Ce travail concerne la modification de surface de nanoparticules magnétiques (MNP) par des copolymères réactifs renfermant des cycles azlactone, aux fins de l’élaboration de nano-supports destinés à l’immobilisation de biomolécules. Trois stratégies basées sur des techniques de polymérisation radicalaire contrôlée ont été mises en œuvre.Dans la première, un copolymère poly(méthacrylate de poly(éthylène glycol)-stat-2-vinyl-4,4-diméthylazlactone) (poly(PEGMA-stat-VDM)) a été préparé par polymérisation radicalaire par transfert d’atome (ATRP) selon la technique « grafting from » à partir des MNP et utilisé pour la bioconjugaison de thymine peptide nucleic acid (PNA). La présence de l’écorce polymère et l’immobilisation du PNA ont été confirmées par différentes techniques complémentaires (FTIR, VSM).La deuxième stratégie est basée sur l’élaboration de MNP greffées pour la bioconjugaison de l’acide folique, via l’ARTP du PEGMA et de la VDM. L’analyse par microscopie électronique à transmission (TEM) a montré qu’après bioconjugaison les MNP possèdent une très bonne aptitude à la dispersion en milieu aqueux.La troisième stratégie met en œuvre la technique «grafting onto » de copolymères poly(oxyde d’éthylène)-bloc-poly(2-vinyl-4,4-dimethylazlactone) (PEO-b-PVDM) pour la préparation de nanosupports magnétiques recyclables. Des copolymères à blocs PEO-b-PVDM ont été préparés par la technique de polymérisation RAFT puis greffés sur des MNP fonctionnalisées amino-silane. Les analyses en TEM et par spectroscopie de corrélation de photons ont révélé l’aptitude à la dispersion aqueuse et à la formation de nanoclusters. Les clusters ainsi obtenus ont été utilisés en tant que nanosupports magnétiques recyclables pour l’adsorption d’anticorps. / We herein report the surface modification of magnetite nanoparticle (MNP) with copolymers containing active azlactone rings via a grafting ‘from’ and grafting ‘onto’ controlled radical polymerization (CRP) for use as a nano-solid support for immobilization with biomolecules. Three different approaches were presented as following. First, synthesis of poly(poly(ethylene glycol) methyl ether methacrylate-stat-2-vinyl-4,4-dimethylazlactone) (PEGMA-stat-VDM)-grafted MNP via a grafting ‘from’ atom transfer radical polymerization (ATRP) and its application as a platform for conjugating thymine peptide nucleic acid (PNA) monomer were presented. The presence of polymeric shell and the immobilization of thymine PNA on MNP core were confirmed by fourier transform infrared spectroscopy (FTIR) and vibrating sample magnetometry (VSM) techniques. The second strategy is based on the synthesis of MNP grafted with PEGMA and VDM via ATRP for conjugation with folic acid (FA). The existence of PEGMA and VDM in the structure was characterized by FTIR, TGA and VSM. After the FA conjugation, Transmission Electron Microscopy (TEM) results indicated that the FA-conjugated MNP having high VDM content exhibited good dispersibility in water.Third, the synthesis of MNP grafted with poly(ethylene oxide)-block-poly(2-vinyl-4,4-dimethylazlactone) (PEO-b-PVDM) block copolymer via a grafting ‘onto’ strategy and its application as recyclable magnetic nano-support for adsorption with antibody were studied. PEO-b-PVDM diblock copolymers were first synthesized using reversible addition-fragmentation chain transfer (RAFT) polymerization and then grafted onto amino-functionalized MNP. TEM images and photo correlation spectroscopy (PCS) indicated an improvement in the particle dispersibility in water after coating with the copolymers. The nanoclusters with PEO-b-PVDM copolymer coating were used as recyclable magnetic nano-supports for adsorption with antibody.

