In chemical analyses, it is crucial to distinguish between chemical species. This is often accomplished via chromatographic separations. These separations are often pushed to their limits in terms of the number of analytes that can be sufficiently resolved from one another, particularly when a quantitative analysis of these compounds is needed. Very often, complicated methods or new technology is required to provide adequate separation of samples arising from a variety of fields such as metabolomics, environmental science, food analysis, etc. An often overlooked means for improving analysis is the use of chemometric data analysis techniques. Particularly, the use of chemometric curve resolution techniques can mathematically resolve analyte signals that may be overlapped in the instrumental data. The use of chemometric techniques facilitates quantitation, pattern recognition, or any other desired analyses. Unfortunately, these methods have seen little use outside of traditionally chemometrics focused research groups. In this dissertation, we attempt to show the utility of one of these methods, multivariate curve resolution-alternating least squares (MCR-ALS), to liquid chromatography as well as its application to more advanced separation techniques. First, a general characterization of the performance of MCR-ALS for the analysis of liquid chromatography-diode array detection (LC-DAD) data is accomplished. It is shown that under a wide range of conditions (low chromatographic resolution, low signal-to-noise, and high similarity between analyte spectra), MCR-ALS is able to increase the number of quantitatively analyzable peaks. This increase is up to five-fold in many cases. Second, a novel methodology for MCR-ALS analysis of comprehensive two-dimensional liquid chromatography (LC x LC) is described. This method, called two dimensional assisted liquid chromatography (2DALC), aims to improve quantitation in LC x LC by combining the advantages of both one-dimensional and two dimensional chromatographic data. We show that 2DALC can provide superior quantitation to both LC x LC and one dimensional LC under certain conditions. Finally, we apply MCR-ALS to an LC x LC analysis of fourteen furanocoumarins in three apiaceous vegetables. The optimal implementation of MCR-ALS and subsequent integration was determined. For these data, simply performing MCR-ALS on the two dimensional chromatogram and manually integrating the results proved to be the superior method. These results demonstrate the usefulness of these curve resolution techniques as a compliment to advanced chromatographic techniques.

Mass spectrometry coupled with liquid chromatography and gel electrophoresis enables separation and detection of components in a complex mixture. During the last two decades, its applications were dramatically extended and remarkable progress has been made in many fields, in particular, environmental and biological analyses. This dissertation focuses on identification and characterization of biologically active compounds and comparative analysis of protein expression changes. The first two projects (Chapters 2 and 3) focus on the application of LC/MS approach to profile the bioactivated intermediates of 4, 4'-methylenedianiline (DAPM) from rat vascular smooth muscle cells (VSMCs) and bile. In our study, several DAPM metabolites were detected and characterized in detail by liquid chromatography-electrospray tandem mass spectrometry. The structural assignments of these metabolites from VSMCs and rat bile significantly improve our understanding of DAPM biotransformations and toxicity. The third project described in Chapter 4 focuses on using electrospray tandem mass spectrometry (ES-MS/MS) and theoretical calculation (GAUSSIAN 03 program) to investigate the unusual methyl radical loss and consecutive fragment ions that dominate the low-energy collision induced dissociation (CID) mass spectra of prodiginine compounds. Structures of the fragment ions are proposed and explanations are given to rationalize the observed competition between the formation of even-electron ions and radical ions. Our study shows that the lower apparent threshold associated with methyl radical loss points to a lower kinetic barrier. In Chapter 5, hypoxia-induced changes of zebrafish skeletal muscle were studied using two-dimensional difference in-gel electrophoresis (2D-DIGE) in vivo after 48 h in hypoxia vs. normoxia. The results showed that proteins involved in mitochondrial oxidative metabolism are down-regulated, whereas glycolytic enzymes are up-regulated to compensate for the loss of ATP synthesis in aerobic metabolism. The up-regulation of two spots identified as hemoglobin variants was also observed. These protein expression changes are consistent with a hypoxic response that enhances anaerobic metabolism or O2 transport to tissues.

