Die Verknüpfung einer exergonischen mit einer endergonischen Reaktion zur Ermöglichung der letzteren ist eine in biologischen Systemen weit verbreitete Strategie. Energetisch benachteiligte Elektronenübertragungsreaktionen im Rahmen der reduktiven Aktivierung von Nitrogenasen, Radikal-abhängigen β,α-Dehydratasen, der zu diesen verwandten Benzoyl-CoA-Reduktasen und diversen Cobalamin-abhängigen Methyltransferasen sind gekoppelt an die Hydrolyse von ATP. Der Methylgruppentransfer des reduktiven Acetyl-CoA-Weges von Carboxydothermus hydrogenoformans erfordert den Co(I)-Zustand des Corrinoid/Eisen-Schwefel Proteins (CoFeSP). Um diese superreduzierte Form nach einer oxidativen Inaktivierung zu regenerieren ist ein „Reparaturmechanismus“ erforderlich. Ein offenes Leseraster (orf7), welches möglicherweise für eine reduktive Aktivase von Corrinoid Enzymen (RACE) kodiert, wurde in dem Gencluster der am reduktiven Acetyl-CoA-Weg beteiligten Proteine entdeckt. Im Rahmen dieser Arbeit wurde dieses potenzielle RACE Protein biochemisch und strukturell charakterisiert und die ATP-abhängige reduktive Aktivierung von CoFeSP untersucht. Auf Grundlage der in dieser Arbeit gewonnenen Ergebnisse wurde ein Mechanismus für die ATP-abhängige Aktivierung entworfen. Dieser gibt Einblicke wie die durch ATP-Hydrolyse bereitgestellte Energie einen energetisch ungünstigen Elektronentransfer ermöglichen kann. Hierzu kombiniert RACo das Ausgleichen von Bindungsenergien mit Modulationen am Elektronenakzeptor. Eine vergleichbare Strategie wurde bisher in keinem anderen ATP-abhängigen Elektronenübertragungssystem wie dem von Nitrogenasen, Radikal-abhängigen β,α-Dehydratasen oder Benzoyl-CoA-Reduktasen beobachtet und könnte ein für RACE Proteine allgemein gültige Eigenschaft darstellen. / The principle of coupling an exergonic to an endergonic reaction to enable the latter is a widespread strategy in biological systems. Unfavoured electron transfer reactions in the reductive activation of nitrogenases, radical-dependent β,α-dehydratases and the related benzoyl- CoA reductases, as well as different cobalamin-dependent methyltransferases are coupled to the hydrolysis of ATP. The reductive acetyl-CoA pathway of Carboxydothermus hydrogenoformans relies on the superreduced Co(I)-state of the corrinoid/iron-sulfur protein (CoFeSP) that requires a “repair mechanism” in case of incidental oxidation. An open reading frame (orf7) coding for a putative reductive activase of corrinoid enzymes (RACE) was discovered in the gene cluster of proteins involved in the reductive acetyl-CoA pathway. In this work, this putative RACE protein was biochemically and structurally characterised and the ATP-dependent reductive activation of CoFeSP was investigated. Based on the results of this study, a mechanism for the ATP-dependent reactivation of CoFeSP was deduced providing insights into how the energy provided by ATP could trigger this unfavourable electron transfer. The reductive activator of CoFeSP combines balance of binding energies and modulations of the electron acceptor to promote the uphill electron transfer to CoFeSP. A comparable strategy has not been observed in other ATP-dependent electron transfer systems like nitrogenases, radical-dependent β,α-dehydratases and benzoyl- CoA reductases and could be a universal feature of RACE proteins.

