La poudre de boehmite, précurseur de l’alumine utilisée pour la production de supports catalytiques variés, est produite suite à une étape de séchage par atomisation. Cet oxyhydroxyde d’aluminium est composé de particules anisotropes chargées. Ces dernières années, il a été ont montré que les propriétés physico-chimiques des suspensions avaient une influence sur les caractéristiques des poudres. La morphologie des grains (sphère creuse, tore…) ainsi que les propriétés des poudres (dispersibilité, résistance mécanique…) peuvent être modifiées en jouant sur les caractéristiques des suspensions.Ce travail de thèse porte sur la compréhension des liens entre les propriétés physico-chimiques et rhéologiques de suspensions de deux boehmites industrielles et les propriétés des grains obtenus après séchage. Dans une première partie, nous avons caractérisé les particules en suspension de ces deux boehmites grâce à une approche multi-technique (SAXS, DLS, MET). Une étude rhéologique des suspensions obtenues par stress osmotique a ensuite permis de construire précisément le diagramme de phase de ces deux boehmites dans le plan force ionique – fraction volumique. La diffusion des rayons X aux petits angles et la diffusion dynamique de la lumière ont été utilisées pour déterminer l’organisation structurale des suspensions. Enfin, des expériences de séchage suivies optiquement et par micro-tomographie X ont été réalisées sur des suspensions de boehmite afin d’étudier l’impact de la concentration et de la force ionique sur l’évolution de la goutte au cours du séchage. / In the catalyst supports industry, one important step of alumina powder production is the spray-drying of boehmite suspensions. This aluminium oxyhydroxyde is often used as precursor for various types of alumina catalyst supports. Boehmite is composed of anisotropic particles bearing an electric charge. In the past decade, it has been shown that the properties of the dry powder depend not only on the drying conditions but on physico-chemical properties of the sprayed suspensions. For instance, different grains morphologies (doughnut-shaped grains, hollow spheres,…) as well as various dry grains properties (textural properties, mechanical resistance,…) may be obtained by tuning the physico-chemical properties of the suspension.This study focus on the understanding of the links between the physico-chemical properties of suspensions of two industrial boehmites and the characteristics of the final dry grain. Since these two boehmites were not well-known, the first step of the study was to characterize boehmite particles present in suspension thanks to a multi-technical approach. A rheological study was performed on boehmite suspensions obtained thanks to osmotic stress to build precisely the phase diagrams of the both boehmites according to the system of ionic strength vs. volume fraction. Small angle X ray scattering and dynamic light scattering were used to investigate the existence of an orientational order of boehmite particles in suspension. Finally, drying experiments of boehmite suspensions were conducted on an optical set-up and on fast micro-tomography X, to examine the impact of both concentration and ionic strength on the evolution of the droplet during drying.

