No / The isolation and characterisation of antiplatelet antibodies in autoimmune thrombocytopenia purpura patients (ITP) is described. Autoimmune thrombocytopenia purpura is an autoimmune disease, clinically defined by low platelet counts, normal or increased megakaryocytopoiesis and antiplatelet antibodies in serum. This study used phage display to isolate Fab antiplatelet antibodies to study the structure-function relationships of pathogenic antibodies in ITP. Out of six randomly selected colonies, four colonies reacted strongly with whole platelets in enzyme-linked immunosorbent assay (ELISA). Sequence analysis showed that all four colonies had the same DNA sequence and were the same antibody. Results of Western blotting against non-reduced human platelet lysate showed that the Fab reacted with platelet proteins with apparent molecular weights of 116, 92 and 39 kD. Furthermore, Western blotting assay against purified membrane glycoprotein IIIa demonstrated reactivity against a band with a molecular weight of 92 kD. Results from Western blotting against platelet lysate and pure platelet glycoprotein confirmed the Fab fragment recognised the platelet glycoprotein IIIa. Three out of the four phage colonies produced soluble Fab, which demonstrated reactivity against platelet autoantigens in ELISA. Further sequence analysis showed that the Fab was somatically mutated suggesting antigen drive and therefore T-cell assistance was important in the development of this antibody. One of the somatic mutations introduced an RSD amino acid sequence in the complementary determining region 1(CDR1) of the light chain, which may mimic the RGD motif of fibrinogen which binds integrin GPIIb/IIIa. This raises the possibility that somatic mutation and antigen drive have produced a pathogenic autoantibody.

Les anticorps sont des molécules de reconnaissance du non-soi permettant de distinguer spécifiquement des marqueurs antigéniques appelés épitopes. Deux types d’anticorps ont été découverts jusqu’à présents : les anticorps « classiques » et les anticorps de camélidés, également appelés nanobody. Cette thèse porte sur des études structurales de fragments d’anticorps d’intérêt thérapeutique et biotechnologique. Au cours d’un premier volet, j’ai résolu la structure par cristallographie aux rayons X du fragment d’un anticorps d’intérêt thérapeutique, MIC12, à une résolution de 1,5 Å. Dans le but de résoudre la structure des complexes de MIC12 avec des protéines homologues du CMH de classe I, j’ai obtenu des cristaux diffractant à une résolution allant jusqu’à 7 Å. En parallèle, des analyses par diffusion de rayons X aux petits angles (SAXS) combinées à des prédictions d’interaction par docking ont été conduites afin d’obtenir une première description de la région globale d’interaction de MIC12 sur l’une de ses cibles. Concernant le second volet, 4 nanobody ont été obtenus contre la protéine périplasmique PorM (pPorM) du système de sécrétion de type 9 de Porphyromonas gingivalis. J’ai résolu la structure par cristallographie du nanobody nb02, à une résolution de 1,5 Å. Leur utilisation comme chaperonne de cristallisation m’a permis de résoudre la structure de la partie N-terminale de pPorM. J’ai également mené une étude par SAXS de la protéine pPorM entière. L’ensemble des résultats que j’ai obtenu par cristallographie et par SAXS, combinés aux résolutions des structures des autres domaines de pPorM, ont permis de proposer un modèle structural de la protéine pPorM entière. / Antibodies are non-self recognition molecules wich help to specifically distinguish antigenic markers called epitopes. Two types of antibody were discovered so far: “classic” antibodies and camelid antibodies, also called nanobodies. This PhD deals with structural studies of antibodies fragments of therapeutic and biotechnological interest. During the first part, I solved the structure of the therapeutic antibody targeting homologous proteins of the MHC class I, MIC12, using X-ray crystallography at a resolution of 1.5 Å. In order to solve the structure of the complexes of MIC12 with its MIC antigens, I obtained crystals of the complexes diffracting at a resolution of up to 7 Å. In parallel, analyses by Small Angles X-ray Scattering (SAXS) combined with in silico docking predictions were led to obtain a first description of the global binding region of MIC12 on one of its targets. Concerning the second part, 4 nanobodies were obtained against the periplasmic protein PorM (pPorM) of the secretion system type 9 from Porphyromonas gingivalis. I solve the structure of the nanobody nb02, at a resolution of 1.5 Å. Their use as chaperones of crystallization helped me to solve the structure of the N-terminal part of pPorM. I also conducted a study by SAXS of the whole pPorM protein. All these results, obtained by crystallography and SAXS studies, combined with the solving of the structures of the other domains of pPorM, made it possible to propose a structural model of the entire pPorM protein.

