Monitoring of public areas and their automatic real-time processing became increasingly significant due to the changing security situation in the world. However, the problem is an analysis of low-quality records, where even the state-of-the-art methods fail in some cases. This work investigates an important area of image similarity – biometric identification based on face image. The work deals primarily with the face super-resolution from a sequence of low-resolution images and it compares this approach to the single-frame methods, that are still considered as the most accurate. A new dataset was created for this purpose, which is directly designed for the multi-frame face super-resolution methods from the low-resolution input sequence, and it is of comparable size with the leading world datasets. The results were evaluated by both a survey of human perception and defined objective metrics. A hypothesis that multi-frame methods achieve better results than single-frame methods was proved by a comparison of both methods. Architectures, source code and the dataset were released. That caused a creation of the basis for future research in this field.

L'arterial spin labelling (ASL) est une technique d'imagerie par résonance magnétique de la perfusion cérébrale. Les travaux présentés dans cette thèse ont d'abord consisté à standardiser les acquisitions ASL dans le contexte d'études de neuro-imagerie multicentriques. Un processus de contrôle de la qualité des images a par la suite été proposé. Les travaux se sont ensuite orientés vers le post-traitement de données ASL, en évaluant la capacité d'algorithmes existants à y corriger les distorsions. Des méthodes de super-résolution adaptées aux acquisitions ASL mono et multi-TI ont finalement été proposées et validées sur des données simulées, de sujets sains, ou de patients imagés pour suspicion de tumeurs cérébrales. / Arterial spin labelling (ASL) is a brain perfusion magnetic resonance imaging technique. The objective of this thesis was first to standardize ASL acquisitions in the context of multicenter neuroimaging studies. A quality control procedure has then been proposed. The capacity of existing algorithms to correct for distortions in ASL images has then been evaluated. Super-resolution methods, developed and adapted to single and multi-TI ASL data in the context of this thesis, are then described, and validated on simulated data, images acquired on healthy subjects, and on patients imaged for brain tumors.

Neisseria meningitidis, ou méningocoque, est une bactérie responsable de méningites et de septicémies, dont la forme la plus grave, purpura fulminans, est souvent fatale. Cette bactérie, qui réside naturellement dans le rhinopharynx de l’Homme, est pathogène lorsqu’elle atteint la circulation sanguine et entre en contact avec les cellules endothéliales. L’établissement d’une interaction étroite entre le méningocoque et les cellules endothéliales est essentiel à la résistance des bactéries au flux sanguin et à la colonisation vasculaire. Cette interaction peut conduire à une désorganisation massive des endothéliums périphériques et cérébraux permettant la dissémination de la bactérie. Ces processus dépendent de la primo-interaction des pili de type IV du méningocoque avec le récepteur endothélial CD147 et de l’activation du récepteur β2-adrénergique (β2AR). L’activation de voies de signalisation en aval du β2AR dans les cellules hôtes permet l’adhérence efficace et intime des bactéries à la surface des cellules endothéliales. Toutefois, comment les récepteurs CD147 et β2AR coopèrent pour promouvoir une interaction initiale efficace et rapide n’était pas connu. Au cours de ma thèse, j’ai donc analysé les éventuelles interactions et liens fonctionnels existants entre les récepteurs CD147 et β2AR et suivi, à l’aide de nouvelles approches d’imagerie à haute résolution, leur organisation moléculaire aux sites de contact bactéries/cellule. Mes travaux ont permis de révéler l’existence d’une interaction fonctionnelle entre les récepteurs CD147 et β2AR, et d’identifier un nouveau partenaire cytosolique interagissant directement avec ces récepteurs, l’α-actinine4 (Actn4). L’expression de l’Actn4 est requise pour l’assemblage organisé de ces récepteurs en complexes multimoléculaires aux sites de contact bactéries/cellule endothéliale. Cette organisation est déterminante pour générer une force suffisante à l’interaction initiale du méningocoque aux cellules endothéliales, et promouvoir l’activation rapide des voies de signalisation nécessaires à la consolidation de cette interaction. L’infection