Cette thèse porte sur l’étude de la relation entre la structure et la fonction chez les cotransporteurs Na+/glucose (SGLTs). Les SGLTs sont des protéines membranaires qui se servent du gradient électrochimique transmembranaire du Na+ afin d’accumuler leurs substrats dans la cellule. Une mise en contexte présentera d’abord un bref résumé des connaissances actuelles dans le domaine, suivi par un survol des différentes techniques expérimentales utilisées dans le cadre de mes travaux. Ces travaux peuvent être divisés en trois projets. Un premier projet a porté sur les bases structurelles de la perméation de l’eau au travers des SGLTs. En utilisant à la fois des techniques de modélisation moléculaire, mais aussi la volumétrie en voltage imposé, nous avons identifié les bases structurelles de cette perméation. Ainsi, nous avons pu identifier in silico la présence d’une voie de perméation passive à l’eau traversant le cotransporteur, pour ensuite corroborer ces résultats à l’aide de mesures faites sur le cotransporteur Na/glucose humain (hSGLT1) exprimé dans les ovocytes. Un second projet a permis d’élucider certaines caractéristiques structurelles de hSGLT1 de par l’utilisation de la dipicrylamine (DPA), un accepteur de fluorescence dont la répartition dans la membrane lipidique dépend du potentiel membranaire. L’utilisation de la DPA, conjuguée aux techniques de fluorescence en voltage imposé et de FRET (fluorescence resonance energy transfer), a permis de démontrer la position extracellulaire d’une partie de la boucle 12-13 et le fait que hSGLT1 forme des dimères dont les sous-unités sont unies par un pont disulfure. Un dernier projet a eu pour but de caractériser les courants stationnaires et pré-stationaires d’un membre de la famille des SGLTs, soit le cotransporteur Na+/myo-inositol humain hSMIT2 afin de proposer un modèle cinétique qui décrit son fonctionnement. Nous avons démontré que la phlorizine inhibe mal les courants préstationnaires suite à une dépolarisation, et la présence de courants de fuite qui varient en fonction du temps, du potentiel membranaire et des substrats. Un algorithme de recuit simulé a été mis au point afin de permettre la détermination objective de la connectivité et des différents paramètres associés à la modélisation cinétique. / This thesis is about the structure/function relationship in Na+/glucose cotransporters (SGLTs). SGLTs are membrane proteins which use the Na+ transmembrane electrochemical gradient to accumulate their substrates within the cell. As an introduction, a short review of the current state of the field will be followed by a presentation of the different technics used in this work. This work can be divided in three main projects. In the first project, we investigated the structural basis of water permeation through SGLTs. By using molecular modeling technics, we have identified, in silico, a passive permeation pathway used by water to go through the cotransporter across the membrane. Using voltage-clamp volumetric measurement, we were able to corroborate these findings for hSGLT1 expressed in oocytes. A second project allowed elucidation of some of hSGLT1 structural characteristics through the use of dipicrylamine (DPA), a fluorescence acceptor whose repartition in the lipid membrane is voltage-dependant. Use of DPA concomitantly with voltage-clamp fluorescence and FRET (fluorescence resonance energy transfer) has clearly demonstrated the extracellular localisation of part of the 12-13 loop which was previously assumed to be intracellular. In addition, we have shown that hSGLT1 forms a dimeric structure where the subunits are linked by a disulfide bridge. A last project aimed at characterizing the steady-state and pre-steadystate currents of a member of the SGLT family named hSMIT2 (human Na/myo-inositol transporter 2). We showed that phlorizin is a poor inhibitor of pre-steady state currents following depolarisation, and the presence of a time, membrane potential and substrate dependent leak current. A simulated annealing algorithm was developed in order to allow objective determination of both the connectivity and the parameters associated with the optimal kinetic model.