Les astrocytes protoplasmiques de la matière grise corticale sont des cellules gliales dont les prolongements très fins et ramifiés sont en contact avec les éléments neuronaux pré- et post-synaptiques d’une part, et les vaisseaux sanguins d’autre part. Ils expriment plusieurs récepteurs des neurotransmetteurs, entre autres des récepteurs purinergiques dont l'activation facilite l’activité calcique astrocytaire et la libération de gliotransmitters (par exemple, le glutamate, le GABA, l'ATP, et la D sérine) qui régulent l’activité des neurones et des cellules gliales situées au voisinage. L’objectif de ma thèse était d’étudier in situ l’activité calcique des astrocytes et de leurs prolongements en réponse à l’application des agonistes purinergiques. Lors de ma thèse, j’ai tout d'abord testé la possibilité d’induire l’expression spécifique de gènes d’intérêt par les astrocytes corticaux de souris adultes par la technique de recombinaison Cre-LoxP. J’ai comparé les performances d’un virus adeno-associé de type 5 (AAV5) flexé (AAV5.FLEX.EGFP) et d’une souris qui exprime un indicateur calcique (GCaMP3) sous contrôle de la recombinase (souris Rosa-CAG-LSL-GCaMP3). L’injection d’AAV5.FLEX.EGFP dans le cortex d’une souris hGFAPcre n’a pas permis l’expression spécifique d’EGFP. La combinaison des souris exprimant le cre recombinase sous contrôle d’un promoteur sélectif des astrocytes (GLAST-CreERT2 et Cx30-CreERT2) avec le AAV5.FLEX.EGFP ou avec une lignée des souris Rosa-CAG-LSL-GCaMP3 permet l’expression spécifique des gènes d’intérêt (EGFP et GCaMP3) par les astrocytes corticaux. J’ai ensuite analysé l’activité calcique des astrocytes qui expriment GCaMP3. J’ai utilisé la microscopie biphotonique et enregistré l’activité calcique spontanée et évoquée par application d’agonistes purinergiques sur des tranches de cortex somatosensoriel primaire de souris adultes GLAST-CreERT2. L’activité calcique spontanée est complexe, généralement locale et désynchronisée, répartie dans les prolongements et la région somatique. Les régions actives ont été identifiées à partir d’une carte de corrélation temporale calculée en MATLAB, et leurs caractéristiques (amplitude, durée, position, fréquence) mesurées grâce à des routines établies sous IGOR. La fréquence et l’amplitude de l’activité calcique paraissent augmenter lors de l’enregistrement, ce qui suggère une sensibilité significative et une photoactivation des astrocytes, en imagerie biphotonique. La durée des impulsions laser modulerait ce phénomène. En présence d'adénosine (1-100 µM) et d’ATP (100 µM), et de façon marginale en présence d’un agoniste P2X7 non sélectif (BzATP 50-100 µM), une activité calcique synchronisée accrue est visible dans le soma et les prolongements astrocytaires en présence de tétrodotoxine qui bloque les potentiels d'action et minimise l’activité synaptique. Le mécanisme de ces réponses synchronisées reste à étudier. Aucun effet significatif n’a été observé en présence d’un agoniste spécifique P2Y1 (MRS2365 50 uM). Mon travail a permis le développement : i) de modèles murins pour l’adressage sélectif de protéines d’intérêt au niveau des astrocytes protoplasmiques ; ii) d’outils d’analyse des signaux calciques astrocytaires au niveau sub-cellulaire. Il a mis en évidence des limites possibles des protocoles standards d'enregistrement de l’activité calcique des astrocytes en imagerie biphotonique. Il confirme l’importance de l’ATP et de l’adénosine pour la signalisation astrocytaire. / Grey matter protoplasmic astrocytes are compact glial cells with highly branched processes, enwrapping synapses, and one or two endfeet contacting the blood vessels. Several neurotransmitter receptors are expressed