Un système d'échangeur à contre-courant, utilisant un carburant –endothermique-, est envisagé pour répondre au refroidissement d’un super statoréacteur. La mesure du débit de carburant décomposé et chauffé ainsi que celle de la composition chimique est nécessaire. Ceci permettra de contrôler et optimiser le fonctionnement d’un tel moteur, puisque sa poussée est principalement déterminée par la nature et le débit des espèces brûlées. La mise en place d’une méthode de caractérisation du fluide fait l’objet de cette étude. L’utilisation d’un spectromètre infrarouge à transformée de Fourier permet une analyse non intrusive des produits de pyrolyse. Cette technique est associée à un banc expérimental permettant la dégradation thermique d’un hydrocarbure en condition supercritique (jusque 60 bar, de 550°C à 900°C). Une méthode est réalisée pour obtenir une mesure quantitative et en ligne des produits majoritaires de la phase gazeuse. Celle-ci est basée sur un étalonnage avec les gaz majoritaires (méthane, éthylène, éthane, propylène et propane) et un traitement quantitatif de type CLS (Classical Least Square). La méthode est ensuite appliquée à l’étude de la pyrolyse en condition supercritique de différents hydrocarbures (alcanes linéaires purs et kérosènes du commerce). Les résultats de cette méthode sont comparés à ceux donnés par une technique chromatographique, et présentent de bonnes concordances (généralement moins de 5 % d'écart). Une approche en transitoire de la décomposition du n-dodécane par spectroscopie infrarouge a permis de suivre qualitativement l’évolution des produits de pyrolyse. / A system of heat hexchanger against the current, using an endothermic fuel, is considered to answer the cooling of a SCRAMJET engine. The measure of the mass flow rate of pyrolysed and heated fuel as well as the chemical composition is necessary. This will allow to control and to optimize the functioning of such engine, as his thrust is in most cases determined by the nature and the flow rate of burned species. This study bears on the development of a method of characterization of fluid. The use of a Fourier transformed infrared spectrometer allows a not interfering analysis of the products of pyrolysis. This technology is linked to an experimental bench allowing the thermal degradation of a hydrocarbon in supercritical condition (to 60 bar, from 550°C to 900°C). A method is accomplished to acquire a quantitative and online measure of the majority products of gaseous phase. This one is based on a calibration with majority gases (methane, ethylene, ethane, propylene and propane) and a quantitative treatment of CLS type (Classical Least Square). Method is then applied to study pyrolysis in supercritical condition of different hydrocarbons (linear alkanes and kerosenes of trade). The results of this method are compared with those given by a chromatographic technique, and introduce good agreement (less than 5 % of distance). An approach in transitional of the decomposition of the n-dodécane by infrared spectroscopy allowed to follow qualitatively the evolution of the pyrolysis products.

Os indicadores de desempenho têm papel fundamental na gestão das organizações, pois mostram aos decisores a situação da organização e como ela se encontra em relação a seus objetivos. Entre os sistemas de indicadores de desempenho utilizado nas organizações, têm tido destaque a conjugação de métricas financeiras e não financeiras, baseada na crença de que a melhora nos resultados não financeiros irá ocasionar a melhora nos índices financeiros. Entretanto, não há uma metodologia consagrada para testar se esses relacionamentos (relações de causa-e-efeito) existem na realidade. O objetivo desse trabalho foi propor e validar parcialmente uma metodologia para testar e quantificar as relações causais entre indicadores de desempenho. A seqüência de passos foi definida a partir da literatura através da implementação de ferramentas do Desdobramento da Função Qualidade, Gerenciamento pelas Diretrizes, Pensamento Sistêmico e Ferramenta para Seleção de Planos de Ação. O método escolhido para sua validação parcial foi o estudo de caso. A unidade de análise foi uma organização que já utiliza métricas financeiras e não financeiras e possui base histórica de dados. A pesquisa utilizou como fontes de evidência a observação participante e entrevista estruturada. Para a análise dos dados foram utilizadas técnicas estatísticas e representação escrita. Os resultados permitem concluir que a metodologia consegue quantificar as relações causais entre métricas de desempenho. A aplicação também gerou grande aprendizado organizacional. A principal contribuição desse trabalho é o modelo conceitual parcialmente validado o qual pode ser utilizado para transformar o sistema de indicadores de desempenho em fonte de informações para a tomada de decisão através da quantificação das relações de causa-e-efeito. / Performance metrics have a fundamental role in organizations, because they show to decision makers the situation of the organization in relation to its objectives. Most of the metrics systems used have financial and non-financial indicators, based on the belief that if a non-financial performance is increased, it will cause the same behavior in financial results. On the other hand, there is not a consecrated