L'imagerie TEP permet de mesurer et visualiser les changements de la distribution biologique des radiopharmaceutiques au sein des organes d'intérêt au court du temps. Ce suivi temporel offre des informations très utiles concernant les processus métaboliques et physiologiques sous-jacents, qui peuvent être extraites grâce à différentes techniques de modélisation cinétique. De plus, un autre avantage de la prise en compte de l'information temporelle dans les acquisitions TEP pour les examens en oncologie thoracique concerne le suivi des mouvements respiratoires. Ces acquisitions permettent de mettre en place des protocoles et des méthodologies visant à corriger leurs effets néfastes à la quantification, et les artefacts associés. L'objectif de ce projet est de développer une méthode de reconstruction permettant de combiner et mettre en oeuvre d'une part les corrections nécessaires à la quantification des données en TEP, et d'autre part la modélisation de la biodistribution du radiotraceur au cours du temps permettant d'obtenir des images paramétriques pour l'oncologie thoracique. Dans un premier temps, une méthodologie de correction des effets de volume partiel intégrant, dans le processus de reconstruction, une déconvolution de Lucy-Richardson associée à un débruitage dans le domaine des ondelettes, a été proposée. Une seconde étude a été consacrée au développement d'une méthodologie combinant une régularisation temporelle des données par l'intermédiaire d'un ensemble de fonctions de base temporelles, avec une méthode de correction des mouvements respiratoires basée sur un modèle élastique. Enfin, dans une troisième étape, le modèle cinétique de Patlak a été intégré dans un algorithme de reconstruction dynamique, et associé à la correction de mouvement afin de permettre la reconstruction directe d'images paramétriques de données thoraciques soumises au mouvement respiratoire. Les paramètres de transformation élastique pour la correction de mouvement ont été calculés à partir des images TEP d'intervalles synchronisés par rapport à l'amplitude de la respiration du patient. Des simulations Monte-Carlo d'un fantôme 4D géométrique avec plusieurs niveaux de statistiques, et du fantôme anthropomorphique NCAT intégrant des courbes d'activités temporelles réalistes pour les différents tissus, ont été réalisées afin de comparer les performances de la méthode de reconstruction paramétrique développée dans ce travail avec une approche 3D standard d'analyse cinétique. L'algorithme proposé a ensuite été testé sur des données cliniques de patients présentant un cancer bronchique non à petites cellules. Enfin, après la validation indépendante de l'algorithme de correction des effets de volume partiel d'une part, et de la reconstruction 4D incorporant la régularisation temporelle d'autre part, sur données simulées et cliniques, ces deux méthodologies ont été associées afin d'optimiser l'estimation de la fonction d'entrée à partir d'une région sanguine des images reconstruites. Les résultats de ce travail démontrent que l'approche de reconstruction paramétrique proposée permet de conserver un niveau de bruit stable dans les régions tumorales lorsque la statistique d'acquisition diminue, contrairement à l'approche d'estimation 3D pour laquelle le niveau de bruit constaté augmente. Ce résultat est intéressant dans l'optique d'une réduction de la durée des intervalles de la reconstruction 4D, permettant ainsi de réduire la durée totale de l'acquisition 4D. De plus, l'utilisation des fonctions d'entrée estimées avec les méthodes de régularisation temporelle proposées ont conduit à améliorer l'estimation des paramètres de Patlak. Enfin, la correction élastique du mouvement amène à une diminution du biais d'estimation des deux paramètres de Patlak, en particulier sur les tumeurs de petites dimensions situées dans des régions sensibles au mouvement respiratoire. / Positron emission tomography (PET) is now considered as the gold standard and the main tool for the diagnosis and therapeutic monitoring of oncology patients, especially due to its quantitative aspects. With the advent of multimodal imaging in combined PET and X-ray CT systems, many methodological developments have been proposed in both pre-processing and data acquisition, image reconstruction, as well as post-processing in order to improve the quantification in PET imaging. Another important aspect of PET imaging is its high temporal resolution and ability to perform dynamic acquisitions, benefiting from the high sensitivity achieved with current systems. PET imaging allows measuring and visualizing changes in the biological distribution of radiopharmaceuticals within the organ of interest over time. This time tracking provides valuable information to physicians on underlying