Os problemas de corte de estoque consistem em cortar um conjunto de objetos dispon´veis em estoque para produzir um conjunto de itens em quantidades e tamanhos especificados, de modo a otimizar uma fun¸cao objetivo. Tais problemas tem in´umeras aplica¸coes industriais e tem sido bastante estudados na literatura. Tipicamente, problemas de corte tem como principal objetivo a minimiza¸cao das sobras. Entretanto, como a qualidade dos padroes de corte depende diretamente dos tamanhos e quantidades dos itens a serem produzidos, nesta tese, consideramos que se a demanda presente gerar sobras indesej´aveis (nem tao grandes para serem aproveit´aveis, nem tao pequenas para serem perdas aceit´aveis), entao conv´em gerar retalhos (nao comput´aveis como perda) que serao utilizados para produzir itens de demandas futuras. Desta forma, algumas caracter´sticas desej´aveis para uma boa solu¸cao sao definidas e altera¸coes em m´etodos heur´sticos cl´assicos sao apresentadas, de modo que os padroes de corte com sobras indesej´aveis sao alterados. Para os problemas de corte unidimensionais, desenvolvemos procedimentos heur´sticos que consideram o aproveitamento de sobras, mantendo como o principal objetivo a minimiza ¸cao das perdas. Outra abordagem para este problema, considera o caso em que al´em da minimiza¸cao das perdas, os retalhos dispon´veis em estoque devem ter prioridade de uso em rela¸cao aos demais objetos durante o processo de corte. A an´alise do desempenho dos procedimentos heur´sticos propostos quando somente a minimiza¸cao das perdas ´e considerada, ´e realizada com base em exemplos da literatura, exemplos pr´aticos e exemplares gerados aleatoriamente. Para os procedimentos heur´sticos que priorizam o corte dos retalhos do estoque, al´em de exemplares da literatura, simulamos uma situa¸cao em m´ultiplos per´odos na qual problemas de corte de estoque em sucessivos per´odos sao resolvidos. A cada per´odo, um problema para o per´odo seguinte ´e gerado considerando atualiza¸coes do estoque, os retalhos gerados nos per´odos anteriores e uma nova demanda de itens que ´e v gerada aleatoriamente. No caso bidimensional, tamb´em consideramos problemas em que, al´em da perda m´nima, os retalhos dispon´veis em estoque devem ter prioridade de corte em rela¸cao aos demais objetos. Para resolver este problema, altera¸coes foram realizadas na abordagem grafo E/OU e em procedimentos heur´sticos da literatura. A an´alise do desempenho dos procedimentos heur´sticos propostos considera problemas pr´aticos retirados da carteira de pedidos de uma pequena empresa de esquadrias met´alicas. Devido `a dificuldade na an´alise dos procedimentos heur´sticos desenvolvidos que consideram o aproveitamento de sobras (as solu¸coes apresentam caracter´sticas importantes e conflitantes), tamb´em apresentamos neste trabalho uma estrat´egia fuzzy para facilitar a analise das solu¸coes obtidas. Os testes computacionais sao realizados considerando os procedimentos heur´sticos desenvolvidos para os problemas de corte unidimensionais com sobras aproveit´aveis e problemas gerados aleatoriamente / Cutting stock problems consist of cutting a set of available objects in order to produce ordered items in specified amounts and sizes, in such way to optimize an objective function. Such problems have a great number of industrial applications and are widely studied in the literature. Typically, cutting problems have as main objective the minimization of the leftovers. However, since the cutting patterns quality depends directly of the sizes and amounts of the items that will be produced, in this tesis, we consider that if the present demand to generate undesirable waste (not large enough to be used, nor too small to be acceptable waste), then it is better to generate retails (not computed as waste) that will be used to produce items to meet future demands. In this way, some desirable characteristics for a good solution are defined and alterations in classical heuristic methods are presented, such that the cutting patterns with undesirable waste are altered. To the one-dimensional cutting stock problems, we developed heuristic procedures that consider the usable leftovers and preserve as main objective the minimization of the waste. Other approach for this problem considers the case in witch, beside minimal waste, the available retails in stock must be used with priority in relation to the other objects during the cutting process. The performance of the modified heuristics procedures, when only the minimal waste is considered, is observed by solving instances from the literature, practical instances and randomly generated instances. For heuristic procedures that prioritize the cut of retails of the stock, beside the instances from the literature, we simulated a situation in multiple periods in that cutting stock problems in successive periods are solved. In each period, a problem to the next period is generated considering updating of the stock, the retails generated in previous periods and a new demand of items that is randomly generated. For the two-dimensional cutting problems, we also consider problems in that, beside minimization of the