Le virus de la stomatite vésiculaire (VSV) et le virus Nipah (NiV) appartiennent respectivement aux familles des Rhabdoviridae et des Paramyxoviridae. VSV est un modèle du virus de la rage tandis que NiV est un virus émergeant, appartenant à la sous-famille des Paramyxovirinae, pour lequel les données moléculaires et structurales sont limitées. Ces sont des virus enveloppés dont le génome code pour cinq à neuf protéines. Le complexe de réplication de ces virus est constitué de trois protéines : la phosphoprotéine (P), la nucléoprotéine (N) et la polymérase virale (L). La N encapside le génome viral et l'ensemble N-ARN sert de matrice pour la transcription et la réplication. La P joue deux rôles : elle sert de cofacteur pour la polymérase et forme le complexe N0-P qui maintient la N sous une forme soluble, compétente pour l'encapsidation des génomes néo-synthétisés. Un premier objectif de mon travail de thèse consistait à étudier la structure et la dynamique des protéines P de VSV et de NiV. Ce sont des protéines modulaires qui contiennent des domaines structurés, séparés par des régions flexibles. A mon arrivée au laboratoire un travail important avait été déjà réalisé sur la P de VSV et j'ai participé à l'achèvement de cette étude. Je me suis ensuite intéressé à la protéine P de NiV. J'ai cristallisé et résolu par diffraction des rayons X les structures du domaine C-terminal et du domaine central (codes PDB : 4F9X et 4GJW). La combinaison de ces modèles cristallographiques avec des données de SAXS sur la P entière et des données de résonance magnétique nucléaire (RMN, collaboration IBS) va permettre d'obtenir un modèle atomique de la P entière sous la forme d'un ensemble de conformères. Un deuxième objectif était d'étudier les complexes N0-P. J'ai activement participé au développement de la méthode de reconstitution et à la caractérisation structurale du complexe N0-P de VSV, entre un mutant de la N (NΔ21) et un peptide N-terminal de la P (code PDB : 3PMK). J'ai ensuite reconstitué, cristallisé et résolu la structure de complexe N0-P de NiV entre la N (tronquée de son domaine C-terminal) et la partie N-terminale de la P. Ces structures montrent par quel mécanismes moléculaires la P maintien la N sous forme monomérique, en empêchant sa polymérisation et son interaction avec l'ARN. Les résultats présentés ici ont permis de générer de nouvelles hypothèses pour expliquer les mécanismes d'encapsidation et d'initiation de la synthèse d'ARN chez ces virus. Le complexe N0-P étant essentiel pour la réplication du virus, l'information structurale obtenue au cours de ce travail devrait permettre d'envisager l'utilisation de ce complexe comme cible pour le développement de composés antiviraux. / Abstract Vesicular stomatitis virus (VSV) and Nipah virus (NiV) belong to the Rhabdoviridae and Paramyxoviridae families, respectively. VSV serves as model system for rabies virus while NiV is an emerging pathogen of the Paramyxovirinae subfamily, for which molecular and structural data are scarce. Both viruses are enveloped and their genomes encode five to nine proteins. Three proteins form their replication complex: the phosphoprotein (P), the nucleoprotein (N) and the viral polymerase (L). N encapsidates the viral genome and this N-RNA complex serves as template for transcription and replication. P has two functions: it serves as a polymerase cofactor and forms an N0-P complex, which keeps the N protein in a soluble and monomeric state, competent for the encapsidation of the newly synthesized genomes. The first goal during the PhD work was to study the structure and dynamics of the VSV and NiV P proteins. These proteins are modular, containing structured domains separated by flexible regions. Before my arrival, a large amount of work was already done on the VSV P protein in the lab and I was involved in the final stages of this work. Then this I studied the NiV P protein, crystallizing and solving the structures of its Central and C-terminal domains by X-ray crystallography (PDB codes: 4F9X and 4GJW). Combining these structures with small angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR, collaboration with IBS group) data obtained for the entire protein will allow the construction of an atomic model of the phosphoprotein in the form of a conformational ensemble. The second goal was to study the N0-P complex. I actively participated in the development of the method which permitted the reconstruction of the VSV N0-P complex, using a truncation mutant of the N protein (NΔ21) and an N-terminal peptide from P, and to its structural determination (PDB code: 3PMK). Then I reconstructed, crystallized and solved the structure of the NiV N0-P complex using a C-terminally truncated N protein and the N-terminal region of the P protein. Both structures yielded insights into the molecular mechanisms used by the phosphoproteins in order to maintain the corresponding nucleoproteins in their monomeric state, thus inhibiting their polymerization and interaction with RNA. The results presented here also offered new hypothesis about mechanisms of encapsidation and of RNA synthesis initiation. Given that the N0-P complex is an essential component of the replication complex, the structural information gained from this work allow us to consider this complex as a potential antiviral target.