des cellules endothéliales par le méningocoque s’accompagne de la désorganisation des jonctions intercellulaires et l’ouverture d’une voie paracellulaire favorisant la dissémination tissulaire des bactéries. Ces évènements dépendent de l’activation de la petite GTPase Cdc42 et, en aval, de la relocalisation du complexe de polarité Par3/Par6/aPKC au site d’adhérence bactérien. Ce complexe moléculaire très conservé est impliqué dans la mise en place de la polarité baso-apicale des cellules endothéliales. La perte de la polarité cellulaire constituant un élément déterminant de la perte de l’intégrité vasculaire, dans une seconde partie de ma thèse, j’ai donc entrepris une analyse des événements de signalisation précoces conduisant au remodelage de l’organisation apico-basale des cellules endothéliales par N. meningitidis. Mes travaux montrent que rapidement après adhésion aux cellules endothéliales, le méningocoque induit la ré-orientation de l’axe de polarité noyau-centrosome des cellules en direction des bactéries et mettant en jeu un mécanisme original indépendant de l’activation de Cdc42 par le β2AR. Le recrutement des ERM (Ezrine et Moesin) et la polymérisation d’actine corticale au site d’infection semblent constituer des facteurs clé de cette étape précoce de modification de la polarité endothéliale induite par le méningocoque. Ainsi, ces études ont permis une avancée majeure dans notre compréhension du mécanisme d’adhésion du méningocoque aux cellules endothéliales et des événements moléculaires précoces conduisant à l’altération de l’intégrité vasculaire, deux étapes clés au cœur de la pathogénèse des infections invasives à méningocoque. / Neisseria meningitidis or meningococcus is a commensal bacterium of the human nasopharynx responsible for septicemia and meningitis. Establishment of a close interaction between meningococcus and endothelial cells is an important step in meningococcal pathogenesis as it promotes bacterial resistance to blood flow and vascular colonization, leading to major endothelial dysfunctions and bacterial dissemination into perivascular tissues. This process depends on the interaction of meningococcal type IV pili with the endothelial receptor CD147 and the activation of the β2 adrenergic receptor (β2AR). Activation of a cellular response downstream of β2AR activation is important to allow the efficient adhesion of meningococci at the endothelial cell surface. However, how CD147 and the β2AR cooperate to promote a rapid and efficient initial adhesion remained to be explored. During my thesis, I have analyzed the interaction and the functional link between these two receptors and, using super resolution microscopy, I have investigated their molecular organization at sites of bacterial adhesion. My work revealed a functional interaction in cis between CD147 and the β2AR and binding of these receptors complexes to the molecular scaffold protein α-actinin 4 (Actn4). Actn4 expression is required for the organized assembly of these receptors in highly-ordered complexes at bacterial adhesion sites. This specific organization is decisive to provide a sufficient binding strength of meningococcal type IV pili with endothelial receptors and to promote a rapid activation of downstream signaling events in a short time frame. Endothelial cell infection by N. meningitidis is associated with the disruption of intercellular junctions and the opening of a paracellular route favoring bacterial dissemination into tissues. These events are dependent on Cdc42 activation and on the relocalization of the Par3/Par6/aPKC polarity complex at bacterial adhesion sites. This molecular complex is conserved and involved in baso-apical polarity establishment in endothelial cells. Since endothelial polarity is essential in maintaining junction integrity, in a second part of my thesis work I have analyzed the early signaling events triggered by meningococcal infection with a particular emphasis on endothelial cell polarity modifications. I observed that bacterial adhesion rapidly induced a re-orientation of the nucleus-centrosome axis toward bacterial adhesion sites. Unexpectedly, this re-orientation was independent of Cdc42 activation downstream of the β2AR. In place, the ERM proteins (Ezrin and Moesin), along with cortical actin polymerization seem to be key factors in this process. This work contributes to the understanding of the meningococcal adhesion mechanism to endothelial cells and the early molecular events leading to the loss of vascular integrity. These two key steps are very important in the meningococcal pathogenesis.

