The thesis describes state of the art volumetric measurement techniques and applies a 3D measurement technique, 3D Scanning Particle Tracking Velocimetry, to the transitional backward facing step flow. The measurement technique allows the spatial and temporal analysis of coherent structures apparent at the backward facing step. The thesis focusses on the extraction and interaction of coherent flow structures like shear layers or vortical structures.

The effect of in-situ or redistributed stress on solute transport in fractured rocks is one of the major concerns for many subsurface engineering problems. However, it remains poorly understood due to the difficulties in experiments and numerical modeling. The main aim of this thesis is to systematically investigate the influences of stress on solute transport in fractured rocks, at scales of single fractures and fracture networks, respectively. For a single fracture embedded in a porous rock matrix, a closed-form solution was derived for modeling the coupled stress-flow-transport processes without considering damage on the fracture surfaces. Afterwards, a retardation coefficient model was developed to consider the influences of damage of the fracture surfaces during shear processes on the solute sorption. Integrated with particle mechanics models, a numerical procedure was proposed to investigate the effects of gouge generation and microcrack development in the damaged zones of fracture on the solute retardation in single fractures. The results show that fracture aperture changes have a significant influence on the solute concentration distribution and residence time. Under compression, the decreasing matrix porosity can slightly increase the solute concentration. The shear process can increase the solute retardation coefficient by offering more sorption surfaces in the fracture due to gouge generation, microcracking and gouge crushing. To study the stress effects on solute transport in fracture systems, a hybrid approach combing the discrete element method for stress-flow simulations and a particle tracking algorithm for solute transport was developed for two-dimensional irregular discrete fracture network models. Advection, hydrodynamic dispersion and matrix diffusion in single fractures were considered. The particle migration paths were tracked first by following the flowing fluid (advection), and then the hydrodynamic dispersion and matrix diffusion were considered using statistic methods. The numerical results show an important impact of stress on the solute transport, by changing the solute residence time, distribution and travel paths. The equivalent dispersion coefficient is scale dependent in an asymptotic or exponential form without stress applied or under isotropic compression conditions. Matrix diffusion plays a dominant role in solute transport when the hydraulic gradient is small. Outstanding issues and main scientific achievements are also discussed. / QC 20111011

Les adhérences focales (AF), structures adhésives reliant la cellule à la matrice extra-cellulaire (MEC), constituent de véritables plateformes de signalisation biochimique et mécanique qui contrôlent l'adhérence, la migration, la différenciation et la survie cellulaire. Les récepteurs transmembranaires intégrines sont au coeur des AF, où elles connectent la MEC au cytosquelette d'actine. Au début des années 2000, la protéine intracellulaire taline, qui se lie aux parties cytoplasmiques bêta des intégrines, était considérée comme le principal activateur des intégrines. Néanmoins, il a depuis été montré que la kindline, autre protéine intracellulaire se liant aux parties bêta cytoplasmiques, jouait également un rôle essentiel dans l'activation des intégrines. Ainsi,plusieurs études ont mis en évidence que la kindline et la taline étaient complémentaires et avaient une action synergique durant l'activation des intégrines. Les bases moléculaires de ces phénomènes restent à déterminer. De plus, la plupart des données sur lerôle de la kindline dans l'adhérence et l'activation des intégrines provient d'expériences menées sur des cellules en suspension et/ou avec l'intégrine plaquettaire αIIbβ3. Ainsi, la régulation de ces processus par la kindline dans les cellules adhérentes est encore peu comprise. Dans cette étude, nous combinons la microscopie PALM et le suivi de protéines individuelles pour révéler le rôle et le comportement de la kindline à l'intérieur et à l'extérieur des AF au cours des événements moléculaires clés se déroulant au niveau de la membrane plasmique, et qui mènent à l'activation des intégrines. Nous avons observé que les intégrines bêta1 etbêta3 portant une mutation ponctuelle inhibant l'interaction avec la kindline montrent un défaut d'immobilisation dans les AF. Nous avons également observé que la kindline-2, qui est enrichie dans les AF, diffusait librement au niveau de la