Orientador: Jorg Kobarg / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T17:04:45Z (GMT). No. of bitstreams: 1 Meirelles_GabrielaVaz_D.pdf: 11269390 bytes, checksum: bfd22a079ffc68a78b96b3e50dd60b1c (MD5) Previous issue date: 2011 / Resumo: A proteína NIMA foi identificada e caracterizada funcionalmente em Aspergillus nidulans como sendo uma serina/treonina cinase critica para a progressão do ciclo celular. As Neks (NIMA-related kinases) constituem uma família de cinases composta por 11 membros em mamíferos, que compartilham 40-45% de identidade com a proteína NIMA no domínio catalítico N-terminal. As Neks estão associadas a funções do ciclo celular e diversas patologias, o que as torna potenciais alvos quimioterápicos. Mutações no gene da Nek1 levam ao desenvolvimento da doença renal policistica e ao aparecimento de diversos efeitos pleiotrópicos, sugerindo sua participação em vias reguladoras de vários processos celulares. A Nek6, por sua vez, e ativada durante a mitose, e a super-expressão de mutantes inativos ou a sua depleção por RNAi produz células exibindo defeitos no fuso, anormalidades nucleares, parada na metáfase e apoptose. A Nek6 humana foi recentemente associada a carcinogênese, mas, assim como para a maioria das Neks, sua estrutura molecular, parceiros de interação e vias de sinalização permanecem ainda desconhecidos. Nesse trabalho, introduzimos a hNek6 como uma hub no interactoma humano. Uma extensa comparação de bancos de dados baseada em analises de conectividade mostrou que o quinoma humano e enriquecido em hubs. Nossas redes de interação incluem um amplo espectro de novos parceiros de interação para a hNek6 identificados em screenings de duplo - hibrido em levedura, classificados em 18 categorias funcionais. Alguns novos parceiros de interação da hNek6 são também possíveis substratos e, ainda, colocalizam com a hNek6 e ?-tubulina em células humanas, apontando para uma possível interação centrossomal. Os diversos parceiros de interação conectam a hNek6 a novas vias, como a sinalização de Notch e a regulação do citoesqueleto de actina, ou fornecem novas pistas de como a hNek6 poderia regular vias previamente propostas, como ciclo celular, reparo de DNA e sinalização do NF-?B. Alem disso, obtivemos o primeiro modelo estrutural de baixa resolução para a hNek6 a partir de SAXS. Analises estruturais revelaram que a hNek6 e um monômero em solução, apresentando uma conformação predominantemente globular, mas levemente alongada. Particularmente, a curta região N-terminal desordenada da hNek6 e importante para mediar as interações com seus parceiros. No caso da hNek1, observamos que ela interage com Fez1 e Clasp2 através de seus motivos coiled-coil, e colocaliza com essas proteínas em uma região candidata ao centrossomo / Abstract: NIMA was identified and functionally characterized in Aspergillus nidulans as a critical Ser/Thr kinase for cell cycle progression. The mammalian Neks (NIMA-related kinases) represent an evolutionarily conserved family of 11 serine/threonine kinases that share 40-45% identity with NIMA N-terminal domain. Neks are associated to cell cyclerelated functions and diverse pathologies, which highlight them as potential chemotherapeutic targets. Nek1 gene mutations lead to the development of polycystic kidney disease and the emergence of several pleiotropic effects, suggesting its involvement in pathways regulating various cellular processes. Nek6, in turn, is activated during mitosis, and overexpression of inactive mutants or its depletion by iRNA produces cells exhibiting mitotic spindle defects, nuclear abnormalities, metaphase arrest and apoptosis. Human Nek6 was recently found to be linked to carcinogenesis, but as for the majority of Neks, the molecular structure, interacting partners and signaling pathways remain elusive. Here we introduce hNek6 as a hub kinase in the human interactome. We performed a broad databank comparison based on degree distribution analysis and found that the human kinome is enriched in hubs. Our networks include a large set of novel hNek6 interactors identified in our yeast two-hybrid screens, classified into 18 functional categories. Some novel interactors are also putative substrates and colocalized with hNek6 and ?-tubulin in human cells, pointing to a possible centrosomal interaction. The interacting proteins link hNek6 to novel pathways, e.g. Notch signaling and actin cytoskeleton regulation, or give new insights on how hNek6 may regulate previously proposed pathways such as cell cycle, DNA repair and NF-?B signalings. Furthermore, we obtained the first low-resolution structural model of hNek6 by SAXS. Structural analysis revealed that hNek6 is a monomer in solution with a mostly globular, though slightly elongated conformation. Notably, we found that hNek6 unfolded short N-terminal region is important to mediate the interactions with its partners. In the case of hNek1, we found that it interacts with Fez1 and Clasp2 through coiled-coil motifs and colocalizes with these proteins in a candidate centrosomal region / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

