Small angle x-ray scattering (SAXS) with synchrotron radiation was used to characterize nano-sized voids in different nominally pure aluminum (Al) alloys produced by quenching. The scattering signal from nano-voids is shown to be predictable from SAXS theory, and the information related to the void population confirm past experiments and reveal new details about quench-void formation in Al. Specifically, voids were produced in 99.97 at.% to 99.9994 at.% Al alloys by infrared heating to 450 – 625 °C followed by controlled rapid quenching at 10^3 to 10^5 °C/s. For changing processing conditions, the size of voids varied between 5 to 11 nm, and the density of voids varied by over an order of magnitude. Results from SAXS were consistent with TEM observations performed on the same specimens, indicating that synchrotron SAXS can be reliably used to characterize nano-voids produced in quenched Al. Factors determined to affect voids were consistent with previous studies, except that the present nano-voids dissolved after only 3 min. at 145 °C, indicating that quenched nano-voids are less stable than previously determined. SAXS also showed that void size is sensitive to quench temperature and quench rate. The activation energies for void nucleation and growth were determined to be 0.75 ± 0.10 and 0.19 ± 0.03 eV/at., respectively, confirming that hydrogen and di-vacancies take part in nucleation and growth during quenching. It was concluded that the non-linear tail of the quench curve plays a crucial role in void formation, and that voids form when long range diffusion is inhibited. This information can be utilized to design new Al alloys that limit incipient void formation, which is detrimental to properties such as formability. / Thesis (Master, Mechanical and Materials Engineering) -- Queen's University, 2008-04-25 15:17:30.211 / Natural Sciences and Engineering Research Council of Canada; General Motors of Canada Limited

5 ABSTRACT The mitogen-activated protein kinase (MAPK) cascade is an essential member of the cell defense system against stressors. The capability and efficiency of the cell reactions to different stress signals depend on signal transduction pathway, where signals from MAPK kinase kinase (MAP3K) are transferred through phosphorylation to downstream MAPK kinase (MAP2K) and finally to MAPK. Apoptosis signal-regulating kinase 1 (ASK1) is a member of a MAP3K family and its activation and inhibition has a significant participation in a regulation of cell response to stress stimuli. The regulation of ASK1 has a strong influence in pathogenesis of several diseases, the excessive activation of human ASK1 or failure in the control of its function are associated with cardiovascular diseases, neurodegenerative disorders, inflammatory diseases, infectious diseases, tumorigenesis, asthma, diabetes and ageing. The activity of ASK1 is regulated by its interaction with several proteins, the attention is focused on two physiological inhibitors, mammalian thioredoxin (TRX) and the 14-3-3 protein. ASK1 in its inactive form is inhibited by bonds formation with TRX and 14-3-3, however the explicit mechanism of this interaction is unclear due to the absence of structural data. This work is a part of an extensive research about...

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This dissertation is aimed at understanding the microstructure evolution in styrene — diene block copolymer — based pressure-sensitive adhesive compositions in melt. The work also focuses on determining the microphase separation mechanism in adhesive melts containing various amounts of low molecular weight resin (tackifiers) blended with styrene — diene block copolymers. To understand the correlation between adhesive morphology and their dynamic mechanical behavior, small angle X-ray scattering (SAXS) and rheological analysis were performed on blends with different compositions. A modified Percus — Yevick model combined with Gaussian functions was used fit the liquid like disordered and bcc — ordered peaks of the SAXS intensity profiles. The morphological parameters derived from SAXS analysis corresponded to features such as the size and extent of ordering of the microphase separated polystyrene domains. The variation in these parameters with respect to temperature and adhesive composition correlated reasonably well with the trends observed in the shear modulus measured using rheological analysis. It was found that the ordering of polystyrene domains was influenced by the tackifier content in the adhesive blends. Polymer chain mobility was determined to be the dominant factor governing ordering kinetics, which depended on both the quench temperature and tackifier content in the blends. The addition of increasing amounts of tackifier eventually leads to a shift from a nucleation and growth type mechanism to a spinodal decomposition mechanism for phase separation and ordering. The compatibility of the tackifier with the polystyrene chains had a significant impact on the morphological transitions and microphase separation in adhesive blends. The blends containing a styrene — incompatible tackifier showed ordering over a broader range of temperatures compared to the blends containing a polystyrene — compatible tackifier. / Ph. D.