methodology to test if these relationships (causal relations) exist in the real world. The objective of this paper is to propose and partially validate a methodology to test and quantify the causal relations among performance metrics. A sequence of steps was defined from literature research, using tools from Quality Function Deployment, Policy Deployment, System Dynamics and Tool for Action Planning Selection. The research method chosen was case study. The research unit was an organization that already uses financial and non-financial metrics and has historic data of it. As font of evidences, were used participant observation and structure interviews. Data analysis was made with statistical techniques and written representation. From the results, it can be concluded using the methodology it’s possible to quantify the causal relations between performance metrics. The application of this methodology also contributed the organizations learning. The mainly contribution of this paper is the partially validated conceptual methodology, that can be used to make the performance metric system a information source to decision making, trough the quantification of causal relations.

L’eau constitue un des défis majeurs de notre époque quelque soit le continent, et à l’instar de nombreux pays de la rive sud de la méditerranée, le Maroc est particulièrement affecté par une pénurie d’eau. Il apparaît que pour pallier ce manque d’eau il est fait appel à la grande hydraulique (128 barrages au Maroc) pour alimenter, à partir des régions les plus arrosées, des bassins versants les plus propices ou des régions riches en eau souterraine, les villes principales ou des périmètres agricoles irrigués de grande surface. Néanmoins cette politique de grande hydraulique qui prévaut actuellement se heurte à plusieurs limites (durée limitée des réservoirs, nombres de sites propices, investissements) si bien qu’un des leviers prometteurs reste une gestion efficiente des ressources en eau. Cependant, pour bien les gérer il est essentiel de connaître la quantité d’eau disponible dans chaque bassin ainsi que ses différentes utilisations. Ce travail de thèse est une contribution à la connaissance de la ressource en eau sur un territoire clef. Le Moyen-Atlas, que l’on peut considérer comme le "château d’eau" du Maroc. Une démarche géographique est mise en place, tant au niveau de l’évaluation de la ressource que celle de l’utilisation, sur une des trois grandes unités hydrologiques qui draine le Moyen-Atlas : le bassin versant du Sebou à l’amont de la station hydrométrique d’Azzaba. Organisé en 4 parties ce travail, inscrit dans un cadre de coopération franco-marocain, démontre l’hétérogénéité des processus hydrologiques en relation avec un contexte géographique montagnard complexe. Tant l’analyse des phases hydrologiques que l’apport de la modélisation démontrent une contribution importante des eaux souterraines aux débits des cours d’eau mais également l’importance de sa redistribution à l’intérieur du bassin du Haut Sebou et vers les bassins contigus. D’autre part une enquête auprès des agriculteurs démontre les mutations agricoles en cours dans ce secteur du Moyen-Atlas et met en évidence les changements des modes de prélèvement d’eau dans l’hydrosystème : le système traditionnel de prélèvement au fil de l’eau étant de plus en plus concurrencé par des prélèvements à même la nappe par pompage / Water is one of the major issues of our time in all continents. Like many countries on the southern shore of the Mediterranean Sea, Morocco is particularly affected by water shortages. It seems that to overcome this lack of water, the country uses large hydro (128 dams in Morocco) to power, from the regions that receive the largest amounts of rain, or regions richest in groundwater, major cities and agricultural areas irrigated large area. However, this large and predominant hydraulic policy faces such several limitations (limited life of reservoirs, number of sites suitable, and investment) that promising levers remain efficient ways of managing water resources. However, to effectively manage this, it is essential to know the amount of water available in each basin and its different uses. This thesis is a contribution to the knowledge of water resources in a key area. The Middle Atlas can be considered the "water tower" of Morocco. A geographical approach is in place, both in terms of the resource assessment and use of one of three major hydrological units that drains the Middle Atlas: the watershed of the Sebou up stream of the hydrometric station Azzaba. set in 4 parts, this work- being of the Franco-Moroccan academic cooperation- demonstrates the heterogeneity of hydrological processes in relation to a complex mountainous geographical context. Both the analysis of hydrological phases and the contribution of modeling show a significant contribution of groundwater to stream flow, the importance of redistribution within the basin of the Upper Sebu and from adjacent basins. In this regard, research in the form of survey aimed at apopulation farmers shows agricultural changes forming in the Middle Atlas sector, and highlights the changes in water sampling modes in river systems, the traditional sampling system over water being increasingly challenged by levies same groundwater pumping.