metabolic and physiological processes, which can be extracted using pharmacokinetic modeling. The objective of this project is, by taking advantage of dynamic data in PET/CT imaging, to develop a reconstruction method combining in a single process all the correction methodology required to accurately quantify PET data and, at the same time, include a pharmacokinetic model within the reconstruction in order to create parametric images for applications in oncology. In a first step, a partial volume effect correction methodology integrating, within the reconstruction process, the Lucy-Richardson deconvolution algorithm associated with a wavelet-based denoising method has been introduced. A second study focused on the development of a 4D reconstruction methodology performing temporal regularization of the dataset through a set of temporal basis functions, associated with a respiratory motion correction method based on an elastic deformation model. Finally, in a third step, the Patlak kinetic model has been integrated in a dynamic image reconstruction algorithm and associated with the respiratory motion correction methodology in order to allow the direct reconstruction of parametric images from dynamic thoracic datasets affected by the respiratory motion. The elastic transformation parameters derived for the motion correction have been estimated from respiratory-gated PET images according to the amplitude of the patient respiratory cycle. Monte-carlo simulations of two phantoms, a 4D geometrical phantom, and the anthropomorphic NCAT phantom integrating realistic time activity curves for the different tissues, have been performed in order to compare the performances of the proposed 4D parametric reconstruction algorithm with a standard 3D kinetic analysis approach. The proposed algorithm has then been assessed on clinical datasets of several patients with non small cell lung carcinoma. Finally, following the prior validation of the partial volume effect correction algorithm on one hand, and the 4D reconstruction incorporating the temporal regularization on the other hand, on simulated and clinical datasets, these two methodologies have been associated within the 4D reconstruction algorithm in order to optimize the estimation of image derived input functions. The results of this work show that the proposed direct parametric approach allows to maintain a similar noise level in the tumor regions when the statistic decreases, contrary to the 3D estimation approach for which the observed noise level increases. This result suggests interesting perspectives for the reduction of frame duration reduction of 4D reconstruction, allowing a reduction of the total 4D acquisition duration. In addition, the use of input function estimated with the developed temporal regularization methods led to the improvement of the Patlak parameters estimation. Finally, the elastic respiratory motion correction led to a diminution of the estimation bias of both Patlak parameters, in particular for small lesions located in regions affected by the respiratory motion.

A presença de antibióticos no ecossistema representa um sério risco à saúde humana e animal em virtude do desenvolvimento crescente de resistência bacteriana. Uma vez que a maioria dos antibióticos é persistente à degradação biológica, os processos oxidativos avançados são apontados como uma das tecnologias mais efetivas para decompor esses compostos em águas residuárias. A bacitracina zíncica (Bc-Zn) é um potente antibiótico constituído por uma mistura complexa de peptídeos não-biodegradáveis, conjugados ao zinco. Apesar de ser um antibiótico amplamente consumido na medicina humana e animal, é preocupante a escassez de estudos que investigam sua degradação e destino ambiental. O presente trabalho analisou a degradação da Bc-Zn através dos processos de fotólise direta e UV/H2O2 em diferentes condições de radiação UVC e concentração inicial de H2O2. Os parâmetros cinéticos rendimento quântico da fotólise, constantes cinéticas de pseudo-primeira ordem e constante cinéticas de segunda ordem foram satisfatoriamente estimados pela modelagem do sistema fotoquímico experimental. Os resultados revelaram que a fotólise direta permitiu degradar todos os congêneres da mistura de Bc-Zn nas maiores doses de radiação UVC empregadas. No entanto, não houve remoção de TOC após 120 minutos de irradiação. A adição de H2O2 acelerou substancialmente a fotodegradação do antibiótico, apresentando constantes cinéticas de pseudo-primeira ordem uma ordem de grandeza superiores às obtidas por fotólise direta. Além disso, remoção considerável de até 71% do TOC foi alcançada. A análise estatística demonstrou que a radiação UV foi um fator bem mais significativo para a fotodegradação da Bc-Zn em relação à concentração