L’objectif de ce travail est d’étudier les transitions de phases sol-gel et isotrope-nématique dans des suspensions de smectites dioctaédriques en fonction de la morphologie et de la nature minéralogique des argiles. Bien que tous les systèmes étudiés présentent une transition sol-gel à de faibles fraction volumique, la transition cristal-liquide isotrope-nématique n’a pu être identifiée que dans le cas de suspensions de smectites ayant un déficit de charge tétraédrique. L’effet de la localisation de la charge sur le comportement colloïdal a été déterminée à l’aide de la diffusion des rayons X aux petits angles (SAXS) et par des mesures rhéologiques. La nature des interactions électrostatiques dans ces suspensions est purement répulsive et rejette l’idée d’une structure tridimensionnelle de type « château de carte ». Cependant, les smectites ayant un déficit de charge tétraédrique sont plus répulsives et ont des propriétés viscoélastiques plus faibles que celles ayant un déficit octaédrique. Il a également été montré que la dépendance en taille de particules de la position de la transition sol-gel était liée à une statistique de piégeage hydrodynamique des plaquettes d’argile. Finalement, l’application de champs externes (électrique et magnétique) a permis d’obtenir l’alignement de la phase nématique tandis que dans la phase isotrope, le champ électrique induit un ordre antinématique parfait. Afin de préserver l’ordre induit, ces suspensions ont été polymérisées sous champ permettant l’obtention de nanocomposites orientées et structurés / The aim of this work is to study sol-gel and isotropic-nematic phases transitions in suspensions of dioctahedral smectites depending on the morphology and mineralogical nature of clays. Although all the systems studied exhibit a sol-gel at low volume fraction, the liquid-crystalline isotropic-nematic transition could be identified only in the case of smectites with tetrahedral charge deficit. The effect of charge location on the colloidal behavior was determined using small-angle X-ray scattering (SAXS) and rheological measurements. The nature of electrostatic interactions in these suspensions is purely repulsive and rejects the idea of the so-called “house of card” network. However, smectites with a charge deficit located in the tetrahedron are more repulsive and their viscoelastic properties are lower than octahedrally substituted clays. It was also shown that the particle size dependence of the volume fraction corresponding to the sol-gel transition c was related to a simple statistical hydrodynamic trapping of clay platelets. Finally, the application of external fields (electric and magnetic) has resulted in the alignment of the nematic phase while in the isotropic phase, the electric field induces a perfect antinematic order. To preserve the induced alignment, these suspensions were polymerized under the field to obtain perfectly aligned and patterned nanocomposites

Neste trabalho estudou-se o comportamento de soluções dos hidrótropos toluenossulfonato de sódio (TSS) e n-butilbenzeno sulfonato de sódio (NBBSNa) quanto a suas características de agregação/associação por Ressonância Magnética Nuclear 1H (RMN 1H), espalhamento de raios-X à baixo ângulo (SAXS), espalhamento de luz estático (SLS) e espalhamento de luz a médios ângulos (WAXS). Medidas realizadas com outros hidrótropos tais como estirenossulfonato de sódio (ESS), salicilato de sódio (SalS) e benzoato de sódio (BS) foram realizadas para comparação dos resultados. O NBBSNa foi sintetizado, purificado e caracterizado. Os experimentos de RMN revelam mudanças no deslocamento químico com a concentração, indicando mudança de ambiente a que as moléculas estão submetidas. Pela técnica de SLS pode-se avaliar o segundo coeficiente do virial das soluções de TSS com e sem a adição de um soluto. Pelas análises de SAXS determinou-se o tamanho do agregado de NBBSNa utilizando ajustes pelo programa SASfit, sendo o resultado comparado com um surfactante clássico dodecilbenzenossulfonato de sódio (DBSNa). O NBBSNa é um sistema de três densidades eletrônicas e esférico. Os demais hidrótropos estudados apresentam comportamento de líquido, sendo observado a correlação das moléculas a curta distância nos experimentos de WAXS. / In this work the behavior of solutions of the hydrotropes sodium toluene sulfonate (TSS) and sodium n-butylbenzene sulfonate (NBBSNa) was studied, concerning the aggregation/association characteristics of this molecules by the use of Nuclear Magnetic Resonance (NMR 1H), small angle x ray scattering (SAXS), static light scattering (SLS) and wide angle x ray scattering (WAXS). Measurements with other hydrotropic molecules like sodium styrene sulfonate (ESS), sodium salicylate (SalS), sodium benzoate (BS) were realized in order to discuss the results. The NBBSNa molecule was synthesized, purified and characterized. The experiments of NMR revealed changes in the chemical shift with the concentration, indicating the changes of the surround environment in what molecules are embedded. For the technique of SLS, the second virial coefficient of the solutions of TSS could be evaluated with and without the addition of a solute. By the SAXS data the size of the aggregate of NBBSNa was determined using the SASfit software. The result was compared with a classic surfactant sodium dodecylbenzene sulfonate (DBSNa). NBBSNa is a spherical system of three electronic densities. All the other hydrotropes studied shows a liquid scattering behavior. By WAXS experiments a correlation of the molecules at short distance was observed.

