Connaître en temps réel la réponse et le comportement des cellules et organismes modèles suite à des changements de leur environnement, ou à des modulations de leurs fonctions biologiques est devenu essentiel dans les sciences du vivant. Ces réponses nous permettent ensuite de comprendre les mécanismes qui régissent le fonctionnement des cellules vivantes, avec des implications en recherche fondamentale, appliquée et biomédicale. Un des plus gros défis technologiques reste le contrôle des paramètres environnementaux en microscopie haute résolution. De nos jours, aucun système ne permet de réguler un ensemble complexe de paramètres de manière précise, dynamique et simultanée tout en observant les cellules dans leur environnement. L’objectif de ma thèse est de mettre au point un tel dispositif permettant a minima une régulation fine de la température, de la composition du milieu, et notamment de la concentration de divers drogues. Ce système doit être compatible avec les applications les plus poussées en microscopie photonique. Mon approche au cours de ma thèse pour élaborer un tel système est l’utilisation de la microfluidique. En effet, c’est la seule technologie qui puisse de réaliser un tel multiplexage. Elle permet de manipuler des petites quantités de fluide à travers un système contenant des canaux de dimensions allant du micromètre au centimètre. Cet ordre de grandeur des canaux constitue un atout majeur (réduction de la consommation des réactifs, réduction des couts, cinétiques des réactions chimiques et biologiques élevées, temps de diffusion court, etc.) et permet d’allier les expériences biologiques à la microscopie. Mon objectif est de concevoir une puce microfluidique qui représentera un pas technologique majeur et ouvrira de nouvelles possibilités de recherche. / Monitoring in real-time the response of cells and model organisms to the changes in their environment or to modulations of their biological functions has become essential in life sciences. One of the main technical challenges for biologists is the precise and dynamic control of various environmental parameters while doing high-resolution microscopy. My thesis consists of building a robust and versatile system, dedicated to live-cell imaging that will be compatible with adherent and non adherent models, that could provide a precise and simultaneous control of 1) the temperature, 2) the media exchanges and 3) the drug concentration while doing photonic microscopy. My approach is to use microfluidics, which is the best candidate in order to achieve this system and provides all the necessary controls of micro-scaled volumes for culturing, maintaining or analyzing cells. It produces miniaturized systems used as tools for biological experiments, in which channels of a micro-scaled dimension are used for the fluid circulation. The laminar flow in these chips allows fast molecule diffusion as well as fast temperature diffusion. Because of the high surface to volume ratio, the consumption of reagents is reduced, and media switches can be fast. This system will represent a major technical and beneficial step and will open new possibilities of research in biology.

A funcionalização da matriz celulósica é um ponto essencial para o aprimoramento dos dispositivos microfluídicos baseados em papel (µPADs). Ela permite minimizar o preparo de amostras e a interferência do usuário, principais fontes de erro no processo analítico. A oxidação da celulose durante uma hora com m-periodato de sódio e a imobilização química de enzimas a partir da formação de bases de Schiff (iminas), via a adição direta da enzima ao substrato oxidado sem a necessidade de outras etapas, é um processo rápido e de baixo custo, apresentando grande potencialidade de aplicação nos dispositivos microfluídicos em papel. A enzima glicose oxidase imobilizada na celulose, com a adição do estabilizante trealose, apresentou elevada atividade catalítica - de 31,9 ± 5,5 mmol L-1 para a enzima não imobilizada a 14,8 ± 2,0 mmol L-1 para a enzima imobilizada e com o estabilizante - além de apresentar maior homogeneidade de sinal, condições desejáveis em testes rápidos em papel. A confecção de dispositivos em papel via impressão em cera alia rapidez e baixo custo de produção, e o arranjo em camadas para originar dispositivos tridimensionais (3D) permite ampliar as funcionalidades dos dispositivos em duas dimensões, tal como o tratamento individualizado de camadas e o armazenamento de reagentes no próprio dispositivo. O método da adição de padrão para obtenção de curvas analíticas no próprio microchip