O objetivo deste estudo foi avaliar a resistência de união, a penetração e a adaptação à dentina radicular de cimentos obturadores à base de resina epóxica e de silicato de cálcio com e sem ativação sônica e ultrassônica. Raízes de caninos superiores com 16 mm de comprimento foram submetidas a preparo biomecânico com instrumentos rotatórios de níquel titânio sob irrigação constante com NaOCl 2,5% e irrigação final com EDTA 17%. As raízes foram distribuídas em grupos de acordo com os cimentos obturadores: AH Plus e MTA Fillapex. Cada grupo foi subdividido em função da ativação do cimento obturador (n=13): sem ativação; ativação sônica por 30 segundos; ativação ultrassônica por 30 segundos. Em cada subgrupo, a manipulação dos cimentos obturadores foi realizada de acordo com as instruções dos fabricantes para 10 espécimes, para os 3 espécimes restantes, o cimento foi acrescido de Rodamina B 0,1% previamente à manipulação. A obturação foi realizada de acordo com o protocolo de cada subgrupo em relação ao cimento e ativação, utilizando-se a técnica de condensação lateral. Após três vezes os tempos de endurecimento dos cimentos, os espécimes foram seccionados obtendo-se 3 slices de 1,0 mm de espessura de cada terço radicular. Dois slices de cada terço foram submetidos ao teste de push-out e o padrão de falha foi determinado. O terceiro slice de cada terço radicular foi submetido à análise qualitativa da adaptação do cimento à dentina radicular e presença de vazios, por meio de microscopia confocal de varredura a laser. Nos espécimes em que o cimento foi acrescido de Rodamina B 0,1%, avaliou-se a penetração do cimento na dentina radicular por microscopia confocal de varredura a laser com fluorescência. Os dados foram analisados por testes estatísticos paramétricos e não-paramétricos. O cimento AH Plus (2,58±1,21) apresentou maior resistência de união comparado ao MTA Fillapex (1,45±0,71) (p<0,122). A ativação ultrassônica (2,64±1,44) propiciou maior resistência de união comparada aos grupos sem ativação (1,58±0,73) (p<0,0001) e com ativação sônica (1,83±0,85) (p<0,0001), não havendo diferença entre estes (p=0,3303). O terço cervical apresentou maior resistência de união (2,48±1,49) comparado ao médio (2,08±0,94) (p=0,0003) e apical (1,48±0,58) (p<0,001). Para o cimento MTA Fillapex houve predomínio de falhas adesivas mistas em todos os grupos, enquanto que para o cimento AH Plus houve predomínio de falhas adesivas mistas para os grupos sem ativação e ativação ultrassônica e falhas adesivas mistas e adesivas ao material obturador para o grupo de ativação sônica. A ativação ultrassônica promoveu, independente do cimento obturador, maior número, densidade e extensão de tags, com massa obturadora mais homogênea e adaptada às paredes do canal radicular. A ativação sônica, por sua vez, apresentou número, densidade e extensão de tags similares ao grupo sem ativação, no entanto a massa obturadora apresentou desadaptações e formação de bolhas. A ativação ultrassônica impactou na maior resistência de união, penetração, densidade e extensão de tags e melhor adaptação do cimento a dentina radicular, ao contrário da ativação sônica que não aumentou a resistência de união e penetração de tags e proporcionou maior desadaptação e inclusão de vazios na massa obturadora. / The aim of this study was to evaluate the bond strength, penetration and adaptation to the root dentin sealers based on epoxy resin and calcium silicate with and without sonic and ultrasonic activation. Maxillary canine roots with 16 mm length were submitted to biomechanical preparation with nickel titanium rotary files, under constant irrigation with 2.5% NaOCl and final irrigation with 17% EDTA. The roots were divided into groups according to the sealers: AH Plus and MTA Fillapex. Each group was subdivided based on sealer activation (n = 13): no activation; sonic activations (30 seconds); ultrasonic activation (30 seconds). In each subgroup, the sealer manipulation was performed according to the manufacturers instructions for 10 specimens, for the 3 remaining specimens, the Rhodamine B 0.1% was incorpored to the sealer prior to manipulation. The root canal filling was performed according to the protocol of each group relative to the sealer and activation, using the lateral condensation technique. After three times the sealer setting times, the specimens were sectioned and obtained 3 slices of 1.0 mm thickness of each root third. Two slices of each third were submitted to the push-out test and the failure pattern was determined. The third slice of each root third was submitted to qualitative analysis of sealer and root dentin adaptation and the presence of voids, by means of confocal laser scanning microscopy. In specimens in which Rhodamine B 0.1% was added in the sealer was evaluated the sealer penetration in root dentin by confocal laser scanning fluorescence. The data were analyzed using parametric and nonparametric statistical tests. The AH Plus (2.58 ± 1.21) showed higher bond strength compared to MTA Fillapex (1.45 ± 0.71) (p <0.122). The ultrasonic activation (2.64 ± 1.44) provided higher bond strength compared to no activation groups (1.58 ± 0.73) (p <0.0001) and sonic activation (1.83 ± 0.85) (p <0.0001), with no difference between them (p = 0.3303). The cervical third showed higher bond strength (2.48 ± 1.49) compared to the middle (2.08 ± 0.94) (p = 0.0003) and apical thirds (1.48 ± 0.58) (P < 0.001). For the MTA Fillapex there was a predominance of mixed adhesive failures in all groups, whereas for AH Plus there was a predominance of mixed adhesive failures for groups without activation and ultrasonic activation and mixed adhesive failures and adhesives to the filling material for the sonic activation group. Ultrasonic activation promoted, regardless of the sealer, the greater number, density and length tags with more homogenous and adapted root canal filling to the root canal walls. On the other hand, the sonic activation, presented number, density and length of tags similar to the group without activation, however the root canal filling showed mismatches and blistering. Ultrasonic activation impacted the highest bond strength, penetration, density and length tags and better adaptation of the sealer root dentin, unlike the sonic activation that not increased bond strength and penetration tags and provided greater mismatch and the inclusion of voids in the root canal filling.