Cette thèse a pour objectif de mieux comprendre et d’accompagner les stratégies d’innovation des entreprises face aux défis liés à l’intensification de la dynamique et des changements de l’environnement économique. Or, afin de survivre à ces rythmes économiques, la littérature préconise aux organisations de concilier leurs activités d’exploitation et leurs activités d’exploration de façon concomitante. Dans ce cadre, notre travail étudie spécifiquement une stratégie d’innovation conférant une importance particulière aux initiatives des individus dans les activités d’exploration : l’ambidextrie contextuelle. Simultanément, notre recherche examine un objet de recherche jusque là inédit dans le milieu académique : le Fab Lab d’entreprise (ou Fab Lab interne). A la croisée des chemins de ces deux sujets, cette thèse pose la question du développement de l’ambidextrie contextuelle à l’aide d’un Fab Lab d’entreprise.L’étude de cas menée au sein de l’une des premières structures à développer cette pratique de Fab Lab d’entreprise – le Groupe Renault – définit cet outil de gestion comme une quasi-structure propice au développement de l’ambidextrie contextuelle. Elle présente ensuite le pilotage à mener afin de développer l’ambidextrie contextuelle à l’aide d’un Fab Lab d’entreprise. / This thesis aims to better understand and support corporate innovation strategies to help them coping with the challenges associated with the intensification of the dynamics and changes in the economic environment. However, in order to survive these economic rhythms, previous research recommends organizations to reconcile their exploitation and their exploration activities, concomitantly. In this context, our work specifically examines an innovation strategy giving particular attention to the initiatives of individuals in exploration activities : contextual ambidexterity. Simultaneously, our research examines an object of research previously unreleased in academic studies : the corporate Fab Lab (or internal Fab Lab). At the crossroads of these two subjects, this thesis raises the question of the development of contextual ambidexterity through a corporate Fab Lab. Our case study is conducted in Renault Group, one of the first structures to develop this practice of corporate Fab Lab. Our analysis permits to define this management tool as a quasi-structure conducive to the development of contextual ambidexterity. It then presents the guidance to lead in the objective of developing contextual ambidexterity through a corporate Fab Lab.

A digoxina é um dos medicamentos indicados para o tratamento de falência cardíaca. Possui janela terapêutica estreita, sendo responsável por casos de intoxicação. O único antídoto disponível para a desintoxicação é o anticorpo policlonal DigiFab®, no formato Fab. O seu uso é eficaz, porém de custo elevado. Clones bacterianos produtores de fragmento Fab monoclonal anti-digoxina foram obtidos previamente pelo nosso grupo, pela técnica de phage display. Neste trabalho as variantes Fab dos 4 clones foram expressas em E.coli para estabelecer o método para a purificação. Com a obtenção dos fragmentos Fab purificados, foi caracterizada a sua afinidade ao antígeno e especificidade, em ensaios de inibição por digoxina, digitoxina, digoxigenina e ouabaina. Os parâmetros cinéticos da ligação dos fragmentos Fab dos 4 clones e do DigiFab® foram avaliados por SPR. Nas condições experimentais, não foram verificadas diferenças significativas entre os produtos dos 4 clones e o comercial, demonstrando o potencial dos fragmentos Fab monoclonais obtidos como antídoto à digoxina. / Digoxin is a medication indicated for heart failure treatment. Its therapeutic window is narrow, being responsible for intoxication cases. The only antidote available for the detoxification is a polyclonal antibody - DigiFab® in Fab format. Its use is effective, but costly. Bacterial clones producing anti-digoxin monoclonal Fab fragments were previously obtained by our group using phage display technology. In this work the Fab variants of the 4 clones were expressed in E.coli to establish the purification method. The purified fragments were characterized regarding the affinity to the antigen and the specificity through inhibition assays with digoxin, digitoxin, digoxigenin and ouabain. The binding kinetic parameters of Fab fragments of the 4 clones and the commercial product to Dig-BSA conjugate were assessed by SPR. Under the experimental conditions no significant differences were observed among the 4 clones and the commercial product, demonstrating the potential of monoclonal Fab fragments as an antidote to digoxin.

