Athabasca asphaltenes were characterized using small-angle X-ray scattering (SAXS) with synchrotron radiation. Two methods were used to separate asphaltenes from Athabasca bitumen. Conventional chemical separation by precipitation with n-pentane, and physical separation realized by passing bitumen through a zirconia membrane with a 20 nm average pore size. The Athabasca permeates and chemically separated samples were dispersed in 1-methylnaphtalene and n-dodecane, with temperature and asphaltene concentration ranges of 50-310 C and 1-8 wt. %, respectively. Two approaches were also taken in the analysis of the SAXS emissions. A model-independent approach provided radii of gyration and scattering coefficients. A model-dependent fit provided size distributions for asphaltenes aggregates assuming that they are dense and spherical. Physically and chemically separated asphaltenes showed significant differences in nominal size and structure, and their structural properties exhibited different temperature dependencies. The results challenge the merits of using chemically separated asphaltene properties as a basis for asphaltene property prediction in crude oil/bitumen.

X-Linked Retinoschisis (XLRS) is a currently incurable, progressive retinal degeneration that affects approximately 1:20,000 males. Sufferers have a loss of retinal structure and visual acuity, leading to blindness. The condition is caused by mutation of the RS1 gene encoding the retinal-specific protein retinoschisin. Retinoschisin is critical in maintaining the normal, ordered retinal architecture, with deletion in mice models leading to loss of both structure and visual processing, analogous to XLRS sufferers. However, re-introduction of retinoschisin using adeno-associated viral vectors leads to complete rescue in these models. Despite the importance of retinoschisin in maintaining retinal architecture, the mechanism by which it maintains this structure remains unknown. As a result, this study aimed to structurally characterise retinoschisin and XLRS-associated point mutants R141H and H207Q to gain insight into the mechanism of retinoschisin action. To this end, retinoschisin was expressed and purified from HEK 293-EBNA cells and the structure of both monomeric and octameric retinoschisin was investigated using Small-Angle X-Ray Scattering (SAXS) and Cryo-electron microscopy (Cryo-EM). Monomeric retinoschisin was found to adopt an elongated structure that allowed for the tight association of the subunits into a planer propeller structure. However, in solution conditions the octamer also stably self-assembled into a dimer of octamers, for which the structure was solved using cryo-EM. This allowed for construction of a quasi-atomic model, enabling mapping of XLRS-associated point mutations on the complex. Two major classes of mutation were identified, in the intra-octamer and inter-octamer interfaces, suggesting a mechanism of pathology for these mutants. Observation of clustered conservative mutations at the inter-octamer interface suggested the dimer of octamers may be physiologically relevant. Furthermore, comparison of the R141H mutant to the wild-type revealed an additional mutated site in the propeller tips. Here, R141H was suggested to induce a small conformational change and alter an interaction site. Another mutant, H207Q, however, induced a destabilization of the assembled retinoschisin molecule. In conclusion, we purified and structurally characterised human retinoschisin, identifying a new hexadecameric oligomer. The structure of this allowed for identification of distinct classes of mutations on the assembled molecule and a hypothesis of the mechanism of retinoschisin action in the retina.