O bagaço de cana-de-açúcar é um abundante coproduto obtido a partir da produção convencional de etanol. No entanto, o bagaço vem se mostrando como uma importante fonte para a produção de etanol de segunda geração. No primeiro capítulo da tese é apresentado um estudo referente à distribuição espacial dos compostos na matriz de bagaço de cana-de-açúcar. A investigação foi realizada utilizando microscopia de fluorescência confocal e espectroscopia por excitação com um e dois fótons. Imagens de autofluorescência em combinação com as medidas de fluorescência e tempos de vida forneceram uma gama de informações necessárias para a caracterização de amostras de bagaço. Além disso, a técnica permite o acompanhamento de processos relacionados com a remoção de lignina. A nanocelulose cristalina (NCC) é um material promissor devido as suas propriedades intrínsecas, tais como seu formato alongado, medindo de 1 a 100 nm de diâmetro e seu comprimento variando de algumas dezenas a centenas de nanômetros. No capítulo 2, a nanocelulose cristalina foi obtida através da hidrólise da celulose cristalina (de Avicel&reg;) com ácido sulfúrico. Em seguida, o material foi caracterizado por técnicas de microscopia SEM e TEM, confirmando a morfologia em forma de haste e a estrutura de tamanho nanométrico. A microscopia de campo largo convencional foi utilizada como ferramenta na caracterização da NCC dispersa em soluções poliméricas de PVA e PVP. A última parte do capítulo 2 descreve a caracterização de estruturas de NCC usando a microscopia de super-resolução de fluorescência STED (depleção de emissão estimulada). As imagens mostraram uma resolução de até 50 nm, permitindo a comparação com resultados de TEM e AFM. No capítulo 3, a nanocelulose cristalina foi covalentemente marcada com o corante ATTO-532, através da chamada reação \"click\". As propriedades relacionadas com o coeficiente de difusão da NCC foram determinadas por espectroscopia de correlação de fluorescência (FCS). Em uma etapa posterior, a NCC foi colocada em diferentes soluções do polímero PEG, contendo quantidades diferentes. As propriedades dinâmicas foram analisadas por métodos de FCS e WFM. O uso de técnicas de espectroscopia e microscopia revelou detalhes relacionados à heterogeneidade das dispersões de NCC, as quais estão relacionadas com as propriedades hidrofílicas e hidrofóbicas das soluções poliméricas. / The sugarcane bagasse is an abundant co-product obtained from the conventional production of ethanol. However, sugarcane bagasse has been proving to be an important source to the production of second-generation ethanol. In the first chapter, the spatial distribution of compounds in the sugarcane bagasse matrix was investigated by confocal fluorescence microscopy and spectroscopy with one and two-photon excitation. Autofluorescence images in combination to spectral emission and lifetime measurements provided a tool for the characterization of natural bagasse samples. Moreover, the technique allows the following of processes related to the lignin removal. Nanocrystalline cellulose (NCC) is a promisor material because of its properties, such as rod-shape with 1-100 nm in diameter, and tens to hundreds of nanometres in length. In the Chapter 2, NCC was obtained via sulphuric acid hydrolysis from Avicel&reg;. Afterwards, the material was characterized by classic electronic microscopy SEM and TEM, confirming the rod-shaped morphology and the nano-sized structure. Conventional wide field microscopy was used as fluorescence microscopy tool in the characterization of NCC, when dispersed in polymeric solutions of PVA and PVP. The last part of the chapter 2 describes the characterization of NCC structures by using the super-resolution fluorescence microscopy STED (Stimulated Emission Depletion). The STED images showed a resolution down to 50 nm, allowing the comparison with TEM and AFM microscopy results. In the Chapter 3, the NCC was covalently labelled, by a click-chemistry reaction, with the ATTO-532 dye. Properties related to diffusion coefficient of NCC were determined by Fluorescence Correlation Spectroscopy (FCS) method. Afterwards, NCC was placed into a solution of PEG, containing different amounts polymer. The dynamic properties were evaluated by FCS and WFM methods. The use of spectroscopy and microscopy imaging techniques revealed heterogeneity details of NCC dispersions, which are related to the hydrophilic and hydrophobic properties of the polymer solution. A better understanding of polymer systems is achieved by investigation of diffusion properties, that allows the comprehension of rheological parameters, and, consequently, in polymer processing and assembly of plastics, films, and fibres. In the Chapter 4 is presented a study where fluorescence correlation spectroscopy (FCS) and wide-field fluorescence microscopy (WFM) were used to follow changes in the diffusion coefficients of growing polymer chains, during the controlled radical polymerization process. Linear and star-shaped polystyrene were grown via nitroxide-mediated polymerization (NMP) from alkoxyamine-based initiators containing a highly fluorescent perylene diimide moiety. This study demonstrates that direct investigation of heterogeneity emerging during a controlled radical polymerization process by means of fluorescence of single-molecule chain initiator allows unravelling information related to the diffusion processes of the growing polymer chain.

