Les anticorps à visée thérapeutique sont en pleine expansion. Le développement de ces protéines issues de la bioingénierie est toutefois entravé par leur tendance naturelle à l'agrégation, particulièrement critique au cours des étapes de repliement. L'association réversible (non covalente) de protéines avec des polymères hydrosolubles pourrait permettre de résoudre ce problème. Nous avons ici étudié les interactions entre des protéines modèles et des dérivés hydrophobes du poly(acrylate de sodium) (PAA) grâce à diverses techniques expérimentales, incluant la spectroscopie de corrélation de fluorescence, le dichroïsme circulaire sous radiation synchrotron, la diffusion de lumière, l'électrophorèse capillaire et la calorimétrie différentielle à balayage. Comme preuve de concept, nous avons démontré que les dérivés du PAA augmentent le rendement de renaturation d'une enzyme modèle, l'anhydrase carbonique bovine, grâce à la protection de conformères partiellement repliés. La stabilisation colloïdale au cours du repliement a été étendue à des fragments d'anticorps de type scFv, d'importants modèles de biothérapeutiques sensibles à l'agrégation. L'efficacité des polymères a aussi été validée pour des immunoglobulines G soumises à un stress thermique. La prédominance de l'association hydrophobe est remise en cause dans cette étude. L'ancrage électrostatique des chaines de PAA s'avère suffisant pour minimiser l'agrégation des protéines, grâce à des associations dynamiques avec les intermédiaires partiellement repliés. Un avantage majeur de ces polymères est la faible quantité nécessaire comparé aux osmolytes conventionnels et l'absence de modification chimique des protéines. / Antibodies constitute the fastest growing class of human biopharmaceuticals. The development of these engineered proteins is yet hampered by their natural propensity towards irreversible aggregation particularly critical during refolding steps. Reversible (non-covalent) association of proteins with water-soluble polymers could circumvent this issue. In the present study, we investigated the interactions between model proteins and hydrophobically-modified poly(sodium acrylate) (PAA) chains with experimental techniques including fluorescence correlation spectroscopy, synchrotron-radiation circular dichroism, light scattering, capillary zone electrophoresis and differential scanning calorimetry. As a proof of concept, we demonstrated that PAA derivatives enhanced the renaturation yield of a single-domain model enzyme, bovine carbonic anhydrase, because of protection of partly-folded conformers against aggregation. Colloidal stabilisation during refolding was extended to two-domain artificial antibody fragments, single chains Fv fragments, as important models of aggregation-prone biotherapeutics. On similar basis, efficient protection was also validated with full-length immunoglobulins G under heat-stress. Prevalence of hydrophobic association between polymers and proteins is questioned in this work. Rather, electrostatic binding with PAA chains turns out to be sufficient to minimize in vitro protein aggregation, most-likely because of dynamic association with partly-folded conformers. A major advantage of these polymers is their use at extremely low amount compared to conventional osmolytes and absence of chemical modification of proteins.

Structural properties of phosphoglucose isomerases isolated from a variety of species have been compared by peptide fingerprinting, predicted amino acid sequence homologies and by denaturation and renaturation studies. The enzymes are more readily denatured in guanidinium chloride than in urea, and the isomerase isolated from yeast is more stable toward acid pH than the rabbit muscle enzyme. The rates of guanidinium chloride-induced denaturation are markedly increased by ionic strength and decreased by substrates, competitive inhibitors or glycerol. The enzyme can be renatured, but only in the presence of glycerol. The renaturation process is dependent on protein concentration and temperature and provides a method for the formation of mixed species heterodimers.