Orientador: Iris Concepcion Kinares de Torriani / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Fisica Gleb Wataghin / Made available in DSpace on 2018-08-08T07:14:14Z (GMT). No. of bitstreams: 1 Silva_JulioCesarda_M.pdf: 3602925 bytes, checksum: ebc541d863211845fb7a5a80d72ed5b5 (MD5) Previous issue date: 2007 / Resumo: Durante as últimas décadas, o estudo de mudanças conformacionais de macromoléculas biológicas tem se tornado um grande desafio para os cientistas, além de ser um tema de interesse biotecnológico e de engenharia de proteínas. O processo de enovelamento (desenovelamento) de proteínas tem sido intensivamente estudado, pois isso pode contribuir para o conhecimento do processo de síntese de proteínas, além de ajudar a entender o desenvolvimento de algumas doenças associadas ao mau enovelamento ou agregação de certas proteínas. Nesse contexto, o Espalhamento de Raios-X a Baixo Ângulo (SAXS) aparece como uma técnica valiosa para esse estudo, pois ela permite obter informações estruturais da molécula em solução. Além de permitir estudos dinâmicos, as experiências de SAXS possibilitam a observação das moléculas em condições fisiológicas. Neste trabalho, a potencialidade da técnica de SAXS foi evidenciada no estudo de mudanças conformacionais de biomoléculas. O processo de desnaturação da proteína lisozima em solução foi estudado através de experiências em equilíbrio. Mudanças conformacionais foram observadas durante o processo de desnaturação por ação de uréia na solução e por altas temperaturas. Os resultados mostraram que a lisozima é uma proteína com certa resistência para se desenovelar completamente, mesmo em condições extremas de concentração de uréia e de altas temperaturas. A molécula tende a não perder totalmente sua compacidade. Além disso, foram observados somente dois estados conformacionais (enovelado e desenovelado). Um estado intermediário reportado na literatura, mas contestado por vários autores, não foi observado. Isso mostra a alta cooperatividade dessa proteína no processo de desnaturação. Outro processo estudado foi a oligomerização da proteína -Lactoglobulina sob ação de irradiação com radiação gama. A proteína foi estudada na forma sólida, com diferentes atividades de água, e em solução, em diferentes concentrações. As amostras foram irradiadas com radiação gama em diferentes doses e as mudanças foram registradas através de experiências de SAXS. Os dados experimentais foram usados para o cálculo de modelos dos oligômeros formados por ação da radiação. Concluindo, este estudo mostrou que a técnica de SAXS é uma ferramenta versátil e muito útil para o estudo de processos de mudanças nas estruturas terciária e quaternária de proteínas em solução / Abstract: During the last decades, the study of conformational changes in biological macromolecules has been a great challenge for the scientists, and continues to be an important subject of biotechnological interest and protein engineering. The process of folding (unfolding) of protein molecules has been intensively studied, because this investigation can contribute to the knowledge of the process of protein synthesis, thus helping to understand the development of some illnesses associated with misfolding or aggregation processes of certain proteins. In this context, the technique of Small Angle X-ray Scattering (SAXS) appears as a valuable technique, because it provides structural information of the molecules in solution. This technique allows dynamical studies and makes possible the study of the protein in physiological conditions. In this work the potentiality of the SAXS technique was evidenced in the study of conformational changes of biological molecules. The process of denaturation of the protein lysozyme in solution was studied using SAXS measurements in equilibrium conditions. Conformational changes were observed during the process of denaturation by the action of urea in the solution and for high temperatures. The results showed that lysozyme is a protein with certain resistance to unfold completely. Even in extreme conditions of high concentration of urea and high temperatures, this protein does not totally lose its compactness. Moreover, only two conformational states (folded and unfolded) were observed. An intermediate state was not observed. This study showed the high cooperativity of the unfolding process of this protein during its denaturation process. Another process studied was the oligomerization of the protein -Lactoglobulin under the effect of gamma irradiation. The protein was studied in the solid form, in different water activities, and in solution, in different concentrations. The samples were exposed to several doses of -radiation. The SAXS technique was used to obtain dimensional parameters of the proteins and models were calculated from the experimental scattering data. Finally, this study showed that the SAXS technique as a versatile and very useful tool for the study of changes in the tertiary and quaternary structures of proteins in solution / Mestrado / Física / Mestre em Física

The objective of this work is the synthesis of highly functionalized hybrid organic/inorganic materials by the polycondensation of bis[3-(trimethoxysilyl) propyl]amine in the presence of surfactants. High contents of amine groups were achieved by carrying out the syntheses without an inorganic cross-linker. The silsesquioxanes obtained had a mesoporous structure. The stability of their porous system in the absence of an inorganic cross-linker was enhanced by the precursor's bridged structure. The material structures were studied by FT-IR spectroscopy, Porosimetry, X-Ray Diffraction and Small Angle X-Ray Scattering methods. A material prepared in the presence of dodecylamine as a template had a higher surface area and narrower pore size distribution while the use of sodium dodecyl sulfate resulted in the formation of mesopores with a wide size distribution. Surface amine groups in silsesquioxanes were accessible for adsorption of small molecules of acidic nature.