waste, the available retails in stock must be used with priority vii in relation to the other objects. To solve this problem, alterations were realized in an AND/OR graph approach and in heuristic procedures of the literature. The performance of the proposed heuristics procedures is observed by solving practical instances provided by a small metallic frameworks industry. Due to difficulty in analyze the heuristic procedures developed for the cutting stock problem with usable leftover (the solutions present important and conflicting characteristics), we also present a fuzzy strategy to facilitate the analysis of the obtained solutions. The computational results are realized considering the developed heuristic procedures to the one-dimensional cutting stock problem with usable leftover and randomly generated instances

Metodologia proteômica baseada em eletroforese bi-dimensional (2D-PAGE) foi usada para auxiliar no entendimento dos mecanismos moleculares envolvidos na injúria renal induzida pelo flúor (F) e definir biomarcadores potenciais para fluorose. Três grupos de ratos Wistar machos recém-desmamados (21 dias de vida) foram tratados com água de beber contendo 0 (controle), 5 ou 50 ppm F, por 60 dias (n=6/grupo). Durante o período experimental, os animais foram mantidos individualmente me gaiolas metabólicas, a fim de que o consumo de água e ração fosse avaliado, bem como as excreções urinária e fecal de F. Os animais foram mortos e o rim esquerdo e o soro foram coletados para análises histopatológica e de F, respectivamente. Para análise proteômica foram coletados o rim direito e a urina (no dia anterior ao sacrifício, num coquetel contendo inibidores de protease em gelo). Após o isolamento das proteínas, os perfis proteômicos renal e urinário foram examinados usando 2D-PAGE e coloração com azul de Coomassie brilhante. Foi possível detectar uma doseresposta em relação à ingestão e excreção de F, bem como em relação aos níveis de F presentes no soro e nos rins dos animais. As análises histológicas não revelaram danos aos rins induzidos pelo F, com exceção de uma congestão vascular no grupo de 50 ppm F. Para os rins, a análise quantitativa de intensidade (software Image Máster Platinum, alterações de 2 vezes) revelou 30 e 17 proteínas diferencialmente expressas, respectivamente, entre os grupos controle X 50 ppm F e controle X 5 ppm F. Para a urina, 9, 10 e 13 proteínas aumentaram ou diminuíram nos grupos controle X 5 ppm F, 5 ppm F X 50 ppm F e controle X 50 ppm F, respectivamente. Nove proteínas foram identificadas satisfatoriamente por MALDI-TOF TOF MS. As proteínas identificadas estão relacionadas principalmente ao metabolismo, desintoxicação e housekeeping. Esses dados indicam que a análise proteômica de rim e urina de animais tratados com F é capaz de identificar proteínas diferencialmente expressas, mesmo em casos de baixas doses de F. Assim, essa ferramenta pode contribuir para o entendimento dos mecanismos envolvidos na fluorose, apontando proteínas-chave que deveriam ser melhor investigadas, bem como potenciais biomarcadores de toxicidade. / Two-dimensional gel electrophoresis (2D-PAGE) based proteomics approach was used to better understand the molecular mechanisms of renal injury induced by fluoride (F) and define potentials biomarkers of fluorosis. Three groups of weanling male Wistar rats (21 days old) were treated with drinking water containing 0 (control), 5, or 50 ppm F for 60 days (n=6/group). During the experimental period, the animals were kept individually in metabolic cages, in order to analyze the water and food consumption, as well as fecal and urinary F excretion. Animals were killed and left kidney and serum were collected for histopathological examination and F analysis, respectively. For proteomic analysis, right kidney and urine (one day before sacrifice, in protease-inhibitors cocktail for 8 hours in ice box) were collected. After protein isolation, renal and urinary proteome profiles were examined using 2D-PAGE and coomassie brilliant blue staining. It was possible to detect a dose-response regarding F intake and F excretion, as well as F levels in serum and kidneys. The histological analysis revealed no damage in kidneys induced by F, except for a vascular congestion in the 50 ppm F group. For kidney, quantitative intensity analysis (Image Master Platinum software, 2-fold changes) revealed 30 and 17 differentially expressed proteins between control X 50 ppm F, and control X 5 ppm F groups, respectively. As for urine, 9, 10 and 13 proteins increased or decreased in control X 5 ppm F, 5 ppm F X 50 ppm F and control X 50 ppm F groups, respectively. Nine proteins were successfully identified by MALDI-TOF TOF MS. The identified proteins are mainly related with metabolism, detoxification and housekeeping. These data indicate that proteomic analysis in kidney and urine of F-treated animals is able to identify differentially expressed proteins, even in cases of low F doses. Thus, this approach can contribute for the understanding of the mechanisms underlying fluorosis, by indicating key-proteins that should be better addressed, as well as potential toxicity biomarkers.