La protection des zones littorales contre les agressions naturelles provenant de la mer, et notamment contre le risque de submersion marine, est essentielle pour sécuriser les installations côtières. La prévention de ce risque est assurée par des protections côtières qui sont conçues et régulièrement vérifiées grâce généralement à la définition du concept de niveau de retour d’un événement extrême particulier. Le niveau de retour lié à une période de retour assez grande (de 1000 ans ou plus) est estimé par des méthodes statistiques basées sur la Théorie des Valeurs Extrêmes (TVE). Ces approches statistiques sont appliquées à des séries temporelles d’une variable extrême observée et permettent de connaître la probabilité d’occurrence de telle variable. Dans le passé, les niveaux de retour des aléas maritimes extrêmes étaient estimés le plus souvent à partir de méthodes statistiques appliquées à des séries d’observation locales. En général, les séries locales des niveaux marins sont observées sur une période limitée (pour les niveaux marins environ 50 ans) et on cherche à trouver des bonnes estimations des extrêmes associées à des périodes de retour très grandes. Pour cette raison, de nombreuses méthodologies sont utilisées pour augmenter la taille des échantillons des extrêmes et réduire les incertitudes sur les estimations. En génie côtier, une des approches actuellement assez utilisées est l’analyse régionale. L’analyse régionale est indiquée par Weiss (2014) comme une manière très performante pour réduire les incertitudes sur les estimations des événements extrêmes. Le principe de cette méthodologie est de profiter de la grande disponibilité spatiale des données observées sur différents sites pour créer des régions homogènes. Cela permet d’estimer des lois statistiques sur des échantillons régionaux plus étendus regroupant tous les événements extrêmes qui ont frappé un ou plusieurs sites de la région (...) Cela ainsi que le caractère particulier de chaque événement historique ne permet pas son utilisation dans une analyse régionale classique. Une méthodologie statistique appelée FAB qui permet de réaliser une analyse régionale tenant en compte les données historiques est développée dans ce manuscrit. Élaborée pour des données POT (Peaks Over Threshold), cette méthode est basée sur une nouvelle définition d’une durée d’observation, appelée durée crédible, locale et régionale et elle est capable de tenir en compte dans l’analyse statistique les trois types les plus classiques de données historiques (données ponctuelles, données définies par un intervalle, données au-dessus d’une borne inférieure). En plus, une approche pour déterminer un seuil d’échantillonnage optimal est définie dans cette étude. La méthode FAB est assez polyvalente et permet d’estimer des niveaux de retour soit dans un cadre fréquentiste soit dans un cadre bayésien. Une application de cette méthodologie est réalisée pour une base de données enregistrées des surcotes de pleine mer (données systématiques) et 14 surcotes de pleine mer historiques collectées pour différents sites positionnés le long des côtes françaises, anglaises, belges et espagnoles de l’Atlantique, de la Manche et de la mer du Nord. Enfin, ce manuscrit examine la problématique de la découverte et de la validation des données historiques / The protection of coastal areas against the risk of flooding is necessary to safeguard all types of waterside structures and, in particular, nuclear power plants. The prevention of flooding is guaranteed by coastal protection commonly built and verified thanks to the definition of the return level’s concept of a particular extreme event. Return levels linked to very high return periods (up to 1000 years) are estimated through statistical methods based on the Extreme Value Theory (EVT). These statistical approaches are applied to time series of a particular extreme variable observed and enables the computation of its occurrence probability. In the past, return levels of extreme coastal events were frequently estimated by applying statistical methods to time series of local observations. Local series of sea levels are typically observed in too short a period (for sea levels about 50 years) in order to compute reliable estimations linked to high return periods. For this reason, several approaches are used to enlarge the size of the extreme data samples and to reduce uncertainties of their estimations. Currently, one of the most widely used methods in coastal engineering is the Regional Analysis. Regional Analysis is denoted by Weiss (2014) as a valid means to reduce uncertainties in the estimations of extreme events. The main idea of this method is to take advantage of the wide spatial availability of observed data in different locations in order to form homogeneous regions. This enables the estimation of statistical distributions of enlarged regional data samples by clustering all extreme events occurred in one or more sites of the region. Recent investigations have highlighted the importance of using past events when estimating extreme events. When historical data are available, they cannot be neglected in order to compute reliable estimations of extreme events. Historical data are collected from different sources and they are identified as data that do not come from time series. In fact, in most cases, no information about other extreme events occurring before and after a historical observation is available. This, and the particular nature of each historical data, do not permit their use in a Regional Analysis. A statistical methodology that enables the use of historical data in a regional context is needed in order to estimate reliable return levels and to reduce their associated uncertainties. In this manuscript, a statistical method called FAB is developed enabling the performance of a Regional Analysis using historical data. This method is formulated for POT (Peaks Over Threshold) data. It is based on the new definition of duration of local and regional observation period (denominated credible duration) and it is able to take into account all the three typical kinds of historical data (exact point, range and lower limit value). In addition, an approach to identify an optimal sampling threshold is defined in this study. This allows to get better estimations through using the optimal extreme data sample in the FAB method.FAB method is a flexible approach that enables the estimation of return levels both in frequentist and Bayesian contexts. An application of this method is carried out for a database of recorded skew surges (systematic data) and for 14 historical skew surges recovered from different sites located on French, British, Belgian and Spanish coasts of the Atlantic Ocean, the English Channel and the North Sea. Frequentist and Bayesian estimations of skew surges are computed for each homogeneous region and for every site. Finally, this manuscript explores the issues surrounding the finding and validation of historical data