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Cell wall mannans of Candida albicans mask β-(1,3)-glucan from recognition by Dectin-1, contributing to innate immune evasion. Glucan exposures are predominantly single receptor-ligand interaction sites of nanoscale dimensions. Candida species vary in basal glucan exposure and molecular complexity of mannans. We used super-resolution fluorescence imaging and a series of protein mannosylation mutants in C. albicans and C. glabrata to investigate the role of specific N-mannan features in regulating the nanoscale geometry of glucan exposure. Decreasing acid labile mannan abundance and α-(1,6)-mannan backbone length correlated most strongly with increased density and nanoscopic size of glucan exposures in C. albicans and C. glabrata, respectively. Additionally, a C. albicans clinical isolate with high glucan exposure produced similarly perturbed N-mannan structures and elevated glucan exposure geometry. Thus, acid labile mannan structure influences the nanoscale features of glucan exposure, impacting the nature of the pathogenic surface that triggers immunoreceptor engagement, aggregation, and signaling. Graus et al. find that N-mannan structural features regulated by Candida mannosyltransfersases control glucan exposure. Loss of mannan increased the frequency and size of glucan exposures and changed multivalent receptor engagement. Changes to mannan structure in a bloodstream isolate are associated with elevated glucan exposure at the nanoscale.

No description available.

Mitochondria are the power plant of most non-green eukaryotic cells. In order to understand mitochondrial functions and their regulation, knowledge of the spatiotemporal organization of their proteins is important. Mitochondrial membrane proteins can diffuse within membranes. They are involved in diverse functions e.g. protein import, cell respiration, metabolism, metabolite transport, fusion, fission or formation of the mitochondrial architecture. Furthermore, mitochondria compose of different subcompartments with different tasks. Especially, the inner mitochondrial membrane (IM), where the oxidative phosphorylation (OXPHOS) takes places, has a complex architecture with cristae extending into the matrix. The present work revealed the restricted localization of some mitochondrial proteins to specific membrane sections and linked it to their function or bioenergetic circumstances in the living cell. 
Single particle tracking (SPT) techniques like tracking and localization microscopy (TALM) allow to localize proteins with a precision below 20 nm. Additionally, tracking single proteins provides information about their mobility, dynamic and their spatiotemporal organization. TALM uses proteins, which were genetically tagged either with the HaloTag® (HaloTag) or the fSnapTag® (fSnapTag). These tags can be orthogonally and posttranslationally stained with specific and self-marking dyes. If the dyes are conjugated to the respective substrate of the tag. Single molecule labeling of mitochondrial proteins was performed substoichiometrically using membrane permeable rhodamine dyes, either tetramethylrhodamine (TMR) or silicon rhodamine (SiR). TALM allowed to localize proteins in different mitochondrial subcompartments. The gained trajectories and trajectory maps of mitochondrial proteins revealed their spatiotemporal organization. In the case of IM proteins like F1FO ATP synthase (Complex V - CV) a restricted diffusion in the CM, which is part of the continuous IM, was determined. The unimpeded diffusion of mitochondrial proteins in the outer mitochondrial membrane (OM) was compared with the mobility of IM proteins. The diffusion of mitochondrial IM proteins was restricted by the IM architecture and their diffusion coefficients were lower. Furthermore, significant differences of different mitochondrial IM proteins were compared, showing different localizations in the IM often coupled to their function, accompanied by different spatiotemporal organization and diffusion coefficients. Furthermore, a distinction was made between diffusion of proteins in the inner boundary membrane (IBM) and proteins that preferentially diffuse in the cristae membrane (CM). Evaluating trajectory maps, the different subcompartments in the IM were revealed by trajectories and the trajectory directionality, allowing the identification of mitochondrial proteins, which mark these subcompartments.
The morphology of mitochondria / mitochondrial networks and their bioenergetic parameters are linked to the metabolic states of the cell. In this work, the connection of the spatiotemporal protein organization of CV and the IM architecture was uncovered on the micro- and nanoscopic level and linked to the metabolic state of the cell. It was determined that the spatiotemporal organization of the CV was altered, when CV was inhibited. In addition, the bioenergetic influence of cells on the spatiotemporal behavior of CV and the reorganization of the IM architecture was investigated by TALM and compared with results of electron microscopy images. It was shown that starvation of cells led to a loss of cristae and thus to an increased mobility and spatiotemporal reorganization of CV. Taken together, the results presented in this work showed that a correctly functioning and active CV helps to maintain the IM architecture and both, the spatiotemporal organization of CV and the IM architecture were coupled to the metabolic state.. 