inicial de H2O2, sendo as melhores condições do processo alcançadas para a maior taxa específica de emissão de fótons (1,11×10-5 Einstein L-1 s-1). Ensaios biológicos com soluções tratadas por fotólise direta e UV/H2O2 indicaram remoção completa da atividade antimicrobiana residual, ainda que os produtos da fotodegradação tenham se mostrado não-biodegradáveis. Análises de toxicidade indicaram que o metal zinco presente no antibiótico é responsável pela a toxicidade no micro-organismo-teste Vibrio fischeri. Estudos adicionais devem ser realizados para identificar os sub-produtos formados, bem como para investigar a degradação da Bc-Zn em efluentes industriais reais. / The presence of antibiotics in ecosystems represents a serious risk to human and animal health, caused by the increase in bacterial resistance. Since most antibiotics resist to biological degradation, advanced oxidation processes are pointed out as the most effective technologies for degrading these compounds in wastewater. Zinc bacitracin (Bc-Zn) is a potent antibiotic with a complex mixture of non-biodegradable peptides conjugated to zinc. Despite being a widely used antibiotic in human and animal medicine, the scarcity of studies dealing with its degradation and environmental fate is a matter of concern. In this work, Bc-Zn degradation by direct photolysis and the UV/H2O2 process was investigated for different UVC radiation conditions and initial H2O2 concentrations. Kinetic parameters, namely the photolysis quantum yield, pseudo-first order kinetic constants and second-order kinetic constants, were satisfactorily estimated from experimental data by modeling the photochemical system. The results showed that all the congeners of the Bc-Zn mixture were photolyzed at the highest UVC doses applied, while no TOC removal was observed after 120 minutes of irradiation. The addition of H2O2 substantially accelerated Bc-Zn photodegradation, with pseudo-first order kinetic constants of one order of magnitude higher than those observed under direct photolysis. In addition, a remarkable removal of up to 71% of TOC was achieved. Statistical analyses showed that UV radiation had a much more important effect on Bc-Zn photodegradation in comparison with initial H2O2 concentration, with the best process conditions achieved for the highest specific photon emission rate (1.11×10-5 Einstein L-1 s-1). Biological assays carried out with the solutions treated by direct photolysis and UV/H2O2 revealed no residual antimicrobial activity, though photodegradation products remained non-biodegradable. In addition, toxicity analyses indicated that the zinc metal present in the antibiotic is responsible for the toxic effect on the test microorganism Vibrio fischeri. Finally, further studies should be performed to identify the by-products formed and to investigate Bc-Zn degradation in real industrial wastewater.

A presença de antibióticos no ecossistema representa um sério risco à saúde humana e animal em virtude do desenvolvimento crescente de resistência bacteriana. Uma vez que a maioria dos antibióticos é persistente à degradação biológica, os processos oxidativos avançados são apontados como uma das tecnologias mais efetivas para decompor esses compostos em águas residuárias. A bacitracina zíncica (Bc-Zn) é um potente antibiótico constituído por uma mistura complexa de peptídeos não-biodegradáveis, conjugados ao zinco. Apesar de ser um antibiótico amplamente consumido na medicina humana e animal, é preocupante a escassez de estudos que investigam sua degradação e destino ambiental. O presente trabalho analisou a degradação da Bc-Zn através dos processos de fotólise direta e UV/H2O2 em diferentes condições de radiação UVC e concentração inicial de H2O2. Os parâmetros cinéticos rendimento quântico da fotólise, constantes cinéticas de pseudo-primeira ordem e constante cinéticas de segunda ordem foram satisfatoriamente estimados pela modelagem do sistema fotoquímico experimental. Os resultados revelaram que a fotólise direta permitiu degradar todos os congêneres da mistura de Bc-Zn nas maiores doses de radiação UVC empregadas. No entanto, não houve remoção de TOC após 120 minutos de irradiação. A adição de H2O2 acelerou substancialmente a fotodegradação do antibiótico, apresentando constantes cinéticas de pseudo-primeira ordem uma ordem de grandeza superiores às obtidas por fotólise direta. Além disso, remoção considerável de até 71% do TOC foi alcançada. A análise estatística demonstrou que a radiação UV foi um fator bem mais significativo para a fotodegradação da Bc-Zn em relação à concentração inicial de H2O2, sendo as melhores condições do processo alcançadas para a maior taxa específica de emissão de fótons (1,11×10-5 Einstein L-1 s-1). Ensaios biológicos com soluções