Analisam-se problemas de interação solo-estrutura através de uma formulação tridimensional obtida da combinação entre o Método dos Elementos de Contorno (MEC) e o Método dos Elementos Finitos (MEF). Os elementos estruturais que interagem com o solo são modelados pelo Método dos Elementos Finitos. E o solo, considerado como um meio semi-infinito, homogêneo, elástico linear e isotrópico, é modelado pelo Método dos Elementos de Contorno, empregando a solução fundamental de Mindlin. A solução fundamental de Mindlin é particularmente adequada para o tipo de problemas em análise, ou seja, problemas envolvendo sólidos tridimensionais semi-infinitos, já que é necessário discretizar apenas a superfície carregada do solo e/ou a linha de carga, e não todo o sólido tridimensional. A discretização da estaca em vários elementos finitos de viga tridimensional, permitindo a consideração de estacas de qualquer tamanho e submetidas a qualquer tipo de carregamento é uma das principais contribuições do trabalho. Outra contribuição diz respeito à consideração da ação horizontal no sistema placa-estaca-solo, diretamente aplicada na placa, e não redistribuída no topo das estacas. Por isso, os elementos estruturais laminares (radiers, sapatas e blocos de fundação) são modelados por elementos finitos de casca plana, possibilitando a consideração dos efeitos de flexão e de membrana. Essa abordagem permite a análise tanto de um grupo de estacas com bloco de capeamento rígido quanto de um radier estaqueado de qualquer rigidez. O acoplamento entre o MEC e o MEF é feito através de uma formulação mista em que a matriz dos coeficientes de influência do solo obtida pelo MEC é adicionada à matriz de rigidez dos elementos estruturais obtida pelo MEF, resultando em uma matriz de rigidez equivalente. Exemplos numéricos de interação estaca-solo, placa-solo e casca plana-estaca-solo são resolvidos para verificar, validar e demonstrar a eficiência das formulações desenvolvidas e implementadas. / Soil-structure interaction problems are analyzed by a tridimensional formulation obtained combining the Boundary Element Method (BEM) and the Finite Element Method (FEM). Structural elements that interact with the soil are modeled by the Finite Element Method. And the soil, considered as a semi-infinite, homogeneous, linear elastic and isotropic medium, is modeled by the Boundary Element Method, using Mindlin\'s fundamental solution. Mindlin\'s fundamental solution is particularly suitable for the type of problems under analysis, viz. problems involving semi-infinite three-dimensional solids, since it is necessary to discretize only the loaded surface of the soil and/or the line-load, and not all the three-dimensional solid. The discretization of the pile in several three-dimensional beam finite elements, allowing the consideration of piles of any size and subjected to any type of loading, is one of the main contributions of this work. Another contribution is about the consideration of the horizontal load in the plate-pile-soil system, directly applied to the plate, and not redistributed on the top of the piles. Therefore, the laminar structural elements (rafts, footings, and foundation blocks) are modeled by flat shell finite elements, making possible the consideration of the effects of flexion and membrane. This approach allows the analysis of both a capped pile group and a piled raft of any stiffness. The coupling between the BEM and the FEM is performed through a mixed formulation in which the matrix of the soil\'s influence coefficients obtained by the BEM is added to the stiffness matrix of the structural elements obtained by the FEM, resulting in an equivalent stiffness matrix. Numerical examples of pile-soil, plate-soil and flat shell-pile-soil interaction are solved to verify, validate and demonstrate the efficiency of the developed and implemented formulations.

