La technique de diffraction de monocristaux est la plus utilisée pour déterminer une structure tridimensionnelle de protéine, permettant ainsi la compréhension de sa fonction biologique.Cependant, cette technique nécessite l'obtention d'un monocristal.La détermination d'une structure par diffraction de poudre ne requiert que l'obtention d'un précipité cristallin, souvent obtenu lors de la recherche d'une condition de cristallisation.Nous présentons dans cette thèse plusieurs protéines étudiées par diffraction de poudre. Le développement de nouvelles méthodes pour la préparation des échantillons et l'acquisition des données sont présentées, étant donnée leur importance cruciale dans ces études.Nous avons étudié le polymorphisme de l'urate oxidase par la détermination des différentes phases cristallines résultant des modifications des conditions de cristallisation. Cette étude a débouché sur l'identification d'une forme cristalline nouvelle d'intérêt pharmaceutique. Une des phases d'urate oxidase à permis l'obtention d'un cliché de diffraction de qualité suffisante à la redétermination et l'affinement de sa structure.Un protocole pour le refroidissement cryogénique de poudre de protéineest présenté, offrant une durée de vie accrue de l'échantillon lors de l'acquisition de données.Ce protocole a permis l'affinement de deux structures d'insuline humaine. Nous présentons également la détermination d'une structure préliminaire du domaine macro du virus Mayaro, basé uniquement sur des données acquises sur un unique échantillon polycristallin en forme d'oursin. Une étude de la matrice de protection de deux baculovirus illustre les limites auxquelles se heurte actuellement la technique de diffraction de poudre de protéines.En annexes, les étapes nécessaires pour la préparation et l'analyse des données sont présentées. / Knowledge of the structure of proteins helps in understanding theirbiological function.The main technique used, single crystal diffraction, requires thelimiting step of growing a single crystal.On the other hand, powder diffraction requires only a crystallineprecipitate made of many microcrystals such as those often discardedduring the search for suitable single crystal growth conditions.We present in this thesis different studies of proteins by powder diffraction.Development of new methods for sample preparation and data acquisitionare also presented, as they have been crucial steps to obtain highquality diffraction data.We studied the polymorphism of Urate oxidase by observing thedifferent crystallographic phases resulting from the changes in thecrystallisationconditions.A crystallographic phase of pharmaceutical interest has been identified.Also one phase of urate oxidase complexed with its inhibitor gave apowder pattern sufficient to re-determine and refine its structure.A protocol for cryocooling protein powder samples has been found,extending the lifespan of the sample in the intense X-ray beam.This allowed the refinement from powder data of two forms ofcryocooled human insulin.We present also the determination of a preliminary structure of themayaro virus macro domain, based on a powder diffraction patternobtained on a single urchin-like bundle of needles.A study of the protective protein matrix of two baculoviruses ispresented, showing some current limits of the method.In annexes, the steps for preparing and analysing protein powders are described.

