Powerful sequential optimization techniques can drastically change the Integrated Circuit (IC) design paradigm. Due to the limited capability of sequential verification tools, aggressive sequential optimization is shunned nowadays as there is no efficient way to prove the preservation of equivalence after optimization. Due to the fact that the number of transistors fitting on single fixed-size die increases with Moore's law, the problem gets harder over time and in an exponential rate. It is no surprise that functional verification becomes a major bottleneck in the time-to-market of a product. In fact, literature has reported that 70% of design time is spent on making sure the design is bug-free and operating correctly. One of the core verification tasks in achieving high quality products is equivalence checking. Essentially, equivalence checking ensures the preservation of optimized product's functionality to the unoptimized model. This is important for industry because the products are modified constantly to meet different goals such as low power, high performance, etc. The mainstream in conducting equivalence checking includes simulation and formal verification. In simulation approach, golden design and design under verification (DUV) are fed with same stimuli for input expecting outputs to produce identical responses. In case of discrepancy, traces will be generated and DUV will undergo modifications. With the increase in input pins and state elements in designs, exhaustive simulation becomes infeasible. Hence, the completeness of the approach is not guaranteed and notions of coverage has to be accompanied. On the other hand, formal verification incorporates mathematical proofs and guarantee the completeness over the search space. However, formal verification has problems of its own in which it is usually resource intensive. In addition, not all design can be verified after optimization processes. That is to say the golden model and DUV are vastly different in structure which cause modern checker to give inconclusive result. Due to this nature, this thesis focuses in improving the strength and the efficiency of sequential equivalence checking (SEC) using formal approach. While there has been great strides made in the verification for combinational circuits, SEC still remains rather rudimentary. Without powerful SEC as a backbone, aggressive sequential synthesis and optimization are often avoided if the optimized design cannot be proved to be equivalent to the original one. In an attempt to take on the challenges of SEC, we propose two frameworks that successfully determining equivalence for hard-to-verify circuits. The first framework utilizes arbitrary relations between any two nodes within the two sequential circuits in question. The two nodes can reside in the same or across the circuits; likewise, they can be from the same time-frame or across time-frames. The merit for this approach is to use global structure of the circuits to speed up the verification process. The second framework introduces techniques to identify subset but yet powerful multi-node relations (involve more than 2 nodes) which then help to prune large don't care search space and result in a successful SEC framework. In contrast with previous approaches in which exponential number of multi-node relations are mined and learned, we alleviate the computation cost by selecting much fewer invariants to achieve desired conclusion. Although independent, the two frameworks could be used in sequential to complement each other. Experimental results demonstrate that our frameworks can take on many hard-to-verify cases and show a significant speed up over previous approaches. / Master of Science

Yes / Brackish water can be considered an important source of fresh water, via desalination, especially for arid districts. Reverse Osmosis (RO) process has been successfully used to produce fresh water from brackish water sources. However, there is still the challenge of improving the performance of multistage RO desalination plants. From the selection of the RO configurations to the selection of the appropriate type of membranes and the operating conditions at the end determines the performance of RO process in terms of recovery, salt rejection, energy consumptions and ultimately the cost of production of freshwater. Using model-based simulation, this work attempts to investigate the most suitable types of membranes for an industrial scale RO plant from a set of different membrane brands that would attain the highest-performance at lowest specific energy consumption (SEC). As a case study, we considered a multistage multi-pass medium-scale RO plant (1200 m3/day) of Arab Potash Company (APC, Jordan) which produces high quality water for the boilers after pre-treatment stage. The simulation results confirmed that employment of the Filmtec BW30LE-440 would increase water recovery by about 22% besides reducing the product salinity and SEC by about 15% and 10%, respectively compared to the existing membrane.