em papel surge como alternativa às curvas analíticas externas, minimizando a manipulação e o preparo de amostras. O uso do ácido 2,2’-azino-bis (3-etilbenzotiazolina-6-sulfônico) - ABTS como indicador redox para as reações enzimáticas e o método de adição de padrão nos µPADs apresentou boa correlação com um modelo de crescimento e saturação de Michaelis-Menten (r2 = 0,8723) na faixa de 0 a 10 mmol L-1, e a utilização da faixa linear para quantificação de glicose (0 a 3 mmol L-1) apresentou grande correlação linear com a concentração estimada pelas curvas de adição de padrão (r2 = 0,959), demonstrando a potencialidade do método. A união da tecnologia desses dispositivos em papel com a de um software automatizado de reconhecimento de imagens (PAlizer) torna instantânea a obtenção de resultados, eliminando-se a necessidade de intervenção humana no processo, tornando os testes em papel mais robustos, reprodutíveis e rápidos. Com o contínuo aperfeiçoamento das funcionalidades e potencialidades dos dispositivos microfluídicos em papel espera-se que os testes diagnósticos de baixo custo atinjam àqueles que deles necessitam, contribuindo para a saúde da população. / Functionalization of a cellulosic matrix is essential for the success of the paper-based microfluidic analytical devices (µPADs). It allows minimization of sample preparation and user interference, both being major sources of errors in the analytical process. Cellulose oxidation with sodium m-periodate during one hour and the direct chemical immobilization of enzymes on it by Schiff-base (imines) formation, which is made by direct insertion of the enzyme on the oxidized substrate without subsequent steps, is a fast and low cost process of immobilization, presenting great potential of application in paper-based microfluidic analytical devices. The glucose oxidase enzyme immobilized on cellulose, with the addition of trehalose stabilizer presented enhanced catalytic activity - from 31.9 ± 5.5 mmol L-1 for the non-immobilized enzyme to 14.8 ± 2.0 mmol L-1 for the immobilized enzyme with the stabilizing agent - also presenting greater signal homogeneity, which are ideal characteristics in a paper-based rapid test. Wax printing is a simple, inexpensive and fast method by which micro-devices can be fabricated. Additionally, the stacking of layers originating tridimensional devices (3D) allow for the improvement of functionalities of 2-dimensional ones, such as individualized layer treatment and reagent storage at different layers in the same device. Standard addition to analytical curves in paper-based microchips is an alternative to external analytical curves, minimizing handling/sample preparation. The use of 2,2\'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid - ABTS redox indicator with the enzymatic reactions and the standard addition method in µPADs presented a good correlation in a growth and saturation Michaelis-Menten model (r2 = 0.8723), in the range of 0 to 10 mmol L-1, and the usage of the linear range to the glucose quantification (0 to 3 mmol L-1) presented a high linear correlation with the estimated concentration from the standard addition curves (r2 = 0.959), showing the potentiality of the method. The coupling of such paper-based devices to automated image analysis software, such as \'PAlizer\', turns the data acquisition process instantaneous, eliminating the need of human intervention during the process, making it more robust, reproducible and rapid. Expectations lie in improving the devices functions and potential so that these low-cost diagnostic devices can one day reach those who need them, contributing significantly to public health.

Neste trabalho são apresentados processos de microfabricação de estruturas contendo microcanais e sistemas de manipulação hidrodinâmica e eletroosmótica de fluídos. Foram desenvolvidos processos de microfabricação utilizando toner sobre poliéster, toner sobre vidro, toner como resiste, além de métodos alternativos de perfuração de lâminas e selagem de microestruturas em vidro, desenvolvimento de microestruturas para eletroforese capilar e espectrometria de massas com ionização por eletronebulização. A caracterização dos materiais e processos permitiu uma ampla visão das potencialidades e alternativas dos processos de microfabricação, tendo sido demonstrado que os dispositivos produzidos em toner-poliéster são quimicamente resistentes às substâncias tipicamente utilizadas em eletroforese capilar. Neste trabalho, um detector condutométrico sem contato foi implementado em microestruturas de