<div>Recently, atomically thin two-dimensional (2D) layered materials such as graphene and transition metal dichalcogenides (TMDCs) have emerged as a new class of materials due to their unique electronic structures and optical properties at the nanoscale limit. 2D materials also hold great promises as building blocks for creating new heterostructures for optoelectronic applications such as atomically thin photovoltaics, light emitting diodes, and photodetectors. Understanding the fundamental photo-physics process in 2D semiconductors and heterostructures is critical for above-mentioned applications. </div><div>In Chapter 1, we briefly describe photo-generated charge carriers in two-dimensional (2D) transition metal dichalcogenides (TMDCs) semiconductors and heterostructures. Due to the reduced dielectric screening in the single-layer or few-layer of TMDCs semiconductors, Columbo interaction between electron and hole in the exciton is greatly enhanced that leads to extraordinary large exciton binding energy compared with bulk semiconductors. The environmental robust 2D excitons provide an ideal platform to study exciton properties in TMDCs semiconductors. Since layers in 2D materials are holding by weak van de Waals interaction, different 2D layers could be assembled together to make 2D heterostructures. The successful preparation of 2D heterostructures paves a new path to explore intriguing optoelectronic properties.</div><div>In Chapter 2, we introduce various optical microscopy techniques used in our work for the optical characterization of 2D semiconductors and heterostructures. These optical imaging tools with high spatial and temporal resolution allow us to directly track charge and energy flow at 2D interfaces.</div><div>Exciton recombination is a critical factor in determining the efficiency for optoelectronic applications such as semiconductor lasers and light-emitting diodes. Although exciton dynamics have been investigated in different 2D semiconductor, large variations in sample qualities due to different preparation methods have prevented obtaining intrinsic exciton lifetimes from being conclusively established. In Chapter 3, we study exciton dynamics in 2D TMDCs semiconductors using ultrafast PL and transient absorption microscopy. Here we employ 2D WS2 semiconductor as a model system to study exciton dynamics due to the low defect density and high quantum yield of WS2. We mainly focus on how the exciton population affects exciton dynamics. At low exciton density regime, we demonstrate how the interlayer between the bright and dark exciton populations influence exciton recombination. At high exciton density regime, we exhibit significant exciton-exciton annihilation in monolayer WS2. When comparing with the bilayer and trilayer WS2, the exciton-exciton annihilation rate in monolayer WS2 increases by two orders of magnitude due to enhanced many-body interactions at single layer limit. </div><div>Long-range transport of 2D excitons is desirable for optoelectronic applications based on TMDCs semiconductors. However, there still lacks a comprehensive understanding of the intrinsic limit for exciton transport in the TMDCs materials currently. In Chapter 4, we employ ultrafast transient absorption microscopy that is capable of imaging excitons transport with ~ 200 fs temporal resolution and ~ 50 nm spatial precision to track exciton motion in 2D WS2 with different thickness. Our results demonstrate that exciton mobility in single layer WS2 is largely limited by extrinsic factors such as charge impurities and surface phonons of the substrate. The intrinsic phonon-limited exciton transport is achieved in WS2 layers with a thickness greater than 20 layers.</div><div>Efficient photocarrier generation and separation at 2D interfaces remain a central challenge for many optoelectronic applications based on 2D heterostructures. The structural tunability of 2D nanostructures along with atomically thin and sharp 2D interfaces provides new opportunities for controlling charge transfer (CT) interactions at 2D interfaces. A largely unexplored question is how interlayer CT interactions contribute to interfacial photo-carrier generation and separation in 2D heterostructures. In Chapter 5, we present a joint experimental and theoretical study to address carrier generation from interlayer CT transitions in WS2-graphene heterostructures. We use spatially resolved ultrafast transient absorption microscopy to elucidate the role of interlayer coupling on charge transfer and photo-carrier generation in WS2-graphene heterostructures. These results demonstrate efficient broadband photo-carrier generation in WS2-graphene heterostructures which is highly desirable for atomically thin photovoltaic and photodetector applications based on graphene and 2D semiconductors.</div><div>CT exciton transport at heterointerfaces plays a critical role in light to electricity conversion using 2D heterostructures. One of the challenges is that direct measurements of CT exciton transport require quantitative information in both spatial and temporal domains. In order to address this challenge, we employ transient absorption microscopy (TAM) with high temporal and spatial resolution to image both bright and dark CT excitons in WS2-tetrance and CVD WS2-WSe2 heterostructure. In Chapter 6, we study the formation and transport of interlayer CT excitons in 2D WS2-Tetracene vdW heterostructures. TAM measurements of CT exciton transport at these 2D interfaces reveal coexistence of delocalized and localized CT excitons. The highly mobile delocalized CT excitons could be the key factor to overcome large CT exciton binding energy in achieving efficient charge separation. In Chapter 7, we study stacking orientational dependent interlayer exciton recombination and transport in CVD WS2-WSe2 heterostructures. Temperature-dependent interlayer exciton dynamics measurements suggest the existence of moiré potential that localizes interlayer excitons. TAM measurements of interlayer excitons transport reveal that CT excitons at WS2-WSe2 heterointerface are much more mobile than intralayer excitons of WS2. We attributed this to the dipole-dipole repulsion from bipolar interlayer excitons that efficiently screen the moiré potential fluctuations and facilitate interlayer exciton transport. Our results provide fundamental insights in understanding the influence of moiré potential on interlayer exciton dynamics and transport in CVD WS2-WSe2 heterostructures which has important implications in optoelectronic applications such as atomically thin photovoltaics and light harvesting devices. </div><div><br></div>