Ce travail porte sur la construction d'un modèle réduit en hydraulique fluviale avec une méthode de décomposition en polynôme du chaos. Ce modèle réduit remplace le modèle direct afin de réduire le coût de calcul lié aux méthodes ensemblistes en quantification d'incertitudes et assimilation de données. Le contexte de l'étude est la prévision des crues et la gestion de la ressource en eau. Ce manuscrit est composé de cinq parties, chacune divisée en chapitres. La première partie présente un état de l'art des travaux en quantification des incertitudes et en assimilation de données dans le domaine de l'hydraulique ainsi que les objectifs de la thèse. On présente le cadre de la prévision des crues, ses enjeux et les outils dont on dispose pour prévoir la dynamique des rivières. On présente notamment la future mission SWOT qui a pour but de mesurer les hauteurs d'eau dans les rivières avec un couverture globale à haute résolution. On précise notamment l'apport de ces mesures et leur complémentarité avec les mesures in-situ. La deuxième partie présente les équations de Saint-Venant, qui décrivent les écoulements dans les rivières, ainsi qu'une discrétisation numérique de ces équations, telle qu'implémentée dans le logiciel Mascaret-1D. Le dernier chapitre de cette partie propose des simplifications des équations de Saint-Venant. La troisième partie de ce manuscrit présente les méthodes de quantification et de réduction des incertitudes. On présente notamment le contexte probabiliste de la quantification d'incertitudes et d'analyse de sensibilité. On propose ensuite de réduire la dimension d'un problème stochastique quand on traite de champs aléatoires. Les méthodes de décomposition en polynômes du chaos sont ensuite présentées. Cette partie dédiée à la méthodologie s'achève par un chapitre consacré à l'assimilation de données ensemblistes et à l'utilisation des modèles réduits dans ce cadre. La quatrième partie de ce manuscrit est dédiée aux résultats. On commence par identifier les sources d'incertitudes en hydraulique que l'on s'attache à quantifier et réduire par la suite. Un article en cours de révision détaille la validation d'un modèle réduit pour les équations de Saint-Venant en régime stationnaire lorsque l'incertitude est majoritairement portée par les coefficients de frottement et le débit à l'amont. On montre que les moments statistiques, la densité de probabilité et la matrice de covariances spatiales pour la hauteur d'eau sont efficacement et précisément estimés à l'aide du modèle réduit dont la construction ne nécessite que quelques dizaines d'intégrations du modèle direct. On met à profit l'utilisation du modèle réduit pour réduire le coût de calcul du filtre de Kalman d'Ensemble dans le cadre d'un exercice d'assimilation de données synthétiques de type SWOT. On s'intéresse précisément à la représentation spatiale de la donnée telle que vue par SWOT: couverture globale du réseau, moyennage spatial entre les pixels observés. On montre notamment qu'à budget de calcul donné les résultats de l'analyse d'assimilation de données qui repose sur l'utilisation du modèle réduit sont meilleurs que ceux obtenus avec le filtre classique. On s'intéresse enfin à la construction du modèle réduit en régime instationnaire. On suppose ici que l'incertitude est liée aux coefficients de frottement. Il s'agit à présent de juger de la nécessité du recalcul des coefficients polynomiaux au fil du temps et des cycles d'assimilation de données. Pour ce travail seul des données in-situ ont été considérées. On suppose dans un deuxième temps que l'incertitude est portée par le débit en amont du réseau, qui est un vecteur temporel. On procède à une décomposition de type Karhunen-Loève pour réduire la taille de l'espace incertain aux trois premiers modes. Nous sommes ainsi en mesure de mener à bien un exercice d'assimilation de données. Pour finir, les conclusions et les perspectives de ce travail sont présentées en cinquième partie.