O objetivo deste estudo foi comparar os ensaios de microtração (MI), ?push-out? (PS), ?push-out? modificado (PSM) e ?pull-out? (PL) na habilidade de mensurar a resistência de união (RU) entre pino de fibra e dentina radicular. Para tanto, além dos resultados de cada ensaio mecânico foram analisados fatores como: sensibilidade quanto às diferentes regiões radiculares, quantidade de falhas prematuras (FP), coeficiente de variação (CV), padrão de fratura (PF) pela microscopia confocal de varredura e distribuição das tensões pela análise de elementos finitos (AEF). Foram utilizados 40 dentes anteriores superiores endodonticamente tratados, divididos em quatro grupos de acordo com o tipo de ensaio realizado. Para os ensaios de PS e MI foi feita a cimentação do pino e posteriormente os cortes para a obtenção dos corpos-de-prova (cps). Para os outros ensaios (PSM e PL), a cimentação antecedeu aos cortes tanto das fatias de dentina como dos pinos. O sistema adesivo Scotbond Multi Uso Plus e o cimento Variolink II foram usados para a cimentação do pino de fibra de vidro FRC Postec Plus em todos os casos. Os cps do ensaio de PS foram obtidos pelos cortes seriados da raiz em fatias de 1±0,1mm, para os cps do ensaio de MI estas fatias sofreram ainda a confecção de entalhes laterais diametralmente opostos, que proporcionando um formato final de ampulheta. Para os testes de PL e PSM foram feitos cortes de fatias de dentina de 1±0,1mm e o pino foi dividido em três partes iguais para, então, cada fatia de uma região ser cimentada com o pino correspondente. Os cps do ensaio de PSM tinham 1mm de pino sobressalente para cima e para baixo da fatia de dentina, enquanto que os cps do PL o pino sobressalente estava apenas para cima da dentina. Todos os cps foram submetidos aos testes de RU, à velocidade de 0,5mm/min em máquina de ensaios universais. O PF foi avaliado em um aumento de cem vezes e foi feita uma distribuição de freqüência. Já a AEF obteve modelos tridimensionais de todos os ensaios, e estes foram analisados quanto à tensão de Von Mises. Para os resultados dos testes mecânicos foram feitas análises de variância de cada ensaio variando o fator região, com ou sem as FP, e análise comparando todos os ensaios. O teste Tukey foi feito quando necessário e as FP foram submetidas ao teste estatístico de Fisher. A inclusão ou não destas falhas mostra alteração dos resultados no ensaio de MI, uma vez que possui um maior número delas neste ensaio. O ensaio de PL mostrou as maiores médias de RU, seguido pelo ensaio de PSM, e ambos também tiveram o PF com maior quantidade de fraturas adesivas. O ensaio de PS conseguiu apontar diferença estatística entre as três regiões, mas mostrou a maior freqüência de falhas coesivas, e maior CV. O menor CV foi encontrado para o PSM que também mostrou uma adequada distribuição das tensões. A MI apresentou concentração de tensões nas bordas dos entalhes. Concluiu-se que o tipo de ensaio influencia diretamente nos resultados obtidos. / The aim of this study was to compare microtensile (MI), \"push-out\" (PS), modified \"push-out\" (PSM) and \"pull-out\" (PL) tests in the ability to accurately measure the bond strength of fiber posts luted inside root canals, at different root dentin levels, cervical, middle and apical. The evaluated parameters were: amount of premature failure (PF), coefficient of variation (CV), failure patterns (FP) and the stress distribution by finite elements analysis (FEA). Forty human intact single-rooted and endodontically treated teeth were divided into four groups according to the type test used. For PS and MI specimens, first the post was luted and then the cuts were done, for PSM and PL, first was done the cuts and then fiber post?s pices were luted. Scotbond Multi Use Plus adhesive system and Variolink II cement had been used for luting FRC Postec Plus glass fiber post, in all cases. PS samples were disc shaped of 1±0,1mm. Bond strength between post and dentin was measured in the root slice and the force applied on the center of the specimen. For the Mi test hourglass-shaped samples were obtained by trimming the proximal surfaces of each slice using a diamond bur until it touched the post. For PL and PSM tests cuts of 1±0,1mm of dentine slices were made and the fiber was divided into three parts, and then each slice were luted with a fiber post?s piece. Specimens for PSM had 1mm of post up and down the dentin slice and for PL all of the post was up the dentine slice. All specimens were submitted to a bond strength test at 0.5 mm/minute crosshead speed in a universal testing machine. For FP, each sample was examined under confocal microscopy at 100 times and a frequency distribution was made. For the AEF, three-dimensional models of each tests had been made and analyzed by Von Mises stress. Bond strength data were analyzed by ANOVA and when necessary was used Tukey?s test. For PF data the Fisher test was made. The inclusion of PF, only shows variation on MI, since these test showed the greater number of them. PL provided higher values of bond strength for root dentin and fiber post, followed by PSM. Both of them had also FP with greater amount of adhesive failures. The PS test was able to point statistically differences between root regions, but it showed the highest frequency of cohesive failure, and greater CV. Minor CV was found for the PSM that also showed adequate stress distribution. The MI showed concentration of tensions in the edges of the trimming area. One concluded that the type of test influences directly in the results obtained.