tratadas por fotólise direta e UV/H2O2 indicaram remoção completa da atividade antimicrobiana residual, ainda que os produtos da fotodegradação tenham se mostrado não-biodegradáveis. Análises de toxicidade indicaram que o metal zinco presente no antibiótico é responsável pela a toxicidade no micro-organismo-teste Vibrio fischeri. Estudos adicionais devem ser realizados para identificar os sub-produtos formados, bem como para investigar a degradação da Bc-Zn em efluentes industriais reais. / The presence of antibiotics in ecosystems represents a serious risk to human and animal health, caused by the increase in bacterial resistance. Since most antibiotics resist to biological degradation, advanced oxidation processes are pointed out as the most effective technologies for degrading these compounds in wastewater. Zinc bacitracin (Bc-Zn) is a potent antibiotic with a complex mixture of non-biodegradable peptides conjugated to zinc. Despite being a widely used antibiotic in human and animal medicine, the scarcity of studies dealing with its degradation and environmental fate is a matter of concern. In this work, Bc-Zn degradation by direct photolysis and the UV/H2O2 process was investigated for different UVC radiation conditions and initial H2O2 concentrations. Kinetic parameters, namely the photolysis quantum yield, pseudo-first order kinetic constants and second-order kinetic constants, were satisfactorily estimated from experimental data by modeling the photochemical system. The results showed that all the congeners of the Bc-Zn mixture were photolyzed at the highest UVC doses applied, while no TOC removal was observed after 120 minutes of irradiation. The addition of H2O2 substantially accelerated Bc-Zn photodegradation, with pseudo-first order kinetic constants of one order of magnitude higher than those observed under direct photolysis. In addition, a remarkable removal of up to 71% of TOC was achieved. Statistical analyses showed that UV radiation had a much more important effect on Bc-Zn photodegradation in comparison with initial H2O2 concentration, with the best process conditions achieved for the highest specific photon emission rate (1.11×10-5 Einstein L-1 s-1). Biological assays carried out with the solutions treated by direct photolysis and UV/H2O2 revealed no residual antimicrobial activity, though photodegradation products remained non-biodegradable. In addition, toxicity analyses indicated that the zinc metal present in the antibiotic is responsible for the toxic effect on the test microorganism Vibrio fischeri. Finally, further studies should be performed to identify the by-products formed and to investigate Bc-Zn degradation in real industrial wastewater.

Cette thèse porte sur l’étude de la relation entre la structure et la fonction chez les cotransporteurs Na+/glucose (SGLTs). Les SGLTs sont des protéines membranaires qui se servent du gradient électrochimique transmembranaire du Na+ afin d’accumuler leurs substrats dans la cellule. Une mise en contexte présentera d’abord un bref résumé des connaissances actuelles dans le domaine, suivi par un survol des différentes techniques expérimentales utilisées dans le cadre de mes travaux. Ces travaux peuvent être divisés en trois projets. Un premier projet a porté sur les bases structurelles de la perméation de l’eau au travers des SGLTs. En utilisant à la fois des techniques de modélisation moléculaire, mais aussi la volumétrie en voltage imposé, nous avons identifié les bases structurelles de cette perméation. Ainsi, nous avons pu identifier in silico la présence d’une voie de perméation passive à l’eau traversant le cotransporteur, pour ensuite corroborer ces résultats à l’aide de mesures faites sur le cotransporteur Na/glucose humain (hSGLT1) exprimé dans les ovocytes. Un second projet a permis d’élucider certaines caractéristiques structurelles de hSGLT1 de par l’utilisation de la dipicrylamine (DPA), un accepteur de fluorescence dont la répartition dans la membrane lipidique dépend du potentiel membranaire. L’utilisation de la DPA, conjuguée aux techniques de fluorescence en voltage imposé et de FRET (fluorescence resonance energy transfer), a permis de démontrer la position extracellulaire d’une partie de la boucle 12-13 et le fait que hSGLT1 forme des dimères dont les sous-unités sont unies par un pont disulfure. Un dernier projet a eu pour but de caractériser les courants stationnaires et pré-stationaires d’un membre de la famille des SGLTs, soit le cotransporteur Na+/myo-inositol humain hSMIT2 afin de proposer un modèle cinétique qui décrit son fonctionnement. Nous avons démontré que la phlorizine inhibe mal les courants préstationnaires suite à une dépolarisation, et la présence de courants de fuite qui varient en fonction du temps, du potentiel