membrane plasmique,à l'intérieur et à l'extérieur des AF. Ceci constitue une distinction majeure par rapport à la taline, qui, au niveau de la membrane plasmique, est essentiellement observée dans les AF où elle est immobile, montrant qu'elle est recrutée dans les AF directement depuis le cytosol sans diffusion latérale membranaire (Rossier et al. 2012). Afin d'identifier les bases moléculaires du recrutement et de la diffusion membranaire de la kindline, nous avons utilisé différents variants mutés de kindline précédemment décrits. Le mutant kindline-2-QW614/615AA (liaison aux intégrines inhibée) montre une diffusion membranaire accrue, ce qui suggère que la kindline peut diffuser au niveau de la membrane plasmique sans être associée aux intégrines. Par ailleurs, la baisse d'immobilisation au niveau des AF observée avec ce mutant montre qu'une partie de l'immobilisation de la kindline est due aux intégrines, suggérant l'existence d'un complexe intégrine-kindline immobile dans les AF. La délétion du domaine PleckstrinHomology (PH) de la kindline diminue considérablement son recrutement et sa diffusion membranaire. Nous avons évalué le rôle fonctionnel du recrutement et de la diffusion membranaire de la kindline en réexprimant ces mutants dans des cellules déplétéesen kindline-1 et -2 (cellules KO kindline-1 -/-, kindline-2 -/-). Ces expériences montrent que le recrutement et la diffusion membranaire de la kindline sont cruciaux pour l'activation des intégrines durant l'étalement cellulaire et favorisent la formation d’adhérences. Cela suggère que la kindline utilise un chemin différent de celui de la taline pour atteindre et activer les intégrines,ce qui pourrait expliquer au niveau moléculaire comment la kindline complémente la taline durant l'activation des intégrines. / Focal adhesions (FAs) are adhesive structures linking the cell to the extracellular matrix (ECM) and constitute molecular platforms for biochemical and mechanical signals controlling cell adhesion, migration, differentiation and survival. Integrin transmembrane receptors are core components of FAs, connecting the ECM to the actin cytoskeleton. During the early 2000s, the intracellular protein talin, which directly binds to the cytoplasmic tail of β-integrins, was considered as the main integrin activator. Nevertheless, it has been shown that kindlin, another intracellular protein that bind to β-integrin, is also a critical integrin activator. In fact, several studies have shown that kindlin and talin play complementary and synergistic roles during integrin activation. The molecular basis of these phenomena remains to determine. Moreover, most studies focusing on the role of kindlin during integrin activation and cell adhesion have been performed with suspended cells and/or with the platelet integrin αIIbβ3. Here we combined PALM microscopy with single protein tracking to decipher the role and behavior of kindlin during key molecular events occurring outside and inside FAs at the plasma membrane and leading to integrin activation, as we have done previously for talin (Rossier et al., 2012). We found that beta1 and beta3-integrins with a point mutation inhibiting binding to kindlin show reduced immobilization inside FAs. We also found that kindlin-2, which is enriched inside FAs, displayed free diffusion at the plasma membrane outside and inside FAs. This constitutes a major difference with talin, which, at the plasma membrane level, is observed almost exclusively in FAs, where it is immobile, which shows that talin is recruited into FAs directly from the cytosol without lateral diffusion along the plasma membrane (Rossier et al. 2012). To determine the molecular basis of kindlin membrane recruitment and diffusion, we used a kindlin variant known to decrease binding to integrins (kindlin-2- QW614/615AA). This mutant displayed increased membrane diffusion, suggesting that kindlin-2 can freely diffuse at the plasma membrane without interacting with integrins. Moreover, the kindlin-2-QW mutant showed decreased immobilization inside FA, showing that part of kindlin immobilization depends on interaction with integrins. This suggests that kindlin can form an immobile complex with integrins inside focal adhesions. Deletion of the kindlin pleckstrin homology (PH) domain strongly reduced the membrane recruitment and diffusion of kindlin. We assessed the functional role of kindlin membrane recruitment and diffusion by re-expressing different kindlin-2 mutants in kindlin-1/kindlin-2 double KO cells. Those experiments demonstrated that kindlin-2 membrane recruitment and diffusion are crucial for integrin activation during cell spreading and favor adhesion formation. This suggests that kindlin uses a different route from talin to reach integrins and trigger their activation, providing a possible molecular basis for their complementarity during integrin activation.