In order to investigate putative protein-protein interactions by colocalization and co-locomotion studies on single molecule level, dual color SPT is needed. Therefore, posttranslational and substoichimetric labeling as performed in TALM was tested for its potential of protein-protein interaction studies of mitochondrial membrane proteins. Here, a genetically double tagged translocase of the outer membrane subunit-20 (Tom20) (Tom20:HaloTag:fSnapTag) acted as a positive control. It turned out that substoichimetric, posttranslational labeling of mitochondrial proteins was not suitable for protein-protein interaction studies on mitochondrial proteins, because it was restricted by the low labeling degrees needed for TALM. However, dual-color TALM still allowed to study effects of proteins influencing the IM architecture and to study their influence on the spatiotemporal organization of CV. The co-transfection of Mic10, as the central protein of the mitochondrial inner membrane organizing system / mitochondrial contact site complex / mitochondrial organizing structure (MINOS / MICOS / MitOS (MINOS/MICOS)), altered the regular and aligned organization of the cristae. This was measured by a changed spatiotemporal organization of the CV, such as the loss of the perpendicular oriented of CV subunit-γ (CV-SUγ) cristae trajectories. In contrast to this, co-transfection of CV subunit-e (CV-SUe), important for dimerization of CV, increased the number of cristae trajectories. 
Mitochondria are three-dimensional (3D) cell organelles. Consequently, subcompartments like the IBM and CM are a 3D space in which CV is localized and diffuses. Thus, the diffusion of mitochondrial proteins is underestimated by two-dimensional SPT e.g. lateral confined diffusion can result from mitochondrial proteins diffusing along the z-axis of the microscope. In order to reveal the 3D spatiotemporal organization of CV, the potential of TALM to be extended to a 3D-SPT technique was investigated. Therto a cylindrical lens was installed in the emission path of a total internal reflection fluorescence (TIRF) microscope. This leads to an astigmatically distorted point spread function (PSF) of the fluorescent single molecule signals. This distortion allowed the reconstruction of single molecule localizations of CV to a superresolved image of the IM, in living cells. In addition, 3D-TALM enabled to display the 3D architecture of the IM by 3D trajectories of CV. 3D-TALM was able to detect whether CV diffuses in the IBM or in the CM, and extended the information about its mobility in the CM that it takes place in a disc-like manner. In this way it could be shown that CV is mobile within the cristae in all directions. Finally, 3D-TALM revealed an altered IM architecture caused by the metabolic state of the cell. As performed in two-dimensional TALM, the cells were kept under starving conditions. Here the now tubular IM architecture was revealed by 3D-TALM. The reversed metabolic state under improved respiratory conditions unexpectedly led to a more diverse IM architecture. These ultrastructural changes were also revealed by electron microscopy. Consequently, 3D-TALM enabled the study of IM architecture by tracking CV under different metabolic conditions, allowing an ultrastructural analysis of mitochondria in living cells. In addition, 3D TALM provided the spatiotemporal organization of CV under different metabolic conditions, so that the diffusion coefficients of CV could be related to changes in IM architecture caused by the metabolic condition.

Les neutrophiles sont les leucocytes les plus nombreux et les premières cellules à arriver au site de l’infection où elles internalisent les pathogènes par phagocytose. Dès le début du processus, la NADPH oxydase s’assemble au phagosome où elle permet la production de formes réactives de l’oxygène contribuant ainsi à la destruction du pathogène. La sous-unité catalytique membranaire de la NADPH oxydase, Nox2, est donc présente à la coupe phagocytaire puis au phagosome. Le dessein de cette étude était de déterminer quelles sont les sources subcellulaires de la protéine Nox2, de savoir si la protéine s’accumule au phagosome et le cas échéant selon quelle cinétique. Dans le but de comprendre la dynamique de la protéine Nox2, la protéine d’échafaudage IQGAP1 qui est associée au cytosquelette a également été étudiée. Enfin l’étude de l’organisation spatiale de la protéine Nox2 à la synapse phagocytaire a également été abordé.En utilisant des cellules neutrophil-like (PLB-985) ainsi que des neutrophiles humains, notre étude a montré par immunofluorescence la présence de la protéine Nox2 dans des endosomes de recyclage ou dans des endosomes précoces. Lors de la phagocytose ils avoisinent le phagosome suggérant leur implication dans l’apport de la protéine Nox2 à la membrane de ce dernier. L’utilisation de cellules PLB-985 pour lesquelles l’expression de Nox2 a été supprimée puis réintroduite avec un transgène codant pour la protéine GFP-Nox2 montre que la sous-unité Nox2 s’accumule au phagosome pendant les vingt