Les lubrifiants à base aqueuse sont très largement utilisés dans les procédés de mise en forme des métaux comme le tréfilage, car ils combinent d’excellentes capacités calorifiques à de bonnes propriétés tribologiques. Pour répondre à des exigences d’augmentation de la productivité, la compréhension des mécanismes de lubrification est nécessaire. L’objectif de ces travaux est d’établir le lien entre l’organisation des molécules en solution dans le lubrifiant et son pouvoir lubrifiant. La démarche expérimentale consiste à déterminer les propriétés structurales et tribologiques d’un système modèle aqueux, composé d’acides gras et d’éthylène diamine, principaux ingrédients des lubrifiants. Le diagramme de phases du système modèle est établi en s’appuyant sur des techniques de microscopie optique de polarisation, diffusion des rayons X aux petits angles (DXPA) et cryofracture. Pour un rapport molaire entre la diamine et les acides gras supérieur à 1, une succession de phases lamellaire, hexagonale et micellaires, biréfringente sous écoulement et isotrope, est observée avec la dilution. Une attention particulière est accordée à la phase lamellaire qui présente des défauts à l’approche de la transition vers la phase hexagonale. Les analyses réalisées en DXPA, cryofracture et RMN relient leur existence à une modulation de l’épaisseur de la bicouche et une augmentation de la courbure liées au couplage entre les acides gras et les contre-ions amines. Les propriétés lubrifiantes de l’ensemble de ces phases sont également comparées à l’aide d’un tribomètre qui réalise un contact entre un disque et une bille sous une cinématique de contact contrôlée, en régime de lubrification élastohydrodynamique. En plus des mesures de frottement, l’observation simultanée du contact permet de suivre la formation du film lubrifiant entre les surfaces et son évolution. L’influence de l’organisation supramoléculaire du lubrifiant sur son comportement est mise en évidence : les échantillons présentant une organisation lamellaire fournissent les meilleurs résultats en termes de réduction du frottement. L’organisation en bicouches de la phase lamellaire au sein du contact et ses propriétés piézovisqueuses peuvent expliquer sa portance accrue et sa prédisposition naturelle à la réduction du frottement. / Water based lubricants are widely used in metal forming processes due to their good cooling and lubrication capabilities. The understanding of the lubrication mechanisms is necessary to improve the current lubricant efficiency. The goal of this work is to correlate the structural properties of the lubricant to its lubricating behaviour. As mixtures of ethylene diamine and fatty acids in water are some of the main ingredients of such lubricants, their phase behaviour and tribological properties are investigated for different compositions. The phase diagram of the model system is established by using optical microscopy of polarisation, small angle X-rays scattering (SAXS) and freeze-fracture transmission electron microscopy (FF-TEM). For a molar ratio between diamine and fatty acids upper to 1, a succession of lamellar, hexagonal and micellar phases is observed with the dilution. A particular attention is turned to the lamellar phase which presents defects close the transition towards the hexagonal phase. According to SAXS, nuclear magnetic resonance and FF-TEM analyses, we propose that their existence is due to a modulation of the bilayer thickness and an increase of the curvature resulted from the evolution of the coupling between the fatty acids polar heads and the amine counter-ions. The lubricating ability of these phases is then investigated experimentally using an EHL tribometre that simultaneously enables contact visualisation, film thickness and friction measurements in controlled kinematic conditions. The influence of the lubricant structure on its tribological behaviour is revealing: the lowest friction coefficients are obtained with lamellar samples. The organisation in bilayers of the lamellar phase within the contact and its piezoviscous properties can explain its greater load-bearing capacity and its natural predisposition to the reduction of the friction.

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.

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.

Indiana University-Purdue University Indianapolis (IUPUI) / Large protein molecules are abundant in biological cells but are very difficult to study in physiological conditions due to molecular disorder. For large proteins, most structural information is obtained in crystalline states which can be achieved in certain conditions at very low temperature. X-ray and neutron crystallography methods can then be used for determination of crystalline structures at atomic level. However, in solution at room or physiological temperatures such highly resolved descriptions cannot be obtained except in very few cases. Scattering methods that can be used to study this type of structures at room temperature include small-angle x-ray and neutron scattering. These methods are used here to study two distinct proteins that are both classified as glycoproteins, which are a large class of proteins with diverse biological functions. In this study, two specific plasma glycoproteins were used: Fibrinogen (340 kDa) and Alpha 1-Antitrypsin or A1AT (52 kDa). These proteins have been chosen based on the fact that they have a propensity to form very large molecular aggregates due to their tendency to polymerize. One goal of this project is to show that for such complex structures, a combination of scattering methods that include SAXS, SANS, and DLS can address important structural and interaction questions despite the fact that atomic resolution cannot be obtained as in crystallography. A1AT protein has been shown to have protective roles of lung cells against emphysema, while fibrinogen is a major factor in the blood clotting process. A systematic approach to study these proteins interactions with lipid membranes and other proteins, using contrast-matching small-angle neutron scattering (SANS), small angle x-ray scattering (SAXS) and dynamic light scattering (DLS), is presented here. A series of structural reference points for each protein in solution were determined by performing measurements under osmotic stress controlled by the addition of polyethylene glycol-1,500 MW (PEG 1500) in the samples. Osmotic pressure changes the free energy of the molecular mixture and has consequences on the structure and the interaction of molecular aggregates. In particular, the measured radius of gyration (Rg) for A1AT shows a sharp structural transition when the concentration of PEG 1500 is between 33 wt% and 36 wt%. Similarly, a significant structural change was observed for fibrinogen when the concentration of PEG 1500 was above 40 wt%. This analysis is applied to a study of A1AT interacting with lipid membranes and to a study of fibrinogen polymerization in the presence of the enzyme thrombin, which catalyzes the formation of blood clots. The experimental approach presented here and the applications to specific questions show that an appropriate combination of scattering methods can produce useful information on the behavior and the interactions of large protein systems in physiological conditions despite the lower resolution compared to crystallography.

No description available.