A análise de proteínas como redes é uma ferramenta poderosa para compreender as suas propriedades e a importância relativa de seus resíduos. Nesta análise, os resíduos que interagem entre si, covalentemente ou não, são chamados conectados. Nesta abordagem, alguns resíduos contribuem mais fortemente para manter as propriedades da rede, sendo chamados de centrais. Diversos trabalhos têm apontado que resíduos centrais da Rede de Estrutura Proteica também são importantes nas propriedades das proteínas, desempenhando papéis na catálise, estabilidade térmica e alosteria. No entanto, existe falta de trabalhos desenhados de forma sistemática para confirmar esta hipótese. Neste sentido, esta tese tem como objetivo avaliar se existe correlação entre a centralidade dos resíduos de uma enzima, a beta-glicosidase de Spodoptera frugiperda, Sfβgli, e a importância destes resíduos na determinação das suas propriedades. Para isso, foram utilizadas duas abordagens (capítulo 1): Na primeira, os resíduos centrais foram diretamente perturbados substituindo-os, através de mutação sítio-dirigida, por alanina. Na segunda, perturbações no resíduo central foram feitas modificando a vizinhança deste resíduo através de mutações que introduziram ou removeram volume de seu entorno. A partir disso, foi avaliado se estas perturbações afetaram as propriedades Sfβgli. De forma geral, foi observado (capítulo 2) que as perturbações nos resíduos centrais por ambas abordagens afetam significativamente a termoestabilidade da proteína, reduzindo a sua Tm em até 15°C e aumentando a velocidade de sua desnaturação térmica em até mais de 20 vezes. Além disso, a atividade catalítica de Sfβgli é reduzida por estas perturbações (capítulo 3), sendo que este efeito e a perda da termoestabilidade parecem resultar da mesma causa, a perturbação do resíduo central. No capítulo 4, a investigação do estado oligomérico da Sfβgli por SAXS revelou que esta ocorre preponderantemente como dímero em citrato-fosfato 100 mM pH 6,0, mas como um grande oligômero, possivelmente um dodecâmero, em fosfato 10 mM pH 6,0. Paralelamente foi demonstrado que Sfβgli passa por uma ativação quando em tampão fosfato 10 mM, convergindo para as propriedades cinéticas de Sfβgli em citrato-fosfato 100 mM. Redes de Estrutura Proteica foram produzidas considerando-se também a interação entre as cadeias polipeptídicas constituintes de oligômeros de Sfβgli (dímeros, tetrâmeros e hexâmeros). Assim, observou-se que cinco resíduos são sempre centrais por betweeness, mesmo considerando diferentes oligomêros da Sfgli. Destes, E187, P188 e N329 desempenham papéis conhecidos na catálise e S247 e N249 foram caracterizados nesta tese. Por fim, no capítulo 5, analisando a centralidade dos resíduos da Rede Estrutural da Sfβgli, observa-se uma preponderante presença de resíduos centrais por CΔLp, closeness e betweeness no topo do beta-barril, demonstrando que esta região é muito próxima dos demais resíduos da proteína. Além disso, uma análise da centralidade dos resíduos de 21 beta-glicosidases GH1 revelou que resíduos centrais por closeness são bastante conservados, sendo encontrados predominantemente no sítio ativo destas enzimas, enquanto que dentre os centrais por betweeness há variabilidade. Portanto os resultados apresentados nesta tese suportam experimentalmente a hipótese de que a centralidade dos resíduos na Rede de Estrutura Proteica é correlacionada com propriedades funcionais das proteínas. / Analysis of protein structures as networks has been shown a powerful tool to understand their properties and to identify important residues. In the network analysis, residues that interact with each other are called connected. Some residues are essential to shorten the connection pathways between distant residues in the protein structure, being called central. Central residues have been proposed to have important roles in catalysis, thermal stability and allostery. In order to experimentally assess the correlation between the residue centrality and its importance in the protein properties, we use two approaches (chapter 1): The first one is to make single mutations at the central residues of a betaglucosidase Sfβgly, changing those residues to alanine. The second one is to perturb a central residue (F251) by changing its environment through single mutations that introduces voids or additional volume. Next, we evaluate how those mutations affect the protein thermostability