Esta tese apresenta uma abordagem para a criação rápida de modelos em diferentes geometrias (complexas ou de alta simetria) com objetivo de calcular a correspondente intensidade espalhada, podendo esta ser utilizada na descrição de experimentos de es- palhamento à baixos ângulos. A modelagem pode ser realizada com mais de 100 geome- trias catalogadas em um Banco de Dados, além da possibilidade de construir estruturas a partir de posições aleatórias distribuídas na superfície de uma esfera. Em todos os casos os modelos são gerados por meio do método de elementos finitos compondo uma única geometria, ou ainda, compondo diferentes geometrias, combinadas entre si a partir de um número baixo de parâmetros. Para realizar essa tarefa foi desenvolvido um programa em Fortran, chamado de Polygen, que permite modelar geometrias convexas em diferentes formas, como sólidos, cascas, ou ainda com esferas ou estruturas do tipo DNA nas arestas, além de usar esses modelos para simular a curva de intensidade espalhada para sistemas orientados e aleatoriamente orientados. A curva de intensidade de espalhamento é calculada por meio da equação de Debye e os parâmetros que compõe cada um dos modelos, podem ser otimizados pelo ajuste contra dados experimentais, por meio de métodos de minimização baseados em simulated annealing, Levenberg-Marquardt e algorítmicos genéticos. A minimização permite ajustar os parâmetros do modelo (ou composição de modelos) como tamanho, densidade eletrônica, raio das subunidades, entre outros, contribuindo para fornecer uma nova ferramenta para modelagem e análise de dados de espalhamento. Em outra etapa desta tese, é apresentado o design de modelos atomísticos e a sua respectiva simulação por Dinâmica Molecular. A geometria de dois sistemas auto-organizado de DNA na forma de octaedro truncado, um com linkers de 7 Adeninas e outro com linkers de ATATATA, foram escolhidas para realizar a modelagem atomística e a simulação por Dinâmica Molecular. Para este sistema são apresentados os resultados de Root Mean Square Deviations (RMSD), Root Mean Square Fluctuations (RMSF), raio de giro, torção das hélices duplas de DNA além da avaliação das ligações de Hidrogênio, todos obtidos por meio da análise de uma trajetória de 50 ns. / This thesis presents an approach to the fast creation of models in different geometries (complex or high symmetry) in order to calculate the scattering intensity, which can be used for the description of small angles scattering experiments. The modeling can be performed using more than 100 geometries cataloged in a database, besides the possibility to build structures from random positions distributed on the surface of a sphere. In all cases the models are generated using the finite element method composing a single geometry, or composing different geometries combined with each other, using a small number of parameters. To accomplish this task it was developed a program called Polygen, written in Fortran language, which allows the modeling of convex polyhedrons in different geometries, as solids, shells, with aligned beads or DNA-like structures at the edges. To simulate the scattering intensity curve, these models are used and is possible simulate oriented and randomly oriented systems. The scattering intensity curve is calculated using the Debye equation and the main parameters describing the models, can be optimized by the fitting of the calculated curves against experimental data. The optimization is performed by the use minimization methods based on simulated annealing, Levenberg-Marquardt and genetic algorithmic. The minimization procedures allows the optimization of themodel parameters (or models of composition) as size, electron density, gyration radius, among others, contributing to provide a new tool for modeling and scattering data analysis. In a further step of this thesis, the design of atomistic models is presented and therespective simulation by Molecular Dynamics. Two geometries for DNA self-assembly

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

In this work, microfluidic and millifluidic droplets are utilized to study and control complex fluid behavior with high composition resolution. Different techniques are used on two length scales to create unique approaches towards the same goal of merging droplet-based experiments with classical colloidal characterization experiments. First, a microfluidic dehydrating droplet device is characterized and a procedure established by concentrating a phase separating organic-inorganic system on chip and using geometric calculations to determine composition. The device is then expanded to a more complex, particle-polymer system to investigate suspension stability and interparticle behavior. A model system containing silica particles and PEO polymer is found to transition from a bridging flocculation mechanism to polymer-coated particle jamming based on the mass ratio of polymer to particle. Lastly, a phase separating particle-polymer system consisting of polystyrene particles and hydroxyethyl cellulose is concentrated on-chip. Interparticle interactions are controlled by varying particle size, polymer size, and polymer type and the effects on phase behavior are examined. Droplet experiments are scaled-up to millifluidic droplets and concentration gradients are used to produce high composition resolution in place of time, used in the dehydrating microfluidic experiments. A novel, millifluidic containment device is created to study aggregation and sedimentation in droplets containing carbon black and OLOA surfactant suspended in dodecane. A slow increase in stabilization behavior is observed as opposed to the previously observed sharp “on-off” effect. The droplet production technique is then improved to achieve more complex composition paths and the device is expanded for a small angle neutron scattering (SANS) application. SANS is performed on flowing droplets with varying concentration to map interparticle interactions and phase behavior of complex particulate systems. Feasibility of device is demonstrated and preliminary model systems of silica particles and polymer, salt, and surfactant are analyzed and characterized.