As imagens de Angiografia por Ressonância Magnética (angio-RM) e Tomografia Computadorizada (angio-TC) são ferramentas amplamente usadas em processos de quantificação vascular e no diagnóstico de doenças cardiovasculares, as quais são consideradas entre as principais causas de morte. Contudo, a análise dos vasos em larga escala a partir das imagens é dificultada, tanto pela variabilidade natural dos vasos no corpo humano, quanto pela grande quantidade de dados disponíveis. Além disso, os métodos de quantificação existentes, usualmente extraem as características a partir dos esqueletos, ou até mesmo das próprias imagens de angiografia, razão pela qual tais métodos podem fazer necessária a reanálise das imagens repetidas vezes. Com o intuito de facilitar a análise e de fornecer uma ferramenta de apoio ao diagnóstico, neste trabalho são apresentados um modelo de representação textual de redes vasculares e uma metodologia de quantificação vascular automática, que é feita a partir dessa representação. A representação é obtida a partir da segmentação de imagens volumétricas de angio-RM e angio-TC, seguida da extração de trajetórias e diâmetros de redes vasculares. Tal representação é híbrida, combinando grafos e uma sequência textual de instruções, e permite não apenas a extração de caraterísticas morfológicas da rede vascular, como também a compressão das imagens e, ainda, a reconstrução de imagens similares às imagens originais. A partir das características extraídas, foram realizados estudos comparativos entre arquiteturas vasculares, o que é feito tanto por meio do uso de imagens sintéticas, como por meio de imagens reais, imagens nas quais foi possível encontrar diferenças entre arquiteturas, além de viabilizar a caracterização de aneurismas em um indivíduo. Paralelamente, desenvolvemos um método que permite identificar similaridade entre segmentos vasculares, o que por sua vez possibilita o reconhecimento e rotulação de segmentos em um conjunto de redes vasculares. A metodologia por nós desenvolvida deve também auxiliar no desenvolvimento de processos de classificação de vasos sanguíneos, de ferramentas para o diagnóstico automático de doenças vasculares, e para a melhora de técnicas utilizadas na prática clínica. / Magnetic Resonance Angiography (angio-MR) and Computed Tomography Angiography (angio-TC) are widely used imaging techniques used for vascular quantification and the diagnosis of cardiovascular diseases, considered one of the main causes of death. Blood vessel analysis using angiographic images is intrisically difficult because of the natural human vessel variability and the large amount of information. Additionally, most quantification methods perform the analysis of entire datasets, which can be very time consuming when they need to be reanalyzed. With the aim of reducing these problems and to provide a tool to aid diagnosis, we propose a textual representation model for vascular networks and an automatic vascular quantification methodology. The representation is obtained from volumetric image segmentation of angio-MR and angio-TC, followed by the extraction of the trajectory and the blood vessel diameters. This representation is hybrid in nature, combining graphs and a sequence of textual instructions, allowing for the extraction of morphological features, image compression, and the synthesis of angiographic images. Using extracted features derived from the model, comparative studies of vascular architecture can be performed. Experiments were made using synthetic and real images, in which was possible to find structural differences that make feasible to characterize abnormalities such as aneurysms. Also, a vessel similarity identification method was developed, which makes it possible to recognize vessel segments and label them in a set of vessel networks. The proposed methodology should aid in blood vessel classification processes, automatic diagnosis of cardiovascular diseases, and allow development of methods and applications that could be used in the clinical practice.