and function. In general, we have observed (chapter 2) that mutations at central residues reduce the Tm in 2 - 15°C and increase the unfolding rate up to 20 times, suggesting that damages in the central residues make the protein more unstable. Moreover, we have observed (chapter 3) that the perturbation of the central residues reduces Sfβgly catalysis, which seems to arise from the same cause that lead to the loss of thermal stability. Besides that, in chapter 4, the investigation of oligomeric state of Sfgli using SAXS indicated that this protein is mainly a dimer in 100 mM citrate-phosphate pH 6,0, whereas it forms large oligomers, possibly dodecamers, in 10 mM phosphate pH 6,0. In parallel it was shown that Sfβgly undergoes an activation process in 10 mM phosphate and its kinetic parameters converge to those observed for Sfβgly in 100 mM citrate-phosphate. Protein Structural Networks were built considering also that there are links between the polypeptidic chains of the Sfβgly oligomers. We observed 5 residues that are central in all kind of oligomeric structures here analyzed. Three of these residues, E187, P188 and N329, play important roles in the catalysis of this enzyme, and two of them (S247 and N249 are described in this thesis. Lastly, in the chapter 5, we observed that central residues by closeness, betweeness and CΔLp are concentrated at the top of the beta-barrel (C-terminal end of the beta-strands and subsequent loops), suggesting that this region, where the active site is placed, is close, in terms of contacts, to the whole Sfβgly structure. Moreover, we have built the Protein Structural Network of 21 beta-glucosidases of the Glucoside Hydrolases family 1, revealing that the closeness central residues are highly conserved, being located in the active site of these enzymes. On the other hand, betweeness central residues are located in the same sites in the structure of different beta-glucosidases, but they are not always conserved. Shortly, these data experimentally support the hypothesis that the residue centrality in Protein Structural Network is correlated with the protein properties, as catalysis and stability.

Le domaine de recherche concerne l’endommagement par cavitation des élastomères exposés à de fortes pressions de gaz diffusant. Ce phénomène résulte de l’expansion locale du gaz préalablement absorbé, lorsque la désorption hors du polymère est trop lente par rapport au chargement imposé. Dans le cas de l’hydrogène qui nous intéresse ici, l’enjeu est le développement de matériaux polymères performants pour les structures de stockage et de distribution d’hydrogène gazeux hyperbare. En conditions d’usage, ces matériaux sont exposés à de fortes pressions d’hydrogène qui diffuse en leur sein et génèrent ensuite de forts endommagements lorsque la pression hydrostatique est relâchée. Les études de laboratoire sur ce sujet restent peu nombreuses, a fortiori sous environnement hydrogène. Sur le plan expérimental, ceci s’explique par la délicate manipulation de l’hydrogène et par le contexte des fortes pressions. Sur le plan de la simulation numérique, un verrou important est lié aux couplages forts entre diffusion et mécanique dans la résolution de l’équilibre de la cavité à chaque instant de son évolution. Cette thèse vise à mieux comprendre le mécanisme élémentaire de formation, puis de croissance et de coalescence des cavités, isolées ou en proche voisinage. Dans ce dernier cas, une éventuelle interaction doit effectivement être caractérisée pour éclairer la coalescence et la transition vers des fissures macroscopiques. Le travail a été mené sur série d’Ethylène Propylène Diène Monomer (EPDM) non-renforcés, avec une densité de points de réticulation variable, exposés à des pressions allant jusqu’à 30 MPa. Le volet expérimental s'appuie sur deux des techniques expérimentales in situ les plus récentes. La diffusion des rayons X aux petits angles (SAXS) vise à caractériser les hétérogénéités du système réseau élastomère – hydrogène à l'échelle submicronique, et éventuellement à détecter les premiers stades de cavitation. Dans la gamme des faibles pressions accessible sous environnement hydrogène, les hétérogénéités ne sont pas assez marquées pour définir plus qu'une distance de corrélation, qui varie très peu comparativement au matériau non exposé. Après exposition à une pression plus élevée (30 MPa), une augmentation de la distance de corrélation est observée, révélant une modification de l'hétérogénéité de la matrice, irréversible même après désorption complète de l'échantillon. À l’échelle micronique, des expériences de tomographie X in-situ (sous des pressions allant jusqu’à 12 MPa) fournissent des vues 3D résolues en temps des cavités, pendant et après décompression. Ces expériences