Specifically binding ions induce the transition of anionic polyacrylate coils from extended conformation to collapsed globules passing through a cascade of intermediate states when solution conditions approach the L-type precipitation threshold. It is the conformation of these intermediate states on surfaces and in solution which is at the focus of this thesis. In comparing the surface and solution conformations of intermediate states, we were able to qualitatively and quantitatively underline the effects of sample history. Two types of quantitative comparisons have been emphasized. In real space, the radius of gyration values of adsorbed molecules have been evaluated incorporating fully the x, y and z axes. These values have been compared with radius of gyration values of the very same sample solution obtained using SLS. In reciprocal space, a novel image processing protocol has been used to generate the 2D form factor curve wherein the correlation maxima have been compared with corresponding maxima obtained for the very same sample solution using small angle scattering techniques like the SANS. The influence of bivalent ions, respectively, Strontium, Lead and Calcium, on the shape of polyacrylate coils is studied. In the last case, temperature has been introduced as a secondary parameter to shed further light on the mechanism by which polyelectrolytebivalent ion complexation takes place. Both scattering and AFM experiments reveal formation of necklace-like structures as intermediates for NaPA-Sr2+ system. Since the mol. wt. of the NaPA coils used was relatively large in this case, adsorption on mica surfaces was strong. Under such conditions, the molecules undergo a z collapse upon flux drying but do not get altered in x and y directions. The ratio Rg(AFM)/Rg(SLS) was found to be in the range 0.7-0.9. The remaining (insignificant) differences in the Rg Abstract ii values arise due to the fact that AFM gives the square root of number averaged mean squared radius of gyration while SLS gives the square root of z-averaged mean squared radius of gyration. The differences in radius of gyration values observed in solution and on surfaces were more prominent for NaPA-Pb2+ system. Again, although both scattering and AFM reveal necklace-like structures as intermediates, the ratio Rg(AFM)/Rg(SLS) was now found to be nearly 0.6. The fact that Rg(AFM) is the square root of number averaged and Rg(SLS) is the square root of z-averaged mean squared radius of gyration, alone cannot explain this low value. Since the mol. wt. of NaPA coils used in this case was quite low, adsorption on mica surfaces was weak. Under such conditions, the molecule does not only undergo a z collapse upon flux drying, but also shrinks in the x and y directions due to capillary forces. Finally, with NaPA-Ca2+ system, the picture did not show a one-to-one correspondence between solution and surface conformations at all. In fact, it showed a one-step-ahead correspondence. As already stated, the coil to globule transition was induced by increasing the equilibration temperature from 15°C to 30°C in this case. SANS could not identify any necklace-like intermediates in solution at the equilibration temperature of 15°C while AFM scans at this temperature showed the beginning of formation of pearls. Likewise, at the equilibration temperature of 30°C, SANS could identify necklace-like intermediates in solution with a large majority of dumbbells while AFM scans at this temperature showed a mix of dumbbells, sausage-like structures and globules. Indeed, we were witnessing an accelerated coil to globule transition on surfaces as compared to the situation in solution resulting in a pre-emption in the formation of intermediate states on surfaces. Since the ratio Rg(AFM)/Rg(SLS) (given the square root of number averaged and the square root of z-averaged mean Abstract iii squared values respectively) at the equilibration temperature of 30°C showed a range of 0.7-0.9 indicating strong adsorption of the relatively high mol. wt. NaPA coil on mica surfaces, our suspect were the substrate-sample interaction forces. The AFM scans were therefore analysed with 2D form factor curves, a better protocol when no assumptions about the shape of adsorbed molecules are made a priori, to trace the effects of sample history. The thesis establishes the general utility of AFM to capture the essential features of a collapsing coil which the very coil exhibits in solution. The shape of the coil on surface and in solution may not be exactly the same, yet reveal the same characteristics. The comparative advantages and disadvantages of salt pre-treated mica surfaces and chemically modified mica surfaces have been brought out. Finally, a definitive new insight is gained as regards the mechanism of coil collapse induced by specifically binding ions. The entropic nature of the process as well as the visualized shape of the collapsing intermediates does not support a mechanism along an electrostatically driven shrinking with linear, rod-like arrays of pearls as intermediates. On a molecular level, it is the liberation of water molecules and Na+ ions which promotes binding of bivalent ions to COO- residues. This binding in turn increases the hydrophobicity of the polyacrylate chains. As a consequence, the chains shrink due to an increased propensity for polymerpolymer contacts (and finally precipitate).