Made available in DSpace on 2014-07-29T15:18:23Z (GMT). No. of bitstreams: 1 Dissertacao Denise Vaz de Campos.pdf: 1915158 bytes, checksum: a102b2411e2e162cde4e66eeca8bc624 (MD5) Previous issue date: 2011-08-22 / The structural sizing pile caps on piles has traditionally been run in simplified manner, disregarding the three-dimensional effect of structural piece. Use the concept of piled cap in calculating enriches scalability providing an understanding of internal efforts on the basis of a distribution of more realistic reactions in the block. The goal of this work was to check the differences in structural sizing pile caps using traditional methods and two numerical methods, one based on finite elements and other hybrid that combines finite elements with elements of the boundary. The main comparison between methods was the internal moment generated in the block after the application of vertical load, for being one of the main parameters of scaling of the armature. In Brazil the scaling manual methods most used are based on the theory of rods or theory of beams. The method of connecting rods, and rods considers inside the block, at the bottom, tensile stress that are combated by frame, and method of the beams is calculated as an internal moment found by reaction of piles and the lever arms to the section you want, and then the block is sized like a beam. The numerical methods presented using the concept of piled raft in scale of blocks. In numerical methods have been used DIANA and GARP programs, which provide internal moments throughout the block of three-dimensional shape and thus were made a comparison with the manual method by the theory of beams. Were simulated blocks with 2, 4, 9, 25, 64 and 81 piles with loads applied in different ways for an assessment of the effect of the size of blocks, of the comparisons performed can be observed that in the manual method and numerical methods total found moments are very close, but with a differentiated distribution along the sections of the blocks, which becomes more apparent as it increases the amount of piles per block. For the manual methods in the center of the block the internal moments are inferior to scaled as piled raft, necessitating an additional frame in this region. / Tradicionalmente, o dimensionamento estrutural de blocos sobre estacas tem sido executado de forma simplificada, desconsiderando o efeito tridimensional da peça estrutural. Utilizar o conceito de radier estaqueado no cálculo enriquece o dimensionamento proporcionando um conhecimento dos esforços internos com base numa distribuição de reações mais realistas no bloco. O objetivo deste trabalho foi de verificar as diferenças no dimensionamento estrutural de blocos sobre estacas utilizando os métodos tradicionais e dois métodos numéricos, um baseado em elementos finitos e outro híbrido que combina elementos finitos com elementos de contorno. A principal comparação entre os métodos foi o momento interno gerado no bloco após a aplicação de carga vertical, por ser um dos principais parâmetros de dimensionamento da armação. No Brasil, os métodos manuais de dimensionamento mais utilizados são baseados na teoria das bielas ou teoria das vigas. O método das bielas e tirantes considera, no interior do bloco, na parte inferior, tensões de tração que são combatidas pela armação, e no método das vigas é calculado um momento interno encontrado pela reação das estacas e os braços de alavanca até a seção desejada e o bloco é dimensionado como uma viga. Os métodos numéricos apresentados utilizam o conceito de radier estaqueado no dimensionamento dos blocos. Nos métodos numéricos foram utilizados os programas DIANA e GARP, que fornecem os momentos internos em todo o bloco de forma tridimensional. E os resultados foram comparados com o método manual pela teoria das vigas. Foram simulados blocos com 2, 4, 9, 25, 64 e 81 estacas com cargas aplicadas de distintas formas para uma avaliação do efeito do tamanho dos blocos. Das comparações executadas observou-se que no método manual e nos métodos numéricos os momentos totais encontrados são muito próximos, porém com uma distribuição diferenciada ao longo das seções dos blocos, que se torna mais evidente à medida que se aumenta a quantidade de estacas por bloco. Para os métodos manuais no centro do bloco os momentos internos são inferiores aos dimensionados como radier estaqueado, necessitando de uma armação complementar nesta região.