minutes suivant sa fermeture. Dans notre étude, la protéine IQGAP1 ne semble pas avoir d’effet sur la phagocytose ou sur la production de FRO par la NADPH oxydase. Enfin, grâce à une technique de microscopie super-résolution (le dSTORM) l’évolution du pattern de Nox2 dans la membrane a été évalué au cours du temps en phagocytose frustrée. En dix minutes, le nombre de clusters de protéine Nox2 augmente mais leur taille reste inchangée. / Neutrophils are the most numerous leukocytes and the first cells to arrive at the site of infection where they internalize pathogens by phagocytosis. From the beginning of the process, the NADPH oxidase is assembled at the phagosome, where it allows the production of reactive oxygen species (ROS), thus contributing to the destruction of the pathogen. The membrane bound catalytic subunit of the NADPH oxidase, Nox2, is therefore recruited at the phagocytic cup and then at the phagosome. The purpose of this study was to determine, which are the subcellular sources of the Nox2 protein, whether the protein accumulates at the phagosome and if so, according to which kinetics. In order to modify the dynamics of the Nox2 protein, the scaffold protein IQGAP1 that is associated with the cytoskeleton was also studied. Finally, the spatial organization of the Nox2 protein in the phagocytic synapse was also investigated.Using neutrophil-like cells (PLB-985) as well as human neutrophils, our study showed by immunofluorescence the presence of the Nox2 protein in recycled or early endosomes. During phagocytosis, they are close to the phagosome, suggesting their involvement in the contribution of the Nox2 protein to the phagosome membrane. The use of PLB-985 for which Nox2 expression has been suppressed and then reintroduced with a transgene encoding the GFP-Nox2 protein shows that the Nox2 subunit accumulates at the phagosome during the first twenty minutes after its closure. In our study, the protein IQGAP1 does not appear to have any effect on phagocytosis or on the production of ROS by NADPH oxidase. Finally, using super resolution microscopy (dSTORM) the evolution of the Nox2 pattern in the membrane has been evaluated over time in frustrated phagocytosis. Within ten minutes, the number of Nox2 protein clusters increases but their size remains unchanged.

Ce travail de thèse propose d’étudier les propriétés photophysiques de systèmes moléculaires à la fois photochromes et fluorescents. Sous l’effet de la lumière, ces molécules peuvent subir une désexcitation radiative, non radiative ou une photo-isomérisation. Dans la première partie de ce travail, la compétition entre ces différentes voies a été étudiée en détail grâce à deux photochromes proches sur le plan structurel et présentant une émission de fluorescence de couleur différente pour chaque forme du photochrome. Les efficacités relatives et les mécanismes de désexcitation ont été étudiés à l’échelle macroscopique. Cette comparaison a été accompagnée d’une étude sous microscope pour laquelle des nano-bâtonnets d’or ont été intégrés au système photochrome-fluorescent de manière à étudier l’influence d’un champ plasmon de surface localisé sur les différents phénomènes de désexcitation à une échelle proche de celle de la nanoparticule individuelle. Une deuxième partie de ce travail consiste en la préparation et l’étude des propriétés photophysiques de nanoparticules organiques constituées de dyades fluorescentes et photochromes. La fluorescence de ce type de nano-objets peut être efficacement contrôlée par la lumière : la conversion de quelques unités photochromes peut permettre, par transfert d’énergie intermoléculaire, d’éteindre la totalité de la fluorescence d’une nanoparticule. L’observation de cet effet pour des nanoparticules individuelles a été mise en application pour le développement d’une méthode de microscopie optique super-résolution. / The main purpose of this PhD work is the study of fluorescent and photochromic molecular systems. Under light illumination, such molecules may undergo radiative and non-radiative deactivation or photoisomerization. In the first part of this work, this competition has been investigated for structurally related photochromic compounds with different fluorescence colors corresponding to both isomers. Efficiency and mechanisms of the deactivation pathways were unraveled at the macroscopic scale. This competition was further studied under microscope with gold nanorods included in the sample, in order to study the effect of localized surface plasmon resonance on the different deactivation processes.The second part of this work consists in the preparation and the photophysical study of organic nanoparticles composed of fluorescent-photochromic dyads. Fluorescence of such nano-objects can be efficiently driven by light : the switching of a few photochromic units is enough to turn off the entire fluorescence of a nanoparticle by intermolecular energy transfer. Observation of this effect at the level of individual nanoparticles allowed the developement of a super-resolution method for optical microscopy.