Virtuellement tous les habitats terrestres sont dominés par les angiospermes, ou plantes à fleurs. Leur capacité à coloniser de nouveaux habitats et supplanter une autre espèce est due à l'avènement d'une nouvelle structure reproductrice – la fleur. La fleur uni les organes mâles et femelles dans une structure compacte et contient la graine. Les plantes à fleurs ne sont pas seulement le type dominant des plantes terrestres, mais sont également la principale source de nourriture et l'habitat de tous les animaux, y compris les humains. En termes d'évolution, les fleurs sont considérées comme un développement récent. Elles ont fait l'objet de spéculations depuis l'époque de Charles Darwin qui à nommé l’évolution dominante et la diversification des plantes à fleurs comme «un abominable mystère» en raison de l'absence d'une transition en douceur de la non-floraison vers la floraison des plantes dans le registre fossile. Avec le séquençage de plusieurs génomes de gymnospermes (semences de plantes non-florales), d’angiospermes basals et de plantes à fleurs supérieures, certaines familles de gènes jouant un rôle central dans le développement et l'évolution de la fleur ont été identifiées. Notre recherche se concentre sur une de ces familles de régulateurs de niveau supérieur appelée « famille de facteur de transcription MADS » (TF). Cette famille de TF permet d'orchestrer le développement des fleurs. Nous nous sommes intéressés à la compréhension des mécanismes moléculaires de la famille des MADS et à la façon dont ces protéines sont capables de contrôler les fonctions de reproduction complexes.Ce projet intègre différentes techniques biophysiques comme la cristallographie aux rayons X, la diffusion des rayons X aux petits angles (SAXS) et la microscopie à force atomique (AFM) afin d’étudier les interactions protéine-protéine et protéine-ADN des FT MADS. Aucune étude n’a, à ce jour, porté sur les mécanismes moléculaires des FT MADS en utilisant cette approche structurale intégrée.Un obstacle important dans l'étude des FT MADS a été l’expression des protéines recombinantes et leur purification. Dans ce projet, les protocoles de purification de plusieurs recombinants FT MADS entières ont été établis, permettant la caractérisation structurale et biochimique des protéines dans leurs intégralités. La structure aux rayons X du domaine d'oligomérisation de la protéine de la famille MADS, SEPALLATA3 (SEP3) est présenté et utilisé comme modèle pour comprendre les motifs d'oligomérisation de la famille élargie et les bases moléculaires des interactions protéine-protéine. Des solutions de structures9provenant d'études SAXS de AGAMOUS (AG) et de la phase végétative courte (SVP) sont présentées et complétés par la caractérisation biochimique de leur état d'oligomérisation.Afin d'étudier les interactions protéine-ADN, des procédés complémentaires ont été utilisés. Une propriété importante des FT MADS est leur capacité à modifier la structure de l'ADN grâce à la formation de boucles d'ADN. De manière hypothétique, les FT MADS oligomérisent et fixent l'ADN sur deux sites différents, bouclant potentiellement l'ADN. En utilisant l'AFM, la première preuve directe de la formation de boucle d'ADN par SEP3 est obtenue. Les caractéristiques de liaison d'ADN de SVP ont été étudiées par analyse de décalage de mobilité électrophorétique (EMSA), par thermophorèseà échelle microscopique (MST) et par AFM. Contrairement au cas de SEP3, l’EMSA et l’AFM ont montrés que SVP est un dimère et présente différents modes de liaison à l'ADN.Ces données fournissent une base atomique et structurale de la fonction des FT MADS. Sur la base de ce travail, nous commençons à comprendre l’oligomérisation et certaines spécificités déterminantes de liaison à l'ADN. Ces études montrent comment les FT MADS s’oligomérisent. / Virtually all terrestrial habitats are dominated by angiosperms, or flowering plants. Their success in colonizing new habitats and supplanting other species is due to the advent of a complex reproductive structure – the flower. The flower unites the male and female organs into one compact structure and encloses the seed. Flowering plants are not only the dominant type of land plants, but also are the primary source of food and habitat for all animals, including humans. In evolutionary terms, flowers are considered a recent development and have been a subject of speculation from the time of Charles Darwin who termed the dominant rise and diversification of flowering plants as “an abominable mystery”* due to the lack of a smooth transition from non-flowering to flowering plants in the fossil record. With the sequencing of multiple genomes from gymnosperms (non-flowering seed plants), basal angiosperms and higher flowering plants, certain gene families