Este trabalho teve o objetivo de avaliar a capacidade da bactéria Enterococcus faecalis em induzir periodontite apical no modelo experimental canino; verificar se o preparo químico-mecânico afeta a sobrevivência dessa bactéria no sistema de canais radiculares e avaliar o reparo desses dentes por meio de radiografia periapical e tomografia computadorizada cone beam. Dentes com e sem tratamento endodôntico foram avaliados por meio de microscopia confocal de varredura a laser. Para tal, foram utilizados 2 cães. A bactéria Enterococcus faecalis foi inoculada nos canais de 4 pré-molares superiores, 11 prémolares inferiores e 9 incisivos superiores. As câmaras pulpares foram seladas e, após 60 dias, os canais das raízes distais dos pré-molares e de 7 incisivos superiores foram submetidos a tratamento endodôntico em sessão única e os canais das raízes mesiais dos pré-molares e 2 incisivos superiores foram deixados sem tratamento (controle). Amostras microbiológicas foram feitas antes e após o preparo químico-mecânico. O reparo foi avaliado após 6 meses do tratamento mediante radiografias periapicais e por tomografia computadorizada cone beam. A comparação entre as imagens obtidas após o período experimental pelos 2 métodos foi feita por medições da área em mm2 de cada lesão encontrada, utilizando o software ImageTool Os resultados mostraram que a presença de periodontite apical crônica foi verificada em todos os dentes inoculados, independentemente da colonização pela bactéria Enterococcus faecalis ou pela flora mista. O preparo químico-mecânico reduziu significativamente o número de bactérias no interior dos canais radiculares (p<0.05). Os resultados radiográficos e tomográficos demonstraram lesões de menor diâmetro nos dentes tratados endodonticamente em comparação ao grupo controle (p<0.05). A comparação entre os métodos demonstrou diferença estatística entre eles sendo evidenciada áreas radiolúcidas maiores utilizando a tomografia cone beam em comparação com a radiografia periapical (p<0.05). Concluiu-se que a bactéria Enterococcus faecalis induziu periodontite apical crônica similarmente à flora mista; o tratamento endodôntico reduziu o número de bactérias cultiváveis significativamente embora sem relação com o reparo radiográfico. Os dentes tratados em sessão única apresentavam lesões menores em comparação aos não-tratados e as áreas das lesões observadas na TC cone beam foram maiores do que as áreas encontradas nas radiografias periapicais. A microscopia confocal e o método proposto neste estudo se mostraram eficazes para determinar, qualitativamente, a viabilidade bacteriana e a distribuição de ácidos nucléicos bacterianos dentro dos túbulos dentinários. Houve diferença entre a dentina infectada in vitro com o padrão de infecção in vivo, caracterizada pela presença de biofilmes aderidos à parede do canal radicular. / The aims of this study were to evaluate the Enterococcus faecalis ability to induce apical periodontitis in dogs root canals, to verify the bacterias ability to survive to the cleaning and shaping procedures and to assess the healing of the induced apical periodontitis by periapical radiograph and cone beam computed tomography. Also, endodontically treated and non-treated teeth were evaluated by confocal laser scanning microscope. Two mongrel dogs were used in the experiment. Enterococcus faecalis strain was inoculated into the root canals of 4 maxillary premolars, 11 mandibular premolars and 9 maxillary incisors. After 60 days the root canals of the distal roots of the mandibular and maxillary pre-molars and 7 maxillary incisors were endodontically treated. The premolars mesial root canal and 2 maxillary incisors were used as control (no treatment). Microbiologic samples were done after and before the cleaning and shaping procedure. The healing was evaluated after 6 months by periapical radiographs and cone beam computed tomography. The comparison between obtained images after the experimental period by the two methods was done using measures of the lesion area in mm2 with ImageTool software. The results showed the presence of chronic apical periodontitis in every inoculated teeth, with Enterococcus faecalis or mixed infection. The cleaning and shaping procedures reduced the number of bacteria of the root canals (p<0.05). The radiographic and tomographic results showed the lower diameter lesion in endodontically treated teeth than in the control group (p<0.05). The comparison between the methods showed statistical difference and greater radiolucent areas were evident in cone beam computed tomography images (p<0.05). It is possible to conclude that the bacteria Enterococcus faecalis induced chronic apical periodontitis as well as the mixed microflora; the endodontic treatment reduced the number of cultivable bacteria in a significant way, but with no relation to the radiographic healing. The treated teeth in only one session presented smaller lesions in comparison to the non-treated teeth and the lesions area in cone beam computed tomography were bigger than the areas found in periapical radiograph. The confocal microscopy and the proposed method of this study showed to be efficient to determine the bacterial ability and the distribution of bacterial nucleic acids inside the dentin tubules. There was difference between in vitro infected dentin with the in vivo infection pattern, which presents biofilm attached to the root canal walls.