This Dissertation consists of two essays. The first essay studies the causal impacts of compensation disclosure on executive compensation, turnover, and executives’ job responsibilities. We find that, after the SEC mandates the disclosure of Chief Financial Officers (CFOs)’ compensation in 2006, CFO pay increases significantly relative to CEO pay, particularly in firms most affected by the mandate. CFOs are more likely to leave their firms following poor performance. The results are absent for the CEO or other executives, suggesting they are unique outcomes of enhanced CFO compensation disclosures. The evidence is consistent with more intense monitoring following the disclosure mandate. CFOs require additional compensation for the loss of private benefits due to greater monitoring and are subject to greater internal discipline. There is also some evidence that the CFOs hide bad news and lower corporate reporting quality after the mandate, suggesting that CFOs engage in more short-term behavior to boost their performance and avoid termination. The second essay of my dissertation focuses on the idiosyncratic volatility puzzle - the negative relation between estimated idiosyncratic volatility and the subsequent month returns documented by Ang et al (2006). We document a systematic pattern of temporary increases in the estimated idiosyncratic volatility for the quintile of stocks with the highest estimated idiosyncratic volatility in a given month. A large portion of this temporary increase in the estimated idiosyncratic volatility is reversed in the subsequent month. This temporary increase in the idiosyncratic volatility for the quintile of stocks with the highest estimated idiosyncratic volatility is associated with relatively large positive returns (positive abnormal returns) in the estimation month and relatively low returns (negative abnormal returns) in the subsequent month. Our evidence shows that these temporary increases in the estimated idiosyncratic volatility and the related positive and negative abnormal returns in the estimation and subsequent months, respectively, create a negative relation between the estimated idiosyncratic volatility and subsequent month returns documented in the prior literature (Ang et al. 2006). We find no significant relation between idiosyncratic volatility and subsequent returns for eighty percent of the stocks that do not exhibit large changes in idiosyncratic volatility despite large differences in the levels of their idiosyncratic volatility. Finally, there is no relation between the estimated idiosyncratic volatility and subsequent returns after a lag of 3 months when the abnormal returns associated with temporary changes are no longer present. Overall, our results are consistent with the notion that there is no relation between the true underlying idiosyncratic volatility and expected returns, and that the previously documented negative relation between estimated idiosyncratic volatility and subsequent month’s returns is being driven by temporary one-month increases in the estimated idiosyncratic volatility and the associated abnormal returns for a subset of stocks. / Ph. D. / The disclosure of executive compensation is an important issue because it affects the investors’ ability to monitor the firms’ compensation practices. Properly designed compensation contracts, in turn, incentivize the executives to make decisions that serve the investors’ interests. The SEC has made continuous regulatory efforts to monitor the executive compensation and has adopted several disclosure rules. However, the impacts of such enhanced compensation disclosure has not been well understood. My first essay studies the impacts of compensation disclosure on executive compensation, turnover, and executives’ job responsibilities. We find that, after the SEC mandates the disclosure of Chief Financial Officers (CFOs)’ compensation in 2006, CFO pay increases significantly relative to CEO pay, particularly in firms most affected by the mandate. CFOs are more likely to leave their firms following poor performance. There is also some evidence that the CFOs hide bad news and lower corporate reporting quality after the mandate, suggesting that CFOs engage in more short-term behavior to boost their performance and avoid termination. Traditional asset pricing models in which investors hold well-diversified portfolios imply that there should be no relation between firm specific risk (the idiosyncratic volatility) and the expected returns. However, Ang et al (2006) document that stocks with high firm specific risks earn low subsequent returns. The significant negative relation between firm specific risk and subsequent returns has puzzled many researchers. The second essay of my dissertation provides a possible resolution to this puzzle.