Notre recherche interroge la portée de la nouvelle politique environnementale de la capitale chinoise, dont la mise en oeuvre a été précipitée dans le cadre de l'accueil des Jeux Olympiques.Si la trame urbaine de Pékin était encore il y a peu l'un des derniers héritages des préceptes traditionnels d'aménagement de l'espace chinois entre "montagne et eau" et se référant au “souffle - qi”, qui anime toute chose et tout être dans la cosmogonie chinoise et qui est à la base de l'implantation de la ville sur son site, les politiques urbaines menées au cours du XXe siècle ont détruit en grande partie cet héritage.La prise de conscience environnementale amorcée dans les années 90 a généré une nouvelle approche au territoire qui s'est accélérée en 2001 dans l'optique de l'accueil des green Olympic Games en 2008. La reforestation de la capitale chinoise a alors été considérée à toutes les échelles, de celle du pays à celle de la rue.A l'échelle de son territoire périurbain, Pékin s'est dotée de deux ceintures vertes. Pour autant, le gouvernement municipal a choisi la voie de l'originalité en planifiant des "ceintures vertes habitées", à 50%pour la première et à 30% pour la seconde. A l'échelle de sa zone urbaine, la municipalité a mis en place de nouvelles promenades paysagères qui s'étirent le long des routes et des canaux et qui font apparaitre une nouvelle typologie d'espaces publics qui trouve son origine dans la réhabilitation de l'axe historique nord-sud et qui a permis outre la réhabilitation de l'histoire ancienne, celle de la réhabilitation du paysage ancien.Des ceintures vertes aux coulées vertes, la qualité de vie est sans nul doute améliorée à Pékin. Ce souffle vert est-il pour autant porteur d'une nouvelle identité urbaine? Attendons encore pour voir. Pékin 2050. Alors ce souffle vert? / Our research examines the scope of the new environmental policy for the Chinese capital, whoseimplementation was precipitated by the hosting of the Olympic Games.If the Beijing urban fabric was one of the last vestiges of the traditional Chinese precepts of spatialplanning between "mountain and water" and referring to "breath – qi" that animates all things and allbeings in the Chinese cosmogony, and that is the basis of the establishment of the city on its site, theurban policies implemented during the twentieth century have destroyed much of this heritage. Theenvironmental awareness that emerged in the 90s has generated a new approach to the territory, whichwas expedited in 2001 in view of hosting the green Olympic Games in 2008. The reforestation of theChinese capital was then considered at all scales, from that of the country to that of the street.At the scale of its periurban area, Beijing has built two green belts. However, the municipal governmenthas chosen an original approach by planning two "inhabited green belts ", 50% for the first and 30% for thesecond. At the scale of its urban area, the municipality set up new scenic walks along roads and canals,revealing a new typology of public spaces that finds its origin in the rehabilitation project of the historicnorth-south axis and that has also allowed the rehabilitation of ancient history and of the ancientlandscape.From green belts to green corridors, the quality of life is undoubtedly better in Beijing. Has, however, thisnew breath of green created a new urban identity? Let's wait and see. Beijing 2050. So, what about this new breath of green?

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 97-104).

La « restauration » est un concept majeur de la gestion de cours d’eau. En France, comme à l’étranger, il a aujourd’hui un ancrage législatif, opérationnel et scientifique fort. La démarche de recherche engagée est destinée à mieux comprendre comment est définie, est pratiquée et est évaluée la « restauration de cours d’eau ». Les matériaux utilisés sont documentaires (publications scientifiques, dossiers administratifs des Agences de l’Eau, documents techniques d’appui à l’action, documents relatifs aux projets réalisés). Ils font l’objet d’analyses textuelles (bibliométrie, analyse de contenu, statistique textuelle, analyse qualitative).Les résultats sont organisés en cinq chapitres. Le Chapitre I concerne le positionnement des sciences dans le champ de la « restauration ». Sont abordés successivement les dynamiques scientifiques, les éléments de définitions et les thématiques