Quantentechnologien sind im Begriff sich von Laborversuchen zu effizienten Anwendungen zu entwickeln. Die Quantenzustände einzelner Photonen spielen dabei die Rolle als Bindeglied zwischen stationären Quantensystemen. Ein hybrider Ansatz wird verfolgt, um die Wechselwirkung gezielt zwischen im Wellenleiter geleiteten Photonen und gekoppelten Quantenemittern zu erreichen. Die Dissertation untersucht zwei zentrale Aspekte solcher hybriden photonischen Quantentechnologien: die effiziente Erzeugung von Photonen und die Optimierung von photonischen Strukturen. Der erste Teil dieser Arbeit behandelt die Entwicklung einer optischen Mikrotechnology. Integrierte nano-photonische Wellenleiter aus Siliziumnitrid wurden für die Kopplung zu Quantenemittern entworfen, optimiert und optisch untersucht. Das finale Design wurde erfolgreich in Kopplungsexperimenten verwendet, bei denen 42 % der Fluoreszenz eines einzelnen Moleküls an einen Wellenleiter gekoppelt wurde. Der zweite Teil der Arbeit untersucht zwei Einzelphotonenquellen. Zunächst wurde ein neuartiger Einzelphotonenemitter basierend auf Defektzentren in Zinkoxid optisch bei tiefen Temperaturen untersucht. Es konnte im Zuge dieser Arbeit erstmals gezeigt werden, dass die Photonen von nano-strukturiertem Zinkoxid sehr schmalbandige Emission aufweisen. Im letzten Teil, wird eine Einzelphotonenquelle bestehend aus einem organischen Molekül untersucht. Bei kryogenen Temperaturen wurden Lebenszeit-limitierte Linienbreiten auf den Molekülproben detektiert. Die Rabi-Oszillationen zwischen den Molekülzuständen konnten akkurat durch eine quantenmechanische Theorie beschrieben werden, wodurch die Vermessung der Dephasierung des Quantensystems durch die nanoskopische Umgebung präzise studiert werden konnte. Die Ergebnisse dieser Arbeit zur Kopplung von Einzelphotonenquellen stellen die Grundlage für weitere Anwendungen durch eine photonische Quantentechnologie dar. / Quantum technologies are on the verge to transition from laboratory experiments to efficient integrated applications. The quantum states of photons are the connecting link between individual stationary quantum systems. A hybrid approach is employed to tailor the interaction of routed photons with optically coupled quantum systems. The thesis investigates two core aspects of a hybrid photonic quantum technology: efficient single photon generation and optimized photonic micro-structures, suitable to form a hybrid system. In the first part of this work, nano-photonic integrated structures were optimized for efficient coupling to quantum emitters. Optical waveguides based on silicon nitride (SiN) were designed, fabricated, and optically characterized. The final design was successfully employed in coupling experiments, where up to 42% of the fluorescence from a single molecule was coupled to a waveguide. In the second part of this thesis two single photon sources are investigated towards their implementation into a hybrid photonic system. First, a novel single photon source based on a defect center in zinc oxide was optically investigated at room-temperature and cryogenic temperature. Spectrally narrow zero-phonon lines of the fluorescence from nano-structured zinc oxide were measured for the first time during this work. A second emitter system, based on an organic dye molecule was investigated in the final part of this research. At cryogenic temperatures, single molecules showed lifetime-limited linewidths of <50MHz. A resonant laser source drives Rabi oscillations, which are accurately described by the quantum mechanical theory of a two-level system. The system's decoherence was mapped, illustrating the quantum sensing capabilities of the system. The results presented in this thesis on coupling efficiencies and single emitter performance provide the necessary background of the elements composing a future hybrid technology.

La gestion et transmission d’information au sein d’organismes vivants implique la production et le trafic de molécules via des voies de signalisation sutructurées en réseaux de réactions chimiques. Ces derniers varient selon leur forme, taille ainsi que la nature des molécules mises en jeu. Parmi eux, les réseaux de régulation génétiques nous ont servi de modèle pour le développement et la mise en place d’un système de programmation moléculaire in vitro. En effet, l’expression d’un gène est majoritairement dominé par des facteurs de transcription, autres protéines ou acides nucléiques, eux-mêmes exprimés par d’autres gènes. L’ensemble forme l’interactome de la cellule, carte globale des interactions entre gènes et sous-produits, où la fonction du réseau est relié à sa topologie. L’observation des noeuds et sous-architectures dénote trois mécanismes récurrents : premièrement, la nature des interactions est de type activation ou inhibition, ce qui implique que tout comportement non trivial est obtenu par une combinaison de noeuds plutôt que le développement de nouvelles interactions. Ensuite, la longévité du réseau est assurée par la stabilité chimique de l’ADN couplée à la chimiosélectivié des réactions enzymatiques. Enfin, l’aspect dynamique est maintenu par le constant anabolisme/catabolisme des intermédiaires et donc l’utilisation de combustible/énergie. C’est suivant ces observations que nous avons développé un ensemble de trois réactions enzymatiques élémentaires : la «PEN-DNA toolbox». L’architecture du réseau, à savoir les connections entre les noeuds est médiée par la séquence de brins d’ADN synthétiques (appelés matrice), et trois enzymes (polymérase, nickase, et exonucléase) assurent la catalyse des réactions chimiques. La production et dégradation des intermédiaires consomme des désoxyribonucléotides triphosphates et rejette des désoxyribonucléotides monophosphate, dissipant ainsi le potentiel chimique. Les réactions sont suivies grâce au greffage d’un fluorophore sur le brin matriciel et au «nucleobase quenching» qui intervient lorsqu’une base d’un intermédiaire se rapproche du fluorophore après hybdridation sur le brin matriciel. L’activation correspond alors à la synthèse d’un brin output en réponse à un brin input, alors que l’inhibition survient lorsqu’un brin output s’hybride sur un brin matriciel, empêchant ainsi à l’input correspondant de s’y fixer. Oscillations, bistabilité et mémoire sont des exemples de comportements implémentés en PEN-DNA toolbox, faisant appel à des architectures de plus en plus complexes. Pour cela, un réglage fin des concentrations en effecteurs (ADN et enzymes) est nécessaire, ce qui sous-tend l’existence de plusieurs comportements pour un même circuit, dépendant des conditions paramétriques. L’établissement d’une carte de chaque combinaison de paramètres avec le comportement global associé permettrait de comprendre le fonctionnement du réseau dans son ensemble, et donnerait accès à tous les comportements disponibles. Dans le cas d’un système dynamique non linéaire, une telle carte est un diagramme de bifurcation du système. Pour explorer de manière exhaustive les possibilités d’un réseau dans un cadre expérimental raisonable, nous avons développé une plateforme microfluidique capable de générer des goutelettes d’eau dans