A esquizofrenia é uma doença crônica, grave e incapacitante que afeta cerca 24 milhões de pessoas em âmbito mundial. É caracterizada por uma desorganização no pensamento que prejudica a funcionalidade do indivíduo. Existem intervenções que são efetivas e contribuem para a diminuição da prevalência do transtorno, pois ajudam o portador a levar uma vida produtiva e integrada à sociedade, porém devem ser ministradas nos estágios iniciais da doença. No entanto, existe uma grande dificuldade em se diagnosticar a esquizofrenia precocemente devido a sua complexidade e às sutilezas dos seus sintomas apresentados antes do surgimento da psicose. O cérebro não é acessível a exames invasivos in vivo e por esse motivo a exploração de fluidos periféricos é de grande importância. As plaquetas e neurônios serotonérgicos possuem características bioquímicas e morfológicas em comum que possibilitam a comparação entre a estrutura e a função de ambos e, por causa dessa similaridade, muitos trabalhos utilizam plaquetas como modelo para o estudo de doenças neuropsiquiátricas, inclusive a esquizofrenia. A detecção precoce da esquizofrenia é um objeto de investigação atual e relevante não somente para revolucionar os meios atuais de diagnóstico, mas também para desenvolver novos tratamentos aplicados aos estágios iniciais da doença, diferenciar os subgrupos de doentes e monitorar as intervenções preventivas. A proposta do presente trabalho é fazer o estudo da expressão de proteínas em plaquetas de pacientes esquizofrênicos e controles com o objetivo de identificar proteínas candidatas a biomarcadores utilizando técnicas proteômicas quantitativas e confiáveis, como 2D-DIGE e a espectrometria de massas. / Schizophrenia is a disabling, serious, and chronic illness, which affects about 24 million people worldwide. It is characterized by a severe disorganization of the thoughts that harms the social life of patients becoming them dependent of the family and/or government. There are effective treatments that contribute to decrease the prevalence of the disorder because they improve the life and social conditions of the patients, but they are only advantageous if the intervention is made in the early stages of the disease. It is difficult to obtain early diagnosis due to the complexity of the disease and its insidious symptoms before the beginning of the psychosis. The brain is not easily accessed in vivo and, because of this, it is very important to study the peripheral tissues like blood, which makes the use of the platelets very interesting. Furthermore, platelets and serotonergic neurons share biochemical and morphological characteristics that allows the comparison between structure and function of both. From these similarities many authors has used platelets as a neuron model to study many neurodegenerative diseases including schizophrenia. The early detection of schizophrenia is a current and suitable goal, not only to improve the early diagnosis but also to develop new treatments, differentiate the subtypes, and monitor the preventive interventions. The purpose of this project is to do a comparative screening of expressed proteins in platelets from schizophrenics and controls with the objective of finding differently expressed proteins that could be candidates to biomarkers using 2D-DIGE and mass spectrometry.

La cognition sociale a récemment connu un intérêt croissant en neuropsychologique clinique. Cet engouement s’explique notamment par le caractère transdiagnostique mais aussi invalidant des troubles. Cependant, ce construit se heurte encore à de nombreux écueils, tant conceptuels que méthodologiques. Ainsi, dans la schizophrénie, les données de la littérature internationale divergent. Nous faisons l’hypothèse que ces contradictions peuvent être sous-tendues par : 1) le manque de considération pour le caractère multidimensionnel de la cognition sociale, et 2) l’hétérogénéité interindividuelle du fonctionnement. L’objectif général de ce travail de thèse était de réaliser une étude comparée des profils de fonctionnement en cognition sociale dans la population générale et dans la schizophrénie. Pour ce faire, une première étape a été consacrée à une revue systématique des études sur la structure factorielle de la cognition sociale en neuropsychologie clinique (Article 1). Cette revue a permis de proposer un modèle bidimensionnel de la cognition sociale, où les processus de bas et de haut niveau croisent les processus affectifs et cognitifs. Une deuxième étape a consisté à fournir des éléments de validation supplémentaires au PECS-B, une batterie d’évaluation de la cognition sociale composée de sept tâches évaluant cinq dimensions (Article 2). Les résultats ont révélé des qualités psychométriques satisfaisantes en population générale (N = 131) comme dans la schizophrénie (N = 101). Enfin, la troisième étape a consisté à réaliser des analyses en clusters pour explorer l’hétérogénéité interindividuelle dans ces mêmes populations (Article 3). Trois profils différents dans l’échantillon population générale ont été mis en évidence, dont deux caractérisés par des faiblesses spécifiques. En revanche, les trois profils obtenus dans l’échantillon schizophrénie étaient caractérisés par trois niveaux de fonctionnement en cognition sociale : capacités efficientes (similaires aux témoins), moyennes et faibles. Ces deux derniers niveaux étaient également caractérisés par des difficultés d’ordre alexithymique ou neurocognitive. Dans une perspective de pratique basée sur la preuve, ces résultats ont des implications importantes tant pour l’évaluation de la cognition sociale que pour l’orientation des interventions thérapeutiques. / Social cognition has received a growing interest in clinical neuropsychology. This popularity can be explained by the transdiagnostic and negative outcome of social cognition disorders. However, this construct faces many difficulties, conceptual as well as methodological. Thus, concerning schizophrenia, data from the international literature is inconsistent. We hypothesize that these contradictions may be underpinned by: 1) a lack of consideration for the multidimensionality of social cognition, and 2) interindividual heterogeneity of functioning. The overall goal of this thesis was to explore and compare the social cognition functioning profiles in the general population with those for schizophrenia. To do this, a first step was devoted to a systematic review of studies that explored the factor structure of social cognition in clinical neuropsychology (Article 1). This review allowed the proposition of a two-dimensional model of social cognition, in which low and high-level processes cross affective and cognitive ones. A second step was to provide additional validation elements to the PECS-B, a social cognition assessment battery with seven tasks assessing five dimensions (Article 2). The results reported satisfactory psychometric qualities in the general population (N = 131) as well as in schizophrenia (N = 101). Finally, the third step was to carry out cluster analyses to explore interindividual heterogeneity in these same populations (Article 3). Three different profiles in the general population sample were highlighted, of which two were characterized by specific weaknesses. In contrast, the three profiles in the schizophrenia sample were characterized by three levels of social cognition functioning: good (similar to controls), medium and low. The last two levels were also characterized by alexithymic or neurocognitive difficulties. From an evidence-based practice perspective, these findings have important implications for both the assessment and the direction of therapeutic interventions.