abstract: Membrane proteins are very important for all living cells, being involved in respiration, photosynthesis, cellular uptake and signal transduction, amongst other vital functions. However, less than 300 unique membrane protein structures have been determined to date, often due to difficulties associated with the growth of sufficiently large and well-ordered crystals. This work has been focused on showing the first proof of concept for using membrane protein nanocrystals and microcrystals for high-resolution structure determination. Upon determining that crystals of the membrane protein Photosystem I, which is the largest and most complex membrane protein crystallized to date, exist with only a hundred unit cells with sizes of less than 200 nm on an edge, work was done to develop a technique that could exploit the growth of the Photosystem I nanocrystals and microcrystals. Femtosecond X-ray protein nanocrystallography was developed for use at the first high-energy X-ray free electron laser, the LCLS at SLAC National Accelerator Laboratory, in which a liquid jet would bring fully hydrated Photosystem I nanocrystals into the interaction region of the pulsed X-ray source. Diffraction patterns were recorded from millions of individual PSI nanocrystals and data from thousands of different, randomly oriented crystallites were integrated using Monte Carlo integration of the peak intensities. The short pulses ( 70 fs) provided by the LCLS allowed the possibility to collect the diffraction data before the onset of radiation damage, exploiting the diffract-before-destroy principle. At the initial experiments at the AMO beamline using 6.9- Å wavelength, Bragg peaks were recorded to 8.5- Å resolution, and an electron-density map was determined that did not show any effects of X-ray-induced radiation damage. Recently, femtosecond X-ray protein nanocrystallography experiments were done at the CXI beamline of the LCLS using 1.3- Å wavelength, and Bragg reflections were recorded to 3- Å resolution; the data are currently being processed. Many additional techniques still need to be developed to explore the femtosecond nanocrystallography technique for experimental phasing and time-resolved X-ray crystallography experiments. The first proof-of-principle results for the femtosecond nanocrystallography technique indicate the incredible potential of the technique to offer a new route to the structure determination of membrane proteins. / Dissertation/Thesis / Ph.D. Chemistry 2011

The rapid advancement of drug delivery systems (DDS) has raised the possibility of using functional engineered nano/micro-particles as drug carriers for the administration of active pharmaceutical ingredients (APIs) to the affected area. The major goals in designing these functional particles are to control the particle size, the surface properties and the pharmacologically active agents release in order to achieve the site-specification of the drug at the therapeutically optimal rate and dose regimen. Two different equipment (i.e. glass capillary microfluidic device and micro-engineered membrane dispersion cell) were utilised in this study for the formation of functional nano/micro-particles by antisolvent precipitation method. This method is based on micromixing/direct precipitation of two miscible liquids, which appear as a straightforward method, rapid and easy to perform, does not require high stirring rates, sonication, elevated temperatures, surfactants and Class 1 solvents can be avoided. Theoretical selection of a good solvent and physicochemical interaction between solvent-water-polymer with the aid of Bagley s two-dimensional graph were successfully elucidated the nature of anti-solvent precipitation method for the formation of desired properties of functional pharmaceutical nano/micro-engineered particles. For the glass capillary microfluidic experiment, the organic phase (a mixture of polymer and tetrahydrofuran/acetone) was injected through the inner glass capillary with a tapered cross section culminated in a narrow orifice. The size of nanoparticles was precisely controlled by controlling phase flow rates, orifice size and flow configuration (two- phase co-flow or counter-current flow focusing). The locations at which the nanoparticles would form were determined by using the solubility criteria of the polymer and the concentration profiles found by numerical modelling. This valuable results appeared as the first computational and experimental study dealing with the formation of polylactide (PLA) and poly(ε-caprolactone) (PCL) nanoparticles by nanoprecipitation in a co-flow glass capillary device. The optimum formulations and parameters interactions involved in the preparation of paracetamol encapsulated nanoparticles (PCM-PCL NPs) using a co-flow microfluidic device was successfully simulated using a 25-full factorial design for five different parameters (i.e. PCL concentration, orifice size, flow rate ratios, surfactant concentration and paracetamol amount) with encapsulation efficiency and drug loading percentage as the responses. PCM-loaded composite NPs composed of a biodegradable poly(D,L-lactide) (PLA) polymer matrix filled with organically modified montmorillonite (MMT) nanoparticles were also successfully formulated by antisolvent nanoprecipitation in a microfluidic co-flow glass capillary device. The incorporation of MMT in the polymer matrix improved the drug encapsulation efficiency and drug loading, and extended the rate of drug release in simulated intestinal fluid (pH 7.4). The encapsulation of MMT and PCM in the NPs were well verified using transmission electron microscopy (TEM), energy dispersive x-ray spectroscopy (EDS), x-ray diffraction (XRD), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR). PCL drug-carrier nanoparticles were also produced by rapid membrane micromixing combined with nanoprecipitation in a stirred cell employing novel membrane dispersion. The size of the NPs was precisely controlled by changing the aqueous-to-organic volumetric ratio, stirring rate, transmembrane flux, the polymer content in the organic phase, membrane type and pore morphologies. The particle size decreased by increasing the stirring rate and the aqueous-to-organic volumetric ratio, and by decreasing the polymer concentration in the aqueous phase and the transmembrane flux. The existence of the shear stress peak within a transitional radius and a rapid decline of the shear stress away from the membrane surface were revealed by numerical modelling. Further investigation on the PCL nanoparticles loaded immunosuppressive rapamycin (RAPA) drug were successfully synthesised by anti-solvent nanoprecipitation method using stainless steel (SS) ringed micro-engineered membrane. Less than 10 μm size of monohydrate piroxicam (PRX) micro-crystals also was successfully formed with the application of anti-solvent precipitation method combined with membrane dispersion cell that has been utilised in the formation of functional engineered nanoparticles. This study is believed to be a new insight into the development of integrated membrane crystallisation system.