Os indicadores de desempenho têm papel fundamental na gestão das organizações, pois mostram aos decisores a situação da organização e como ela se encontra em relação a seus objetivos. Entre os sistemas de indicadores de desempenho utilizado nas organizações, têm tido destaque a conjugação de métricas financeiras e não financeiras, baseada na crença de que a melhora nos resultados não financeiros irá ocasionar a melhora nos índices financeiros. Entretanto, não há uma metodologia consagrada para testar se esses relacionamentos (relações de causa-e-efeito) existem na realidade. O objetivo desse trabalho foi propor e validar parcialmente uma metodologia para testar e quantificar as relações causais entre indicadores de desempenho. A seqüência de passos foi definida a partir da literatura através da implementação de ferramentas do Desdobramento da Função Qualidade, Gerenciamento pelas Diretrizes, Pensamento Sistêmico e Ferramenta para Seleção de Planos de Ação. O método escolhido para sua validação parcial foi o estudo de caso. A unidade de análise foi uma organização que já utiliza métricas financeiras e não financeiras e possui base histórica de dados. A pesquisa utilizou como fontes de evidência a observação participante e entrevista estruturada. Para a análise dos dados foram utilizadas técnicas estatísticas e representação escrita. Os resultados permitem concluir que a metodologia consegue quantificar as relações causais entre métricas de desempenho. A aplicação também gerou grande aprendizado organizacional. A principal contribuição desse trabalho é o modelo conceitual parcialmente validado o qual pode ser utilizado para transformar o sistema de indicadores de desempenho em fonte de informações para a tomada de decisão através da quantificação das relações de causa-e-efeito. / Performance metrics have a fundamental role in organizations, because they show to decision makers the situation of the organization in relation to its objectives. Most of the metrics systems used have financial and non-financial indicators, based on the belief that if a non-financial performance is increased, it will cause the same behavior in financial results. On the other hand, there is not a consecrated methodology to test if these relationships (causal relations) exist in the real world. The objective of this paper is to propose and partially validate a methodology to test and quantify the causal relations among performance metrics. A sequence of steps was defined from literature research, using tools from Quality Function Deployment, Policy Deployment, System Dynamics and Tool for Action Planning Selection. The research method chosen was case study. The research unit was an organization that already uses financial and non-financial metrics and has historic data of it. As font of evidences, were used participant observation and structure interviews. Data analysis was made with statistical techniques and written representation. From the results, it can be concluded using the methodology it’s possible to quantify the causal relations between performance metrics. The application of this methodology also contributed the organizations learning. The mainly contribution of this paper is the partially validated conceptual methodology, that can be used to make the performance metric system a information source to decision making, trough the quantification of causal relations.

Quimicamente, o ecstasy é a 3,4-metilenodioximetanfetamina (MDMA), um composto sintético com propriedades estimulante central e alucinogênicas. Algumas substâncias análogas à MDMA, já identificadas em comprimidos de ecstasy, são principalmente: 3,4-metilenodioxietilanfetamina (MDEA), 3,4-metilenodioxianfetamina (MDA), metanfetamina e anfetamina. Os adulterantes mais comuns, normalmente encontrados são: cafeína e efedrinas. O objetivo deste trabalho foi a validação de um método analítico para quantificar a MDMA em comprimidos e cápsulas de ecstasy, através da cromatografia em fase gasosa com detector de nitrogênio/fósforo (GC/NPD). Substâncias análogas à MDMA e adulterantes também foram identificados. Amostras de comprimidos e cápsulas de 25 diferentes lotes, apreendidos como ecstasy em São Paulo (SP), foram analisadas pelo método proposto. Desse total de amostras, 21 continham somente MDMA (84%) e apenas 1 delas apresentou MDMA associada com cafeína (4%). A concentração total de MDMA nessas amostras variou entre 30,9 e 92,7mg, resultando em uma média aritmética de 63mg. / Chemically, \"ecstasy\" is 3,4-methylenedioxymethamphetamine (MDMA), a synthetic compound with stimulant and hallucinogenic properties. Some MDMA analog substances such as 3,4-methylenedioxyethylamphetamine (MDEA), 3,4-methylenedioxyamphetamine (MDA), methamphetamine and amphetamine have already been identified in \"ecstasy\" tablets. Caffeine and ephedrines are the most common adulterants also found. The aim of this paper is to describe the validation of an analytical method to quantify MDMA in \"ecstasy\" tablets and capsules. Gas chromatography with nitrogen/phosphorus detector was used in the method. Analog substances to MDMA and adulterant compounds were also identified. Samples from 25 lots of tablets seized in the city of São Paulo were analyzed. From that total, 21 showed only MDMA (84%) and just 1 of them presented MDMA plus caffeine (4%). MDMA total concentration in these samples had a variation between 30.9 and 92.7mg, resulting in an arithmetic average of 63mg.