have been identified which play a central role in the development and evolution of the flower. My research focuses on one such family of high-level regulators, the MADS transcription factor (TF) family. This TF family helps to orchestrate flower development among other functions. As such, there is great interest in understanding the molecular mechanisms of the MADS family and how these proteins are able to control complex reproductive pathways.This project integrates different biophysical techniques including x-ray crystallography, small angle x-ray scattering (SAXS) and atomic force microscopy (AFM) to investigate protein-protein and protein-DNA interactions of MADS TFs. No studies to date have investigated the molecular mechanisms of MADS TFs using this integrated structural approach.One important hurdle in the study of the MADS TFs has been recombinant protein expression and purification. In this project, recombinant purification protocols for several full length MADS TFs were established, allowing the structural and biochemical characterisation of the proteins. The crystal structure of the oligomerisation domain of the MADS family protein SEPALLATA3 (SEP3) is presented and used as a template for understanding the oligomerisation patterns of the larger family and the molecular basis for protein-protein interactions. Investigation of solution structures, derived from SAXS studies, of AGAMOUS (AG) and SHORT VEGETATIVE PHASE (SVP) along with biochemical characterisation of their oligomerisation states are also presented.In order to study protein-DNA interactions, complementary methods were used. An important putative property of the MADS TFs is their ability to change the structure of DNA through the formation of DNA loops. MADS TFs are hypothesized to oligomerise and bind DNA at two different sites, potentiating looping of DNA. Using AFM, the first direct evidence of DNA looping by SEP3 is described. The DNA binding characteristics of SVP were studied using electrophoretic mobility shift assay (EMSA), microscale thermophoresis (MST) and AFM. Unlike SEP3, SVP is dimeric and thus exhibits different DNA-binding patterns.The data presented here provide an atomic and structural basis for MADS TF function. Based on this work, we now are beginning to understand some of the oligomerisation and DNA-binding specificity determinants. These studies demonstrate how the MADS TFs oligomerise and the results show that we can disrupt oligomerisation and potentially DNA-binding very specifically through the introduction of point mutations. Future work will investigate the in vivo consequences of altered oligomerisation and how this affects different developmental programs in plant reproduction and floral organ morphogenesis.*Letter from Charles Darwin to Joseph Dalton Hooker, written 22 July 1879 (Source: Cambridge University Library DAR 95: 485 – 488) (Friedman, 2009b).

Dans le but d'accroître nos connaissances sur les poudres pour usage alimentaire ou pharmaceutique, nous proposons d'étudier l'effet des procédés de fabrication, de mise en forme, la granulation sur la réactivité des poudres. La première partie est consacrée à l'effet de pureté sur les interactions-Lactose Lactose et Lactose-API mesurées / quantifiée par des techniques de champ proche (AFM, SMM, MSAFM) sous atmosphère ambiante et dans des conditions de stress stockage. Dérivant une seconde partie de la partie précédente mettra en évidence les effets des interactions sur différentes formulations pharmaceutiques, telles que la compression directe et avec une poudre sèche Inhalation inhalateur. Tout ce travail aidera à comprendre l'impact de la surface de pureté Lactose pharmaceutique qualité et la stabilité des formulations pour inhalation et par compression. / The discovery of the problem will go through training techniques at the nanoscale characterization and chemical analysis, the discovery of the industrial problem in society Armor Proteins, the validation of a pharmaceutical lactose. With the aim to increase our knowledge on powders for food or pharmaceutical use, we propose to study the effect of manufacturing processes, shaping, granulation on the reactivity of powders. The first part is devoted to the effect of purity on interactions-Lactose Lactose and Lactose-API measured / quantified by near-field techniques (AFM, SMM, MSAFM) under ambient atmosphere and under stressful conditions storage. Deriving a second portion of the preceding part will highlight the effects of interactions on different pharmaceutical formulations, such as direct compression and with a Dry Powder Inhalation Inhaler. All of this work will help to understand the impact of purity surface Lactose Pharmaceutical quality and stability of formulations for inhalation and compression.