ont permis de mieux comprendre la cinétique de croissance des cavités dans différentes conditions aux limites locales (dans le volume de l’échantillon, à proximité d'autres cavités, près d'une surface libre) et de les interpréter en regard des propriétés de diffusion de l'échantillon lui-même. Plusieurs populations de cavités, présentant des comportements différents, ont ainsi pu être distinguées en fonction de leur distance à la surface libre de l'échantillon, en lien avec la désorption globale de l’échantillon. Comparativement à cet effet de bord libre, la présence d’une autre cavité en proche voisinage (i.e. à une distance bord à bord supérieure ou égale à 30μm) n’a qu’une influence minime. Les résultats suggèrent que la croissance de la cavité est un processus très local. Dans un contexte diffuso-mécanique fortement couplé, l’interprétation des mécanismes se heurte à l’impossibilité d’accéder expérimentalement aux champs mécaniques et de concentration de gaz. Les codes éléments finis existants rencontrent des problèmes de convergence que le code interne Foxtrot développé à l’Institut Pprime tente de surmonter. Dans une dernière partie exploratoire de la thèse, il a été mis à profit pour comparer les gradients générés par une paire de cavités comparativement à une cavité isolée. / The optimum design and formulation of seals used in hydrogen transport system is crucial for the purposes of safety of operation and well as economic sustainability of hydrogen as energy carrier. The exposure of the sealing materials to hydrogen and subsequent decompression causes cavitation damage. The studies so far on this subject have been few due to the strong limitations arising from the safety issues related to hydrogen testing in laboratory conditions. This study addresses the cavitation in Ethylene Propylene Diene Rubber (EPDM) due to pressure release after exposure to high-pressure hydrogen up to 30 MPa. Three different unfilled EPDM with variable cross-link density were investigated. The study was based on some of the newest in-situ experimental techniques which allow a time-resolved tracking of the evolution of damage. On one side, in-situ SAXS (Small Angle X-ray Scattering) tests of hydrogen-exposed EPDM were aimed at the characterisation of EPDM at submicron scale as a function of network heterogeneity and for tracking the possible onset of distinguishable cavities. At the low pressure range accessible with the device, heterogeneities were not marked enough to define more than a correlation length that was significantly changed compared the unexposed material, whatever the cross-link density. After the exposure at higher pressure (30 MPa) a change in correlation length was observed corresponding to the change in heterogeneity of the matrix which was found to be non-reversible even after full desorption of the sample. At a higher scale, in-situ X-ray tomography was used to provide time-resolved 3D views of damage during and after hydrogen pressure release. These experiments provided insight into the growth kinetics of cavities in different local boundary conditions (within the bulk, close to other cavities, close to a free surface) correlated with the diffusion characteristics of the sample itself. Classification of cavities as bulk and edges cavities was possible with respect with different kinetics depending on their proximity to the free surface of the sample. This could be correlated with the diffusion characteristics of the material. The dependence of kinetics of cavities on the proximity of another cavity was found to be trivial at the scale investigated (above 30 μm between cavity borders) suggesting that growth is a very local process. The previous studies have clarified that the cavitation in rubber is a coupled diffuso-mechanical phenomenon and so far, the numerical tools available have not addressed the problem as such. Therefore, the development of a numerical tool aimed at solving such coupled problems has also been addressed in the present work. This numerical tool called Foxtrot, developed at Institut PPRIME, is in the early stages of development but is a crucial step towards the more realistic simulation of this phenomenon of cavitation. In this fully coupled diffuso-mechanical context, the interpretation of mechanisms is highly limited by the lack of experimental access to the mechanical and gas content fields. Commercial Finite Element codes face convergence problems that the internal code developed at the Pprime Institute (Foxtrot) is trying to overcome. In the last exploratory part of the thesis, the code was used to as a step towards a more realistic simulation of the phenomenon. In particular, gradients around a pair of cavities were compared to those obtained around an isolated cavity.