The phosphatidylinositol-3-kinase α (PI3Kα) is a heterodimeric enzyme that is composed of a p85α regulatory subunit and a p110α catalytic subunit. PI3Kα plays a critical role in cell survival, growth and differentiation, and is the most frequently mutated pathway in human cancers. The PI3Kα pathway is also targeted by many viruses, such as the human immunodeficiency virus (HIV-1), the herpes simplex virus 1 (HSV-1) or the influenza A virus, to create favourable conditions for viral replication. The regulatory p85α stabilizes the catalytic p110α, but keeps it in an inhibited state. Various ligands can bind to p85α and allosterically activate p110α, but the mechanisms are still ill-defined. Intriguingly, p85α also binds to, and activates, the PTEN phosphatase, which is the antagonist of p110α. Previous studies indicated that only p85α monomers bind to the catalytic p110α subunit, whereas only p85α dimers bind to PTEN. These findings suggest that the balance of p85α monomers and dimers regulates the PI3Kα pathway, and that interrupting this equilibrium could lead to disease development. However, the molecular mechanism for p85α dimerization is controversial, and it is unknown why PTEN only binds to p85α dimers, whereas p110α only binds to p85α monomers. Here we set out to elucidate these questions, and to gain further understanding of how p85α ligands influence p85α dimerization and promote activation of p110α. We first established a comprehensive library of p85α fragments and protocols for their production and purification. By combining biophysical and structural methods such as small angle X-ray scattering, X-ray crystallography, nuclear magnetic resonance, microscale thermophoresis, and chemical crosslinking, we investigated the contributions of all p85α domains to dimerization and ligand binding. Contrarily to the prevailing thought in the field, we find that p85α dimerization and ligand recognition involves multiple domains, including those that directly bind to and inhibit p110α. This finding allows us to suggest a molecular mechanism that links p85α dimerization and allosteric p110α activation through ligands.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23520号 / 農博第2467号 / 新制||農||1086(附属図書館) / 学位論文||R3||N5351(農学部図書室) / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 谷 史人, 教授 保川 清, 准教授 中川 究也 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

No description available.

Thermal and morphological studies of the segmented multiblock copolyesters containing 2,2,4,4-tetramethyl-1,3-cyclobutanediol and dimethyl-1,4-cyclohexane dicarboxylate were carried out using differential scanning calorimetry, small angle X-ray scattering, wide angle X-ray diffraction and dynamic mechanical analysis. Molecular origins of the thermal transitions appearing in copolyesters were assigned by the copolyester analysis at different temperatures. The hard segments in copolyesters underwent short-range and long-range ordering (crystallization) during cooling or annealing above glass transition temperature, as concluded from thermal and wide angle X-ray diffraction analysis. Annealing process affected the ordering in hard segments and annealing temperatures of 160 °C and above led to increased microphase mixing. The small angle X-ray scattering studies confirmed the microphase separated morphology of copolyesters and supported the argument of increased microphase mixing in copolyesters annealed at higher temperatures. The amount of sulfonate containing co-monomer and its presence in either hard or soft microphase affected the morphology of the copolyesters. Introduction of the sulfonate groups led to increased microphase mixing in copolyesters as well as destruction of long-range order in the hard segments. / Master of Science

Yes / AB and ABA di- and triblock copolymers where A is the hydrophilic poly(oligoethylene glycol methacrylate) (POEGMA) block and B is a thermo-responsive sulfobetaine block [2-(methacryloyloxy) ethyl] dimethyl-(3-sulfopropyl) ammonium hydroxide (PDMAPS) were synthesised by aqueous RAFT polymerisation with narrow dispersity (ĐM ≤ 1.22), as judged by aqueous SEC analysis. The di- and triblock copolymers self-assembled in salt-free water to form micelles with a PDMAPS core and the self-assembly of these polymers was explored by SLS and TEM analysis. The micelles were shown, by DLS analysis, to undergo a micelle-to-unimer transition at a critical temperature, which was dependent upon the length of the POEGMA block. Increasing the length of the third, POEGMA, block decreased the temperature at which the micelle-to-unimer transition occurred as a result of the increased hydrophilicity of the polymer. The dissociation of the micelles was further studied by SLS and synchrotron SAXS. SAXS analysis revealed that the micelle dissociation began at temperatures below that indicated by DLS analysis and that both micelles and unimers coexist. This highlights the importance of using multiple complementary techniques in the analysis of self-assembled structures. In addition the micelle-to-unimer morphology transition was employed to encapsulate and release a hydrophobic dye, Nile Red, as shown by fluorescence spectroscopy. / Engineering and Physical Sciences Research Council (EPSRC), University of Warwick

Yes / The synthesis and self-assembly of thymine-containing polymers were performed using RAFT dispersion polymerization. A combination of microscopy and scattering techniques was used to analyze the resultant complex morphologies. The primary observation from this study is that the obtained aggregates induced during the polymerization were well-defined despite the constituent copolymers possessing broad dispersities. Moreover, a variety of parameters, including the choice of polymerization solvent, the degree of polymerization of both blocks and the presence of an adenine-containing mediator, were observed to affect the resultant size and shape of the assembly. / University of Warwick, National Science Foundation (U.S.) (NSF), Engineering and Physical Sciences Research Council (EPSRC)