Nous présentons dans cette thèse les différentes étapes de conception d'une plateforme microrobotique dédiée au positionnement sous faisceau de lumière d'objets de dimensions micrométriques. Cette plateforme a pour vocation de mettre en oeuvre des méthodologies d'asservissement et de suivi de position de micro-objets placés au coeur même d'un faisceau de lumière. L'objectif final étant de caractériser des micro/nano-matériaux par diffraction et diffusion des rayons X. Les différentes contraintes technologiques rencontrées par les systèmes de micro-nanomanipulation actuels en environnement synchrotron nous ont amené à concevoir une plateforme microrobotique de manipulation duale utilisant des sondes de microscope à force atomique (AFM). Diverses méthodologies de préhension avec une ou deux sondes AFM avec capteur de force intégré - ont été proposées en vue d'évaluer chacune d'entre-elles dans un contexte de positionnement tridimensionnel. Une stratégie de commande des micromanipulateurs à double étage est mise en oeuvre pour assurer l'asservissement de position des sondes AFM lors des tâches d’approche et de transport du micro-échantillon sous test. Afin d’augmenter la robustesse de positionnement vis-à-vis des erreurs de modélisation, des perturbations extérieures et des bruits de mesure, nous avons proposé une commande robuste de type H∞, avec optimisation des paramètres de pondération à partir d’algorithmes génétiques. De plus, les erreurs aléatoires d’alignement du faisceau de lumière avec l’objet sont corrigées en temps-réel par l’utilisation d’estimateurs de position (filtres de Kalman et particulaire). Finalement, des tâches microrobotiques automatisées de micro-préhension, de transport et de positionnement de microobjets sphériques de 8 μm de diamètre placés sous faisceau de lumière laser ont été réalisées avec succès. Le « benchmark » proposé est en cours de transfert au sein du European Synchrotron Radiation Facility (ESRF) à Grenoble pour validation sous faisceau de rayons X. / We present in this thesis the various stages in the design of a microrobotics platform dedicated to the manipulation of micro/nano objects in a synchrotron environment. It is composed of dual micro/nano manipulators in order to handle and to maintain a micro/nano-sample through the focus of a X-ray or laser beam for material characterization and analysis. The main idea is to control and to drive in a robust way the micro/nanomanipulators by focusing the beam on the center part of the handled micro-object. A microgripper based on two Atomic Force Microscope (AFM) tips with integrated piezoresistive force sensors is proposed. First, the dual manipulators are controlled cooperatively by combining the different actuator dynamics to track a laser beam with nanometer precision. A robust control strategy based on H∞ control ensures a robust microhandling task under the focus of the laser beam whatever the external perturbations involved and parametric model uncertainties. The Genetic Algorithm (GA) approach is used to compute the parameter weighting functions in to obtain an optimal H∞ controller. Then, we propose to compensate the laser beam variations (thermal drift, mechanical variations) by estimating the position of the laser beam using stochastic estimators (Kalman and particle filters). To this aim, the maximum intensity of the laser beam is measured and tracked in real-time by a four-quadrant photodiode sensor. Finally, experimental results performed of micro-spheres (diameter: 8 μm) demonstrate the robustness of the robotic microhandling tasks using the proposed control scheme strategy.

In this work we made a review of how a multi-probes microscope using Atomic Force and Near Field Scanning Microscopy works. Currently, the Nanonics Multiview 4000 instaled at the Materials Caracterization and Microscopy Laboratory (LCMMAT) is not completly working. Nowadays, we are able to do Atomic Force Microscopy (AFM) and reflection and transmission Scanning Near Field Microscopy (SNOM) measurements. This kind of microscope have three probes which are able to do simultaneaous mesurements of AFM, C-AFM, SNOM, Raman Microscopy and nanolitography. It is the first multi-probe microscope to be instaled in Latin America. This work consists in studying the structure of this kind of microscope, how does it make AFM and SNOM measurements and how to analise them. We study the different electronic circuits which are used in this kind of microscopes and we compare both optical and tuning-fork feedback. It was explain step by step how to do and AFM and SNOM measurement. We study the processing and analise of this measurements. Finally, we made some different measurements using this tecniques. Some of this measurements were compared with that found in references in order to try to find some possible aplications which could be useful for future researches at our laboratory. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Neste trabalho realizamos uma revisão do funcionamento do Microscópio Raman Confocal Multipontas com Campo Próximo e Força Atômica modelo Multiview 4000 da empresa Nanonics. Atualmente, o microscópio Multiview 4000 do Laboratório de Caracterização e Microscopia de Materiais (LCMMAT) ainda não se encontra operando aos 100%. Ele encontra-se em fase de montagem, estando disponível hoje em dia para uso só o Microscópio de Força Atômica (AFM) e o Microscópio de Campo Próximo (SNOM) nos modos reflexão e transmissão. Este modelo de microscópio, o qual possui três ponteiras que são capazes de fazer medidas em simultâneo de AFM, C-AFM, SNOM e microscopia Raman Confocal, alem de poder fazer nanolitografia, é o primeiro a ser instalado na America Latina. Durante a realização do presente trabalho, estudamos a estrutura do microscópio, como ele realiza as medidas destas duas técnicas e como elas são feitas. No estudo estrutural do Microscópio foram descritos os princípios físicos que são usados para a formação da imagem, alem dos diferentes tipos de circuitos eletrônicos usados em equipamentos de este tipo. Explicou-se passo a passo como são feitas as medidas de AFM e de SNOM. Estudamos também como é feito o analise e o processamento das imagens. Finalmente foram mostradas algumas imagens que foram feitas usando o microscópio, e comparou-se com alguns resultados encontrados na bibliografia a fim de encontrar possíveis aplicações de cada uma das amostras aqui mostradas.