Orientador: Adriana Vitorino Rossi / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-20T21:18:58Z (GMT). No. of bitstreams: 1 Souza_PatriciaTononde_M.pdf: 1500158 bytes, checksum: 99c73fcb4e4c8143227e13cef0571323 (MD5) Previous issue date: 2012 / Resumo: Capsaicinóides (CAPS) são substâncias responsáveis pela pungência em pimentas do gênero Capsicum. Quantificar CAPS é relevante devido às aplicações desses compostos na indústria alimentícia, farmacêutica e cosmética, além do uso em armas químicas. Nesse trabalho, foi desenvolvido método espectrofotométrico baseado na reação colorimétrica entre CAPS, Co e 4-(2-piridilazo)resorcinol, PAR, para quantificação de CAPS totais (CCAPS) em diversas espécies de Capsicum. O produto da reação tem proporção estequiométrica Co:PAR:CAPS 1:2:2 e absorção em 510 nm proporcional a CCAPS. A curva analítica apresentou faixa linear entre 0,001 e 0,03 U, limite de detecção = 0,0004 U e limite de quantificação = 0,0005 U (U = mg CAPS/g pimenta) e coeficiente de correlação = 0,9979. Os resultados foram comparados com o método de cromatografia líquida de alta eficiência (HPLC) e não apresentaram diferença significativa com 95% de confiança, além disso, ambos resultados são coerentes com as escalas organolépticas disponíveis na literatura. No aperfeiçoamento das condições para obtenção de extratos etanólicos de CAPS, estabeleceu-se o uso de cachaça (teor alcoólico 39%), proporção pimenta:cachaça 30:50 g/mL e agitação de 30 min. O estudo da conservação dos extratos indicou que é favorável armazená-los a 6°C, a estocagem das pimentas in natura a -11 °C por até três meses não leva a perda significativa de CAPS e a CCAPS aumenta a medida que o estágio de maturação de pimenta é completado / Abstract: Capsaicinoids (CAPS) are substances responsible for pungency in Capsicum. CAPS is relevant to quantify due to the applications of these comounds in food, pharmaceutical and cosmetic industries, and the use of chemical weapons. Inthis work, we developed a spectrophotometric method based on the colorimetric reaction between CAPS, Co and 4-(2-pyridylazo)resorcinol, PAR, for quantification of total CAPS (CCAPS) in several species of Capsicum. The product of the reaction is stoichiometric Co:PAR:CAPS 1:2:2 and absorption at 510 nm proportional to CCAPS. The analytical curve linear range between 0,001 and 0,03 U, detection limit = 0,0004 and limit of quantification U = 0,0005 U (U = mg CAPS/g pepper) and correlation coefficient = 0,9979. The results were compared with the method of high performance liquid chromatography (HPLC) and not significantly different at 95% confidence, moreover, both results are consistent with the organoleptic scales available in the literature. The improvement of conditions for obtaining ethanol extracts of CAPS, established the use of cachaça (alcohol content 39%), proportion pepper:cachaça 30:50 g/mL and stirred for 30 min. The study of the conservation of the extracts indicated that it supports storing them at 6 °C, the storage of in nature peppers at -11 °C for up to three months does not lead to significant loss of CAPS and CCAPS increases as the stage of maturation of pepper is added / Mestrado / Quimica Analitica / Mestra em Química

Les biofilms représentent une contrainte technologique dans le secteur industriel et sont à l'origine de nombreux cas d'infections chroniques dans le domaine médical. De ce fait, la compréhension des processus biologiques qui s'opèrent au sein de ces communautés microbiennes est un défi permanent. Des méthodes spécifiques, sensibles et diversifiées s'avèrent essentielles pour apporter une connaissance approfondie de l'organisation des biofilms en réponse à des facteurs environnementaux. La matrice extracellulaire qui englobe les microorganismes constitue un réseau complexe, et la description de sa composition et de sa structure constitue un enjeu majeur. Des outils de caractérisation in situ et non-destructifs des Substances ExoPolymériques (SEP) ont pu être proposés lors de ce travail de thèse : des méthodes de quantification ciblant spécifiquement chaque composant majeur de la matrice extracellulaire ont ainsi été développées et validées sur des biofilms modèles. Ces biofilms, choisis pour leur diversité en terme de matrice extracellulaire sont formés par les souches Pseudomonas