by astrocytes, among them purinergic receptors. Upon activation of these receptors, intracellular calcium (Ca2+) transients can be induced, that, in turn, trigger gliotransmitter release (e.g. glutamate, GABA, ATP, D-serine) and participate in astrocyte-to-astrocyte signaling as well as in the communication between astrocytes and neurons or other glia. During my PhD work, I first implemented and validated several approaches for targeting transgene expression specifically to cortical astrocytes and employed them to study purinergic signaling in astrocytes. To achieve astrocyte-specific transgene expression, I used either floxed adeno-associated viral (AAV) vectors or a Cre-dependent mouse line and several mouse lines expressing the Cre recombinase under astrocyte-specific promoters. Intracerebral injections of a Cre-dependent AAV serotype 5 containing the ubiquitous CAG promoter and an enhanced green fluorescent protein (AAV5.CAG.flex.EGFP) in adult mice expressing Cre recombinase under the human glial fibrillary protein (hGFAP) promoter resulted in a non-astrocyte specific expression in the cortex. Combining inducible mouse lines expressing Cre recombinase under the glutamate aspartate transporter (GLAST) promoter with the same AAV vector resulted in a virtually astrocyte-specific expression of the reporter gene. As an alternative approach for astrocyte-specific transgene expression, we used a Cre-dependent mouse line expressing the genetically encoded Ca2+ indicator GCaMP3. Crossing this mouse line with the above described GLAST-CreERT2 mouse line or a Connexin30 (Cx30)-CreERT2 line led to selective GCaMP3 expression in cortical astrocytes. Second, I investigated both spontaneous and agonist-evoked Ca2+ transients in astrocytic processes, the investigation of which has presented a major challenge in earlier studies, due to the unspecific and weak labeling by membrane-permeable chemical Ca2+ indicators. Using the strategy developed in the first part of my work allowing an astrocyte-specific expression of the genetically encoded Ca2+ indicator GCaMP3. Using two-photon excitation fluorescence (2PEF) imaging in acute slices of the primary somatosensory cortex, I recorded Ca2+ transients in the astrocytic soma and processes. By aid of a custom-made MATLAB routine based on a temporal Pearson correlation coefficient, active regions could be identified in an unbiased manner. Evoked Ca2+ transients were quantified using custom IGOR routines. Spontaneous desynchronized Ca2+ transients occurred in the processes and rarely in the soma. Ca2+ signals appeared localized in distinct microdomains. Their frequency appeared to increase during long recordings of several hundred images, suggesting that fine astrocytes are vulnerable to photodamage under imaging conditions routine in 2PEF microscopy. The possibility to minimize photodamage, by varying the length of the femtosecond laser pulses is under investigation. Bath application of adenosine (1-100 µM) and adenosine-triphosphate (ATP, 100 µM), as well as the application of the non-selective P2X7 receptor agonist (2'(3')-O-(4-Benzoylbenzoyl)adenosine-5'-triphosphate, BzATP, 50-100 µM), in the presence of tetrodotoxin to block neuronal action potentials, evoked synchronized Ca2+ rises in the soma and the processes of astrocytes. The effect of adenosine was dose-dependent. No significant effect of the specific P2Y1 agonist (MRS2365, 50 µM) was seen. Altogether, my work sets up a powerful and versatile toolbox for studying astrocytic Ca2+ signaling at the sub-cellular level. It also pinpoints possible limits of standard two-photon recording protocols to investigate the local Ca2+ signals in fine astrocytic processes.