La chromatographie en phase gazeuse couplée à la spectrométrie de masse simple quadripolaire (GC-qMS) est la technique la plus utilisée dans l’exploration des acides organiques urinaires dans le cadre du diagnostic des aciduries organiques (AO). Cependant, après analyse par GC-MS de plus de 1000 échantillons d'urines recueillis auprès d'enfants atteints de troubles neurologiques, moins de 0,5% d'entre eux ont reçu un diagnostic positif. Ceci témoigne de l’urgence de rechercher de nouveaux biomarqueurs dans le domaine des troubles neurologiques associés à des erreurs innées du métabolisme (EIM).L'objectif principal de cette thèse a été de développer une nouvelle méthode d’analyse des échantillons urinaires par chromatographie bidimensionnelle couplée à la spectrométrie de masse (GCxGC-qMS) et d'évaluer son apport dans le profilage métabolique de ces échantillons. Par ailleurs, dans le cadre du diagnostic des AO, dans un objectif de simplification de la procédure de préparation de l’échantillon, nous avons évalué une autre approche d’analyse directe en temps réel couplée à la spectrométrie de masse « DART-MS » des acides organiques urinaires. La première étape de notre travail a été de développer un protocole de préparation de l’échantillon compatible avec une méthode de séparation bidimensionnelle. Puis, grâce à l’étude systématique des différentes conditions de séparation, nous avons développé une méthode par CGxCG-qMS parfaitement adaptée à la séparation des composés extraits.Après validation analytique, la méthode proposée a été ensuite évaluée par le profilage des composés organiques présents dans des échantillons urinaires prélevés chez des volontaires sains. Par rapport aux profils obtenus par une méthode par GC-qMS classique, optimisée, les profils chromatographiques obtenus par la méthode proposée présentent une sensibilité et une résolution nettement plus élevées. Ceci se traduit par la détection en GCxGC-qMS de nombreux composés supplémentaires, jusqu’à 92, selon les échantillons. Parmi les composés supplémentaires détectés et caractérisés, certains sont d’un intérêt diagnostic reconnu.Malgré les difficultés liées à l’étape de préparation de l’échantillon et au traitement des données, l’ensemble des résultats obtenus par la méthode proposée sur de nombreux échantillons urinaires a confirmé les potentialités et la nécessité d’utiliser la chromatographie bidimensionnelle dans le domaine de la découverte de candidats biomarqueurs. Il s’agit maintenant de poursuivre l’identification des composés supplémentaires détectés dans les échantillons urinaires étudiés et d’appliquer la méthode proposée à un plus grand nombre d’échantillons témoins et pathologiques afin d’essayer d’identifier des candidats biomarqueurs.Enfin, les essais préliminaires par la technique DART-MS, effectués dans un laboratoire de la Direction des Applications Militaires (DAM) du CEA, ont été axés sur la détection d’un panel d’acides organiques représentatifs des différentes anomalies rencontrées lors des AO. Malgré les effets de suppression ionique qui restent à éliminer, les résultats obtenus montrent que cette approche mérite d’être approfondie. / Among inherited metabolic diseases, organic acidemia (OA) or organic aciduria is characterized by urinary excretion of abnormal amounts or types of organic acids. OA is mostly associated with genetic conditions resulting in a specific step of amino acid catabolism dysfunction. Such alterations can produce disease states that range from mild to lethal neurological involvement. Gas chromatography coupled to mass spectrometry (GC-MS) remains the most used analytical technique for detecting specific urinary organic metabolites related to OA. However, after analyzing more than 1,000 urine samples collected from children with neurological disorders, by this technique less than 0.5% of them were positive. Thus, the discovery of new biomarker candidates for other metabolic diseases is urgently needed. The main objective of this thesis was to evaluate a new method using two-dimensional gas chromatography coupled to mass spectrometry (GCxGC-MS) for the both qualitative and quantitative metabolic profiling of children’s urine. As compared to GC-MS, GCxGC-MS shows great resolution power and high peak capacity. For this purpose, we first developed a GCxGC-MS method with an appropriate sample preparation protocol for urinary organic acids profiling. Applied to urine samples of healthy children and children with neurological disorders, the proposed method showed high sensitivity and peak capacity thus opening new possibilities for the characterization of new biomarker candidates. For instance, by this technique, we were able to detect in urines samples more than ninety additional compounds, which are not detected by a conventional GC-MS method. However, taken together the obtained results show that while the GCxGC technique has unmatched power to separate compounds in a complex mixture, the sample preparation protocol remains a limiting step for the precise quantification of the detected compounds. On another hand, in order to reduce the sample preparation step, we evaluated the direct analysis in real time (DART) method for the urinary organic acids screening. The obtained results are very promising.