Ce travail de thèse a permis d’étudier l’influence de l’ajout d’ingrédients fonctionnels laitiers sur l’encapsulation de L. rhamnosus GG (LGG). Deux ingrédients laitiers (ß-lactoglobuline et membrane des globules gras du lait - MFGM) ont été identifiés comme étant capables d’adhérer fortement à LGG par l’intermédiaire de ses pili. Le rôle clé de ces adhésions dans la localisation spatiale des bactéries dans la matrice laitière a été mis en évidence, ainsi que le rôle des constituants de la matrice dans sa structuration. Cela a permis de sélectionner, in vitro, une matrice d’encapsulation capable de protéger de manière efficace les bactéries des conditions gastriques et de libérer les bactéries vivantes au niveau de l’intestin. En parallèle, la MFGM dans l’encapsulation des bactéries s’est révélée prometteuse. Ce travail a également démontré l’importance primordiale du choix de la matrice d’encapsulation. En effet, une compétition entre l’adhésion de LGG aux cellules intestinales et l’adhésion de LGG à certains composants de la matrice laitière a été démontrée. Les deux phénomènes impliquent probablement les mêmes mécanismes : adhésion aux pili glycosylés de LGG. Pour terminer, un procédé de séchage par atomisation a été développé pour encapsuler LGG. Il permet une bonne survie des bactéries après séchage et la production de microparticules présentant des propriétés fonctionnelles innovantes liées à la température du milieu de réhydratation / The aim of this work was to understand how functional dairy components influence L. rhamnosus GG (LGG) encapsulation. First, two dairy components (-lactoglobulin and milk fat globule membrane - MFGM) able to strongly adhere to LGG through their pili are identified. The key role of these adhesions on bacteria spatial location in the matrix is highlighted, as well as the role of matrix dairy components in their structuration. This allowed to select, in vitro, a matrix able to protect bacteria in gastric conditions and to release them viable in the intestine. Simultaneously, the use of MFGM in bacteria encapsulation has proven to be promising. This work demonstrated the importance of the matrix choice in the encapsulation procedure. Results demonstrated that adhesion between LGG and dairy matrix may compete with adhesion of LGG to epithelial intestinal cell. The two phenomena likely involve the same mechanisms: adhesion to glycosylated pili of LGG. To finish, a spray drying encapsulation process is developed to encapsulate bacteria. It leads to a high bacteria survival after drying and the production of microparticles with innovative properties depending on rehydration temperature

A microscopia de fluorescência é uma das técnicas mais poderosas disponíveis atualmente, uma vez que proporciona uma combinação excepcional de alta sensibilidade na detecção, alta especificidade, além de ser consideravelmente não invasiva. Avanços recentes permitiram a detecção em resolução de subdifração, o que eleva sua potencialidade de investigação de um maior número de sistemas e, consequentemente, de avanço científico. O estudo de novas sondas fluorescentes é de fundamental importância para a aplicação em métodos avançados de microscopia óptica. Na primeira vertente da pesquisa, Capítulo 2, foi realizado o estudo fotofísico de uma série de compostos bisarilados derivados do anidrido maleico e de maleimidas sintetizados pela reação de Heck-Matsuda. Visando o aprimoramento do design dessas moléculas, foi realizada a ciclização fotoquímica de tais compostos, resultando em moléculas com anéis condensados, nomeados como derivados de fenantreno, as quais proporcionaram maior estabilidade fotoquímica. A dinâmica do estado excitado remete ao efeito push-pull, em que há um deslocamento de carga notável, mas não completo. Para os compostos com a substituição 4-hidroxifenil foi observado um processo de deslocamento de carga combinado com uma transferência de próton no estado excitado assistida por solvente. Ademais, o estudo dos compostos derivados de fenantreno em microscopia confocal demonstrou que as propriedades locais do solvente afetam a dinâmica de relaxação de fluorescência em diferentes meios condensados e que os mesmos são passíveis de serem aplicados a técnicas avançadas de microscopia de fluorescência. A segunda vertente desta tese, Capítulo 3, explora um sistema biológico em nível de uma única molécula. Especificamente, este capítulo concerne à investigação de uma metodologia ótima para a marcação fluorescente de DNA em sequência específica, através de microscopia de fluorescência com super-resolução. As reações foram conduzidas utilizando uma metodologia de marcação de duas etapas, de acordo com o princípio mTAG. Na primeira etapa, grupamentos contendo alquino