toner-poliéster e a separação eletroforética de alguns metais alcalinos é demonstrada. A microestrutura foi projetada no formato padrão em cruz, tendo o canal de separação 22 mm de comprimento, 12 µm de profundidade e largura típica. A cela condutométrica foi construída sobre o canal de separação utilizando-se fita adesiva de cobre (1 mm de largura) como eletrodos. O sinal aplicado na cela foi de 530 kHz e 10 Vpp . A separação de K+, Na+ e Li+ na concentração de 100 µmol L-1 foi efetuada em torno de 0,8 min, utilizando-se 1 kV como potencial de separação. Foram desenvolvidos microchips para análise por espectrometria de massas com introdução de amostra por eletronebulização, sendo determinado cluster do íon cloreto em concentração de 1 mmol L+. Também solução com 1 mmol/L de glucosamina em água/metanol 1: 1 (v/v), sob corrente de 100 nA gerou sinal estável e livre de descarga corona. Utilizando detecção amperométrica, obteve-se eletroferogramas mostrando a separação de iodeto (10 mmol L-1) e ascorbato (40 mmol L-1) em potencial de separação de 4,0 kV (800 V cm-1 potencial de detecção de 0,9 V (vs. Ag/AgCI), injeção com 1,0 kV/1°s, tampão borato de sódio 10 mmol L+ com CTAH 0,2 mmol L-1, pH 9,2. Obteve-se eficiência de 1,6.104 pratos/m e foi possível obter limites de detecção de 500 nmol L-1 (135 amol) e 1,8 µmol L-1 (486 amol) para iodeto e ascorbato, respectivamente. O processo de fabricação utilizando toner como material estrutural para microchips em vidro foi bem estabelecido, assim como os modos de detecção fotométrico e condutométrico foram demonstrados. Foram obtidos eletroferogramas par detecção condutométrica sem contato de solução 200 µmol L-1 de K+, Na+ e U+, em tampão histidina/ácido lático 30 mmol L-1 9:1 (v/v) água:metanol, injeção eletrocinética de 2,0 kV/5,0 s, potencial de separação de 1 kV, 530 kHz de frequência e tensão de 2,0 Vpp. Também foi implementado um sistema de detecção fotométrico para microchip operando em 660 nm, tendo sido utilizado para a detecção de azul de metileno 1,0 mmol L-1 em tampão de corrida de barato de sódio 20 mmol L-1 (pH 9,2), com o detector posicionado a 40 mm do ponto de injeção e com injeção eletrocinética a 2,0 kV por 12 s com picos bem resolvidos em menos de 1 min. / Microfabrication processes and devices for hydrodynamic and electroosmotic manipulation were developed based on toner-polyester, toner-glass and toner-as-resist techniques. Additionally, techniques to perforate glass slides and sealing of glass devices were introduced. Microdevices for capillary electrophoresis and electrospray for mass spectrometry were developed using these techniques. The characterization of the materiais and the processes demonstrated that the devices obtained by the toner-polyester process are compatible with the media used for capillary electrophoresis. The detection of alkaline ions with capillary electrophoresis with contactless conductivity detection was demonstrated. The typical cross shape microstructure was designed with a 22-mm long and 12-µm deep separation channel. The conductivity cell was implemented with 1-mm wide adhesive copper stripes. The applied signal was 530kHz and 10Vpp . The separation of 100µmo1L-1 K+, Na+, and Li+ was accomplished in 0.8 min under a voltage of 1 kV. Another toner-polyester microchip was developed to demonstrate its usefulness for electrospray/mass spectrometry. Solutions of 1 mmol L-1 potassium chloride and 1 mmol L-1 glucosamine in water/methanol 1:1 (v/v) were introduced with stable current of 100 nA without corona discharge. Capillary electrophoresis with amperometric detection was also demonstrated. The separation of iodide (10 mmol L-1) and ascorbate (40 mmol L-1) was carried out at 4.0 kV (800 V cm-1) with detection potential of 0.9 V (vs. Ag/AgCl), electrokinetic injection at 1.0 kV/10 s, running buffer of sodium borate 10 mmol L-1 with CTAH 0.2 mmol L-1 , pH 9.2. The efficiency was 1.6.104 plates/m and the limits of detection were 500 nmol L-1 (135 9mol) and 1.8 µmol L-1 (486 amol) for iodide and ascorbate, respectively. The toner-glass process was proposed and conductivity and photometric detections were demonstrated for the devices generated by this new technique. The separation of 200 pmol L-1 K+, Na+, and Li+ was achieved in buffer histidine/lactic acid 30 mmol L-1 water/methanol 9: 1 (v/v), electrokinetic injection at 2.0 kV/5.0 s, separation potential of 1 kV, and contactless conductivity detection at 530 kHz and 2.0 Vpp. The photometric detection of methylene blue at 660 nm was carried out in sodium borate 20 mmol L-1 (pH 9.2).