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Dans cette thèse, des particules de polymère fonctionnalisées en surface avec des groupes poly (éthylène glycol) (PEG) ont été synthétisées pour favoriser leur interaction avec les dérivés cellulosique via liaisons hydrogène intermoléculaires. Deux voies de synthèse ont été proposées pour obtenir ses composites cellulose/latex.La première voie est basée sur l'auto-assemblage induit par polymérisation (PISA) pour former des nanoparticules fonctionnalisées avant leur adsorption sur un substrat cellulosique. La PISA tire profit de la formation de copolymères blocs amphiphiles dans l'eau en combinant la polymérisation en émulsion avec les techniques de polymérisation radicalaire contrôlées (RDRP). Ces dernières sont utilisées pour synthétiser des polymères hydrophiles agissant à la fois comme précurseur pour la polymerization en émulsion d'un monomère hydrophobe, et comme stabilisant des particules de latex obtenues. Deux techniques de RDRP ont été étudiées : les polymérisations RAFT et SET-LRP. Des polymères hydrophiles à base de PEG de faible masse molaire ont été synthétisés en utilisant ses deux techniques qui sont ensuite utilisés pour la polymérisation d'un bloc hydrophobe dans l'eau. Le transfert de l'agent de contrôle au site de la polymérisation était difficile en utilisant la SET-LRP en émulsion, conduisant à la formation de larges particules. En utilisant la RAFT en émulsion, des particules nanométriques ont été obtenues, avec un changement morphologique observé en fonction de la taille du segment hydrophobe, puis adsorbées sur des nanofibrilles de cellulose (CNF).La seconde voie utilise la polymérisation en émulsion classique réalisée en présence de nanocristaux de cellulose (CNC) conduisant à une stabilisation Pickering des particules de polymère. L'interaction cellulose/particule est assurée grâce à l'ajout d’un comonomère à type PEG. Une organisation a été visualisé dans laquelle plusieurs particules de polymère recouvrent chaque CNC / In this thesis, polymer particles surface-functionalized with poly(ethylene glycol) (PEG) groups were synthesized to promote their interaction with cellulose derivatives via intermolecular hydrogen bond. Two synthetic routes were proposed to obtain such cellulose/latex composites.The first route was based on the polymerization-induced self-assembly (PISA) to form functionalized polymer nanoparticles prior to adsorption onto cellulosic substrate. PISA takes advantage of the formation of amphiphilic block copolymers in water by combining emulsion polymerization with reversible-deactivation radical polymerization (RDRP) techniques. The latter were used to synthesize well-controlled hydrophilic polymer chains, acting as both precursor for the emulsion polymerization of a hydrophobic monomer, and stabilizer of the final latex particles. Two RDRP techniques were investigated: reversible addition-fragmentation chain transfer (RAFT), and single electron transfer-living radical polymerization (SET-LRP). Low molar mass PEG-based hydrophilic polymers have been synthesized using both techniques, used for the polymerization of a hydrophobic block in water. The transfer of controlling agent at the locus of the polymerization was challenging for SET-LRP in emulsion conditions leading to surfactant-free large particles. Nanometric latex particles were obtained via RAFT-mediated emulsion polymerization, with morphology change from sphere to fibers observed depending on the size of the hydrophobic segment, which were then able to be adsorbed onto cellulose nanofibrils (CNFs).The second route used conventional emulsion polymerization performed directly in presence of cellulose nanocrystals (CNCs) leading to Pickering-type stabilization of the polymer particles. Cellulose/particle interaction was provided thanks to the addition of PEG-based comonomer. Original organization emerged where CNCs were covered by several polymer particles