Les suspensions d'objets microscopiques ayant la faculté de se déplacer par eux-mêmes dans le fluide qui les entoure sont des systèmes qui présentent un intérêt croissant dans la communauté scientifique. Du fait de leur dynamique intrinsèquement hors-équilibre au sens de la physique statistique, ils génèrent des effets particulièrement complexes. Parmi les micro-objets autopropulsés existants, les micro-algues vertes représentent une part importante de la biomasse de la Terre et participent activement au retraitement du CO2 par leur activité photosynthétique. Elles présentent de plus un remarquable potentiel dans les domaines de la production de bio-carburants, du retraitement des déchets, de la fabrication de cosmétiques et de compléments alimentaires. La compréhension de la dynamique de nage de ce type de microorganisme est d'un intérêt primordial d'un point de vue industriel. Cet ouvrage présente l'étude de la dynamique de la micro-algue Chlamydomonas Reinhardtii. En utilisant un système de suivi de particules en imagerie optique que nous avons développé, nous analysons ici le mécanisme fondamental de nage utilisé par cette algue jusqu'à ses implications en terme d'effets collectifs sur la dynamique de nage d'une suspension semi-diluée. / The suspensions of microscopic objects with the ability to propel themselves into the surrounding fluid are systems of growing interest in the scientific community. Due to their intrinsic out-of-equilibrium dynamics in the sense of statistical physics, they generate complex effects. Among the existing self-propelled micro-objects, green micro-algae are an important part of the biomass of Earth and they actively participate to the recycling of CO2 by their photosynthetic activity. Moreover they have remarkable potential for the production of bio-fuels, waste reprocessing, cosmetics and dietary supplements production. From an industrial point of view, understanding the dynamics of this type of swimming microorganism is of primary interest. This work presents the study of the dynamics of microalgae Chlamydomonas Reinhardtii. Using a system of particle tracking with optical imaging that we have developed, we analyze the mechanism of stroke used by the algae up to its implications in terms of collective effects on the dynamics of swimming in a semi-dilute suspension.

Cette thèse présente un travail expérimental dans le domaine de la mécanique des milieux granulaires.Ce travail introduit une nouveauté fondamentale: durant la déformation d’échantillonsde sable en compression triaxiale classique, leur micro-structure est enregistrée par tomographieà rayons-x en environ 15 étapes de déformation différentes.Afin que tous les grains d’un échantillon soient individuellement visibles dans les imagesde tomographie, les échantillons sont considérablement réduits pour qu’ils mesurent 11 mm endiamètre et 22 mm en hauteur. Cela permet d’identifier et d’individualiser tous les plus decinquante mille grains d’un échantillon.Dans ce travail expérimental, une série d’essais triaxiaux a été réalisée sur trois sables naturels(le sable d’Hostun, le sable d’Ottawa et des Caicos ooids, tous à partir des états initiauxrelativement denses), à deux valeurs de pression de confinement différents (100 et 300 kPa).Dans les images 3D résultantes de la reconstruction des acquisitions tomographiques réaliséesau cours de chaque essai, les grains sont identifiés dans chaque état en utilisant un algorithmede type watershed (ligne de partage des eaux) classique. À partir de ces données discrétisées,des techniques ont été mises au point pour caractériser les contacts grain-à-grain mais aussipour mesurer la cinématique de tous les grains identifiés entre les états pour lesquels des imagestridimensionnelles existent. La cinématique des grains est mesurée avec deux outils spécialementdéveloppés: “ID-Track” suit les grains et donne leurs déplacements; cette information est ensuitenécessaire pour une technique hybride de corrélation d’images discrète pour mesurer la rotationde chaque grain.Des mesures à l’échelle du grain sont présentées en détail pour un essai, et sont ensuitecomparées à des essais dans des conditions différentes. L’objectif est de mettre en évidence lesmicro-mécanismes responsables des différents comportements macroscopiques observés pour cesmatériaux. Cette comparaison met en évidence certains micro-mécanismes importants tels que,par exemple, la déformation d’un échantillon. Celle-ci est concentrée dans une bande de cisaillementbien développée qui correspond à une bande de grains avec des rotations intenses. Dans unéchantillon de grains arrondis, cette bande – définie par des grains – est très nette. Par contre,avec