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. (...)

Les écoulements de suspensions dans des milieux poreux sont particulièrement complexes, notamment à cause du couplage d’écoulements de cisaillement et d’écoulements élongationnels (Herzig, Leclerc et Goff, 1970). On les retrouve fréquemment dans les applications industrielles, l’une des applications principales se trouvant lors des opérations de production de pétrole et de gaz. Lors de la construction d'un puits, des additifs polymères anti perte de fluide empêchent à plusieurs étapes l'écoulement de fluide entre le puits et la formation de roche poreuse environnante. La perte de fluide est un problème grave si elle n’est pas maîtrisée ; elle s’ajoute alors au coût total des opérations et pourrait surtout avoir des conséquences néfastes pour l’environnement et les opérateurs. Parmi les technologies disponibles, des additifs polymères connus sous le nom d'additifs anti perte de fluide, tels que des microgels et des particules de latex, sont ajoutés aux fluides injectés pour limiter les pertes de fluide (généralement de l'eau) via un mécanisme de colmatage / blocage de pores du support poreux.Le comportement de blocage de beaucoup de ces additifs anti perte de fluide a été testé par des méthodes conventionnelles qui impliquent l'application d'une différence de pression élevée (typiquement 35-70 bars) sur le fluide formulé comprenant les additifs contre un filtre représentatif de la taille typique des pores de la formation (une grille métallique, de la céramique ou du papier filtre) et la mesure du volume de filtrat en fonction du temps. Cependant, ces méthodes standard ne permettent pas de comprendre le mécanisme sous-jacent de la dynamique de blocage des supports poreux. Par conséquent, une meilleure compréhension du mécanisme de blocage d’un support poreux par des additifs industriels anti perte de fluide est nécessaire.Dans ce travail, nous utilisons des suspensions de microgels réticulés chimiquement comme additifs anti perte de fluide et nous étudions leur comportement de blocage dans des milieux poreux modèles transparents. Nous utilisons des dispositifs à base de polydiméthylsiloxane (PDMS) comme modèle de support poreux permettant l'observation directe du processus de blocage couplée à des mesures quantitatives. Nous fabriquons des dispositifs microfluidiques de filtration frontale et de filtration latérale avec différentes tailles de pores afin de déterminer comment des paramètres tels que la mouillabilité de la surface, la concentration en particules, la taille des particules et le débit affectent la formation du gâteau de filtration.Nous présentons une méthode de contrôle de la taille des particules constituant les suspensions de microgels. Nous décrivons ensuite une approche pour préparer des suspensions à plus haute concentration et étudions la rhéologie des suspensions en fonction de la concentration en particules. De plus, nous présentons un procédé simple pour former un gâteau de filtration à partir de la suspension de microgels sur une membrane et estimons la perméabilité à l’eau du gâteau de filtration formé selon la loi de Darcy. / The flow of suspensions in porous media is a complex phenomenon due to the mechanisms involved such as both shear and extensional flows (Herzig, Leclerc, & Goff, 1970). Their use in industrial applications is quite extensive with one of the major applications being at various stages of oil and gas production operations. At several stages of a well construction, flow of fluid between the well and the surrounding porous rock formation is prevented thanks to the polymeric fluid loss control additives. Fluid loss is a severe problem if not controlled, which would add up to the total cost of operations and more importantly could have hazardous impacts on the environment or operators. Among several technologies industrially available, polymeric additives popularly known as fluid loss additives such as microgels and latex particles are added to the injected fluids to limit the loss of fluid (usually water) via the mechanism of pore clogging/jamming in porous media.Many of these fluid loss additives have been tested for their jamming behaviour by conventional methods which involve the application of a high pressure difference (typically 35-70 bars) on the formulated fluid comprising of the additives against a filter representative of the formation’s typical pore size (either a metallic grid, ceramic or filter paper) and the measurement of the filtrate volume versus time. However, these standard methods do not give any insight in understanding the underlying mechanism of jamming dynamics in porous media, hence, a better understanding of the mechanism of jamming in porous media by industrial fluid loss additives is needed.In this work, we use chemically cross-linked microgel suspensions as the fluid loss additive and study its jamming behaviour in transparent model porous media. We make use of polydimethylsiloxane (PDMS) devices as model porous media which allows direct observation of the jamming process coupled with quantitative measurements. We fabricate microfluidic devices for frontal flow filtration and lateral flow filtration with different pore sizes to see how parameters like surface wettability, particle concentration, particle size and flow rates affect the filter cake formation.We present a method of controlling the size of the microgel suspensions. We then describe an approach for preparing higher concentration suspensions and investigate the rheology of the suspensions as a function of concentration. Furthermore, we present a simple method of forming a filter cake of the microgel suspension on a supporting membrane and estimate the permeability of the filter cake formed for the flow of water using Darcy’s law.