l’huile à partir de quatres canaux aqueux différents. Ce dispositif nous donne accès, grâce à un contrôle fin des contributions de chaque canal aqueux, à des goutelettes monodisperses (volume de l’ordre du picolitre) dont le contenu est différent pour chaque goutelette. Nous avons adapté notre dispositif aux contraintes matérielles (design microfluidique, génération de goutelettes à contenu différents et controllés, observation et stabilité à long terme) et techniques (tracabilité des goutelettes et stabilité/compatibilité chimique). (...) / Information processing within and in between living organisms involves the production and exchange of molecules through signaling pathways organized in chemical reactions networks. They are various by their shape, size, and by the nature of the molecules embroiled. Among them, gene regulatory networks were our inspiration to develop and implement a new framework for in-vitro molecular programming. Indeed, the expression of a gene is mostly controlled by transcription factors or regulatory proteins and/or nucleic acids that are themselves triggered by other genes. The whole assembly draws a web of cross-interacting genes and their subproducts, in which the well controlled topology relates to a precise function. With a closer look at the links between nodes in such architectures, we identify three key points in the inner operating system. First, the interactions either activate or inhibit the production of the later node, meaning that non trivial behaviors are obtained by a combination of nodes rather than a specific new interaction. Second, the chemical stability of DNA, together with the precise reactivity of enzymes ensures the longevity of the network. Finally, the dynamics are sustained by the constant anabolism/catabolism of the effectors, and the subsequent use of fuel/energy. All together, these observations led us to develop an original set of 3 elementary enzymatic reactions: the PEN-DNA toolbox. The architecture of the assembly, i.e. the connectivity between nodes relies on the sequence of synthetic DNA strands (called DNA templates), and 3 enzymes (a polymerase, a nickase and an exonuclease) are taking care of catalysis. The production and degradation of intermediates consume deoxyribonucleoside triphosphates (dNTP) and produce deoxynucleotide monophosphates leading to the dissipation of chemical potential. Reactions are monitored thanks to a backbone modification of a template with a fluorophore and the nucleobase quenching effect consecutive to an input strand binding the template. The activation mechanism is then the production of an output following the triggering of an input strand, and the inhibition comes from the production of an output strand that binds the activator-producing sequence. Various behaviors such as oscillation, bistability, or switchable memory have been implemented, requiring more and more complex topologies. For that, each circuit requires a fine tuning in the amount of chemical parameters, such as templates and enzymes. This underlies the fact that a given network may lead to different demeanors depending on the set of parameters. Mapping the output of each combination in the parameter space to find out the panel of behaviors leads to the bifurcation diagram of the system. In order to explore exhaustively the possibilities of one circuit with a reasonable experimental cost, we developped a microfluidic tool generating picoliter-sized water-in-oil droplets with different contents. We overcame the technical challenges in hardware (microfluidic design, droplet generation and long-term observation) and wetware (tracability of the droplet and emulsion compatibility/stability). So far, bifurcation diagrams were calculated from mathematical models based on the enzymes kinetics and the thermodynamic properties of each reaction. The model was then fitted with experimental data taken in distant points in the parameter space. Here, millions of droplets are created, and each one encloses a given amount of parameters, becoming one point in the diagram. The parameter coordinates are barcoded in the droplet, and the output fluorescence signal is recorded by time lapse microscopy. We first applied this technique to a well-known network, and obtained the first experimental two-dimensional bifurcation diagram of the bistable system. The diagram enlightens features that were not described by the previous mathematical model. (...)

Os materiais restauradores utilizados na região cervical dos dentes podem ser afetados por resultantes de forças oclusais excêntricas, que já podem ter contribuído à formação da lesão, e deveriam ser capazes de resistir às tensões. Não há consenso, porém, quanto ao papel de diferentes materiais, quiçá especialmente no tocante a seus módulos de elasticidade, no sucesso das respectivas restaurações. Avaliou-se, pois, a influência do uso desses materiais, com módulos de elasticidade (E) distintos em essência, e da associação entre eles, na qualidade marginal de restaurações cervicais em pré-molares submetidos a carregamento oclusal excêntrico. Cavidades cervicais em forma de cunha, preparadas em pré-molares unirradiculares superiores humanos, hígidos e extraídos, foram restauradas com uma resina composta (RC: Z250 XT, n=20); com a resina em associação a um ionômero modificado por resina para forramento (Mista/M: Vitrebond, n=20); ou com um ionômero modificado por resina para restauração (CIV: Vitremer, n=20). Metade dos dentes de cada grupo foi submetida a carregamento oclusal excêntrico (coe: inclusão em resina acrílica e ligamento periodontal artificial, 150 N, 106 ciclos, 2,5 Hz), e a outra metade, apenas armazenada por período de tempo equivalente (Controle/c: água destilada, 37º C). Um sistema adesivo marcado por fluoresceína foi utilizado para delimitação dos defeitos formados, que foram avaliados por meio de Microscopia Confocal de Varredura a Laser. Para a presença ou ausência de fendas marginais aplicou-se, num contexto geral, para cada fator em estudo (carregamento oclusal excêntrico - coe / c; material restaurador - RC / M / CIV), o teste do qui-quadrado (?=0,05). Em específico, aplicaram-se, individualmente, em cada nível do fator carregamento oclusal excêntrico, e em cada nível do fator material restaurador, respectivamente, o teste do qui-quadrado e o teste exato de Fisher (?=0,05). Para a localização das fendas em relação às paredes cavitárias (O: oclusal / C: cervical / OC: ambas) aplicou-se, para cada fator em estudo, o teste do qui-quadrado (?=0,05). O teste de Mann-Whitney foi aplicado para a largura e a profundidade das fendas marginais, para comparar os diferentes níveis do fator carregamento oclusal excêntrico, e o teste de Kruskal-Wallis, para comparar os diferentes níveis do fator material restaurador (?