aeruginosa ATCC 15442, Bacillus licheniformis CIP 110824 et Weissella confusa LBAE-UPS C39-2. Des critères communs ont été retenus pour guider le choix des marqueurs retenus pour développer les dosages : spécificité pour la détection de toutes les formes moléculaires d'une même famille biochimique, sensibilité pour la détection de faibles quantités de polymères dans des biofilms en microplaque, et possibilité d'observation en microscopie basée sur la détection de fluorescence. Une stratégie commune a été adoptée pour la quantification des exoprotéines (ePN), exopolysaccharides (ePS) et fibres amyloïdes (FA) de la matrice et a consisté à : (i) définir une molécule standard pour calibrer le test ; (ii) analyser d'éventuelles interférences en solution ; (iii) valider la faisabilité et la fiabilité du test in situ par la méthode des ajouts dosées réalisée sur chacun des biofilms modèles. Un composé naturel pro-fluorescent, l'épicocconone, a été retenu pour la quantification des exoprotéines. Le test a montré une limite de détection de 0,2 µg par puits, sans aucune interférence significative des autres composants majeurs du biofilms (ePS, ADNe, cellules). D'autre part, les protéines sous forme amyloïdes ont pu être détectées avec la même sensibilité que les non amyloïdes par ce marqueur. Par la suite, les exopolysaccharides ont été dosés en exploitant la réaction de Schiff, et la méthode a permis d'obtenir une limite de détection de 0,3 µg par puits. Les protéines amyloïdes bactériennes (FA) ont également été quantifiées au moyen du marqueur Thioflavine T. La k-caséine a été utilisée comme étalon, après fibrillation de la protéine native in vitro. / Biofilms are detrimental in many industrial and medical areas and understanding of biofilms processes has been a challenge for decades. Specific, sensitive and rapid methods for monitoring biofilm formation are essential for a deep knowledge of biofilm organization and response to environmental factors. In such complex network that constitutes the biofilm matrix, it has become a major issue to succeed in describing its composition and local structure to determine the interactions that govern the biofilm formation. Non-destructive and in situ methods for Exopolymeric Substances (EPS) characterization were proposed along this PhD work. The first aim of the study was to propose some quantitative tools that would specifically target each major compound of the biofilm matrix. Some performance criteria were commonly established to guide the choice of each dye and to validate each step of the analytical development. These requirements can be displayed in terms of biochemical specificity, sensitivity and applicability on fluorescence-based microscopy. A common experimental strategy was carried out to quantify the exoproteins (ePN), exopolysaccharides (ePS) and amyloid fibrils (AF) and aimed at (i) choosing a calibration standard; (ii) analyzing in vitro interferences; (iii) validating the practicability and the reliability of each proposed method for an in situ implementation on biofilms. This last criteria was verified by implementing the Standard Addition Method (SAM). For that purpose, a first method was developed to take advantage of a natural pro-fluorescent dye, called epicocconone, to quantify the ePN of three model bacterial biofilms formed by the strains Bacillus licheniformis CIP 110824, Pseudomonas aeruginosa ATCC 15442 and Weissella confusa LBAE-UPS C39.2. The three bacterial models were chosen because of their contrasted EPS matrix composition. This method showed a detection limit of about 0.2µg per well and no significant interference were revealed. Moreover the epicocconone assay was able to quantify the AF with the same sensitivity as the other proteins. Subsequently, exopolysaccharides from the same strains were assayed by applying the Periodic acid-Schiff reaction in a microplate format. This chemical reaction targets the majority of the hydroxyl groups of carbohydrate and allows to give an exhaustive estimation of the sugar content of the matrix with a detection limit of 0.3µg per well. Bacterial amyloids were also specifically quantified by the using of the benzothiazole dye Thioflavine T (ThT). An amyloidogenic protein, the ?-casein, was used as standard after an in vitro fibrillation of the native form.