de travail privilégiées par les chercheurs. Le Chapitre II dessine une chronologie des définitions françaises de la « restauration ». Il présente les permanences et les évolutions observées du XIXème siècle jusqu’à nos jours. Le Chapitre III s’intéresse aux politiques d’intervention de trois Agences de l’Eau. Il offre ainsi une première analyse des pratiques françaises de « restauration de cours d’eau ». Le travail sur les pratiques est ensuite centré, dans le Chapitre IV, sur la « restauration écologique de cours d’eau ». Il prolonge ainsi l’étude de l’action publique française et propose une comparaison avec l’Allemagne. Enfin, le Chapitre V s’intéresse plus particulièrement aux pratiques de suivi et d’évaluation des projets franco-Allemands de « restauration écologique ».Les résultats ont fait l’objet de réflexions opérationnelles et de recommandations afin d’aider l’action publique et les stratégies d’évaluation dans le domaine. Une attention particulière a également été accordée aux perspectives scientifiques dans le cadre d’une thématique stratégique pour la mise en œuvre de la Directive Cadre sur l’Eau. / “Restoration” is a key concept in river management. In France and worldwide, “river restoration” has been provided a strong legislative, operational and scientific anchor. This research aims to better understand “river restoration”: that is, how is “river restoration” defined, practiced and evaluated? Research materials stand on various documents (scientific articles, administrative documents of French Water Agencies, technical documents about “restoration” procedures and documents related to specific “restoration projects”). These documents are analysed with textual analysis methods (bibliometrics, content analysis, textual statistics, qualitative analysis).Results are organized into five chapters. Chapter I deals with the position of sciences in the field of “river restoration”. This chapter covers research dynamics, scientific definitions of “restoration” and scientists’ research themes related to “river restoration”. Chapter II draws a timeline of French definitions of “river restoration”. It analyses permanence and change in the definition of “river restoration” from the late 19th Century until today. In Chapter III, a framework which suggests the establishment of three French Water Agencies in order to analyse French “river restoration” public policy and practice is proposed. Chapter IV is dedicated to the analysis of “ecological river restoration” practices. This Chapter’s objective is to better understand French public action. French practices are also compared to German practices. Finally, the monitoring and evaluation of contemporary “ecological river restoration” projects in France and in Germany is described in Chapter V.Operational reflections and recommendations are provided through the results of this research in order to aid public action and evaluation strategies in the field of “river restoration”. Special attention was also paid to scientific perspectives in the context of the implementation of the Water Framework Directive.

Muitas das proteínas de valor biomédico relevante são encontradas em baixas concentrações em suas fontes nativas. O alto nível de expressão de proteínas recombinantes em E. coli, muitas vezes gera o acúmulo de proteínas como agregados insolúveis no citoplasma e/ou periplasma da bactéria, denominados de corpos de inclusão (CI). A alta pressão tem sido amplamente utilizada no estudo da conformação das proteínas,ela modula as interações proteína-proteína e proteína-solvente através de mudanças no volume das mesmas, promovendo a entrada de água nas cavidades não expostas da molécula e promovendo hidratação e solubilização dos agregados. O presente trabalho teve como objetivo a renaturação de proteínas recombinantes expressas como CI em Escherichia coli usando alta pressão hidrostática como condição branda de dissociação dos agregados. As tiorredoxinas TsnC e TrxA, a proteína YbbN e a proteína comigratória com bacterioferritina (Bcp), todas de Xylella fastidiosa, foram estudadas neste trabalho. As condições de renaturação foram otimizadas, utilizando-se diferentes proporções do par redox, concentrações de GdnHCl, presença de aditivos e esquemas de descompressão. Para a quantificação e análise da eficácia do processo de renaturação das proteínas sob pressão foram utilizadas as