Our goal was to develop a mucoadhesive, oral vaccination delivery platform designed to target Peyer’s patches at ileum. In order to achieve this, we prepared poly(methyl methacrylate) (PMMA) particles of various sizes using W/O/W emulsification solvent evaporation and surface polymerization methods. We then coated and employed mucoadhesive polymers into the carrier system to enhance the residence time in the targeted site. Also we developed our own in vitro mucoadhesion testing ramp as an evaluation tool. Finally, nano- and micro-structured particles were manufactured as two different oral vaccine delivery systems (Solid Lipid Nanoparticles, SLNs; and Protein Coated Microcrystals, PCMC). After the model antigen, bovine serum albumin (BSA) was loaded into the SLNs or PCMC; mucoadhesive polymers were then incorporated and formulated the mixture into pellets. The pellets were then layered with an enteric coating, which was composed of a mixture of Eudragit® FS 30 D/Eudragit® L 30 D-55 for ileum targeted delivery. The in vitro mucoadhesion test ramp was capable of investigating the mucoadhesive properties of tablets and pellets, providing a rank order for study. Most important of all, it was anticipated that this might reduce the burden of testing animals for future proposed mucoadhesive studies. Microcapsules/beads of specific size were manufactured reproducibly by solvent evaporation and surface polymerization. Although we could not specify the cut-off size at the pyloric sphincter in mice, we concluded that the cut-off size at the pyloric sphincter in rats was approximately 2.5-3 mm, which was supported by both the biodistribution data and the direct image results from scintigraphy scanning. Moreover, we found that the particle size significantly alters the gastric emptying time in both rodent models. The small microcapsules/beads were hindered in the folds of the stomach (size 50-100μm for mice and size 0.5-1 mm for rats) and emptied the slowest, followed by the large particles, then the medium particles. Finally, PCMC and SLNs we manufactured were suitable carriers for protein API, such as BSA. These particles were of fitting size for M cell uptake, which would possibly induce mucosal immune responses. Therefore, an antigen containing PCMC and SLNs might be suitable platforms for oral vaccination. / text

Submitted by Luís Fernando da Silva Lopes null (luisfsl@gmail.com) on 2017-11-28T17:52:20Z No. of bitstreams: 1 SÍNTESE E CARACTERIZAÇÃO DO WO3-Ag PREPARADO VIA ROTA HIDROTÉRMICA - Luis Fernando Lopes.pdf: 6363810 bytes, checksum: 39b63795e9ad16d122fbcbe80ebe8719 (MD5) / Submitted by Luís Fernando da Silva Lopes null (luisfsl@gmail.com) on 2017-11-28T19:04:29Z No. of bitstreams: 1 SÍNTESE E CARACTERIZAÇÃO DO WO3-Ag PREPARADO VIA ROTA HIDROTÉRMICA - Luis Fernando Lopes.pdf: 6363810 bytes, checksum: 39b63795e9ad16d122fbcbe80ebe8719 (MD5) / Approved for entry into archive by Lucilene Cordeiro da Silva Messias null (lubiblio@bauru.unesp.br) on 2017-11-29T15:53:51Z (GMT) No. of bitstreams: 1 lopes_lfs_me_bauru.pdf: 6363810 bytes, checksum: 39b63795e9ad16d122fbcbe80ebe8719 (MD5) / Made available in DSpace on 2017-11-29T15:53:51Z (GMT). No. of bitstreams: 1 lopes_lfs_me_bauru.pdf: 6363810 bytes, checksum: 39b63795e9ad16d122fbcbe80ebe8719 (MD5) Previous issue date: 2017-09-29 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / As estruturas hierárquicas do WO3-Ag tridimensional (3D) auto-montadas foram sintetizadas através da rota hidrotérmica utilizando como