Mass spectrometry has increasingly become the method of choice for the analysis of complex biological samples, including proteins and lipids. This thesis describes the development of MS-based analytical methodologies for the analysis of complex proteomic and lipidomic samples. Chapter 3 describes the development of microfluidic proteomic reactors, in the formats of SCX reactor, SCX 96-well plate reactor, and SAX reactor, for the enzymatic digestion of complex proteomic samples for subsequent LC-MS/MS analysis. These microfluidic proteomic reactors greatly simplified the enzymatic digestion of complex proteomic samples by combining multiple processing steps, such as rapid extraction and enrichment of proteins. Furthermore, chemical and enzymatic treatments of proteins were all performed in a few nanoliters effective volume, resulting in an increased protein digestion efficacy. After the protein digestion process, the resulting peptides were eluted in buffers that were compatible with HPLC-MS/MS analysis. In chapter 4, a methodology based on nano-HPLC-ESI-MS/MS for the analysis of PAF and LPC lipid species is described. In this method, lipid extracts from biological samples were separated by nano-flow HPLC prior to being introduced into a Q-TRAP 2000 mass spectrometer, where the lipid species of interest were detected using a precursor ion scan at m/z 184. Absolute quantitation of PAF family lipid species were performed with standard addition method, where 5 standard solutions containing 0.2-1 ng each of C16:0, C18:0 PAF and C16:0, C18:0 lyso-PAF were used in the experiment. Further, the spiking of identical amount of non-endogenous C13:0 LPC at time of extraction allow the relative comparisons of other LPC lipid species of interest between different samples. The developed methods were employed to analyze the changes of PAF and LPC lipid species in NGFdifferentiated PC12 cells, in the posterior/entorhinal cortex of AD patients and TgCRND8 transgenic mice, and over the course of 24 hour exposure of human hNT neurons to Aβ42 treatment, respectively, in comparison to controls. iii Chapter 5 describes the development of a novel shotgun lipidomic methodology for the determination of stereospecificity of diacyl glycerophospholipids including glycerophosphatidic acids (PA), glycerophosphoserines (PS), glycerophosphoglycerols (PG), glycerophosphoinositols(PI), and glycerophosphoethanolamines (PE), which can be conventionally ionized under negative ion mode. The stereospecificity of diacyl glycerophospholipids was determined based on the relative abundance of the lyso-form fragment ions, attributed to the neutral loss of fatty acyl moieties. The fragmentation patterns of a variety of diacyl glycerophospholipid standards were first fully examined over a wide range of collision energy. We observed that lyso-form fragment ions corresponding to the neutral loss of fatty acyl moieties attached to the sn2 position as free fatty acids ([M-Sn2]-) and as ketenes ([M-(Sn2-H2O)]-) exhibited consistently higher intensity than their counter part ions due to the neutral loss of fatty acyl moieties attached to the sn1 position ([M-Sn1]- and [M-(Sn1-H2O)]-). We then examined the product ion spectra of diacyl glycerophospholipids recorded from lipid extracts of rat hepatoma cells, where the stereospecific information of these lipids was conclusively determined.