Neste trabalho estudamos por espalhamento de raios-X a baixos ângulos (SAXS) quatro diferentes sistemas de interesse biológico. Visamos investigar a auto-agregação de proteínas e de complexos proteicos que darão origem a fibras amilóides, interação proteína-proteína, simulando ambientes altamente concentrados, interação proteína-membrana simulando vesículas de matriz extracelular (MVs) de sistemas de biomineralização e interações proteína-agentes desnaturantes. No caso de formação de amilóides, investigamos a agregação do domínio GTPase da septina 6 (SEPT6G) e do complexo formado com o domínio GTPase da septina 2 (SEPT2G-SEPT6G). A temperaturas de até 15°C, tanto SEPT6G quanto SEPT2G-SEPT6G apresentam-se predominantemente diméricas em solução. Já a 25°C, o heterodímero SEPT2G-SEPT6G permanece estável enquanto agregados maiores de SEPT6G evoluem e coexistem em solução com SEPT6G-SEPT6G dimérica, sendo que a proporção de dímeros diminui com a temperatura. No estudo das MVs, mostramos que miméticos lipossomais de DPPC e DPPC:DPPS (9:1) possuem as mesmas características estruturais na ausência e presença de cálcio na solução. A interação da proteína anexina V humana (A5), envolvida em processos de biomineralização, impacta na membrana modelo induzindo a formação de nanoporos. A adição da fosfatase alcalina tecido não-específico (TNAP) não altera as propriedades estruturais do proteolipossomo na presença de A5. A ação do surfactante dodecil sulfato de sódio (SDS) a 30 mM não altera a conformação da albumina soro bovina (BSA), de maneira que é observada a formação de micelas de SDS coexistindo com a proteína livre em solução. Já a adição de 50 mM de SDS induz um desenovelamento parcial da proteína, identificado pela análise das curvas de SAXS via modelo de \"colar de pérolas\". A ação de uréia a 3 M e 8 M promove um desenovelamento parcial e total da BSA, respectivamente, com subsequente agregação de proteína dependente da temperatura (T > 30°C). A adição de 6 mM de SDS em proteínas parcialmente desenoveladas pela ação da uréia promove um desenovelamento mais acentuado. O potencial efetivo resultante da interação entre duas proteínas distintas, BSA e lisozima a concentração total de 100 mg/mL em solução, pH 7.0, foi obtido da análise de curvas de SAXS. Para isto, utilizou-se uma análise simplificada (em primeira aproximação) considerando um potencial efetivo de interação entre BSA-BSA, lisozima-lisozima e lisozima-BSA. Variamos a razão molar BSA:LISO até 1:42. No pH estudado, BSA tem uma carga residual superficial de -11e, enquanto a lisozima possui +9e. Conforme variamos a razão molar BSA:LISO, observamos dois regimes para o potencial efetivo resultante: i) até BSA:LISO 1:2, a carga efetiva do sistema é praticamente nula com um potencial resultante de caráter atrativo e ii) para razões entre BSA:LISO 1:3 a 1:42, a carga efetiva aumenta e o potencial resultante tem caráter repulsivo. Assim, lisozima e BSA coexistem sem agregar, através de um delicado balanço de forças atrativas e repulsivas no sistema. / In this work we have used small-angle x-ray scattering (SAXS) to study four systems of biological interest. We aim to investigate the self aggregation of proteins and protein complexes that would form amyloid fibers; protein/protein interaction, simulating high concentrations; protein/cell-membrane interaction, simulating extracellular matrix vesicles (MVs) from biomineralizing systems; and protein/denaturating-agents interactions. On the case of amyloid formation, we have investigated the aggregation of G-domain of septin-6 (SEPT6G) and the protein complex formed with G-domain of septin-2 (SEPT2G-SEPT6G). At temperatures lower than 15°C, both SEPT6G and SEPT2G-SEPT6G were found predominantly as dimers. At 25°C, SEPT2G-SEPT6G heterodimer is still stable while aggregates of SEPT6G grow. Both coexist in solutions of SEPT2G-SEPT6G dimers, with the percentage of dimers decreasing the higher the temperature. As for the study of MVs, we have shown that DPPC and DPPC:DPPS (9:1) liposomal mimetics have the same structural characteristics at the absence or presence of Calcium. The interaction with human annexin V protein (A5), related to biomineralization processes, affects the model membrane by the creation of nanopores. The addition of tissue-nonspecific alkaline phosphatase (TNAP) does not change the structural properties of the proteoliposome when A5 is present. The addition of SDS surfactant (30 mM) does not alters