des grains anguleux, les rotations des grains dans la bande “polluent” les grains voisins;leur forme facilite l’engrenage entre grains, qui limite aussi les grosses rotations individuelles desgrains. Cette différence de mécanisme de déformation est utilisée pour expliquer la différencedans la contrainte résiduelle observée à macro-échelle. Des signes de déformation localisée sontsystématiquement observés en train de se produire avant le pic de déformation des échantillons etdes systèmes complexes de chaînes de rotations de grains (qui correspondent aussi à une mesurelocale de déformation calculée avec les déplacements des grains) sont remarquées autour du picde la réponse macroscopique de chaque échantillon. / This doctoral thesis presents an experimental investigation into the mechanics of granular media.The novelty that this work brings is that the specimens of sand tested in this work are systematicallyand non-destructively imaged using x-ray tomography. Sample size is considerably reducedfrom standard (specimens measure approximately 22 mm height by 11 mm diameter), allowingentire specimens to be scanned at a sufficiently high resolution to identify all the grains (morethan fifty thousand) in each specimen.A campaign of triaxial compression tests has been run on a series of three different naturalsands with different grain shapes (Hostun sand, Ottawa sand and Caicos ooids – all prepared atrelatively dense initial states), and tested at 100 or 300 kPa cell pressure. In each test around 15x-ray scans are performed. In the 3D images resulting from the reconstruction of the x-ray scansperformed, grains are identified each state using a standard watershed algorithm. Starting fromthese discretised data, techniques are developed in order characterise grain-to-grain contacts,as well as to measure the kinematics of all the identified grains between imaged states. Grainkinematics are measured with two specifically-developed tools: “ID-Track” to track grains yieldingtheir displacements, and a discrete image correlation technique to measure grain rotations.Grain-scale measurements are reported in detail for one test, and are then compared to testsin different conditions, in order to highlight the micro-mechanisms responsible for the observedmacroscopic behaviour. This comparison highlights some important micro-scale mechanisms suchas the increasing rotational frustration of more angular grains when the sample’s deformation isconcentrated in a fully developed shear band; this is used to explain to some extent the highervalue of their residual stress for these materials. Signs of localised deformation are seen to occurwell before the peak in many samples, and complex patterns of rotating grains (which match alocal, grain-based measurement of strain) are noticed around the peak of each sample’s response.

O objetivo deste estudo é compreender a dinâmica das partículas no estuário do rio Araranguá (SC) em termos de transporte em suspensão, deposição e exportação, em diferentes condições físico-químicas da água, com pH baixo (<4,5) e normal (>=7), e sob condições de regime transiente da descarga fluvial. O estuário do rio Araranguá é classificado como altamente estratificado e dominado pela descarga fluvial. Um aspecto fundamental nos processos que controlam a dinâmica de materiais em suspensão no estuário é a atividade de lavra e beneficiamento do carvão, que gera uma drenagem ácida no rio Mãe Luzia. O seu pH baixo (&#732;3) indica que o processo de floculação é intensificado no interior do estuário. Com o objetivo de melhor entender a hidrodinâmica do estuário, um modelo tridimensional foi implementado para a região. O modelo foi calibrado e validado com dados observados. Simulações com diferentes picos de descarga fluvial (100, 400, 700 e 1000 m3.s-1) e baixa descarga (30 m3.s-1) foram realizadas. Para calcular o transporte do material particulado em suspensão foi desenvolvido um modelo Lagrangeano da trajetória da partícula. O modelo da trajetória utiliza os resultados do modelo hidrodinâmico e a velocidade de decantação das partículas. O estuário respondeu rapidamente às variações da descarga fluvial. A cunha salina foi transportada para fora do estuário durante os picos de 700 e 1000 m3.s-1. A descarga fluvial é o fator dominante no controle da intrusão da salinidade no estuário e, em condições de baixa descarga, o transporte advectivo de sal é dominado pelo termo dispersivo da circulação gravitacional. O tempo de residência médio das partículas no estuário foi menor em condições de pH baixo. A exportação das partículas aumentou com o aumento da descarga fluvial (pico de 1000 m3.s-1), sendo que 27% e 30% foram exportadas para