Les carcinomes de la corticosurrénale (CC) sont des tumeurs malignes rares dont le pronostic est globalement sombre et les thérapeutiques encore limitées, la chirurgie étant le seul traitement efficace. Il est donc important de comprendre les mécanismes impliqués dans le développement et l'agressivité des CC. L’activation constitutive de la voie de signalisation Wnt/b-caténine est fréquente dans les CC (40%) et est associée à un caractère agressif. L’objectif de mon projet de thèse était d’étudier l’implication de la voie Wnt/β-caténine dans la tumorigenèse corticosurrénalienne. La lignée cellulaire humaine H295R, issue d’un CC présente une activation de la voie Wnt/β-caténine qui a pour origine une mutation activatrice de la β-caténine. Nous avons montré que l'invalidation de la β-caténine dans les cellules H295R inhibe l'activité transcriptionnelle de la voie Wnt/β-caténine, diminue la prolifération, augmente l'apoptose et bloque la progression du cycle cellulaire. De plus, nous avons montré que la voie Wnt/b-caténine est essentielle au développement tumoral de xénogreffes de ces cellules chez la souris. L’activation de la voie Wnt/b-caténine participe à la tumorigenèse de nombreux organes en régulant l’expression de gènes impliqués par exemple dans la prolifération, la survie cellulaire ou l'adhésion. Afin de mieux comprendre comment la voie Wnt/β-caténine participe à la tumorigenèse corticosurrénalienne, nous avons cherché à identifier les gènes cibles de cette voie dans les CC. L’analyse des transcriptomes de deux cohortes indépendantes de CC et des cellules H295R avec ou sans invalidation de la β-caténine a permis d’identifier des gènes dont l'expression est corrélée à l'activation de la voie Wnt/b-caténine. Nous avons montré que parmi ces gènes, AFF3 est essentiel pour transmettre les effets de l'activation de la voie Wnt/b-caténine dans les carcinomes de la corticosurrénale. En effet, AFF3 est un gène cible direct de la voie Wnt/b-caténine et son invalidation dans les cellules corticosurrénaliennes H295R diminue la prolifération cellulaire et déclenche l'apoptose à l'image de l'invalidation de la b-caténine. AFF3 est une protéine nucléaire, localisée au niveau des speckles qui sont impliqués dans l'épissage des ARNm. De plus, AFF3 interagit avec le P-TEFb (CDK9/CyclineT1/2) au sein du Super elongation complex (SEC) nécessaire à l’élongation de la transcription des ARNm par l'ARN polymérase II. Nous avons ainsi montré dans les cellules corticosurrénaliennes H295R, que la surexpression d'AFF3 altère l’organisation des speckles et la localisation de CDK9 et Cycline T1. En conclusion, ce travail a permis d'identifier une nouvelle cible transcriptionnelle de la voie de signalisation Wnt/b-caténine, AFF3, qui code pour un médiateur important des effets de l'activation de cette voie dans la tumorigenèse corticosurrénalienne. AFF3 agirait notamment en altérant la structure des speckles et en interagissant avec le P-TEFb qui sont importants respectivement pour l'épissage des ARNm et la transcription. Ces résultats conduisent à une meilleure compréhension de la tumorigenèse corticosurrénalienne et permettent d'envisager le P-TEFb et le SEC comme de nouvelles cibles thérapeutiques pour le traitement des CC. / Adrenocortical carcinoma (ACC) is a rare and highly aggressive endocrine neoplasm, with limited therapeutic option. Currently, surgical resection is considered the only effective treatment. It is therefore essential to understand the molecular mechanisms involved in ACC development in order to improve their clinical management. Activation of the Wnt/b-catenin signaling pathway is frequent (40%) in ACC and is associated with poor prognosis. The aim of my thesis was to study the involvement of the Wnt/b-catenin signaling pathway in adrenocortical tumorigenesis. The human cell line H295R, derived from an ACC, carries the S45P β-catenin mutation which leads to constitutive β-catenin/TCF transcriptional activity. In the ACC cell line H295R we show that β-catenin silencing resulted in a decreased transcriptional activity of the Wnt/b-catenin signaling, cell cycle alterations, a decreased cell proliferation and an increased apoptosis. Moreover we show that β-catenin silencing abolish xenograft development of H295R adrenocortical cells. Aberrant activation of the Wnt/b-catenin signaling promotes tumorigenesis of several organs by enhancing expression of genes involved in proliferation, cell survival or cell adhesion. To better understand the role of the Wnt/b-catenin signaling in adrenocortical tumorigenesis, we wanted to identify target genes of this pathway in ACC. Combined transcriptomic analysis on two independent cohorts of ACC and on H295R adrenocortical cells with or without β-catenin silencing allow us to identify alterations of gene expression due to aberrant Wnt/βcatenin pathway activation. Among these genes, we show that AFF3 is essential to mediate the effect of the activation of the Wnt/β-catenin signaling pathway in adrenocortical cancer. Indeed, AFF3 is a direct target gene of the Wnt/b-catenin and its silencing in H295R adrenocortical cells induces a decreased cell proliferation and an increased apoptosis similar to that induced by b-catenin silencing. AFF3 is a nuclear protein located in nuclear speckles, which serve as a reservoir of factors participating in mRNA splicing. Moreover, AFF3 interacts with P-TEFb (CDK9/CyclinT1/2) in the Super elongation complex (SEC) required for transcriptional elongation of mRNA by RNA polymerase II. In H295R adrenocortical cells, we show that strong overproduction of AFF3 altered the structural organization of nuclear speckles and the localization of CDK9 and Cycline T1. In conclusion, this study has identified a new transcriptional target of the Wnt/β-catenin signaling pathway, AFF3, which encodes an important mediator of this pathway in adrenocortical tumorigenesis. AFF3 might especially act by affecting the structural organization of speckles and interacting with the P-TEFb, which are respectively involved in mRNA splicing and transcription. These results provide a better understanding of the biological process involved in ACC development and suggest that P-TEFb and SEC could be new therapeutic targets for the treatment of ACC.