=0,05). Numa conjuntura abrangente, nenhum dos fatores associou-se significativamente com as variáveis de resposta qualitativas nominais ou exerceu influência sobre as quantitativas. Mais especificamente, por exclusivo quando da aplicação do carregamento oclusal excêntrico, o uso dos diferentes materiais restauradores associou-se significativamente com a frequência de fendas marginais (RC>M>CIV). Ainda que a qualidade marginal de restaurações cervicais possa não ser influenciada, de maneira geral, pelo carregamento oclusal ou pelo uso de diferentes materiais, inclusive em associação, o aspecto desfavorável do emprego da resina composta pode tornar-se relevante, em particular, quando da existência de carga oclusal excêntrica. / Restorative materials used in the cervical region of teeth may be impacted by resultants of eccentric occlusal loads that have already contributed to the formation of the lesion, and should be capable of withstanding the stresses. There is no consensus, however, about the role of different materials, maybe especially regarding their moduli of elasticity, in the success of respective restorations. The influence of the use of these materials, with different moduli of elasticity (E) in essence, and of their association, on marginal quality of cervical restorations in premolars subjected to eccentric occlusal loading was evaluated thus. Cervical wedge-shaped cavities, prepared in human upper single-rooted premolars, healthy and extracted, were restored with a resin composite (RC: Z250 XT, n=20); with the composite in combination with a resin-modified lining ionomer (Mixed/M: Vitrebond, n=20), or with a resin-modified restorative ionomer (GIC: Vitremer, n=20). Half of the teeth of each group was subjected to eccentric occlusal loading (eol: embedment in acrylic resin, artificial periodontal ligament, 150 N, 106 cycles, 2.5 Hz), and the other, only stored for an equivalent period of time (Control/c: distilled water, 37°C). A fluorescein-marked adhesive system was used for delimitation of formed defects, which were evaluated by means of Laser Scanning Confocal Microscopy. For the presence or absence of marginal gaps, in a general context and for each factor under study (eccentric occlusal loading - eol / c; restorative material - RC / M / GIC), chi-square test was applied (?=0.05). In specific, individually, at each level of the factor eccentric occlusal loading, and at each level of the factor restorative material, chi-square test and Fisher\'s exact test were respectively applied (?=0.05). For the location of the gaps in relation to the cavity walls (O: occlusal / C: cervical / OC: both), for each factor under study, chi-square test was applied (?=0.05). The Mann-Whitney test was applied to the width and depth of marginal gaps, to compare the different levels of the factor eccentric occlusal loading, and the Kruskal-Wallis test, to compare the different levels of the factor restorative material (?=0.05). In a comprehensive view, none of the factors was significantly associated with qualitative response variables or wielded any influence on the quantitative ones. More specifically, for exclusive when eccentric occlusal loading was applied, the use of the different restorative materials was significantly associated with the frequency of marginal gaps (RC>M>GIC). Though marginal quality of cervical restorations may not be in general influenced by the occlusal loading or the use of different materials, even in association, the unfavorable aspect of the use of the resin composite may become relevant particularly in face of the existence of eccentric occlusal load.

Das menschliche Skelett besteht aus einem dynamischen Material welches in der Lage ist zu heilen, sowie sich durch strukturellen Umbau an mechanische Beanspruchung anzupassen. In dieser Arbeit ist die mechanische Regulierung dieser Prozesse untersucht worden. Hierfür ist ein Computermodell, sowie die dreidimensionale Abbildung des Knochens und die Auswertung dieser Bilder benutzt worden. An dem Heilungsprozesses von Knochen sind verschiedene Gewebetypen beteiligt. Dabei hängt die räumliche und zeitliche Anordnung dieser Gewebe von der mechanischen Belastung ab. Ein Computermodell, welches den vollständigen Verlauf der Heilung beschreibt, wurde mit der dokumentierten Gewebeentwicklung eines Tierexperimentes verglichen. Verschiedene Hypothesen, wie die mechanische Stimulation die Bildung verschiedene Gewebe beeinflusst, wurden getestet. Zwar ließen sich durch den Vergleich mit dem Experiment keine der Hypothesen verwerfen, jedoch konnten wir Vorschläge machen, worauf bei zukünftigen Experimenten verstärkt geachtet werden soll. Es wird angenommen dass der Umbauprozesses des Knochens vom dichten Netzwerk der Osteozyten mechanisch reguliert wird. Diese Zellen sind in den Knochen eingebettet und über ein dichtes Netzwerk aus engen Kanälen, den sogenannten Canaliculi, miteinander verbunden. Dieses Netzwerk mittels konfokaler Mikrokopie dreidimensional abgebildet. Spezielle Routinen zur Auswertung der Netzwerkorientierung sowie dessen Dichte wurden entwickelt. Die Hauptorientierung des Netzwerkes entspricht der Richtung in der Knochengewebe aufgebaut wird. Die Orientierung des zu dieser Richtung senkrechten Anteils des Netzwerkes rotiert abhängig von der Position entlang der Aufbaurichtung. Dies verdeutlicht den Zusammenhang zwischen der Netzwerkorientierung und der Vorzugsrichtung des Kollagens, dem faserigen Bestandteils des Knochens. Darüber hinaus zeigt die Auswertung der Daten weitere strukturelle Unterschiede im Netzwerk. / Our skeleton is composed of a dynamic material that is capable of healing and of adapting to changing mechanical loads through structural remodeling. In this thesis the mechano-regulation of these dynamic processes are addressed using computer modeling and 3-dimensional imaging and image analysis. During bone healing an intricate pattern of different newly formed tissues around the fracture site evolves in time and is influenced by the mechanical loading. Using a computer model which is describing this temporal-spatial evolution of tissue types for the full time-course of healing, this evolution is compared to the documented evolution of an animal experiment. Different hypotheses were tested how the mechanical stimulation results in the formation of different tissues. While the comparison with the outcome of the animal experiments does not allow to falsify any of the hypotheses, it suggests a different design of future animal experiments. Bone remodeling is thought to be mechano-regulated by the dense network of osteocytes. These osteocytes are embedded in bone and are connected to each other via a network of narrow canaliculi. The 3-dimensional structure of the network was imaged using rhodamine staining and laser scanning confocal microscopy. Image analysis tools were developed to determine the network topology and to analyze its density and orientation. The analysis focused on osteons, the building blocks of cortical bone. Within an osteon we found a large variability of the network density with extensive regions without network. Most of the network is oriented radially towards the center of the osteon, i.e.\ parallel to the direction in which the bone material is deposited. The network perpendicular to this direction twists when moving along the direction of bone deposition. A correlation with the main orientation the fibrous constituent of bone, collagen, was detected. Furthermore indicates our data additional structural changes in the network alignment.