Les astrocytes protoplasmiques de la matière grise corticale sont des cellules gliales dont les prolongements très fins et ramifiés sont en contact avec les éléments neuronaux pré- et post-synaptiques d’une part, et les vaisseaux sanguins d’autre part. Ils expriment plusieurs récepteurs des neurotransmetteurs, entre autres des récepteurs purinergiques dont l'activation facilite l’activité calcique astrocytaire et la libération de gliotransmitters (par exemple, le glutamate, le GABA, l'ATP, et la D sérine) qui régulent l’activité des neurones et des cellules gliales situées au voisinage. L’objectif de ma thèse était d’étudier in situ l’activité calcique des astrocytes et de leurs prolongements en réponse à l’application des agonistes purinergiques. Lors de ma thèse, j’ai tout d'abord testé la possibilité d’induire l’expression spécifique de gènes d’intérêt par les astrocytes corticaux de souris adultes par la technique de recombinaison Cre-LoxP. J’ai comparé les performances d’un virus adeno-associé de type 5 (AAV5) flexé (AAV5.FLEX.EGFP) et d’une souris qui exprime un indicateur calcique (GCaMP3) sous contrôle de la recombinase (souris Rosa-CAG-LSL-GCaMP3). L’injection d’AAV5.FLEX.EGFP dans le cortex d’une souris hGFAPcre n’a pas permis l’expression spécifique d’EGFP. La combinaison des souris exprimant le cre recombinase sous contrôle d’un promoteur sélectif des astrocytes (GLAST-CreERT2 et Cx30-CreERT2) avec le AAV5.FLEX.EGFP ou avec une lignée des souris Rosa-CAG-LSL-GCaMP3 permet l’expression spécifique des gènes d’intérêt (EGFP et GCaMP3) par les astrocytes corticaux. J’ai ensuite analysé l’activité calcique des astrocytes qui expriment GCaMP3. J’ai utilisé la microscopie biphotonique et enregistré l’activité calcique spontanée et évoquée par application d’agonistes purinergiques sur des tranches de cortex somatosensoriel primaire de souris adultes GLAST-CreERT2. L’activité calcique spontanée est complexe, généralement locale et désynchronisée, répartie dans les prolongements et la région somatique. Les régions actives ont été identifiées à partir d’une carte de corrélation temporale calculée en MATLAB, et leurs caractéristiques (amplitude, durée, position, fréquence) mesurées grâce à des routines établies sous IGOR. La fréquence et l’amplitude de l’activité calcique paraissent augmenter lors de l’enregistrement, ce qui suggère une sensibilité significative et une photoactivation des astrocytes, en imagerie biphotonique. La durée des impulsions laser modulerait ce phénomène. En présence d'adénosine (1-100 µM) et d’ATP (100 µM), et de façon marginale en présence d’un agoniste P2X7 non sélectif (BzATP 50-100 µM), une activité calcique synchronisée accrue est visible dans le soma et les prolongements astrocytaires en présence de tétrodotoxine qui bloque les potentiels d'action et minimise l’activité synaptique. Le mécanisme de ces réponses synchronisées reste à étudier. Aucun effet significatif n’a été observé en présence d’un agoniste spécifique P2Y1 (MRS2365 50 uM). Mon travail a permis le développement : i) de modèles murins pour l’adressage sélectif de protéines d’intérêt au niveau des astrocytes protoplasmiques ; ii) d’outils d’analyse des signaux calciques astrocytaires au niveau sub-cellulaire. Il a mis en évidence des limites possibles des protocoles standards d'enregistrement de l’activité calcique des astrocytes en imagerie biphotonique. Il confirme l’importance de l’ATP et de l’adénosine pour la signalisation astrocytaire. / Grey matter protoplasmic astrocytes are compact glial cells with highly branched processes, enwrapping synapses, and one or two endfeet contacting the blood vessels. Several neurotransmitter receptors are expressed by astrocytes, among them purinergic receptors. Upon activation of these receptors, intracellular calcium (Ca2+) transients can be induced, that, in turn, trigger gliotransmitter release (e.g. glutamate, GABA, ATP, D-serine) and participate in astrocyte-to-astrocyte signaling as well as in the communication between astrocytes and neurons or other glia. During my PhD work, I first implemented and validated several approaches for targeting transgene expression specifically to cortical astrocytes and employed them to study purinergic signaling in astrocytes. To achieve astrocyte-specific transgene expression, I used either floxed adeno-associated viral (AAV) vectors or a Cre-dependent mouse line and several mouse lines expressing the Cre recombinase under astrocyte-specific promoters. Intracerebral injections of a Cre-dependent AAV serotype 5 