Les carcinomes de la corticosurrénale (CC) sont des tumeurs malignes rares dont le pronostic est globalement sombre et les thérapeutiques encore limitées, la chirurgie étant le seul traitement efficace. Il est donc important de comprendre les mécanismes impliqués dans le développement et l'agressivité des CC. L’activation constitutive de la voie de signalisation Wnt/b-caténine est fréquente dans les CC (40%) et est associée à un caractère agressif. L’objectif de mon projet de thèse était d’étudier l’implication de la voie Wnt/β-caténine dans la tumorigenèse corticosurrénalienne. La lignée cellulaire humaine H295R, issue d’un CC présente une activation de la voie Wnt/β-caténine qui a pour origine une mutation activatrice de la β-caténine. Nous avons montré que l'invalidation de la β-caténine dans les cellules H295R inhibe l'activité transcriptionnelle de la voie Wnt/β-caténine, diminue la prolifération, augmente l'apoptose et bloque la progression du cycle cellulaire. De plus, nous avons montré que la voie Wnt/b-caténine est essentielle au développement tumoral de xénogreffes de ces cellules chez la souris. L’activation de la voie Wnt/b-caténine participe à la tumorigenèse de nombreux organes en régulant l’expression de gènes impliqués par exemple dans la prolifération, la survie cellulaire ou l'adhésion. Afin de mieux comprendre comment la voie Wnt/β-caténine participe à la tumorigenèse corticosurrénalienne, nous avons cherché à identifier les gènes cibles de cette voie dans les CC. L’analyse des transcriptomes de deux cohortes indépendantes de CC et des cellules H295R avec ou sans invalidation de la β-caténine a permis d’identifier des gènes dont l'expression est corrélée à l'activation de la voie Wnt/b-caténine. Nous avons montré que parmi ces gènes, AFF3 est essentiel pour transmettre les effets de l'activation de la voie Wnt/b-caténine dans les carcinomes de la corticosurrénale. En effet, AFF3 est un gène cible direct de la voie Wnt/b-caténine et son invalidation dans les cellules corticosurrénaliennes H295R diminue la prolifération cellulaire et déclenche l'apoptose à l'image de l'invalidation de la b-caténine. AFF3 est une protéine nucléaire, localisée au niveau des speckles qui sont impliqués dans l'épissage des ARNm. De plus, AFF3 interagit avec le P-TEFb (CDK9/CyclineT1/2) au sein du Super elongation complex (SEC) nécessaire à l’élongation de la transcription des ARNm par l'ARN polymérase II. Nous avons ainsi montré dans les cellules corticosurrénaliennes H295R, que la surexpression d'AFF3 altère l’organisation des speckles et la localisation de CDK9 et Cycline T1. En conclusion, ce travail a permis d'identifier une nouvelle cible transcriptionnelle de la voie de signalisation Wnt/b-caténine, AFF3, qui code pour un médiateur important des effets de l'activation de cette voie dans la tumorigenèse corticosurrénalienne. AFF3 agirait notamment en altérant la structure des speckles et en interagissant avec le P-TEFb qui sont importants respectivement pour l'épissage des ARNm et la transcription. Ces résultats conduisent à une meilleure compréhension de la tumorigenèse corticosurrénalienne et permettent d'envisager le P-TEFb et le SEC comme de nouvelles cibles thérapeutiques pour le traitement des CC. / Adrenocortical carcinoma (ACC) is a rare and highly aggressive endocrine neoplasm, with limited therapeutic option. Currently, surgical resection is considered the only effective treatment. It is therefore essential to understand the molecular mechanisms involved in ACC development in order to improve their clinical management. Activation of the Wnt/b-catenin signaling pathway is frequent (40%) in ACC and is associated with poor prognosis. The aim of my thesis was to study the involvement of the Wnt/b-catenin signaling pathway in adrenocortical tumorigenesis. The human cell line H295R, derived from an ACC, carries the S45P β-catenin mutation which leads to constitutive β-catenin/TCF transcriptional activity. In the ACC cell line H295R we show that β-catenin silencing resulted in a decreased transcriptional activity of the Wnt/b-catenin signaling, cell cycle alterations, a decreased cell proliferation and an increased apoptosis. Moreover we show that β-catenin silencing abolish xenograft development of H295R adrenocortical cells. Aberrant activation of the Wnt/b-catenin signaling promotes tumorigenesis of several organs by enhancing expression of genes involved in proliferation, cell survival or cell adhesion. To better understand the role of the Wnt/b-catenin signaling in adrenocortical tumorigenesis, we wanted to identify target genes of this pathway in ACC. Combined transcriptomic analysis on two independent cohorts of ACC and on H295R adrenocortical cells with or without β-catenin silencing allow us to identify alterations of gene expression due to aberrant Wnt/βcatenin pathway activation. Among these genes, we show that AFF3 is essential to mediate the effect of the activation of the Wnt/β-catenin signaling pathway in adrenocortical cancer. Indeed, AFF3 is a direct target gene of the Wnt/b-catenin and its silencing in H295R adrenocortical cells induces a decreased cell proliferation and an increased apoptosis similar to that induced by b-catenin silencing. AFF3 is a nuclear protein located in nuclear speckles, which serve as a reservoir of factors participating in mRNA splicing. Moreover, AFF3 interacts with P-TEFb (CDK9/CyclinT1/2) in the Super elongation complex (SEC) required for transcriptional elongation of mRNA by RNA polymerase II. In H295R adrenocortical cells, we show that strong overproduction of AFF3 altered the structural organization of nuclear speckles and the localization of CDK9 and Cycline T1. In conclusion, this study has identified a new transcriptional target of the Wnt/β-catenin signaling pathway, AFF3, which encodes an important mediator of this pathway in adrenocortical tumorigenesis. AFF3 might especially act by affecting the structural organization of speckles and interacting with the P-TEFb, which are respectively involved in mRNA splicing and transcription. These results provide a better understanding of the biological process involved in ACC development and suggest that P-TEFb and SEC could be new therapeutic targets for the treatment of ACC.