Keratins are important structural components of hair and skin. There has been extensive study of keratins from the health and medical perspectives, although little work has been done to date to investigate their basic physicochemical properties in the form of biomaterials. The work presented in this thesis aimed to study surface and interfacial adsorption and solution aggregation of water soluble keratin polypeptides (made available by previous work within the research group). A range of physical techniques were employed including spectroscopic ellipsometry (SE), neutron reflection (NR), dual polarisation interferometry (DPI), quartz crystal microbalance with dissipation (QCM-D), dynamic light scattering (DLS) and small-angle neutron scattering (SANS).A major technical advantage of the neutron techniques is the use of hydrogen/deuterium substitution to enhance structural resolution. This approach was explored to study the interaction of keratins with both conventional surfactants and novel biosurfactants. The work presented comprises four results chapters. The first examines and compares four widely used interfacial techniques, SE, DPI, QCM-D and NR, by studying the adsorption of C12E6 at the silicon oxide/water interface. Whilst the data exhibits a large degree of consistency in the interfacially adsorbed amount, each technique helped reveal unique structural information with a high degree of complementarity. The second results chapter reports on findings regarding the properties of keratin polypeptides in surface adsorption and solution aggregation. It was found that the keratins adsorbed strongly on the surface of water, and formed rugby-shaped nanoaggregates in solution, the size and shape of which responded to salt concentration. The third results chapter reports on the interfacial behaviour of keratin/surfactants complexes in bulk solution, with cationic DTAB and anionic SDS as model conventional surfactants. It was found that both the electrostatic and hydrophobic forces contributed strongly to the surface adsorption processes. The final results chapter reports on interactions of a coated keratin film with novel biosurfactants including rhamnolipids (R1 and R2 with 1 and 2 sugar head(s), respectively) and Mel-C. The keratin films formed were found to be exceptionally stable and reproducible below pH 8, and these films could be widely used as model keratin substrates for screening their binding with surfactants and bioactive molecules. Both rhamnolipids and Mel-C exhibited strong adsorption onto the keratin substrate and interestingly, whilst R1 exhibited a completely reversible adsorption, R2 showed only a partially reversible adsorption. Mel-C showed some degree of irreversible adsorption similar to R2 and exhibited the strongest adsorption at around pH 4-5. These results show mild interactions with the keratin substrate, but indicate that the extent of adsorption and desorption could be manipulated by surfactant structure or solution conditions. The findings presented in this thesis are fundamental in aiding the development of the use of keratin polypeptides as biomaterials, in applications such as personal care. The work is also highly relevant to the understanding of the interactions between surfactants and keratin molecules at interfaces and in solution.

The work in this PhD thesis covers two strands of x-ray experiments: firstly, the characterisation of large arrays of dense structures by means of x-ray scattering, and, secondly, the investigation of hybrid anisotropy square structures with x-ray microscopy. The ability to accurately characterise large arrays of nanoscale magnetic structures is a key requirement for both scientific understanding and technological advance such as bit patterned recording media (BPM). In this work small angle x-ray scattering (SAXS) was investigated as a characterisation technique for large arrays of patterned structures. Dense arrays of magnetic nanostructures were prepared on x-ray transparent membranes and measured. The SAXS data was then modelled to obtain structure parameters such as the mean structure diameter, the diameter distribution and the mean position variance with statistical significance. Arrays (500 x 500 μm2) of nominally uniform nanostructures with centre-to-centre distances between 250 nm-50 nm were structurally characterised and compared to structure diameters obtained by optical scanning electron microscopy measurements. The mean structure diameter was found to be between 39 nm-15nm and agree within the errors with the diameter obtained from SEM measurements. This work provides accurate data on the distribution (variance) of nanostructure sizes which is key for modelling these arrays for applicationin BPM. In the second part of the work, the static and dynamic properties of patterned hybrid anisotropy square structures ([Co/Pd]-Py) were investigated by using time-resolved scanning transmission X-ray microscopy (STXM). In these patterned structures the magnetisation in the layers change both in magnitude and direction and gives rise to interesting new domain configurations. The reciprocal interaction between magnetic vortices in the Py layer and locally circular stripe domains in the Co/Pd was investigated and a mutual domain imprint between the layer was observed. In dynamic excitation experiments the precession of the vortex core is studied and showed good agreement with micromagnetic simulations made by Hrkac and Bryan. As demonstrated patterned hybrid anisotropy square structures have promising magnetic properties with potential applications in data storage (vortex switching) or spintronics (vortex oscillators).

In dieser Arbeit wurde die durch Bestrahlung mit schnellen Neutronen bedingte Materialalterung von Reaktordruckbehälterstählen untersucht. Das Probenmaterial umfasste unbestrahlte, bestrahlte und ausgeheilte RDB-Stähle russischer und westlicher Reaktoren sowie Eisenbasis-Modelllegierungen. Mittels Neutronen-Kleinwinkelstreuung ließen sich bestrahlungsinduzierte Leerstellen/Fremdatom-Cluster unterschiedlicher Zusammensetzung mit mittlerem Radius um 1.0 nm nachweisen. Ihr Volumenanteil steigt mit der Strahlenbelastung monoton, aber im allgemeinen nicht linear an. Der Einfluss der Elemente Cu, Ni und P auf den Prozess der Clusterbildung konnte herausgearbeitet werden. Eine Wärmebehandlung oberhalb der Bestrahlungstemperatur reduziert den Anteil der Strahlendefekte bis hin zu deren vollständiger Auflösung. Die Änderungen der mechanischen Eigenschaften der Werkstoffe lassen sich eindeutig auf die beobachteten Gefügemodifikationen zurückführen. Die abgeleiteten Korrelationen können als Hilfsmittel zur Vorhersage des Materialverhaltens bei fortgeschrittener Betriebsdauer von Leistungsreaktoren mit herangezogen werden.

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

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