La barrière hémato-encéphalique (BHE) protège le système nerveux central (SNC) en contrôlant le passage des substances sanguines et des cellules immunitaires. La BHE est formée de cellules endothéliales liées ensemble par des jonctions serrées et ses fonctions sont maintenues par des astrocytes, celles ci sécrétant un nombre de facteurs essentiels. Une analyse protéomique de radeaux lipidiques de cellules endothéliales de la BHE humaine a identifié la présence de la voie de signalisation Hedgehog (Hh), une voie souvent liées à des processus de développement embryologique ainsi qu’au niveau des tissus adultes. Suite à nos expériences, j’ai déterminé que les astrocytes produisent et secrètent le ligand Sonic Hh (Shh) et que les cellules endothéliales humaines en cultures primaires expriment le récepteur Patched (Ptch)-1, le co-récepteur Smoothened (Smo) et le facteur de transcription Gli-1. De plus, l’activation de la voie Hh augmente l’étanchéité des cellules endothéliales de la BHE in vitro. Le blocage de l’activation de la voie Hh en utilisant l’antagoniste cyclopamine ainsi qu’en utilisant des souris Shh déficientes (-/-) diminue l’expression des protéines de jonctions serrées, claudin-5, occcludin, et ZO-1. La voie de signalisation s’est aussi montrée comme étant immunomodulatoire, puisque l’activation de la voie dans les cellules endothéliales de la BHE diminue l’expression de surface des molécules d’adhésion ICAM-1 et VCAM-1, ainsi que la sécrétion des chimiokines pro-inflammatoires IL-8/CXCL8 et MCP-1/CCL2, créant une diminution de la migration des lymphocytes CD4+ à travers une monocouche de cellules endothéliales de la BHE. Des traitements avec des cytokines pro-inflammatoires TNF-α and IFN-γ in vitro, augmente la production de Shh par les astrocytes ainsi que l’expression de surface de Ptch-1 et de Smo. Dans des lésions actives de la sclérose en plaques (SEP), où la BHE est plus perméable, les astrocytes hypertrophiques augmentent leur expression de Shh. Par contre, les cellules endothéliales de la BHE n’augmentent pas leur expression de Ptch-1 ou Smo, suggérant une dysfonction dans la voie de signalisation Hh. Ces résultats montrent que la voie de signalisation Hh promeut les propriétés de la BHE, et qu’un environnement d’inflammation pourrait potentiellement dérégler la BHE en affectant la voie de signalisation Hh des cellules endothéliales. / The blood-brain barrier (BBB), composed of tightly bound endothelial cells (ECs), regulates the entry of blood-borne molecules and immune cells into the CNS. Recent studies indicate that the Hedgehog (Hh) signaling pathway in adult tissues plays an important role in vascular proliferation, differentiation and tissue repair. Using a lipid membrane raft-based proteomic approach, I have identified the Hedgehog (Hh) pathway as a signaling cascade involved in preserving and upkeeping BBB functions. My study shows that human astrocytes express and secrete Sonic Hh (Shh) and conversely, that human BBB-ECs bear the Hh receptor Patched-1 (Ptch-1), the signal transducer Smoothened (Smo) as well as transcription factors of the Gli family. Furthermore, activation of the Hh pathway in BBB-ECs restricts the passage of soluble tracers in vitro. By blocking the Hh signaling in vitro and by using Shh knock-out (-/-) embryonic mice, I demonstrate a reduced expression of TJ molecules claudin-5, occludin and ZO-1. Hh activation also decreases the surface expression of cell adhesion molecules ICAM-1 and VCAM-1, and decreases BBB-ECs secretion of pro-inflammatory chemokines IL-8/CXCL8 and monocytes chemoattractant protein 1 MCP-1/CCL2, resulting in a reduction of migrating CD4+ lymphocytes across human BBB-EC monolayers. In vitro treatment with inflammatory cytokines TNF-α and IFN-γ, upregulates the production of astrocytic Shh and the BBB-EC surface expression of Ptch-1 and Smo. In active Multiple Sclerosis (MS) lesions, in which the BBB is disrupted, Shh expression is drastically upregulated in hypertrophic astrocytes, while Ptch-1 and Smo expression is down-regulated or left unchanged, suggesting that a deregulation in the Hh signaling pathway may prevent the barrier stabilizing properties of Hh. Our data demonstrate an anti-inflammatory and BBB-promoting effect of astrocyte-secreted Hh and suggest that a pro-inflammatory environment disrupt the BBB by impacting, at least in part, on Hh signaling in brain ECs.