a plataforma interna durante os experimentos com pH baixo e pH normal, respectivamente. A região de deposição das partículas indica que com a diminuição do pH, as partículas passam a depositar mais à jusante no estuário. O evento de descarga de 1000 m3.s-1 não foi capaz de remover a grande maioria das partículas do estuário, mas foi responsável por redistribuir as partículas ao longo do médio estuário, onde se depositaram em condições favoráveis com baixas velocidades e ausência de salinidade. O trabalho demonstra que apesar da alta estratificação do estuário, a acidez de suas águas, seu padrão de circulação, com baixas velocidades e a presença da cunha salina, possuem um papel fundamental no transporte das partículas, resultando em maior potencial de retenção do material em suspensão no estuário do rio Araranguá. / The present study assessed the dynamics of particles of the Araranguá (SC) river estuary in terns of suspended transport, deposition and exportation in different conditions of physical-chemical of water, with low (<4,5) and normal pH (>=7), and transient of river discharge conditions. The Araranguá river estuary is a highly stratified flash-flood dominated. The river basin of the Mãe Luzia river, one of the main tributaries of the Araranguá river, is performed the activity of mining and coal processing, wich generates an acidic drainage. Its low pH indicates that flocculation process is intensified in estuary inner. With the objective of better understanding the estuarine processes, a three-dimensional hydrodynamics model was implemented. The model was calibrated and verified using observational data. Simulations were conducted with freshet peak ranged over 100, 400, 700 and 1000 m3.s-1. Lagrangian particle tracking algorithms was developed to represent the suspended particle transport at estuary. The particle tracking used the montion resulting from hydrodynamics model and the particle settling velocity. The estuary responded quickly the river discharge variations. During the pulse event the velocities at water column were unidirectional down-estuary is shown the barotropic dominates over baroclinic component. During the highest flash flood event (700 and 1000 m3.s-1) the salt water was pushed out of the estuary. The river discharge is a dominating factor affecting the salinity intrusion in the estuarine system and under low discharge conditions the advective salt transport is dominated by dispersive term of gravitational circulation. The mean residence time of the particles was lowest in pH low conditions. The particles export is great as the river discharge is rising (1000 m3.s-1) with 27% and 30% exported to inner shelf during the experiments with low and normal pH, respectively. The particles deposition local indicates that with the pH decrease the particles become to be deposited further downstream. The discharge event of 1000 m3.s-1 was not able to remove all particles out, but was response to redistribute the particles along of the meddle estuary, where settling in favorable conditions at low velocities and absence of salinity. The study shows that although high stratification of estuary, the acidity of water, the pattern of circulation, at low velocities, and the salt wedge presence play a fundamental role in the transport of particle matter resulting in increased the potential retention of suspended matter in the Araranguá river estuary.

When protection zones for wells are delineated, it is important to acquire good knowledge about possible travel time from different points in the catchment area to the well. Often, simple analytical methods are used for estimating travel times and the assumption is made that the hydraulic conductivity is relatively homogenous within the aquifer. Nevertheless, many aquifers are strongly heterogeneous which may lead to differences between estimates and actual travel times. As a part of the process to develop improved methods for delineating protection zones for groundwater supply wells, a tracer experiment was performed in a glaciofluvial esker formation in Järlåsa. On the basis of the experiment, a numerical flow model was created for the test site. The purpose of this master’s thesis was to apply the flow model to an aquifer where the hydraulic conductivity shows great variability and should be described by a stochastic distribution. The purpose was also to determine the statistical properties of the hydraulic conductivity and to simulate the transport times and their variation from different locations in the aquifer to the pumping well. The hydraulic conductivity was estimated from grain size distributions in soil samples that were taken at various locations within