soluções precursoras o ácido túngstico (H2WO4) e peróxido de hidrogênio (H2O2), adicionando diferentes quantidades de Ag. As amostras foram analisadas por difração de raios X (DRX), Microscopia Eletrônica de Varredura de Campo (MEV-FEG), análise termogravimétrica (TGA), espectroscopia micro Raman, espectroscopia no infravermelho por transformada de Fourier (FTIR), espectroscopia no ultravioleta visível UV-VIS e espectroscopia de fotoelétrons excitados por raios X (XPS). Os estudos de DRX e micro Raman mostraram que à medida que a quantidade de Ag variou de 0 a 10% em peso na solução de crescimento hidrotérmico, a fase do cristal mudou gradualmente de WO3•0,33H2O ortorrômbica para WO3 hexagonal. Os estudos de FTIR e TGA revelaram diferentes níveis de hidratação, apoiando os resultados de DRX e micro Raman. Ao controlar a quantidade de Ag na solução precursora, foram obtidos blocos de construção semelhantes a plaquetas e blocos de construção hexagonais, destacando o papel da prata no crescimento hidrotérmico dos microcristais 3D WO3•0.33H2O e WO3. Além disso, as imagens de MEV-FEG de alta magnificação mostraram que as nanopartículas de Ag estão ancoradas na superfície das estruturas hierárquicas WO3-Ag 3D. Medidas no UV-vis demonstraram que as estruturas hierárquicas 3D absorveram gradualmente mais luz quando a quantidade de Ag foi aumentada. Além disso, a energia do intervalo de banda proibida diminuiu quando a quantidade de Ag aumentou de 0% em peso (Eg = 2,65 eV) para 10% em peso (Eg = 2,26 eV). Esses resultados experimentais demonstram que a quantidade de Ag desempenhou um papel crucial na determinação da morfologia dos blocos de construção e do nível de hidratação, propriedades ópticas e fase cristalina dos microcristais WO3•nH2O. / The self-assembled three-dimensional (3D) WO3-Ag hierarchical structures were synthesized through the hydrothermal route using tungstic acid (H2WO4) and hydrogen peroxide (H2O2) as precursor solutions, adding different amounts of Ag. The samples were analyzed by diffraction X-ray diffraction (XRD), field scanning electron microscopy (FESEM), thermogravimetric analysis (TGA), micro Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR), visible ultraviolet UVVIS spectroscopy and spectroscopy Photoelectrons (XPS). XRD and micro Raman studies showed that as the amount of Ag ranged from 0 to 10% by weight in the hydrothermal growth solution, the crystal phase gradually changed from orthorhombic WO3•0.33H2O to hexagonal WO3. The FTIR and TGA studies revealed different levels of hydration, supporting the XRD and micro Raman results. By controlling the amount of Ag in the precursor solution, platelet-like building blocks and hexagonal building blocks were obtained, highlighting the role of Ag in the hydrothermal growth of the 3D microcrystals WO3•0.33H2O and WO3. In addition, high magnification FESEM images showed that Ag nanoparticles are anchored on the surface of the WO3-Ag 3D hierarchical structures. UV-vis measurements demonstrated that the 3D hierarchical structures gradually absorbed more light when the amount of Ag was increased. In addition, the energy of the band gap was decreased when the amount of Ag increased from 0% by weight (Eg = 2.65 eV) to 10% by weight (Eg = 2.26 eV). These experimental results demonstrate that the amount of Ag played a crucial role in determining the morphology of the building blocks and the level of hydration, optical properties and crystalline phase of WO3• nH2O microcrystals. / FAPESP: 13/07296-2.