terminal, azida ou amina primária são transferidos do cofator análogo ao S-adenosil-L-metionina para o DNA através de uma enzima metiltransferase. Foi utilizada a enzima M.TaqI, a qual tem como alvo a sequência 5\'- TGCA -3\' para modificação. Na segunda etapa é realizado o acoplamento do fluoróforo aos sítios funcionais do plasmídeo (pUC19) através de reações químicas bioortogonais, tais como reação click catalisada por cobre (CuAAC), reação click na ausência de cobre (SPAAC) e acoplamento do grupo amina primária com NHS-éster. Também foi desenvolvida uma metodologia direta de uma etapa, na qual o fluoróforo é diretamente transferido do cofator análogo para o DNA em uma única etapa reacional. Para acompanhar o desempenho das reações foi desenvolvido um ensaio single-molecule para a contagem do número de moléculas de corante ligadas a plasmídeos individuais. A topologia dos plasmídeos após a marcação foi investigada por imagens de AFM em alta resolução. A combinação de ambas as análises demonstrou que a reação SPAAC assim como a reação direta de uma etapa promoveram uma marcação fluorescente quase completa e a técnica de AFM confirmou que o acoplamento de fluoróforos não induziu danos à estrutura dos plasmídeos, os quais preservaram sua morfologia nativa, superenrolada. Além disso, os plasmídeos marcados foram aplicados com sucesso a procedimentos de transfecção em células de mamíferos, indicando que o DNA reteve sua capacidade de codificar informação genética, mesmo na presença de fluoróforos ligados. / Fluorescence microscopy is one of the most powerful techniques currently available, since it provides the unique combination of a high sensitivity in detection, a high specificity, and a considerable non-invasiveness. Recent developments have allowed the detection at a sub-diffraction resolution, which elevates its potentiality to investigate several systems and hence to go further in science. The study of new fluorescent probes is crucial for the application in advanced methods in optical microscopy. In the first extent of this research, Chapter 2, a photophysical study of maleic anhydride and maleimide derivatives, synthesized by the Heck-Matsuda reaction, was performed. Aiming at the improvement of the design of these molecules, a photochemical cyclization was carried out, resulting in molecules with condensed rings, termed as phenanthrene derivatives, which promoted more photochemical stability. The excited state dynamics rely on the push-pull effect, in which a notable, but not complete, charge shift takes place. For the compounds with a 4-hydroxyl substituent, a charge shift combined with an excited state solvent-assisted proton transfer was observed. Additionally, the confocal microscopy study of the phenanthrene derivatives showed that the local properties of the solvent modulate the fluorescence relaxation dynamics in condensed media and hence such dyes can be potential candidates for use in advanced fluorescence microscopy techniques. The second extent of this thesis, Chapter 3, explores a biological system at the single-molecule level. Specifically, this chapter concerns to an investigation of an optimal sequence-specific DNA fluorescent labelling, using super-resolution fluorescence microscopy. The reactions were performed using a two-step methodology, according to the mTAG approach. In the first step, moieties containing a terminal alkyne, azide, or primary amine group are transferred from an S-adenosyl-L-methionine analogue cofactor to the DNA by a methyltransferase enzyme. Herein, the enzyme M.TaqI was used, which targets the 5\'- TCGA -3\' sequence for modification. In the second step, a fluorophore is coupled to the functional sites of the plasmid (pUC19) using bio orthogonal reactions, such as the click reaction catalysed by copper (CuAAC), the copper-free click reaction (SPAAC), and the amino-to-NHS-ester coupling reaction. A direct one-step approach in which the fluorophore is directly transferred to the DNA from the analogue cofactor in a single reaction step, was also developed. A single-molecule assay was developed for counting the number of fluorophores associated with the individual plasmids. The topology of the plasmids after labelling was also investigated by high-resolution AFM imaging. Combining both analysis, the SPAAC as well as the direct one-step reactions were found to promote near-complete labelling and the AFM showed that the fluorophore coupling did not damage the structure of the plasmids and that their native, supercoiled, morphology was preserved. Moreover, labelled plasmids were successfully applied for transfection into mammalian cells, implying that the DNA retained its ability to encode genetic information, even while carrying bound fluorophores.