membranaire et des substrats. Un algorithme de recuit simulé a été mis au point afin de permettre la détermination objective de la connectivité et des différents paramètres associés à la modélisation cinétique. / This thesis is about the structure/function relationship in Na+/glucose cotransporters (SGLTs). SGLTs are membrane proteins which use the Na+ transmembrane electrochemical gradient to accumulate their substrates within the cell. As an introduction, a short review of the current state of the field will be followed by a presentation of the different technics used in this work. This work can be divided in three main projects. In the first project, we investigated the structural basis of water permeation through SGLTs. By using molecular modeling technics, we have identified, in silico, a passive permeation pathway used by water to go through the cotransporter across the membrane. Using voltage-clamp volumetric measurement, we were able to corroborate these findings for hSGLT1 expressed in oocytes. A second project allowed elucidation of some of hSGLT1 structural characteristics through the use of dipicrylamine (DPA), a fluorescence acceptor whose repartition in the lipid membrane is voltage-dependant. Use of DPA concomitantly with voltage-clamp fluorescence and FRET (fluorescence resonance energy transfer) has clearly demonstrated the extracellular localisation of part of the 12-13 loop which was previously assumed to be intracellular. In addition, we have shown that hSGLT1 forms a dimeric structure where the subunits are linked by a disulfide bridge. A last project aimed at characterizing the steady-state and pre-steadystate currents of a member of the SGLT family named hSMIT2 (human Na/myo-inositol transporter 2). We showed that phlorizin is a poor inhibitor of pre-steady state currents following depolarisation, and the presence of a time, membrane potential and substrate dependent leak current. A simulated annealing algorithm was developed in order to allow objective determination of both the connectivity and the parameters associated with the optimal kinetic model.

Ce travail a pour objectif général de développer une compréhension approfondie de l’interaction du CO avec des catalyseurs anodiques dans les piles à combustible de type PEM (PEMFC), et d’évaluer son impact vis-à-vis de leur réactivité et stabilité lors de l’oxydation de l’hydrogène. Premièrement un modèle physique multi-échelle a été conçu pour simuler les performances de piles PEMFC alimentées par de l’hydrogène contenant des traces de CO. Il est basé sur la simulation Monte Carlo et la modélisation cinétique des étapes électrochimiques/chimie élémentaires. Une étude expérimentale de l’adsorption et de l’oxydation de CO simulant la technique d’ « O2 bleeding » a été utilisée pour mieux comprendre les mécanismes. Des catalyseurs de Pt ainsi que des bimétalliques PtxCoy et PtRu, supportés sur du carbone de grande aire spécifique, ont été étudiés. La spectroscopie IR (DRIFTS) et l’analyse QMS ont été utilisées pour l’étude de l’adsorption et oxydation de CO. Les défauts de surface, l’historique du catalyseur dans son interaction avec les différents gaz (H2, O2, CO), la température, la charge en Pt, la taille des particules, l’alliage de Pt avec Co ou Ru se sont révélé des paramètres clés dans la réactivité de CO avec O2. Le modèle multi-échelle a été appliqué aux catalyseurs Pt et PtxCoy. Les catalyseurs PtxCoy se révèlent plus tolérants au CO mais, en fonction du rapport Pt/Co, ils peuvent se dégrader par dissolution de Co comme démontré par nos expériences / The general objective of this work is to develop a deep understanding of the interaction of the CO with anodic catalysts in PEM Fuel Cells (PEMFCs), and to evaluate its impact on the reactivity towards the hydrogen oxidation and their stability. Firstly, a multiscale kinetic model is built up based on Monte Carlo simulation and kinetic modelling of elementary electrochemical/chemical steps as a tool to simulate the performance of PEMFCs fed with H2 containing CO traces. Experiments on CO adsorption and oxidation mimicking O2 bleeding were used to better understand the mechanisms. Monometallic Pt and bimetallic PtxCoy and PtRu catalysts supported on high surface area carbon were studied. CO adsorption and oxidation were investigated by means of DRIFT spectroscopy and QMS analysis. Defect sites (kink, edge), history of interaction with different gases (H2, O2, CO), temperature, Pt loading, particle size, alloying with Co or Ru are key parameters influencing the CO reactivity with O2. The multiscale kinetic model was applied to Pt and PtxCoy. PtxCoy nanocatalysts are shown to be highly CO tolerant but might degrade by Co dissolution in long term operation, depending on the Pt to Co ratio