京都大学 / 0048 / 新制・課程博士 / 博士(人間・環境学) / 甲第22708号 / 人博第958号 / 新制||人||227(附属図書館) / 2020||人博||958(吉田南総合図書館) / 京都大学大学院人間・環境学研究科相関環境学専攻 / (主査)教授 内本 喜晴, 教授 高木 紀明, 教授 中村 敏浩 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DFAM

No description available.

This thesis studies the properties and behaviour of phase-separated hydrogels prepared by interaction of hyaluronan with oppositely charged surfactants. Three representatives of surfactants, namely cetyltrimethylammonium bromide, chloride and p-toluensulfonate (CTAB, CTAC, CTAT), were selected for comparison. Using the method of rheology, the fact that the system of Hya-CTAT forms the most rigid hydrogels has been proved. Higher molecular weight of hyaluronan has also direct influence on the volume and stiffness of the newly formed hydrogels. Preparation methods were compared as well. Mixing the stock solutions of entry components appeard to be the most suitable. Small-angle X-ray scattering was used for determination of shape and size of surfactant's micelles, concluding that it has no effect on the volume of formed gels. The results of this study indicate that given gels are to a certain extent competitive and incorporation of hyaluronan into their structure is desirable with respect to its biological activity. This offers a potential usage of these substances in the field of medical applications.

In dieser Arbeit werden zwei Schwerpunkte behandelt. Zum Einen soll der Einsatz nanoskaliger Materialien als Funktionskomponenten in Elektrolumineszenzbauteilen beschrieben werden. Dabei wird in einem ersten Aufbau ein transparenter Nanokompositfilm als emittierende Schicht in einem, den organischen Leuchtdioden, analogen Aufbau eingesetzt, während in einer zweiten Struktur eine transparente Elektrode, die auf nanoskaligen Kohlenstoffmaterialien (Kohlenstoffnanoröhren bzw. Graphenen) basiert, hinsichtlich ihrer Eignung als Alternative zu etablierten transparenten Elektroden untersucht werden soll. In weiterführenden Arbeiten werden die Erfahrungen aus der Graphensynthese auf die Generierung poröser, funktionalisierter Kohlenstoffmaterialien angewendet. Verbindend, wird die Röntgenkleinwinkelstreuung eingesetzt, um in vergleichenden Untersuchungen möglichst detailierte Informationen über die jeweiligen Systeme zu erhalten.

Lactobacillus kunkeei is the most common and dominant bacterium in the honey stomach of honeybees. L. kunkeei has been isolated from honeybees all over the world. Genome sequencing has identified 5 genes for exceptionally large proteins in the genome of L. kunkeei. These proteins do not show any similarity to sequences of proteins with a known structure. These giant proteins all have a conserved region of 60 amino acids in their C-terminus. This conservation led to the hypothesis that the C-terminal domains of the giant proteins are important for their function with possibly a role in the attachment to the cell wall. In this study, a total of eight different constructs were made for two of these giant proteins. The boundaries for the constructs were determined based on bioinformatic predictions. The eight constructs all have different start positions and all end at the very C-terminal end of the protein. These constructs were cloned into an expression vector. One of the full-length giant protein was cloned into an expression vector as well.  The C-terminal constructs and the full-length proteins were recombinantly produced in Escherichia coli. Expression of six C-terminal constructs was observed and an attempt was made to purify two of the C-terminal constructs. Expression of the full-length giant protein was observed as well and purification was attempted. Neither the C-terminal constructs nor the full-length giant protein could be purified at full length. The results for the C-terminal constructs show that no folded C-terminal domain has been found for the giant proteins. A purified protein construct of the N-terminal of one of the giant proteins was available. This protein was analyzed using biophysical techniques. Circular dichroism was used to test the thermal stability. The construct did not refold after being thermally denatured. Circular dichroism measurements indicated that the N-terminal construct is composed of a mixture of α-helices and ß-sheets. Small-angle X-ray scattering data indicated that the N-terminal construct had an elongated shape with knot-like parts. Protein crystals have been obtained for the N-terminal construct and these will be analyzed using X-ray diffraction.