técnicas de microscopia eletrônica de varredura dos CI e de SDS-PAGE, e ensaios de atividade enzimática das proteínas. A TsnC foi renaturada em Tris HCl 50 mM com proporção de 10GSH:1GSSG em concentração final de 10 mM, 0,75 M GdnHCl, na presença de 0,5 M de Triton-X e a pressão utilizada foi de 2,4 kbar por 1 hora e 30 minutos seguida de descompressão direta e incubação por 16 horas em pressão atmosférica. O rendimento final de obtenção de TsnC solúvel foi altíssimo, de até 89,9%. A renaturação de proteína YbbN, nunca antes descrita, foi obtida em tampão de renaturação Tris HCl 50 mM, na presença de 0,5 M de L-Arginina e a pressão utilizada foi de 2,4 kbar por 1 hora e 30 minutos seguida de descompressão direta e incubação por 16 horas em pressão atmosférica. A proteína YbbN, que apresentou atividade de tioredoxina, foi renaturada com rendimento de até 98% a partir da proteína insolúvel nos CI. Foi possível a solubilização da tiorredoxina TrxA e Bcp sob alta pressão hidrostática em tampão de renaturação Tris HCl 50 mM, utilizando diferentes proporções do par redox na concentração final de 10 mM e 1,5 M de GdnHCl, porém não foi possível obter a atividade biológicas destas proteínas. Mostramos também que a L-Arginina apresenta efeito auxiliar na solubilização dos CI induzida pela alta pressão, e ao mesmo tempo se mostrou altamente protetora contra a inativação da atividade da YbbN promovida pela incubação em altas temperaturas, o que sugere que a presença deste aminoácido pode ter alta aplicabilidade juntamente com a aplicação de altas pressões para elevar os rendimentos de renaturação de proteínas recombinantes a partir de CI. / Many of the relevant proteins are found in low concentrations in their native sources. The high level expression of recombinant heterologous proteins in Escherichia coli often causes their accumulation as insoluble aggregates in the cytoplasm or periplasm of bacteria in a mainly inactive form, known as inclusion bodies (IB). The high pressure has been widely used to study the conformational states of proteins. It modulates the protein-protein and protein-solvent interactions by changing the volume of the system, promoting the entrance of water into the cavities unexposed to water, exposing hydrophobic regions and promoting solubilization of the aggregates. The present study aimed to refold recombinant proteins expressed in E. coli as IB using high hydrostatic pressure as a mild condition for IB dissociation. The thioredoxins TsnC, TrxA and Ybbn and the protein comigratory with bacterioferritin (Bcp) of Xylella fastidiosa were studied in this work. The conditions for refolding were optimized for proportions of the redox pair, GdnHCl concentration, presence of additives and schemes for decompression. To analyze the effectiveness of the process of refolding under pressure we have used, scanning electron microscopy of the IB and SDS-PAGE and enzymatic activity assays for quantification and analysis of the solubilized proteins. The TsnC was renatured in 50 mM TrisHCl at a ratio of 10GSH: 1GSSG in 10 mM final concentration, 0.75 M GdnHCl in the presence of 0.5M Trito X-100 and application of 2.4 kbar for 1.5 hours, followed by direct decompression and incubation for 16 h at atmospheric pressure. The final yield of soluble and biological active TsnC was very high, up to 89,9%. The refolding of protein YbbN was obtained by application of 2.4 kbar for 1.5 h to an IB suspension in buffer 50 mM Tris HCl, in the presence of 0.5 M L-arginine, followed by direct decompression and incubation for 16 h at atmospheric pressure. We also show that L-arginine had a highly protective effect on the inactivation of biological activity of YbbN promoted by incubation at high temperatures.The final yield of refolded YbbN, that present thioredoxin activity, was also very high: 98%. TrxA and Bcp thioredoxins were solubilized at 2.4 kbar and step decompression. However, these proteins did not present biological activity. We also show that L-arginine has an auxiliary effect on the solubilization of IB induced by high pressure, while its presence proved highly protective against inactivation of the enzymatic activity promoted by incubation at high temperatures. These results suggest that the presence of this amino acid can have high applicability to increase the yield of refolding of recombinant proteins from the IC at high pressure.