Le développement des outils analytiques pour l'analyse de la matière organique complexe en géochimie organique a connu de nombreuses avancées ces dernières années. Ce développement a permis de répondre à un grand nombre de questions quant à la composition de la matière organique. Cependant, beaucoup des points restent encore à élucider comme notamment la caractérisation des fractions de hauts poids moléculaires ainsi que le suivi de la réactivité de la matière organique. Ce travail de thèse a eu pour objectif (i) d'adapter les techniques de spectrométrie de masse déjà existantes pour l'analyse de la matière organique fossile (notamment par la sélection de la source d'ionisation atmosphérique la plus adaptée) mais également (ii) de développer un nouveau type de couplage entre la chromatographie d'exclusion stérique (SEC) et la spectrométrie de masse APPI-QTOF pour l'analyse des fractions peu polaires de hauts poids moléculaires. L'adaptation du l'APPI-QTOF a tout d'abord permis de mieux comprendre la réactivité de contaminants organiques polyaromatiques en présence de phases minérales. Le couplage SEC-APPI-QTOF a, quant à lui, permis d'améliorer les connaissances sur la structure des asphaltènes. Cependant, malgré la « simplification » rendue possible par la SEC, la très grande quantité d'informations reste difficile à interpréter et prend beaucoup de temps. Un modèle mathématique a donc été développé basé sur des analyses numériques et statistiques des spectres de masse, permettant de les comparer entre eux afin de distinguer l'origine des échantillons et de suivre l'impact de processus physico-chimiques (altérations naturelles - traitements de remédiation). / The development of analytical tools for organic geochemistry analysis has increased these past years. This development has allowed answering many questions about organic matter composition. However, many issues remain to be clarified including the characterization of high molecular weight fractions and monitoring the reactivity of organic matter. This thesis has focused on both (i) existing method improvements for fossil organic geochemistry analysis but also on (ii) developing a new type of coupling between the size exclusion chromatography (SEC) and the APPI-QTOF mass spectrometry for high molecular weight weakly polar fractions. Adjustments on APPI-QTOF mass spectrometry have allowed a better understanding of polyaromatic organic contaminant reactivity in presence of mineral matrices. The success of this coupling has allowed a better understanding of the structure of asphaltenes. However despite the "simplification" obtained by the SEC, the large amount of information remains difficult to interpret and time-consuming. A mathematical model has been developed based on numerical and statistical analysis of mass spectra, allowing direct comparison of mass spectra and being able to identify several types of information such as origins of samples, monitoring of physico-chemical processes and also the efficiency of soil recovery treatments as well as the identification of analytical protocols.