Im Rahmen der vorliegenden Arbeit, wird ein neuartiges Markierungsschema für die Festkörper-NMR-Spektroskopie vorgestellt, das sogenannte Reduced Adjoining Protonation (RAP) Schema, welches die Protonendetektion sämtlicher Aliphaten erlaubt. Hochaufgelöste, 1H-detektierte 1H,13C Korrelationsspektren wurden erhalten. Des Weiteren wurde der Vorteil von hohen MAS-Frequenzen untersucht. 1H- und 13C-detektierte 3D Zuordnungsexperimente wurden implementiert, welche uns die Zuordnung von 90% aller aliphatischen Resonanzen von alpha-Spektrin SH3 erlaubten. Da die chemische Verschiebung abhängt vom Strukturmotiv, kann sie verwendet werden, um Sekundärstruktur-Informationen abzuleiten. Darüber hinaus wurde ein 1H-detektiertes H(H)CH 3D Experiment entwickelt, um weitreichende 1H,1H Kontakte zu ermitteln, welche für die Bestimmung der Tertiärstruktur genutzt werden können. Um artefaktfreie Relaxationsdaten zu erhalten, wurde das RAP-Markierungsschema modifiziert, um 1H- und 13C-verdünnte Proben zu erhalten, in denen Spindiffusion unterdrückt ist. Für die Untersuchung von Sub-Mikrosekunden-Dynamik werden Experimente vorgestellt zur Bestimmung von 13C T1 Relaxationszeiten und 1H,13C dipolaren Kopplungstensoren für Rückgrat- und Seitenketten-Resonanzen. Des weiteren zeigen wir, dass das RAP-Markierungsschema auf nicht-kristalline Systeme, wie Amyloidfibrillen des Abeta1-40 Peptids der Alzheimer-Krankheit, angewendet werden kann. Unter Verwendung von 1H-Detektion, erhielten wir hochaufgelöste 1H,13C Korrelationsspektren. Schließlich wurde der Perdeuterierungsansatz auf den L7Ae-box C/D Protein-RNA Komplex aus P. furiosus angewendet. Wir erhielten hochaufgelöste, 1H-detektierte 1H,15N, sowie 13C,13C Korrelationsspektren des Protein-RNA Komplexes. Weiterhin haben wir eine Methode zur Bestimmung genauer Abstands- und Winkelinformationen für die Protein-RNA Schnittstelle etabliert und schlagen Ansätze vor, für die Zuordnung der chemischen Verschiebungen von RNA-Resonanzen. / In this thesis, a novel labeling scheme for solid-state NMR spectroscopy, the Reduced Adjoining Protonation (RAP) scheme, is introduced, which allows proton detection of all aliphatic sites, as shown for the microcrystalline SH3 domain of alpha-spectrin. These samples yield high-resolution, 1H-detected 1H,13C correlation spectra. In addition, the benefit of high MAS frequencies was investigated. 1H- and 13C-detected 3D assignment experiments are implemented, which allowed us to assign 90% of all aliphatic resonances of alpha-spectrin SH3. As the chemical shift is dependent on the structural motif, it can be employed to derive secondary structure information. Furthermore, a 1H-detected H(H)CH 3D experiment is introduced, to obtain long-range 1H,1H contacts, which can be used for the determination of the tertiary structure. To obtain artifact-free relaxation data, the RAP labeling scheme was modified to obtain sparsely proton labeled, 13C dilute samples, in which spin diffusion is suppressed. To probe sub-microsecond dynamics, we report experiments to determine 13C T1 relaxation times and 1H,13C dipolar coupling tensors for backbone and side chain resonances, respectively. Furthermore, we show, that the RAP labeling scheme can be applied to non-crystalline systems, such as amyloid fibrils of the Alzheimer’s disease peptide Abeta1-40. Using 1H-detection, we obtained high-resolution 1H,13C correlation spectra. Finally, we applied the perdeuteration approach to the L7Ae-box C/D protein-RNA complex from P. furiosus. We obtained high-resolution, 1H-detected 1H,15N, as well as 13C,13C correlation spectra of the protein-RNA complex. In addition, we established a methodology to determine accurate distance and angular restraints for the protein-RNA interface and propose approaches for the chemical shift assignment of RNA resonances.