the test site. The analysis of the hydraulic conductivity showed a large variation and confirmed the hypothesis that the aquifer is heterogeneous. Using the statistics, a large number of stochastic conductivity fields were created and flow simulation was performed for each realization. From the simulation result, frequency distributions of the transport times were produced describing the probability for the transit time for a water particle between a certain location in the aquifer and the pumping well. A comparison with the tracer experiment shows higher simulated transport times implying the need for improved model calibration. The conclusion is that the method used in this project is suitable for glaciofluvial esker aquifers. When protection zones are delineated, stochastic modeling can be used to express the zone boundaries in statistical terms. / När skyddsområden för grundvattentäkter skapas är det viktigt att ha god kännedom om vattnets transporttider från olika delar i tillrinningsområdet till uttagsplatsen av grundvattnet. Metoderna för att bestämma dessa tider är ofta enkla och vanligen görs antagandet att områdets hydrauliska konduktivitet är relativt homogen. Många akviferer är dock kraftigt heterogena och de verkliga transporttiderna kan då skilja sig från de uppskattade. Som ett led i metodutvecklingen för bättre avgränsning av skyddsområden genomfördes ett spårämnesförsök i en isälvsavlagring i Järlåsa. Med utgångspunkt från försöket har en numerisk flödesmodell konstruerats över försöksområdet. Syftet med examensarbetet var att tillämpa flödesmodellen i en akvifer där den hydrauliska konduktiviteten visar sådana variationer att den beskrivs bäst av en stokastisk fördelning. Vidare var syftet att bestämma den hydrauliska konduktivitetens statistiska egenskaper och att simulera transporttiderna och deras variation från olika punkter i akviferen till pumpbrunnen. Den hydrauliska konduktiviteten uppskattades utifrån kornstorleksfördelningar i jordprover som togs på en mängd platser i försöksområdet. Analysen av den hydrauliska konduktiviteten visar stora variationer i området vilket bekräftar att akviferen är heterogen. Utifrån konduktivitetens statistik genererades ett stort antal stokastiska konduktivitetsfält och transporttiderna beräknades för varje realisering. Resultatet från simuleringarna gav frekvensfördelningar för transporttiderna som beskriver sannolikheten för hur lång uppehållstid en vattenpartikel har i marken mellan en startpunkt och pumpbrunnen. Jämfört med spårämnesförsöket blev de simulerade transporttiderna något större vilket tyder på att flödesmodellen kräver en bättre kalibrering mot fältmätningar. Slutsatsen är att metodiken är lämplig för att studera vattnets transporttider i isälvsavlagringen och när ett skyddsområde skapas för den här typen av akvifer kan stokastisk modellering användas för att beskriva skyddszoner i form av statistiska termer.

In a particle based geometallurgical model, the behavior of the particles can be used for forecast the products and quantify the performance of the different ore types within a deposit. The particle tracking is an algorithm developed by Lamberg and Vianna 2007 whose aim is to balance the liberation data in a mineral processing circuit composed by several processing units. Currently, this tool is being developed for the HSC Chemistry software by Outotec.The objective of this study is to understand and evaluate the particle tracking algorithm in a geometallurgical test for iron ore. To achieve this objective, the liberation data is balanced in a Davis tube test circuit. A total of 13 samples from Leveäniemi iron ore were process in a Davis tube circuit.The magnetite is the main mineral in the Leveäniemi iron ore samples. Its high recovery in the Davis tube circuit along with the V, Ti and Mn suggest that these elements are present in the magnetite lattice. These penalty elements in the iron concentrates cannot be avoided at the stage of mineral concentrations.The washing effect of the Davis tubes controlled by the rotational and longitudinal agitation of the tube perturb the particles agglomeration between the pole tips of the electromagnet. A higher agitation frequency and amplitude will wash away most of the gangue minerals and also fine grained magnetite.In this work, the particle tracking is depicted and implemented in a magnetic separation circuit for high liberated material. The liberation data was balanced in a way that the particle classes can be followed through circuit and their recoveries can be calculated. Nevertheless, the algorithm requires further validation and analysis of its limitations in terms of resolution and reproducibility.