Orientador: Maria Heloísa Martins Dias / Banca: Sônia Helena de Oliveira Raymundo Piteri / Banca: Annie Gisele Fernandes / Resumo: Esta dissertação apresenta confluências entre as poesias da brasileira Orides Fontela (1940-1998) e da portuguesa Natália Correia (1923-1993), a partir das diferenças de suas específicas linguagens, buscando analisar, no posicionamento lírico, um contraste referente à construção do ser como projeção de sentido do poema, sendo essa projeção entendida como "sujeito poético". Se a obra de Orides, aproximando-se do fio condutor do trabalho de João Cabral pela impessoalidade, contenção, busca pela palavra exata e culto a um silêncio esfíngico, constrói um sujeito cognoscente como o resultado do olhar contemplativo da voz lírica, em seu ato de flagrar e registrar as coisas do mundo, transformando a apreensão em conhecimento analítico, a de Natália projeta como sujeito uma afirmação intensa do eu no discurso, em um olhar voltado para a gênese do indivíduo, promovendo, na consubstanciação narcisista da voz com a poeta, uma figura de linguagem necessária tanto para romper com a lógica (o absolutismo racionalista) e com o senso comum em relação a Deus quanto para defender o poeta, a poesia e a urgência de praticá-la valorizando-se o princípio da especificidade da arte, a memória literária em geral. Identificando, por um lado, a fragmentação e a aridez no estilo contido e esfíngico de Orides e, por outro, o enredamento e a força impulsiva de uma "feiticeira" no tom rebelde da linguagem de Natália, evita-se, desse modo, a tendência usual, em muitos dos estudos comparativos, de busca de analogias assentadas na semelhança. Não se constroem binarismos entre os poemas, mas articulações de modo mais aberto, favorecendo a capacidade de estabelecer relações e, assim, uma visão mais ampla da literatura, em uma aproximação intercontinental liberta das limitações dos estudos centrados em um único espaço. / Abstract: This dissertation presents convergence between the poetry of the Brazilian author Orides Fontela (1940-1998) and the Portuguese author Natália Correia (1923- 1993). We seek to analyze from both their specific languages and their poetic positioning, the contrast concerning the construction of the subject as projection of meaning in the poem; this projection seen as "poetic subject". The work of Orides has some similarities with the work of João Cabral due to its impersonal characteristics, self-restraint, quest for the proper word and reverence for a sphinx silence. It also creates a cognoscenti subject as consequence of the contemplative look of the poetic voice, a subject depicted while perceiving and registering the things of the world, changing his perception to analytical knowledge. On the other hand, Natália projects as subject an intense affirmation of the subject in the discourse, a look directed towards the genesis of the individual. Fostering, while gathering narcissist voice and poet, a figurative language required to break both with the logic (rational absolutism) and with what is generally accepted about God. Making use of this language, poet, poetry, literary memory in general, and the eagerness to write valuing the idea of purpose to art, are protected. We focused on one hand on the fragmentation and arid self-constrained sphinx style of Orides, and on the other hand on the entanglement and impulsive power of a "witch" written by Natália in a rebellious language, thus avoiding the search for analogies based on similarities, which is a common tendency in many comparative studies. It was not raised binary oppositions between the poems, but open articulations, favoring the capability to establish connections. Therefore, it was possible to have a clear vision of literature, and an intercontinental approach free from the limitations posed by studies centered in one space. / Mestre

A esterificação do sec-butanol com ácido acético, submetida à catálise homogênea, apresenta poucos resultados experimentais de cinética reacional e equilíbrio químico disponíveis na literatura. A presente dissertação visa determinar os parâmetros cinéticos e de equilíbrio químico desta reação de esterificação, empregando modelo cinético de 2ª ordem baseado em atividades. Os experimentos cinéticos foram realizados em reator batelada com auxílio da metodologia Karl Fischer para análise do teor de água e determinação do perfil de frações molares ao longo da reação. A principal dificuldade na modelagem desta esterificação consistiu em representar satisfatoriamente as não-idealidades da mistura em fase líquida, em espacial nas reações cuja fração molar inicial de ácido acético foi superior a 50%. Netas condições admitiu-se a ocorrência de dissociação do ácido acético levando ao surgimento de interações iônicas. O modelo empregado para obtenção das atividades, com base em frações molares, foi o NRTL. Este modelo representou bem as interações moleculares em condição de baixa acidez, mas apresentou desvios em condições de acidez e teores de água mais elevados. O estudo também visou o desenvolvimento de modelos de calibração para um método analítico on-line visando determinar o perfil de frações molares ao longo da reação. O método empregado foi a espectroscopia de infravermelho próximo (NIR), em vista à capacidade deste método analítico em determinar quantitativamente frações molares de água, além dos demais compostos envolvidos. Outro fator determinante ao emprego do NIR é a seu crescente uso no meio industrial, e o presente trabalho visou também contribuir para o uso futuro desta técnica de monitoramento no processo industrial. Modelos de calibração multivariável com conjuntos de calibração interna por validação cruzada se mostraram adequados ao sistema estudado. Calibrações externas resultaram em modelos imprecisos, não sendo possível determinar claramente os fatores responsáveis pela imprecisão. / There is a lack of literature data on the reaction kinetics and equilibrium of the homogeneously catalyzed esterification of sec-butanol with acetic acid. This work aims at obtaining the kinetic parameters and chemical equilibrium parameters of this esterification reaction by using a second order kinetic model based on activities. The kinetic experiments were performed in a batch reactor and the Karl Fischer analytical method was used to determine the molar fraction of water along the reaction progress. The main challenge of modeling this reaction was to represent the highly non-ideal mixture in liquid phase, specially for reactions runs starting with acetic acid mole fraction higher than 50%. In these conditions, it was assumed the occurrence of acetic acid dissociation that leads to ionic interactions. The thermodynamic model used to calculate the activities was the NRTL. This model predicted well the molecular interactions on low acidity conditions but presented deviations when molar fractions of acid and water were higher. The present study also aims to develop calibration models for analytical on-line method to obtain molar fraction profiles along the reaction. Near infrared spectroscopy (NIR) was used due to the possibility of quantitatively analyze water mole fraction, besides the mole fractions of the other components. Another important reason to choose NIR was its crescent use in industry, and the present work intended to contribute towards the future use of this on line monitoring technique in industrial applications. Multivariate calibration models using internal set of calibration and cross-validation seems to be suitable for this system. External set of calibration leads to less accurate models, and the causes of this lack of accuracy were not clearly identified.