Cette thèse s'inscrit dans la continuité des études menées pour améliorer la fiabilité des relais MEMS ohmiques et comprendre les mécanismes de dégradation se produisant au niveau du contact électrique aux échelles micro et sub-micrométriques. Les deux premiers chapitres de ce manuscrit permettent d'établir l'état de l'art du domaine et de décrire les différentes techniques expérimentales utilisées afin de caractériser les mécanismes physiques se produisant lors de l'ouverture et la fermeture d'un relais MEMS sous courant. Le troisième chapitre étudie qualitativement et quantitativement le transfert de matière aux distances sub-micrométriques. L'utilisation d'un microscope à force atomique (AFM) permet d'identifier les paramètres clés, notamment la tension de contact à l'état ouvert et la vitesse de commutation. L'origine de ce transfert de matière est attribuée à des émissions de courant se produisant dans les derniers nanomètres avant la fermeture du contact. Un plasma métallique est également observé et caractérisé pendant les phases de commutations. Ces observations conduisent à l'élaboration d'un scénario permettant d'expliquer le transfert de matière à ces dimensions. Le quatrième chapitre se consacre en première partie à l'étude des rebonds lors de la fermeture du contact. On montre que des rebonds peuvent apparaître quelques µs après la fermeture du contact au cours des cycles. Ceux-ci semblent être des indicateurs de la fin de vie du composant. D'autres rebonds, liés aux forces électrostatiques de contact, sont également mis en évidence lors de fermetures à faibles vitesses (qq nm/s). L'importance de ces forces est néanmoins du second ordre et ces derniers rebonds n'interviennent pas directement dans la phase de fermeture d'un relais MEMS. L'étude de la quantification de la résistance de contact lors de l'ouverture du contact constitue la deuxième partie de ce dernier chapitre. La nature quantique de ce phénomène est mise en évidence dans deux dispositifs : un interrupteur MEMS et à l'aide d'un AFM. Il est notamment montré que ce phénomène est seulement observable pour des courants inférieurs à 100µA. Finalement, l'ensemble de ces travaux mènent à différentes recommandations, détaillées en conclusion, nécessaires pour assurer le bon fonctionnement des relais MEMS. / This thesis aims to improve the reliability of ohmic MEMS switches and focuses on the degradation mechanisms of the electrical contact at the micro and nano-scales. The two first chapters of the manuscript provide a state-of–the-art of MEMS switches and describe the different experimental techniques used to characterize the physical phenomena involved in the opening and closure of a MEMS switch under current (“hot switching actuation”). The third chapter studies qualitatively and quantitatively the material transfer at sub micrometer scale. An Atomic Force Microscope (AFM) is used to identify the main parameters involved in this phenomenon such as the opening contact voltage and the closing velocity. The origin of the material transfer is attributed to field emission in the last tens of nanometers before the contact closure. A metallic plasma is also observed and characterized during switching operations. According to the different observations, a scenario is suggested to explain material transfer at such small dimensions. The fourth chapter deals with dynamic observation during switching operations. First, bounces can be detected after a few millions of operations, they usually appear a few µs just after the first contact. Such bounces seem to be an early indicator of the lifetime of those devices. Other types of bounces related to the electrostatic contact force can be observed at very low closing velocity (a few nm/s). Nevertheless in a MEMS switch the closing and opening velocity is high enough to avoid such bounces. The second part of this chapter investigates the contact conductance quantization during the opening phase of a contact. We show that this phenomenon can be observed in a MEMS switch and with an AFM when the current is lower than 100µA. As a conclusion, several recommendations are provided to improve the reliability of MEMS switches.