Introdução: A disfunção atrial esquerda está associada a pior prognóstico em diversas situações clínicas. O método de strain bidimensional do átrio esquerdo permite avaliar de forma direta todas as fases da função atrial. Pouco se conhece sobre o comportamento das fases da função atrial esquerda em pacientes com síndrome coronariana aguda. O objetivo desse estudo foi correlacionar as funções de reservatório, conduto e contração do átrio esquerdo com desfechos adversos cardiovasculares em pacientes com síndrome coronariana aguda sem supradesnivelamento do segmento ST. Método: Esse estudo recrutou prospectivamente 109 pacientes com diagnóstico de infarto agudo do miocárdio sem elevação do segmento ST e de angina instável de risco moderado ou alto pelo escore GRACE para realização de ecocardiograma nas primeiras 72 horas. A função atrial foi avaliada por parâmetros ecocardiográficos convencionais e pelo strain bidimensional obtido pela média das janelas apicais 2 e 4 câmaras. O desfecho primário foi avaliado em até um ano de seguimento e foi composto pelos seguintes eventos adversos: óbito, insuficiência cardíaca nova, nova internação por síndrome coronariana aguda ou por insuficiência cardíaca, angina estável com necessidade de nova intervenção coronariana, arritmia (fibrilação atrial ou taquicardia ventricular) e acidente vascular cerebral. Os desfechos secundários foram os combinados desses eventos. Resultados: As médias do strain de reservatório, conduto e contração foram de 25% ± 8, 12% ± 5 e 12% ± 4, respectivamente. O desfecho primário teve uma incidência de 31,8% em até um ano e apresentou uma correlação significativa com o strain de reservatório (HR= 0,92; IC95% 0,88-0,96; p<0,001), de conduto (HR= 0,87; IC95% 0,81-0,94; p<0,001) e de contração (HR= 0,90; IC95% 0,84-0,98; p=0,011). Análise multivariada envolvendo variáveis clínicas e as de função atrial esquerda demonstraram que o strain de reservatório (p=0,03) e de conduto (p=0,046) se mantiveram significativos como preditores do desfecho primário. O strain de conduto se manteve significativo no desfecho combinado de óbito e insuficiência cardíaca (HR= 0,82; IC95% 0,74-0,91; p<0,001) mesmo após análise multivariada com parâmetros clínicos (p<0,001) e ecocardiográficos (p=0,049). Conclusão: A avaliação da função atrial esquerda por meio do strain bidimensional se correlacionou significativamente com desfechos adversos em pacientes com síndrome coronariana aguda sem elevação do segmento ST. O strain de reservatório e de conduto foram marcadores prognósticos independentes para o desfecho primário quando comparados às variáveis clínicas. Já para o desfecho combinado de óbito e insuficiência cardíaca, o strain de conduto foi um preditor independente mesmo após ajustado para variáveis clínicas e ecocardiográficas. / Background: Left atrial dysfunction is associated with worse prognosis in several clinical situations. The left atrial two-dimensional strain method allows direct evaluation of all phases of atrial function. There are few studies on the behavior of the various stages of left atrial function in patients with acute coronary syndrome. The aim of this study was to correlate the functions of reservoir, conduit and contraction of the left atrium with adverse cardiovascular outcomes in patients with non-ST elevation acute coronary syndrome. Method: This study prospectively recruited 109 patients with a non-ST-segment elevation myocardial infarction and unstable angina with moderate or high risk by GRACE score and echocardiography parameters were collected within the first 72 hours of admission. The atrial function was evaluated by conventional echocardiographic parameters and the two-dimensional strain obtained by the mean of the apical two- and four-chamber views. The primary endpoint was assessed during the 1 year follow-up period and was composed of theses adverse events: death, heart failure, rehospitalization for acute coronary syndrome or heart failure, stable angina requiring new coronary intervention, arrhythmia (atrial fibrillation or ventricular tachycardia) and stroke. Secondary outcomes were those combined for these events. Results: The means of reservoir, conduit and contraction strain were 25% ± 8, 12% ± 5 and 12% ± 4, respectively. The primary endpoint occurred in 31.8% patients during the 1 year follow-up period and had a statistically significant correlation with the reservoir strain (HR = 0.92, 95% CI: 0.88-0.96, p <0.001), conduit strain (HR = 0.87, 95% CI: 0.81-0.94, p <0.001) and contraction strain (HR = 0.90, 95% CI: 0.84-0.98, p = 0.011). Multivariate analysis involving clinical variables and left atrial function showed that the reservoir strain (p = 0.03) and conduit (p = 0.046) were independent predictors of endpoint primary. The conduit strain were statistically significant in the combined outcome of death and heart failure (HR = 0.82, 95% CI: 0.74-0.91, p <0.001) even after multivariate analysis with clinical (p <0.001) and echocardiography parametrs (p = 0.049). Conclusion: Evaluation of left atrial function by two-dimensional strain correlated significantly with adverse outcomes in patients with non-ST elevation acute coronary syndrome. The reservoir and conduit strain were independent prognostic markers for the primary endpoint when compared to clinical parametrs. For the combined outcome of death and heart failure, the conduit strain was an independent predictor even after adjusting for clinical and echocardiographic variables.

<div>Amongst the various classes of mass analyzers, the quadrupole ion trap (QIT) is by far the most versatile. Although it can achieve only modest resolution (unit) and mass accuracy (101-102 ppm), it has high sensitivity and selectivity, can operate at pressures exceeding 10-3 torr, is tolerant to various electrode imperfections, and has single analyzer tandem mass spectrometry (MS/MS) capabilities in the form of product ion scans. These characteristics make the QIT ideal for mass spectrometer miniaturization, as most of the fundamental performance metrics of the QIT do not depend on device size. As such, the current drive in miniature systems is to adopt miniature ion traps in various forms – 3D, linear, toroidal, rectilinear, cylindrical, arrays, etc.</div><div><br></div><div>Despite being one of the two common mass analyzers with inherent MS/MS capabilities (the other being the Fourier transform ion cyclotron resonance mass spectrometer), it is commonly accepted that the QIT cannot perform one-dimensional precursor ion scans and neutral loss scans - the other two main MS/MS scan modes - or two-dimensional MS/MS scans. The former two are usually conducted in triple quadrupole instruments in which a first and third quadrupole are used to mass select precursor and product ions while fragmentation occurs in an intermediate collision cell. The third scan can be accomplished by acquiring a product ion scan of every precursor ion, thus revealing the entire 2D MS/MS data domain (precursor ion m/z vs. product ion m/z). This, however, is not one scan but a set of scans. Because the ion trap is a tandem-in-time instrument rather than a tandem-in-space analyzer, precursor ion scans, neutral loss scans, and 2D MS/MS are, at best, difficult.</div><div><br></div><div>Yet miniature mass spectrometers utilizing quadrupole ion traps for mass analysis would perhaps benefit the most from precursor scans, neutral loss scans, and 2D MS/MS because they generally have acquisition rates (# scans/s) an order of magnitude lower than their benchtop counterparts. This is because they usually use a discontinuous atmospheric pressure interface (DAPI) to reduce the gas load on the backing pumps, resulting in a ~1 scan/s acquisition rate and making the commonly-used data-dependent acquisition method (i.e. obtaining a product ion scan for every abundant precursor ion) inefficient in terms of sample consumption, time, and instrument power. Precursor and neutral loss scans targeting specific molecular functionality of interest - as well as 2D MS/MS – are more efficient ways of moving through the MS/MS data domain and thus pair quite readily with miniature ion traps.</div><div><br></div><div>Herein we demonstrate that precursor ion scans, neutral loss scans, and 2D MS/MS are all possible in a linear quadrupole ion trap operated in the orthogonal double resonance mode on both benchtop and portable mass spectrometers. Through application of multiple resonance frequencies matching the secular frequencies of precursor and/or product ions of interest, we show that precursor ions can be fragmented mass-selectively and product ions ejected simultaneously, preserving their relationship, precursor ion -> product ion + neutral, in the time domain and hence allowing the correlation between precursor and product ions without prior isolation. By fixing or scanning the resonance frequencies corresponding to the targeted precursor and product ions, a precursor ion scan or neutral loss scan can be conducted in a single mass analyzer. We further show that 2D MS/MS - acquisition of all precursor ion m/z values and a product ion mass spectrum for every precursor ion, all in a single scan - is possible using similar methodology. These scan modes are particularly valuable for origin-of-life and forensic applications for which the value of miniature mass spectrometers is readily evident.</div>