presented in this work provide the research community with novel and markedly improved repertoire of computational tools and methods that facilitate the systematic analysis of accumulated information obtained from high-throughput studies into novel biological insights.

A wide range of screening strategies have been employed to isolate antibodies and other proteins with specific attributes, including binding affinity, specificity, stability and improved expression. However, there remains no high-throughput system to screen for target-binding proteins in a mammalian, intracellular environment. Such a system would allow binding reagents to be isolated against intracellular clinical targets such as cell signalling proteins associated with tumour formation (p53, ras, cyclin E), proteins associated with neurodegenerative disorders (huntingtin, betaamyloid precursor protein), and various proteins crucial to viral replication (e.g. HIV-1 proteins such as Tat, Rev and Vif-1), which are difficult to screen by phage, ribosome or cell-surface display. This study used the â-lactamase protein complementation assay (PCA) as the display and selection component of a system for screening a protein library in the cytoplasm of HEK 293T cells. The colicin E7 (ColE7) and Immunity protein 7 (Imm7) Escherichia coli proteins were used as model interaction partners for developing the system. These proteins drove effective â-lactamase complementation, resulting in a signal-to-noise ratio (9:1 – 13:1) comparable to that of other â-lactamase PCAs described in the literature. The model Imm7-ColE7 interaction was then used to validate protocols for library screening. Single positive cells that harboured the Imm7 and ColE7 binding partners were identified and isolated using flow cytometric cell sorting in combination with the fluorescent â-lactamase substrate, CCF2/AM. A single-cell PCR was then used to amplify the Imm7 coding sequence directly from each sorted cell. With the screening system validated, it was then used to screen a protein library based the Imm7 scaffold against a proof-of-principle target. The wildtype Imm7 sequence, as well as mutants with wild-type residues in the ColE7- binding loop were enriched from the library after a single round of selection, which is consistent with other eukaryotic screening systems such as yeast and mammalian cell-surface display. In summary, this thesis describes a new technology for screening protein libraries in a mammalian, intracellular environment. This system has the potential to complement existing screening technologies by allowing access to intracellular proteins and expanding the range of targets available to the pharmaceutical industry.

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.

L’ingénierie des protéines fonctionnelles est un processus d’amélioration des propriétés physiques ou catalytiques d’enzyme au travers d’approches rationnelles et d'évolution dirigée, aussi bien que la combinaison des deux méthodes. Malgré le progrès de la modélisation moléculaire des protéines, les méthodes de prédiction restent aléatoires et un grand nombre de variantes restent à tester. De ce fait, le développement et l’utilisation d’un système de criblage d’activité de protéines à très haut débit, comme la microfluidique en gouttes, est indispensable. Cette thèse de doctorat présente trois projets d’évolution dirigée de protéines en trois approches différentes avec expression d’enzyme in vitro et in vivo. Les plateformes microfluidiques ont été développées et validées pour chaque projet. De plus, plusieurs banques de variants ont été criblées avec, dans certains cas, isolement de molécules 5-10 fois que le clone parental. / This work describes the development of high-throughput droplet microfluidic platforms fine-tuned for protein of interest and their employment in directed evolution experiments. When not available, fluorogenic assay for monitoring desired enzyme activity (-ies) in droplets was developed. Moreover, the in vivo expression allowed the successive integration of microfluidic modules on the same chip. After a couple of evolution rounds the initial retro-aldolase variant was significantly improved. In other project, to meet industrial requirements a high-throughput screening platform for protease evolution in detergent has been assembled and validated. Two evolution rounds showed the accumulation of a certain pool of beneficial mutations over the selection rounds. The research described in this work highlighted that in vitro expression systems are sensitive to the amount of supplied DNA and reaction conditions. This observation led to the development of a multistep completely in vitro microfluidic platform.