Neste trabalho, se pretende caracterizar a morfologia de filmes finos poliméricos por meio de técnicas de processamento de imagens, utilizando principalmente a geometria computacional e técnicas de classificação de padrões. Os objetivos principais foram quantificar as grandezas geométricas das estruturas observadas nos filmes finos e descrever padrões de superfície formados nestes filmes. Foram estudadas imagens obtidas por microscopia de força atômica (AFM) de amostras de filmes finos SEBS [poliestireno-poli(etileno-co-butileno)-poliestireno], depositados sobre um substrato de mica por técnicas de imersão. Os filmes finos SEBS são considerados de grande interesse devido à formação de estruturas auto-organizadas na escala nanométrica. A caracterização e a obtenção da morfometria dos filmes são de relevância neste trabalho, pois contribuem para o entendimento da dinâmica de formação destes padrões nas nanoestruturas estudadas. Foram analisadas diferentes morfologias, como forma de gotículas com anéis concêntricos e forma de tiras e pontos regularmente espaçados. Os resultados obtidos permitem caracterizar os padrões observados. / In this work, we intend to characterize the morphology of polymer thin films by techniques of image processing, mainly using computational geometry and pattern classification. The main objectives were to quantify the geometrical structures observed in thin films and describe surface patterns formed in these films. Were studied images obtained by atomic force microscopy (AFM) of SEBS [polystyrene-poly(ethylene-co-butylene)-polystyrene] thin films samples, deposited on a mica substrate by dip-coating technique . SEBS thin film polymers have great interest due to the formation of self-organized structures on the nanometer scale. The characterization and obtaining measurements of the morphology of the thin films are of relevance in this work, because they contribute to the understanding of the formation dynamics of these patterns in nanostructures studied. We analyzed different morphologies, such as droplets form with concentric rings and stripe and regularly spaced points forms. The results allow to characterize the observed patterns.

Neste trabalho foi estudado o desenvolvimento de uma superfície modelo de silício monocristalino, SiO2/Si, modificada com organossilanos derivados de N-heterocíclicos que permitisse a imobilização de um complexo de coordenação, FeTIM. Estas superfícies modificadas poderão ser empregadas em estudos de reatividade frente a analitos de interesse, como o NO. Sob esse aspecto, a síntese desses novos silanos, contendo N-heterocíclicos, e o desenvolvimento de uma metodologia de formação dos filmes finos automontados, sobre a superfície de SiO2/Si, tornou-se de grande relevância na aplicabilidade deste trabalho. Para a obtenção dessas superfícies, fez-se necessária a compreensão dos parâmetros de formação dos filmes de silanos. Os parâmetros estudados foram os efeitos do tempo de adsorção, da concentração da solução dos silanos, da polaridade do solvente e do tamanho da cadeia alquílica do silano no processo de formação dos filmes. Deste modo, foi possível inferir sobre as alterações na morfologia e na estrutura química dos filmes formados, através de medidas de Espectroscopia de Fotoelétrons excitados por Raios-X (XPS), Microscopia de Força Atômica (AFM) e Microscopia Eletrônica de Varredura (MEV). A imobilização do complexo de FeTIM sobre a superfície organomodificada foi comprovada pela variação da linha de fotoemissão do Fe 2p nas medidas de XPS. / This work describes the study of model surfaces on oxidized silicon wafer, SiO2/Si, modified with N-heterocycles rings, that allows the grafting of a macrocycle iron complex, FeTIM, that could be used in reactivity studies, with biologically relevant molecules, as nitrogen monoxide (NO). On this way, the synthesis of these silanes and a new methodology of the formation of self-assembled monolayers had become a relevant question on this work applicability. These thin films contain silanes bearing nitrogenated Lewis bases on silicon surfaces. In order to obtain these modified surfaces, it was necessary a comprehensive study of the adsorption parameters of the thin films. The parameters studied were the effect of: adsorption time, the solution concentration, the role of the solvents polarity and the chain length alkylsilanes in the film formation. Then, it was possible to infer about the film\'s morphology differences and chemical structures by the XPS, AFM and MEV measurements. X-ray photoemission lines of Fe 2p were used to probe the iron chemical environment in the chemically adsorbed macrocycles complexes.