Des particules constituées d’un cœur polymère polylactide (PLA), recouvertes d’une couche de polysaccharide adsorbé physiquement (dextrane hydrophobisé par des chaînes alkyle en C6, DexC6) et contenant des substances actives (SA) de type polyphénolique ont été élaborées par différents procédés physico-chimiques discontinus (nanoprécipitation et émulsion-évaporation de solvant) ou continus (procédé microfluidique d’émulsion-diffusion de solvant). L’extrapolation du procédé de nanoprécipitation de l’échelle laboratoire (25 mL) à l’échelle pilote (1 L) a été examinée. Ces particules étaient destinées au traitement des milieux aquatiques, pour la lutte contre le développement des cyanobactéries et des algues. Dans le but de maîtriser les caractéristiques des particules élaborées (distribution de taille, stabilité colloïdale, rendement d’encapsulation en SA ...), l’influence de paramètres physico-chimiques a été étudiée (concentration du PLA dans la phase organique, concentration du DexC6 dans la phase aqueuse, rapport volumique des deux phases, fraction massique SA/PLA …). Les procédés mis au point ont permis d’obtenir des particules dont les diamètres moyens allaient de 0,1 µm à 1 mm, avec des distributions granulométrique bien maîtrisées. Ces objets ont été caractérisés en termes de taux de recouvrement en dextrane, de quantité de SA encapsulée et de morphologie. La stabilité colloïdale des suspensions de nanoparticules a été examinée dans des milieux de force ionique variable. Par ailleurs, nous avons vérifié la possibilité de redisperser les suspensions de particules après lyophilisation. Pour les nanoparticules, l’addition d’un cryoprotecteur s’est avérée indispensable. Les cinétiques de libération des substances actives à partir des particules nano- et micrométriques ont également été suivies et les phénomènes limitant leur libération ont été identifiés.Enfin, des essais sur des milieux de culture contenant des cyanobactéries ont été réalisés. Ils ont montrés que la libération des SA conduisait à des effets algistatiques ou algicides selon les quantités utilisées. / Polysaccharide-covered polyester particles were prepared. The core of particles was made of polylactic acid (PLA) while their surface was covered by dextran chains via the use of water-soluble randomly hydrophobized dextran (DexC6) as a polymeric stabilizer. Polyphenolic active substances were encapsulated inside those particles. Polyphenol loaded PLA particles were designed for preventing cyanobacterias and algae proliferation in aquatic media. Conventional batch processes (nanoprecipitation or emulsion-solvent evaporation) and continuous processes (emulsion-solvent diffusion in microfluidics) were used to elaborate particles with average diameters ranging between 0.1 µm and 1 mm. The scale up of nanoprecipitation from lab scale (25 mL) to pilot scale (1 L) was also studied. The formulation parameters (PLA concentration in the organic phase, DexC6 concentration in the aqueous phase, aqueous phase to organic phase volume ratio, active substance weight fraction…) were optimized in order to obtain particles with well controlled characteristics (average diameter and size distribution, colloidal stability, encapsulation efficiency …). Loaded particles elaborated by different processes were characterized with regards to DexC6 surface coverage, colloidal stability at various ionic strengths, morphology and encapsulation efficiency. We also investigated the re-dispersion ability of particle suspensions after freeze drying and we showed that the use of a cryoprotectant was required in case of nanoparticles. The release of polyphenolic molecules from the elaborated polymeric nano- and microparticles was studied and limiting steps were identified. Finally, the cytotoxicity of nanoparticles toward cyanobacterias was evaluated. It was demonstrated that anti-algal effects were observed depending on the added quantities.