Muitas das proteínas de valor biomédico relevante são encontradas em baixas concentrações em suas fontes nativas. O alto nível de expressão de proteínas recombinantes em E. coli, muitas vezes gera o acúmulo de proteínas como agregados insolúveis no citoplasma e/ou periplasma da bactéria, denominados de corpos de inclusão (CI). A alta pressão tem sido amplamente utilizada no estudo da conformação das proteínas,ela modula as interações proteína-proteína e proteína-solvente através de mudanças no volume das mesmas, promovendo a entrada de água nas cavidades não expostas da molécula e promovendo hidratação e solubilização dos agregados. O presente trabalho teve como objetivo a renaturação de proteínas recombinantes expressas como CI em Escherichia coli usando alta pressão hidrostática como condição branda de dissociação dos agregados. As tiorredoxinas TsnC e TrxA, a proteína YbbN e a proteína comigratória com bacterioferritina (Bcp), todas de Xylella fastidiosa, foram estudadas neste trabalho. As condições de renaturação foram otimizadas, utilizando-se diferentes proporções do par redox, concentrações de GdnHCl, presença de aditivos e esquemas de descompressão. Para a quantificação e análise da eficácia do processo de renaturação das proteínas sob pressão foram utilizadas as técnicas de microscopia eletrônica de varredura dos CI e de SDS-PAGE, e ensaios de atividade enzimática das proteínas. A TsnC foi renaturada em Tris HCl 50 mM com proporção de 10GSH:1GSSG em concentração final de 10 mM, 0,75 M GdnHCl, na presença de 0,5 M de Triton-X e a pressão utilizada foi de 2,4 kbar por 1 hora e 30 minutos seguida de descompressão direta e incubação por 16 horas em pressão atmosférica. O rendimento final de obtenção de TsnC solúvel foi altíssimo, de até 89,9%. A renaturação de proteína YbbN, nunca antes descrita, foi obtida em tampão de renaturação Tris HCl 50 mM, na presença de 0,5 M de L-Arginina e a pressão utilizada foi de 2,4 kbar por 1 hora e 30 minutos seguida de descompressão direta e incubação por 16 horas em pressão atmosférica. A proteína YbbN, que apresentou atividade de tioredoxina, foi renaturada com rendimento de até 98% a partir da proteína insolúvel nos CI. Foi possível a solubilização da tiorredoxina TrxA e Bcp sob alta pressão hidrostática em tampão de renaturação Tris HCl 50 mM, utilizando diferentes proporções do par redox na concentração final de 10 mM e 1,5 M de GdnHCl, porém não foi possível obter a atividade biológicas destas proteínas. Mostramos também que a L-Arginina apresenta efeito auxiliar na solubilização dos CI induzida pela alta pressão, e ao mesmo tempo se mostrou altamente protetora contra a inativação da atividade da YbbN promovida pela incubação em altas temperaturas, o que sugere que a presença deste aminoácido pode ter alta aplicabilidade juntamente com a aplicação de altas pressões para elevar os rendimentos de renaturação de proteínas recombinantes a partir de CI. / Many of the relevant proteins are found in low concentrations in their native sources. The high level expression of recombinant heterologous proteins in Escherichia coli often causes their accumulation as insoluble aggregates in the cytoplasm or periplasm of bacteria in a mainly inactive form, known as inclusion bodies (IB). The high pressure has been widely used to study the conformational states of proteins. It modulates the protein-protein and protein-solvent interactions by changing the volume of the system, promoting the entrance of water into the cavities unexposed to water, exposing hydrophobic regions and promoting solubilization of the aggregates. The present study aimed to refold recombinant proteins expressed in E. coli as IB using high hydrostatic pressure as a mild condition for IB dissociation. The thioredoxins TsnC, TrxA and Ybbn and the protein comigratory with bacterioferritin (Bcp) of Xylella fastidiosa were studied in this work. The conditions for refolding were optimized for proportions of the redox pair, GdnHCl concentration, presence of additives and schemes for decompression. To analyze the effectiveness of the process of refolding under pressure we have used, scanning electron microscopy of the IB and SDS-PAGE and enzymatic activity assays for quantification and analysis of the solubilized proteins. The TsnC was renatured in 50 mM TrisHCl at a ratio of 10GSH: 1GSSG in 10 mM final concentration, 0.75 M GdnHCl in the presence of 0.5M Trito X-100 and application of 2.4 kbar for 1.5 hours, followed by direct decompression and incubation for 16 h at atmospheric pressure. The final yield of soluble and biological active TsnC was very high, up to 89,9%. The refolding of protein YbbN was obtained by application of 2.4 kbar for 1.5 h to an IB suspension in buffer 50 mM Tris HCl, in the presence of 0.5 M L-arginine, followed by direct decompression and incubation for 16 h at atmospheric pressure. We also show that L-arginine had a highly protective effect on the inactivation of biological activity of YbbN promoted by incubation at high temperatures.The final yield of refolded YbbN, that present thioredoxin activity, was also very high: 98%. TrxA and Bcp thioredoxins were solubilized at 2.4 kbar and step decompression. However, these proteins did not present biological activity. We also show that L-arginine has an auxiliary effect on the solubilization of IB induced by high pressure, while its presence proved highly protective against inactivation of the enzymatic activity promoted by incubation at high temperatures. These results suggest that the presence of this amino acid can have high applicability to increase the yield of refolding of recombinant proteins from the IC at high pressure.