Les carcinomes de la corticosurrénale (CC) sont des tumeurs malignes rares dont le pronostic est globalement sombre et les thérapeutiques encore limitées, la chirurgie étant le seul traitement efficace. Il est donc important de comprendre les mécanismes impliqués dans le développement et l'agressivité des CC. L’activation constitutive de la voie de signalisation Wnt/b-caténine est fréquente dans les CC (40%) et est associée à un caractère agressif. L’objectif de mon projet de thèse était d’étudier l’implication de la voie Wnt/β-caténine dans la tumorigenèse corticosurrénalienne. La lignée cellulaire humaine H295R, issue d’un CC présente une activation de la voie Wnt/β-caténine qui a pour origine une mutation activatrice de la β-caténine. Nous avons montré que l'invalidation de la β-caténine dans les cellules H295R inhibe l'activité transcriptionnelle de la voie Wnt/β-caténine, diminue la prolifération, augmente l'apoptose et bloque la progression du cycle cellulaire. De plus, nous avons montré que la voie Wnt/b-caténine est essentielle au développement tumoral de xénogreffes de ces cellules chez la souris. L’activation de la voie Wnt/b-caténine participe à la tumorigenèse de nombreux organes en régulant l’expression de gènes impliqués par exemple dans la prolifération, la survie cellulaire ou l'adhésion. Afin de mieux comprendre comment la voie Wnt/β-caténine participe à la tumorigenèse corticosurrénalienne, nous avons cherché à identifier les gènes cibles de cette voie dans les CC. L’analyse des transcriptomes de deux cohortes indépendantes de CC et des cellules H295R avec ou sans invalidation de la β-caténine a permis d’identifier des gènes dont l'expression est corrélée à l'activation de la voie Wnt/b-caténine. Nous avons montré que parmi ces gènes, AFF3 est essentiel pour transmettre les effets de l'activation de la voie Wnt/b-caténine dans les carcinomes de la corticosurrénale. En effet, AFF3 est un gène cible direct de la voie Wnt/b-caténine et son invalidation dans les cellules corticosurrénaliennes H295R diminue la prolifération cellulaire et déclenche l'apoptose à l'image de l'invalidation de la b-caténine. AFF3 est une protéine nucléaire, localisée au niveau des speckles qui sont impliqués dans l'épissage des ARNm. De plus, AFF3 interagit avec le P-TEFb (CDK9/CyclineT1/2) au sein du Super elongation complex (SEC) nécessaire à l’élongation de la transcription des ARNm par l'ARN polymérase II. Nous avons ainsi montré dans les cellules corticosurrénaliennes H295R, que la surexpression d'AFF3 altère l’organisation des speckles et la localisation de CDK9 et Cycline T1. En conclusion, ce travail a permis d'identifier une nouvelle cible transcriptionnelle de la voie de signalisation Wnt/b-caténine, AFF3, qui code pour un médiateur important des effets de l'activation de cette voie dans la tumorigenèse corticosurrénalienne. AFF3 agirait notamment en altérant la structure des speckles et en interagissant avec le P-TEFb qui sont importants respectivement pour l'épissage des ARNm et la transcription. Ces résultats conduisent à une meilleure compréhension de la tumorigenèse corticosurrénalienne et permettent d'envisager le P-TEFb et le SEC comme de nouvelles cibles thérapeutiques pour le traitement des CC. / Adrenocortical carcinoma (ACC) is a rare and highly aggressive endocrine neoplasm, with limited therapeutic option. Currently, surgical resection is considered the only effective treatment. It is therefore essential to understand the molecular mechanisms involved in ACC development in order to improve their clinical management. Activation of the Wnt/b-catenin signaling pathway is frequent (40%) in ACC and is associated with poor prognosis. The aim of my thesis was to study the involvement of the Wnt/b-catenin signaling pathway in adrenocortical tumorigenesis. The human cell line H295R, derived from an ACC, carries the S45P β-catenin mutation which leads to constitutive β-catenin/TCF transcriptional activity. In the ACC cell line H295R we show that β-catenin silencing resulted in a decreased transcriptional activity of the Wnt/b-catenin signaling, cell cycle alterations, a decreased cell proliferation and an increased apoptosis. Moreover we show that β-catenin silencing abolish xenograft