O uso da microfluídica na área de análises genéticas possibilita não apenas a diminuição de custos, mas também menor manipulação de amostras e reagentes e ainda maior portabilidade das análises. Com isso aumenta a possibilidade da sua utilização em locais remotos, sem a infraestrutura de um laboratório bem equipado. Dispositivos capazes de usar apenas a força centrifuga para movimentação de fluidos juntamente com a utilização de válvulas passivas para controle dos fluidos pode potencializar a sua utilização nos diagnósticos Point-of-Care. Este trabalho teve como objetivo o desenvolvimento de um microdispositivo descartável de poliéster para análises genéticas, visando a extração e amplificação do DNA alvo, de forma rápida, barata, integrada e automatizada. Os resultados confirmam a viabilidade dos dispositivos poliéster-toner (PeT) e poliéster-fita dupla face (PeDF) automatizados de extração, obtendo por meio da extração dinâmica em fase sólida de amostras complexas, DNA com qualidade compatível à técnica da reação em cadeia de polimerase (PCR). Esses resultados foram confirmados por meio da amplificação por PCR dos genes β-globina nas amostras de sangue e urina, e o gene malB nas amostras de Escherichia coli. Também foi confirmado a compatibilidade dos dispositivos de PeT para amplificação por PCR mediado por infravermelho (IV-PCR) do gene malB presente no DNA genômico de bactéria E. coli. Por fim, os dispositivos de extração e amplificação foram interligados para obtenção de um dispositivo integrado e automatizado formado pela combinação de dispositivos fabricados com diferentes filmes e métodos, PeT e PeDF. O controle de todas as soluções no interior dos dispositivos foi realizado por meio da força centrífuga combinada a válvulas passivas, sem qualquer necessidade de equipamento adicional. Portanto, podemos concluir que o dispositivo integrado PeDF - PeT possui grande potencial para aplicações em análises genéticas de forma mais barata, portátil e com menor manipulação das amostras pelo analista. / The development of microfluidics for genetic analysis allows not only cost reduction but also reduces sample and reagents handling, and increases the chances of a portable analysis. With this, increasing the possibility to use the techniques on remote places without the infrastructure of an equipped laboratory. Microdevices capable of using the centrifugal force in combination with passive valves to fluidic control can promote Point-of-Care analysis. The primary goal of this thesis was to associate these tools for the development of a disposable microdevice for genetic analysis, aiming faster, inexpensive, integrated and automated DNA extraction and amplification. The results confirmed the viability of PeT and PeDF automated microdevices, for DNA dynamic solid phase extraction, in providing high-quality DNA compatible to PCR analysis using complex samples. These results were confirmed by the β-globin PCR amplification using blood and urine samples, and the malB gene amplification in Escherichia coli samples. We have also verified the compatibility of the PeT microdevices with IV-PCR for malB gene amplification in genomic E. coli DNA. The extraction and amplification modules were interconnected to obtain an integrated and automated microdevice by the combination of devices made with different films and microfabrication methods, PeT and PeDF. The fluidic control in the devices was made using the centrifugal force combined to passive valves, with no requirement of any extra equipment. Therefore, we can conclude that the integrated PeDF - PeT microdevice has a great potential for cheaper and portable genetic analysis application, with less operator manipulation.