Este trabalho analisa, mais do que os cantos de guerra do Brasil durante a Segunda Guerra Mundial, o Brasil dos cantos de guerra. Trabalha com as peças criadas pelos combatentes da FEB e da FAB na Itália, com as músicas difundidas pelos músicos militares e pelas transmissões de rádio feitas pela FEB naquele país, e com os hinos que estimularam as ações patrióticas dos brasileiros em território nacional. Também se utiliza de discos comerciais, lançados nos anos em que o país participou efetivamente do conflito (1942-1945), que revelam a matéria brasileira naquela situação de rearranjo internacional. Todas essas canções passaram pelo crivo da teoria crítica brasileira, encontrando na paródia a ferramenta mais comum à época para a estruturação social na música. / This work analyses, over and beyond the Brazilian war songs during the Second World War, the Brazil (itself) in the war songs. It uses the plays created by the FEB and FAB combatants in Italy, the music revealed by the military musicians and by radio transmissions of FEB as well as the hymns that stimulated the patriotic actions of Brazilians throughout the country. Also, commercial records, launched during the years that the country effectively participated in the conflict (1942-1945), illustrating the Brazilian viewpoint during this particular situation of international rearrangement. All these songs have passed through the sieve of Brazilian Critical Theorry, finding in the parody the most common tool at the time for show the social structures in music.

Este trabalho analisa, mais do que os cantos de guerra do Brasil durante a Segunda Guerra Mundial, o Brasil dos cantos de guerra. Trabalha com as peças criadas pelos combatentes da FEB e da FAB na Itália, com as músicas difundidas pelos músicos militares e pelas transmissões de rádio feitas pela FEB naquele país, e com os hinos que estimularam as ações patrióticas dos brasileiros em território nacional. Também se utiliza de discos comerciais, lançados nos anos em que o país participou efetivamente do conflito (1942-1945), que revelam a matéria brasileira naquela situação de rearranjo internacional. Todas essas canções passaram pelo crivo da teoria crítica brasileira, encontrando na paródia a ferramenta mais comum à época para a estruturação social na música. / This work analyses, over and beyond the Brazilian war songs during the Second World War, the Brazil (itself) in the war songs. It uses the plays created by the FEB and FAB combatants in Italy, the music revealed by the military musicians and by radio transmissions of FEB as well as the hymns that stimulated the patriotic actions of Brazilians throughout the country. Also, commercial records, launched during the years that the country effectively participated in the conflict (1942-1945), illustrating the Brazilian viewpoint during this particular situation of international rearrangement. All these songs have passed through the sieve of Brazilian Critical Theorry, finding in the parody the most common tool at the time for show the social structures in music.

A digoxina é um dos medicamentos indicados para o tratamento de falência cardíaca. Possui janela terapêutica estreita, sendo responsável por casos de intoxicação. O único antídoto disponível para a desintoxicação é o anticorpo policlonal DigiFab®, no formato Fab. O seu uso é eficaz, porém de custo elevado. Clones bacterianos produtores de fragmento Fab monoclonal anti-digoxina foram obtidos previamente pelo nosso grupo, pela técnica de phage display. Neste trabalho as variantes Fab dos 4 clones foram expressas em E.coli para estabelecer o método para a purificação. Com a obtenção dos fragmentos Fab purificados, foi caracterizada a sua afinidade ao antígeno e especificidade, em ensaios de inibição por digoxina, digitoxina, digoxigenina e ouabaina. Os parâmetros cinéticos da ligação dos fragmentos Fab dos 4 clones e do DigiFab® foram avaliados por SPR. Nas condições experimentais, não foram verificadas diferenças significativas entre os produtos dos 4 clones e o comercial, demonstrando o potencial dos fragmentos Fab monoclonais obtidos como antídoto à digoxina. / Digoxin is a medication indicated for heart failure treatment. Its therapeutic window is narrow, being responsible for intoxication cases. The only antidote available for the detoxification is a polyclonal antibody - DigiFab® in Fab format. Its use is effective, but costly. Bacterial clones producing anti-digoxin monoclonal Fab fragments were previously obtained by our group using phage display technology. In this work the Fab variants of the 4 clones were expressed in E.coli to establish the purification method. The purified fragments were characterized regarding the affinity to the antigen and the specificity through inhibition assays with digoxin, digitoxin, digoxigenin and ouabain. The binding kinetic parameters of Fab fragments of the 4 clones and the commercial product to Dig-BSA conjugate were assessed by SPR. Under the experimental conditions no significant differences were observed among the 4 clones and the commercial product, demonstrating the potential of monoclonal Fab fragments as an antidote to digoxin.

No description available.

No description available.