development of H295R adrenocortical cells. Aberrant activation of the Wnt/b-catenin signaling promotes tumorigenesis of several organs by enhancing expression of genes involved in proliferation, cell survival or cell adhesion. To better understand the role of the Wnt/b-catenin signaling in adrenocortical tumorigenesis, we wanted to identify target genes of this pathway in ACC. Combined transcriptomic analysis on two independent cohorts of ACC and on H295R adrenocortical cells with or without β-catenin silencing allow us to identify alterations of gene expression due to aberrant Wnt/βcatenin pathway activation. Among these genes, we show that AFF3 is essential to mediate the effect of the activation of the Wnt/β-catenin signaling pathway in adrenocortical cancer. Indeed, AFF3 is a direct target gene of the Wnt/b-catenin and its silencing in H295R adrenocortical cells induces a decreased cell proliferation and an increased apoptosis similar to that induced by b-catenin silencing. AFF3 is a nuclear protein located in nuclear speckles, which serve as a reservoir of factors participating in mRNA splicing. Moreover, AFF3 interacts with P-TEFb (CDK9/CyclinT1/2) in the Super elongation complex (SEC) required for transcriptional elongation of mRNA by RNA polymerase II. In H295R adrenocortical cells, we show that strong overproduction of AFF3 altered the structural organization of nuclear speckles and the localization of CDK9 and Cycline T1. In conclusion, this study has identified a new transcriptional target of the Wnt/β-catenin signaling pathway, AFF3, which encodes an important mediator of this pathway in adrenocortical tumorigenesis. AFF3 might especially act by affecting the structural organization of speckles and interacting with the P-TEFb, which are respectively involved in mRNA splicing and transcription. These results provide a better understanding of the biological process involved in ACC development and suggest that P-TEFb and SEC could be new therapeutic targets for the treatment of ACC.

L’objectif de cette thèse porte sur l’étude de la compartimentalisation cellulaire et de la prise en charge de l’uranium (U) par les protéines cytosoliques des cellules branchiales du poisson zèbre (Danio rerio, espèce modèle en toxicologie aquatique) après exposition contrastées (chronique vs. aiguë, 20 et 250 µg.L-1) par voie directe. Cette étude a nécessité le développement, l’utilisation et le couplage d’outils analytiques de pointe (SEC, IEF hors-gel, RP-UHPLC pour la séparation, ICP-SFMS, ESI-FTMS/MS pour la détection) avec comme défis majeurs la conservation des interactions non-covalentes U biomolécule et une sensibilité maximale pour travailler à des niveaux d’exposition proches de ceux rencontrés dans l’environnement. Après extraction, 24 à 32% de la charge branchiale totale en U est contenue dans le cytosol dans lequel la distribution de l’U sur les biomolécules (en fonction de leur PM mais aussi de leur pI) diffère selon le niveau d’exposition. Enfin, une cartographie des biomolécules cibles de l’U a permis (i) de mettre en évidence une affinité particulière de l’U pour les protéines à caractère acide et/ou contenant du phosphore et (ii) d’identifier 24 protéines candidates pour lier U. / The objective of this thesis is to study the cellular compartmentalization and the chelation of uranium (U) by cytosolic proteins of gill cells of the zebrafish (Danio rerio, model species in aquatic toxicology) under different direct exposure conditions (chronic vs. acute, 20 and 250 µg.L 1). This study required the development of hyphenated techniques (SEC, IEF off-gel, RP-UHPLC for the separation, ICP-SFMS, ESI-FTMS/MS for the detection) with the main challenges of maintaining the non-covalent U-biomolecule interactions and enhancing sensitivity for the analysis of environmentally relevant samples. After extraction, 24% to 32% of the total U detected in the gills were present in the cytosolic fraction, in which the U distribution on the biomolecules (as a function of their MW and pI) varied depending on the exposure level. Finally, U target biomolecules mapping allowed us (i) to highlight a particular affinity of U for acidic and/or P-containing proteins and (ii) to identify 24 protein candidates for U binding.