Les peptides pénétrateurs de cellule (CPP) sont des oligopeptides cationiques faisant parti des vecteurs les plus étudiés dans le cadre du développement du transport ciblé de médicament à l’intérieur de l’organisme. Les applications principales sont par exemple le traitement des cancers ou la thérapie génique. Néanmoins, certaines caractéristiques des CPPs rendent leur utilisation médicale compliquée, tels que leur manque de spécificité à l’égard des cellules cibles ou la perte de leurs propriétés pénétrantes lorsqu’un cargo moléculaire leur est greffé. L’une des solutions envisagées pour résoudre ces problèmes est le greffage sur des polypeptides di-blocs auto-assemblés basés sur de l’élastine (ELPBC), des systèmes développés par l’équipe d’Ashutosh Chilkoti à l’Université de Duke (USA). Des travaux précédents ont montré que ces macromolécules, que l’on appelle CPP-ELPBC, retrouvaient les propriétés pénétrantes du CPP même en présence d’un cargo et permettaient également d’induire une spécificité à l’encontre des cellules cancéreuses. En revanche, le mécanisme de pénétration de ces systèmes restait inconnu.Dans cette thèse, je me suis concentré sur l’étude du mécanisme de pénétration des CPP et des CPP-ELPBC au travers de membranes lipidiques modèles, et en particulier sur l’adsorption de ces molécules à la surface de vésicules unilamellaires géantes (GUV). Le développement d’une nouvelle méthode de quantification de la fluorescence en microscopie confocale m’a permis de réaliser des mesures simples de comptage de peptides à la surface des vésicules, ce qui m’a permis par la suite de procéder à des mesures thermodynamiques de l’adsorption des peptides. / Cell-penetrating peptides (CPP) are cationic oligopeptides currently investigated as potential vectors for targeted drug delivery design, for applications in cancer treatment and/or gene therapy. Nevertheless, some drawbacks make the CPP complex for medical applications, such as their lack of specificity toward target cells or the loss of their penetrating properties once they have been grafted with a molecular cargo. One of the solutions studied to overcome these issues is the binding of the CPP unit on a self-assembling elastin-like diblock polypeptide (ELPBC), a macromolecular system designed by the team of Ashutosh Chilkoti from Duke University (USA). While it has already been proven that these molecules, named CPP-ELPBC, recover the penetrating properties of the CPP despite the presence of a cargo and also induce a selectivity toward tumorous cells, the exact mechanism of translocation is still under debate.In this PhD thesis, I focused on the investigation of the translocation mechanism of the CPP and CPP-ELPBC using model lipid membranes, and specifically the adsorption of these molecules at the surface of giant unilamellar vesicles (GUV). The development of a new quantification method of fluorescence in confocal microscopy allowed me to directly count the peptides adsorbed on the surface of the GUVs, which I used to perform thermodynamic measurements on the peptide adsorption.

Ce travail concerne l’altération du comportement de phase de membranes lipidiques lors de leur interaction avec des molécules hydrophiles ou hydrophobes. L’utilisation de sondes moléculaires de fluorescence sensibles à leur micro-environnement constitue un aspect majeur de ce travail. Les techniques de spectroscopie de fluorescence et de microscopie confocale ont été mises à profit pour l’étude du comportement de ces sondes, donnant accès au degré de compacité et d’ordre dans les membranes.Nos résultats montrent que le polystyrène, un plastique rencontré de façon commune dans les régions polluées des océans, présente la capacité de modifier le comportement de phase des membranes lipidiques, entrant notamment en compétition avec le cholestérol.Nous avons montré que la présence élevée de sucres, tel que l’on peut le rencontrer dans certaines situations relevant de la bio-préservation, a pour effet de rompre la qualité de compaction des lipides, et nous avons proposé un nouveau modèle thermodynamique pour interpréter nos résultats.Enfin, les effets sur la membrane de l’incorporation d’un polymère amphiphile comportant un cholestérol greffé ont été étudiés, dans le cadre de l’élaboration de nouvelles stratégies thérapeutiques à base de lipides. / This work focuses on the alterations of lipid membrane phase behavior upon interaction with hydrophobic and hydophilic molecules. One major aspect of this thesis is the employement of environment sensitive probes to obtain information on the lipid bilayer packing by means of confocal spectral imaging and fluorescence spectroscopy. Our results show that polystyrene, a commonly found plastic in ocean wastes, has the ability to disrupt the lipid bilayer phase behavior and has a competitive interaction with cholesterol. The presence of high concentration of sugars, relevant in the field of biopreservation, has been found to alter the lipid bilayer packing and a new thermodynamics model has been proposed to complement the experimental results. Finally, the effects of an amphiphilic cholesterol-grafted polymer on model membrane was investigated, providing insight into potential new lipid therapeutic strategies.