L’utilisation de la mousse en récupération assistée du pétrole (Enhanced Oil Recovery, EOR) présente un avantage indéniable par rapport à l’injection du gaz seul pour pallier les problèmes de ségrégation gravitaire et de digitations visqueuses. Son utilisation systématique en ingénierie du réservoir nécessite des connaissances plus approfondies sur son comportement en milieu poreux. La littérature montre deux types d’approches expérimentales basées soit sur des études pétrophysiques effectuées sur des systèmes poreux 3D et basées sur des mesures de pressions intégrées sur l’ensemble du milieu poreux, soit sur des études micro-fluidiques qui permettent une visualisation directe de l’écoulement mais qui sont limitées à des systèmes modèles dans des géométries à 1 ou 2 dimensions. L’objectif de cette thèse est de faire le pont entre ces deux approches. La stratégie proposée consiste à caractériser in situ l’écoulement de la mousse dans des milieux poreux 3D à différentes échelles, en utilisant des techniques complémentaires permettant d’accéder à une large gamme de résolutions spatiale et temporelle. Un environnement instrumenté donnant accès aux mesures pétro-physiques classiques a été développé puis couplé à différentes cellules d’observation conçues spécifiquement pour chaque instrument de caractérisation. Dans un premier temps, un scanner X a été utilisé pour décrire et visualiser les écoulements de la mousse à l’échelle de la carotte. La rhéologie de la mousse à cette échelle a pu être étudiée en fonction des conditions d’injections comme la vitesse interstitielle du gaz et la qualité de mousse. Dans un deuxième temps, la technique de diffusion des neutrons aux petits angles (SANS) a permis de sonder la texture de la mousse en écoulement sur trois ordres de grandeurs en taille. Des informations in situ sur la texture de la mousse en écoulement (taille et densité des bulles et des lamelles) ont pu être mesurées pour différentes qualités de mousse puis en fonction de la distance au point d’injection. Une comparaison avec les caractéristiques géométriques du milieu poreux a également été effectuée. Dans un troisième temps, la micro-tomographie X rapide haute résolution sur Synchrotron a été utilisée pour visualiser la mousse en écoulement à l’échelle du pore. Cette technique a permis de confirmer de visu certaines caractéristiques de la mousse mesurées par SANS et de décrire en sus les effets d’intermittence du piégeage de la mousse. Cette étude constitue une étape importante de la caractérisation multi-échelle de l’écoulement des mousses en milieux poreux 3D et apporte des éléments de réponse à certaines hypothèses admises. / Foam has long been used as a mobility control agent in Enhanced Oil Recovery (EOR) processes to enhance sweep efficiency and overcome gravity segregation, viscous fingering and gas channeling, which are gas-related problems when the latter is injected alone in the reservoir. However, the systematic use of foam in reservoir engineering requires more in-depth knowledge of its dynamics in porous media. The literature shows two types of experimental approaches based either on petrophysical studies carried out on 3D porous systems and based on pressure measurements, or on microfluidic studies that allow direct visualization of foam flow but are limited to 1D or 2D model systems. The research investigated in this thesis aims to bridge the gap between these two approaches. The proposed strategy is to characterize in situ the foam flow in 3D porous media with techniques providing a wide range of temporal and spatial resolutions. A coreflood setup giving access to classical petro-physical measurements was developed and then coupled to different observation cells designed specifically for each characterization instrument. First, an X-ray CT scanner was used to describe and visualize the foam flow at the core scale. The rheological behavior of foam on this scale was studied as a function of the injection conditions such as gas velocity and foam quality. Secondly, Small Angle Neutron Scattering (SANS) was used to probe the foam structure in situ during the flow, on a wide length scale, up to three orders of magnitude in size. In situ foam texture (size and density of bubbles and lamellae) was measured for different foam qualities and at different propagation distances from the injection point. A comparison to the geometric characteristics of the porous medium was also realized. Thirdly, High Resolution Fast X-ray Micro-tomography on a Synchrotron was used to visualize the foam flow at the pore scale. This allowed to confirm visually some foam characteristics measured with SANS and to investigate on local intermittent gas trapping and mobilization. This study is an important step in the multi-scale characterization of foam flow in 3D porous media and provides some answers to certain generally accepted assumptions.