Nous avons cherché à concevoir des macromolécules ayant un comportement de chaperonne vis à vis de diverses protéines et notamment en fondant leur activité sur des propriétés stimulables par la lumière. Les interactions hydrophobes constituent un paramètre clé de l'effet chaperonne qui a été mis en évidence dans les chaperonnes biologiques aussi bien que dans les chaperonnes artificielles. Nous avons synthétisé des polymères à amphiphilie photo-stimulable portant des chaînes pendantes azobenzènes. Ces polymères sont capables d'association/dissociation photo-stimulables avec des particules colloïdales à coeur hydrophobe. Différents paramètres peuvent moduler ces associations comme la force ionique, le taux de modification hydrophobe, la nature du greffon... Ces polymères ont montré plusieurs propriétés caractéristiques de chaperonnes artificielles : ils déstabilisent une protéine modèle, le cytochrome C, protègent de l'agrégation et augmentent l'efficacité des procédés de renaturation de l'anhydrase carbonique et d'un fragment d'anticorps surexprimé en bactérie. L'évolution du repliement a été caractérisée par suivi des structures secondaires en dichroïsme circulaire et par suivi de la compacité des protéines en fluorescence. L'association polymère/protéine a été étudiée par électrophorèse capillaire et par diffusion de la lumière.

Le pronostic de certains cancers s'est considérablement amélioré avec l'arrivée sur le marché des anticorps thérapeutiques. Devant l'essor de ces nouveaux médicaments associé à l'identification de nouveaux biomarqueurs, de nouvelles perspectives émergent pour l'imagerie moléculaire in vivo. En effet, disposer de nouveaux traceurs moléculaires spécifiques de ces biomarqueurs permettra de caractériser l'hétérogénéité des cellules cancéreuses, de suivre l'expression de ces marqueurs au cours de l'évolution de la tumeur, mais également de suivre l'efficacité d'un traitement sur la régression tumorale du patient. Pour répondre à cette évolution de diagnostic moléculaire in vivo, il convient de développer de nouvelles sondes moléculaires. L'objectif de ma thèse répond à ce nouveau besoin avec l'ingénierie et le marquage d'un format d'anticorps recombinant adapté à l'imagerie in vivo : le diabody 12G4 dirigé contre le récepteur de l'hormone antimüllérienne (AMH), marqueur de certains cancers de la sphère génitale.

Les récepteurs de chimiokines sont des régulateurs essentiels de la migration cellulaire dans le cadre de la surveillance immunitaire, et le développement. Les récepteurs CCR5 et CXCR4 sont de plus spécifiquement impliqués dans les métastases cancéreuses et l'infection par le VIH. Nous avons développé un système permettant de sur-exprimer ces deux RCPGs. Afin de s'affranchir des problèmes de toxicité inhérents à l'expression des protéines membranaires en bactérie notre approche de production consiste à adresser les protéines vers les corps d'inclusion d'E. coli grâce à une fusion protéique N-terminale permettant de hauts niveaux d'expression. Après purification en conditions dénaturantes, les protéines sont alors repliées en présence de surfactants originaux, les amphipoles. La validation de cette nouvelle approche pour les récepteurs des chimiokines représente un des objectifs principaux de ce travail. Afin de tester la fonctionnalité des protéines repliées, une série d'outils a été développée : des versions modifiées des chimiokines ont été produites (RANTES pour CCR5 et SDF 1a pour CXCR4). La fonctionnalité des chimiokines a été évaluée au niveau moléculaire et cellulaire. L'interaction entre le récepteur replié en amphipole et son ligand a été testé par résonance de plasmons de surface (SPR). Différents types de surfaces fonctionalisées avec le récepteur de chimiokine replié en amphipole ont été explorés au cours de ce travail. A la fin de ce projet, la production des chimiokines et de leur récepteur a été mise au point. L'accès à ces outils ouvre la voie à de futures études moléculaires telles que la compréhension de la dimérisation du récepteur ou la détermination de la stoechiométrie du complexe.