La première partie de ce manuscrit concerne l'étude en monocouches à l'interface air-eau de mélanges lipidiques susceptibles de former des "rafts" dans les membranes cellulaires, c'est-à-dire des domaines enrichis spécifiquement en certains lipides (glycolipides, sphingolipides, cholestérol) et certaines protéines. Du fait de leur composition spécifique, ces domaines sont caractérisés par une phase particulière ("liquid ordered phase") présentant des caractéristiques intermédiaires entre celles des phases fluides et condensées. Les principales méthodes mises en oeuvre dans cette étude sont des mesures de pression de surface (isothermes de compression, analyse des aires moléculaires moyennes, études de désorption en présence de beta-cyclodextrine), la microscopie de fluorescence (après marquage de la monocouche à l'aide d'une sonde fluorescente), ou la microscopie à l'angle de Brewster. Une des parties les plus importantes de ce travail concerne le comportement de monocouches constituées d'un glycolipide, le GM3, en présence de phospholipides (phase fluide), de sphingomyéline ou de cholestérol (phase "raft"). Les mesures montrent que le GM3 n'a pas d'affinité particulière pour la sphingomyéline. De même, son interaction avec le cholestérol induit une condensation de la monocouche similaire à celle observée avec les phospholipides en phase fluide, et ne permet pas le maintien du cholestérol dans la monocouche lors d'une désorption induite par la beta-cyclodextrine. Ce manque d'interaction spécifique du GM3 avec des lipides présents dans les "rafts" permet donc d'expliquer sa répartition assez large dans les membranes d'adipocytes, tant dans la phase fluide que dans les "rafts". La seconde partie concerne l'étude de molécules organiques de type hémicyanine organisées en monocouches de Langmuir et films de Langmuir-Blodgett, étudiés par des méthodes complémentaires (absorption, spectroscopie de fluorescence stationnaire et résolue en temps), mesures de pression de surface et microscopie de fluorescence. Ces hémicyanines comportent une chaîne hydrophobe nécessaire à la réalisation de films organisés stables. Ces derniers peuvent avoir de multiples applications dans la collecte et le transfert de l'énergie lumineuse ou les analyses chimiques ou biochimiques (reconnaissance sélective de cations lors de pollutions par exemple).

El càncer de pàncrees és altament agressiu i representa la cinquena causa de mort al mon occidental. Anteriorment, en el nostre laboratori, vam identificar que el receptor tissular del plasminogen (tPA) hi està sobre-expressat i juga un paper important el la progressió tumoral. En la present tesi hem profunditzat en l'estudi del mecanisme molecular de tPA i seus receptors en aquest càncer. En primer lloc hem caracteritzat en detall la interacció de tPA amb Annexina A2 (principal receptor de tPA en endoteli i altament expressada en pàncrees) demostrant que les dades publicades sobre la seqüència responsable de la interacció no eren correctes. A més a més hem caracteritzat les proteïnes de lisats cel·lulars pancreàtics que interaccionen amb tPA mitjançant un assaig pull down i posterior anàlisi proteòmic. de tot identificant un conjunt de possibles lligands de tPA. D'entre aquests hem seleccionat galectina 1, una lectina que mai s'ha descrit que interaccioni amb tPA, per realitzar la caracterització bioquímica i funcional del seu paper com a nou lligand de tPA en càncer de pàncrees. / Pancreatic cancer is a highly aggressive disease and represents the fifth cause of death in occidental world. Our laboratory has previously reported tissue type plasminogen activator (tPA) over expression in this cancer and its role in tumoral progression. During the present thesis we have studied tPA and its molecular mechanism through its receptors in this tumor.We have first characterized tPA interaction with annexin A2 (its main receptor in endothelium and highly expressed in pancreas). Our results showed that published data about the sequence responsible of this interaction was not correct. We have also identified a set of new putative tPA receptors in pancreatic cell lisates using a pull down assay and proteomic analysis. One of the proteins identified was galectin 1, a lectin with not know relation with tPA. We performed a biochemical and functional characterization of the interaction between these two proteins in pancreatic cancer.