[pt] Alguns projetos pilotos de injeção de polímeros em campos de produção de petróleo mostraram um incremento na recuperação de óleo, embora os mecanismos que governam a dinâmica do escoamento não são bem compreendidos. Recentes investigações experimentais mostraram que as propriedades viscoelásticas de soluções poliméricas podem alterar o comportamento do escoamento na escala de poros e reduzir a saturação residual de óleo. Para entender esses fenômenos em meios porosos, é importante estudar o escoamento de soluções viscoelásticas através das gargantas dos poros. Este trabalho apresenta um estudo experimental do escoamento de uma solução viscoelástica de PEO (0,1 porcento em peso de óxido de polietileno) de alto peso molecular escoando através de uma constrição, utilizado como modelo de uma geometria de garganta de poro de um meio poroso. Medições de queda de pressão e campos de velocidade do escoamento são obtidos utilizando a técnica de velocimetria por imagem de partículas (Micro-PIV). Experimentos com uma solução viscosa de glicerina (45 porcento em peso de glicerina em água) de viscosidade similar à solução de PEO foram também realizados com a finalidade de estimar os efeitos elásticos da solução de PEO. O escoamento da solução de PEO exibiu uma queda de pressão extra (comportamento não linear) acima de uma condição crítica do escoamento, acima da qual os efeitos elásticos passam a ser preponderantes. Para toda a faixa de vazão explorada, os campos de velocidade da solução de glicerina mostraram um regime de escoamento Newtoniano, enquanto a solução de PEO apresenta instabilidade no escoamento a partir de um número de Weissenberg crítico, coincidindo com o aumento da queda de pressão. Esta instabilidade pode ser relacionada ao aumento da viscosidade extensional na entrada da garganta acima de uma determinada taxa de extensão. Os resultados obtidos indicam a variação do padrão do escoamento da solução polimérica de PEO devido à presença dos efeitos elásticos do polímero, e fornecem informações importantes sobre o comportamento das soluções poliméricas viscoelásticas em um meio poroso e que podem impactar sua utilização na recuperação melhorada de óleo. / [en] Some pilot projects of polymer injection in oil fiel ds have shown an increase in oil recovery, although the mechanisms that govern the flow dynamics are still not well understood. Recent experimental investigations have shown that the viscoelastic properties of polymer solutions may change the pore-scale flow behavior and reduce the residual oil saturation. To understand these phenomena in porous media, it is important to understand viscoelastic flow behavior through the pores-throats. This work presents experimental study of the flow of a high molecular viscoelastic PEO solution (0.1 wt percent Polyethylene Oxide) flowing through a constricted capillary, used as model for a pore-throat geometry of a porous media. Pressure drop measurements are performed and velocity fields are obtained using the micro-particle image velocimetry (Micro-PIV) technique. Experiments with a viscous solution of glycerin (45 wt percent glycerin in water), of similar shear viscosity to the PEO solution were also performed in order to isolate the elastic effects of the PEO solution. The flow of the PEO solution exhibited an extra pressure drop (nonlinear behavior) above a critical flow condition beyond which the elastic forces become relevant. For the entire flow rate range explored, the velocity field of the glycerin solution showed a Newtonian flow regime, while the PEO solution shows instability in the flow above a critical Weissenberg number, coinciding with the onset of the extra pressure drop. This instability in the flow is associated with the high extensional viscosity near the constriction at high enough extension rates. The results show the changes in the flow pattern of the PEO polymer solution due to the presence of the elastic effects of polymer, and provide important information on how viscoelastic polymer solutions behave in a porous media and can impact their use in Enhanced Oil Recovery operations.

Les mégacaryocytes sont des cellules de la moelle osseuse, à l’origine de la production des plaquettes sanguines. Quand elles arrivent à maturité, elles grossissent et émettent des prolongements de cytoplasme à travers la paroi des vaisseaux irriguant la moelle. Dans la circulation sanguine, ces prolongements, soumis aux forces de l’écoulement, s’allongent et se rompent pour former des plaquettes. Des techniques microfluidiques capables de produire des plaquettes in vitro existent et sont une alternative prometteuse au don. Mais le rendement reste à améliorer. Pour cela, il est nécessaire de mieux comprendre la fragmentation des mégacaryocytes en plaquettes. Ce travail de doctorat s’inscrit dans ce contexte et sera développé en deux axes principaux dans ce manuscrit. Dans une première partie nous développons une méthode pour trier des cellules en fonction de leur déformabilité, afin de savoir si les propriétés mécaniques d’un mégacaryocyte sont liées à leur stade de maturité. La méthode a d’abord été mise au point avec des microcapsules. Leurs propriétés mécaniques sont déterminées par analyse inverse à partir de la mesure de leur forme en écoulement dans des constrictions droites. Puis le dispositif utilisé a été miniaturisé pour s’adapter à la taille des cellules. Pour la caractérisation de leurs propriétés mécaniques, deux outils ont été utilisés: l’analyse inverse et la microscopie à force atomique sans pointe. Une deuxième partie porte sur l’étude de l’élongation et de la rupture de mégacaryocytes soumis écoulement. Nous avons quantifié les variations spatiotemporelles du taux d’élongation et développé un protocole d’ablation laser pour étudier les mécanismes de rupture de cellules en élongation. / When they mature in the bone marrow, the precursors of platelets, called megakaryocytes, grow and extend protrusions able to join blood circulation. There these protrusions elongate and break into platelets. Microfluidic techniques for in vitro platelet production represent a promising alternative to donation. In order to enhance platelet production and match the needs of clinical applications such as transfusion, we need to better understand the fragmentation of megakaryocytes into platelets. Our contribution will be described in this manuscript in two main axes. First, in order to know if mechanical properties of megakaryocytes can indicate their maturity stage, we develop a cell sorting method based on deformability. The method is first validated with microcapsules. Their mechanical properties are determined by inverse analysis from their shape under flow in straight microchannels. Then the device is downscaled. The characterization of cell mechanical properties are performed using inverse analysis and tipless atomic force microscopy. Second, we study megakaryocyte elongation and rupture in a microfluidic device. We quantify the spatial and temporal variations of the elongation rate and develop a laser ablation protocol to trigger and study the rupture of elongating cells.