L’effet photothermique permet de détecter efficacement des nanoparticules d’or avec un microscope en champ lointain grâce à leur forte absorption de la lumière. L’absence de problème photophysique fait des nanoparticules d’or une alternative au marquage de biomolécules par des sondes fluorescentes. La méthode PhACS (Photothermal Absorption Correlation Spectroscopy) utilise les fluctuations de signal photothermique dues au passage de nanoparticules dans le volume de détection pour étudier leur diffusion. Cette méthode permet également la mesure précise de diamètres hydrodynamiques de nanoparticules fonctionnalisées. La méthode SNaPT (Single Nano-Particle Tracking) réalise le suivi bidimensionnel de nanoparticules individuelles grâce à une localisation effectuée par triangulation. Nous avons appliqué cette méthode pour étudier la diffusion des intégrines alphaV-beta3 marquées par des nanoparticules d’or de 5 nm dans les adhérences focales, points d’ancrage entre le cytosquelette de la cellule et la matrice extracellulaire. Nous observons que ces intégrines ont tendance à former des agrégats qui alternent entre un mouvement diffusif et un mouvement confiné. Ce résultat appelle maintenant à un nouveau modèle où nous aurions une redistribution continue des intégrines au sein des adhérences focales. / Gold nanoparticles may be detected with optical far-field microscopy by use of the photothermal effect due to their strong light absorbance. With no photophysic issues, gold nanoparticles are an alternative to fluorescent probes for use in biological systems. The PhACS method (Photothermal Absorption Correlation Spectroscopy) is used to study diffusion by measuring the autocorrelation of photothermal signal fluctuations due to nanoparticles passing through the detection volume. This method is sensitive enough to mesure the precise hydrodynamic diameter of functionalised nanoparticles. The SnaPT method (Single Nano-Particle Tracking) can track 2-dimensional motion of individual nanoparticles by pinpointing the localization with a triangulation method. The SNaPT method was used to study motion of alphaV-beta3 integrins that were bound to a 5 nm gold nanoparticle inside focal adhesion, where the cell cytoskeleton is linked to the extracullular matrix. The integrin was found to organize into clusters oscillating between the bound and diffuse states. These observations require new working models where integrins would be constantly redistributed.

La compréhension fondamentale de fonctions biologiques est accordée par l’imagerie de molécules uniques dans les cellules vivantes. Cet environnement nanoscopique est compliqué et largement mal compris. La microscopie de localisation permet cet environnement d’être sondé avec le suivi de molécules uniques. Depuis longtemps, l’obstacle principal qui empêcher la compréhension de processus physiques à cette échelle était la pénurie d’information accessible; de fortes hypothèses ne peuvent pas être établies à partir de quelques dizaines de trajectoires de molécules uniques. L’introduction des techniques de suivi de molécules à haute densité a recadré les possibilités. Dans cette thèse, les outils d’inférence Bayesienne ont été développé pour élucider le comportement de molécules uniques à partir la cartographie de leurs paramètres physiques de mouvement. Ces cartes décrivent, entre autre, les hétérogénéités aux échelles locales mais aussi à l’échelle de la cellule entière. Notamment elles dévoilent les détails quantitatifs sur les processus cellulaires de base. En utilisant cette approche cartographique, les interactions entre récepteurs et protéines d’échafaudage chez les neurones et les cellules non-neuronales sont étudiés. Une étude sur les interactions imprimées dans la cellule est également effectuée grace à un système prometteur d’impression de protéine. Les différents types d’interactions entre constructions chimériques du récepteur de glycine et de protéines d’échafaudage de géphyrine sont décrits et distingué in situ. Enfin, les perspectives vis-à-vis l’obtention de cartes tridimensionnelles de dynamiques de molécules uniques sont également commentées. / Imaging of single molecules inside living cells confers insight to biological function at its most granular level. Single molecules experience a nanoscopic environment that is complicated, and in general, poorly understood. The modality of choice for probing this environment is live-cell localisation microscopy, where trajectories of single molecules can be captured. For many years, the great stumbling block in comprehension of physical processes at this scale was the lack of information accessible; statistical significance and robust assertions are hardly possible from a few dozen trajectories. It is the onset of high-density single-particle tracking that has dramatically reframed the possibilities of such studies. Importantly, the consequential amounts of data it provides invites the use of powerful statistical tools that assign probabilistic descriptions to experimental observations. In this thesis, Bayesian inference tools have been developed to elucidate the behaviour of single molecules via the mapping of motion parameters. As a readout, maps describe heterogeneities at local and whole-cell scales. Importantly, they grant quantitative details into basic cellular processes. This thesis uses the mapping approach to study receptor-scaffold interactions inside neurons and non-neuronal cells. A promising system in which interactions are patterned is also examined. It is shown that interactions of different types of chimeric glycine receptors to the gephyrin scaffold protein may be described and distinguished in situ. Finally, the prospects of whole-cell mapping in three-dimensions are evaluated based on a discussion of state-of-the-art volumetric microscopy techniques.