the conformation of bovine serum albumin (BSA), and we have observed the formation of SDS micelles coexisting with free protein in solution. The addition of 50 mM of SDS, on the other hand, induces the partial unraveling of the protein, as seen by the analysis of SAXS data via the pearl necklace\'\' model. The effect of adding 3M and 8M urea is, respectively, the partial and total unraveling of BSA, with ensuing aggregation of the protein dependent on the temperature (T > 30°C). The introduction of SDS 6mM promotes further unraveling in proteins that were previously partially unraveled by urea. The resulting effective potential for the interaction between BSA and lysozyme at total concentration of 100mg/ml and 7.0 pH has been obtained from the analysis of SAXS curves. In order to obtain this result we have used a simplified analysis (first order approximation) in which were considered the effective potentials for the interactions between BSA-BSA, lysozyme-lysozyme and lysozyme-BSA. We have varied the BSA:LISO molar ratio up to 1:42. At the studied pH, BSA has a surface residual charge of -11e, and lysozyme has +9e. As we changed the BSA:LISO molar ratio, we have found two regimens for the resulting effective potential: i) up to BSA:LISO 1:2, the effective charge of the system is virtually zero and the resulting potential is attractive; and ii) for BSA:LISO between 1:3 and 1:42 the effective charge increases, and the resulting potential is repulsive. Therefore, both lysozyme and BSA coexist without forming aggregates, by a delicate balance of attractive and repulsive forces.

O espalhamento de raios X a baixo ângulo tem se mostrado uma poderosa ferramenta na ánalise estrutural de proteínas em solução. Estudos em condições próximas ao estado nativo podem ser realizados, permitindo a visualização tridimensional de proteínas ou complexos formados. A tese apresentada aborda a teoria envolvida para utilização desta ferramenta. Uma nova metodologia foi proposta para a determinação da massa molecular de proteínas em solução, utilizando apenas uma curva de SAXS em unidades arbitrárias, visto que até o momento, este procedimento era realizado em comparação com outra proteína padrão de peso molecular conhecido. Com relação à instrumentação científica, um equipamento de SAXS foi desenvolvido no Instituto de Física de São Carlos, permitindo agora que medidas de SAXS em proteínas em solução sejam realizadas no instituto. Clonagem, expressão e purificação foram realizadas para o domínio de ligação ao DNA da isoforma do receptor tireoideano humano, a caracterização experimental desta proteína foi realizada por anisotropia de fluorescência, crosslink e SAXS. Após formação do complexo DNA-proteína, F2-DBD hTR, o mesmo foi submetido a cristalização, os cristais obtidos para o complexo não apresentaram padrão de difração e modelos de baixa resolução foram gerados utilizando SAXS. Além disso, estudos de baixo ângulo foram realizados linha de SAXS do LNLS para a enzima ferredoxina redutase de leptospira interrogans e para o complexo formado por interleucina-22 e pelo receptor interleucina-22, sendo seus modelos tridimensionais resolvidos. / Small angle X-ray scattering has been proven to be a powerful tool in the structural analysis of proteins in solution. This technique permits the three-dimensional visualization of native proteins envelop at the level of nanometers. In this study we discuss the small angle X-ray scattering theory and we proposed a new methodology to determine the molecular weight of proteins in solution, using only SAXS curve in arbitrary units. Prior the development of this method, the proteins molecular weighs were calculated by comparison with another of known size, usually bovine serum albumin. We also assembled SAXS equipment at the Physics Institute of São Carlos, which will permits in house measurements; as well as the cloning, expression and purification of DBD hTR, followed by the characterization of this protein by fluorescence anisotropy, crosslink and SAXS. The DNA-protein complex, F2-DBD hTR, was subjected to crystallization assays. Although, the crystals obtained for the complex showed no pattern of diffraction we were able to generate low-resolution models for the F2-DBD hTR using SAXS analysis. Moreover, the studies of the protein LepFNR and the complex IL-22/IL-22R1 by small angle X-ray scattering were performed in the line of SAXS of the LNLS, and their threedimensional models were resolved