O uso da microfluídica na área de análises genéticas possibilita não apenas a diminuição de custos, mas também menor manipulação de amostras e reagentes e ainda maior portabilidade das análises. Com isso aumenta a possibilidade da sua utilização em locais remotos, sem a infraestrutura de um laboratório bem equipado. Dispositivos capazes de usar apenas a força centrifuga para movimentação de fluidos juntamente com a utilização de válvulas passivas para controle dos fluidos pode potencializar a sua utilização nos diagnósticos Point-of-Care. Este trabalho teve como objetivo o desenvolvimento de um microdispositivo descartável de poliéster para análises genéticas, visando a extração e amplificação do DNA alvo, de forma rápida, barata, integrada e automatizada. Os resultados confirmam a viabilidade dos dispositivos poliéster-toner (PeT) e poliéster-fita dupla face (PeDF) automatizados de extração, obtendo por meio da extração dinâmica em fase sólida de amostras complexas, DNA com qualidade compatível à técnica da reação em cadeia de polimerase (PCR). Esses resultados foram confirmados por meio da amplificação por PCR dos genes β-globina nas amostras de sangue e urina, e o gene malB nas amostras de Escherichia coli. Também foi confirmado a compatibilidade dos dispositivos de PeT para amplificação por PCR mediado por infravermelho (IV-PCR) do gene malB presente no DNA genômico de bactéria E. coli. Por fim, os dispositivos de extração e amplificação foram interligados para obtenção de um dispositivo integrado e automatizado formado pela combinação de dispositivos fabricados com diferentes filmes e métodos, PeT e PeDF. O controle de todas as soluções no interior dos dispositivos foi realizado por meio da força centrífuga combinada a válvulas passivas, sem qualquer necessidade de equipamento adicional. Portanto, podemos concluir que o dispositivo integrado PeDF - PeT possui grande potencial para aplicações em análises genéticas de forma mais barata, portátil e com menor manipulação das amostras pelo analista. / The development of microfluidics for genetic analysis allows not only cost reduction but also reduces sample and reagents handling, and increases the chances of a portable analysis. With this, increasing the possibility to use the techniques on remote places without the infrastructure of an equipped laboratory. Microdevices capable of using the centrifugal force in combination with passive valves to fluidic control can promote Point-of-Care analysis. The primary goal of this thesis was to associate these tools for the development of a disposable microdevice for genetic analysis, aiming faster, inexpensive, integrated and automated DNA extraction and amplification. The results confirmed the viability of PeT and PeDF automated microdevices, for DNA dynamic solid phase extraction, in providing high-quality DNA compatible to PCR analysis using complex samples. These results were confirmed by the β-globin PCR amplification using blood and urine samples, and the malB gene amplification in Escherichia coli samples. We have also verified the compatibility of the PeT microdevices with IV-PCR for malB gene amplification in genomic E. coli DNA. The extraction and amplification modules were interconnected to obtain an integrated and automated microdevice by the combination of devices made with different films and microfabrication methods, PeT and PeDF. The fluidic control in the devices was made using the centrifugal force combined to passive valves, with no requirement of any extra equipment. Therefore, we can conclude that the integrated PeDF - PeT microdevice has a great potential for cheaper and portable genetic analysis application, with less operator manipulation.