containing the ubiquitous CAG promoter and an enhanced green fluorescent protein (AAV5.CAG.flex.EGFP) in adult mice expressing Cre recombinase under the human glial fibrillary protein (hGFAP) promoter resulted in a non-astrocyte specific expression in the cortex. Combining inducible mouse lines expressing Cre recombinase under the glutamate aspartate transporter (GLAST) promoter with the same AAV vector resulted in a virtually astrocyte-specific expression of the reporter gene. As an alternative approach for astrocyte-specific transgene expression, we used a Cre-dependent mouse line expressing the genetically encoded Ca2+ indicator GCaMP3. Crossing this mouse line with the above described GLAST-CreERT2 mouse line or a Connexin30 (Cx30)-CreERT2 line led to selective GCaMP3 expression in cortical astrocytes. Second, I investigated both spontaneous and agonist-evoked Ca2+ transients in astrocytic processes, the investigation of which has presented a major challenge in earlier studies, due to the unspecific and weak labeling by membrane-permeable chemical Ca2+ indicators. Using the strategy developed in the first part of my work allowing an astrocyte-specific expression of the genetically encoded Ca2+ indicator GCaMP3. Using two-photon excitation fluorescence (2PEF) imaging in acute slices of the primary somatosensory cortex, I recorded Ca2+ transients in the astrocytic soma and processes. By aid of a custom-made MATLAB routine based on a temporal Pearson correlation coefficient, active regions could be identified in an unbiased manner. Evoked Ca2+ transients were quantified using custom IGOR routines. Spontaneous desynchronized Ca2+ transients occurred in the processes and rarely in the soma. Ca2+ signals appeared localized in distinct microdomains. Their frequency appeared to increase during long recordings of several hundred images, suggesting that fine astrocytes are vulnerable to photodamage under imaging conditions routine in 2PEF microscopy. The possibility to minimize photodamage, by varying the length of the femtosecond laser pulses is under investigation. Bath application of adenosine (1-100 µM) and adenosine-triphosphate (ATP, 100 µM), as well as the application of the non-selective P2X7 receptor agonist (2'(3')-O-(4-Benzoylbenzoyl)adenosine-5'-triphosphate, BzATP, 50-100 µM), in the presence of tetrodotoxin to block neuronal action potentials, evoked synchronized Ca2+ rises in the soma and the processes of astrocytes. The effect of adenosine was dose-dependent. No significant effect of the specific P2Y1 agonist (MRS2365, 50 µM) was seen. Altogether, my work sets up a powerful and versatile toolbox for studying astrocytic Ca2+ signaling at the sub-cellular level. It also pinpoints possible limits of standard two-photon recording protocols to investigate the local Ca2+ signals in fine astrocytic processes.

The chromatin structure, consisting of DNA and histones, changes dynamically during the cell cycle and cell differentiation. DNA can only be transcribed and replicated when it is packaged loosely, whereas tight packaging allows for more efficient storage. Chromatin remodelling is therefore one of the tools of gene expression control. The chromatin remodelling factors recognise chromatin with varying specificity and have an effect on the interaction between DNA and the histones. One of these factors is the Smarca5 protein. This study investigates the role of Smarca5; its goal is to create a mouse model with the ability to trigger Smarca5 overproduction in specific tissues. This model will be used to study the effect of a high, unregulated dose of Smarca5 on the physiological function of the protein. Previous studies have shown that non-physiological expression of a chromatin-remodelling factor can lead to malignant transformation. Our model can help to understand this process. Another goal of this study is to investigate some phenotype aspects of the mouse model with conditional deletion of Smarca5 in T and B cells, in particular the effects of this deletion on progenitor cell differentiation. Our results show that Smarca5 has an important role in lymphocyte development, and we have observed that...

Os transportadores da família ABC são proteínas transmembrânicas envolvidas com o tráfego de substâncias endógenas e exógenas do meio intracelular para o extracelular, sendo alvos de estudo na resistência celular a agentes quimioterápicos. O ABCC2 é um transportador transmembrânico que exporta ativamente fármacos aniônicos conjugados e facilita o transporte de agentes anticâncer. O ABCG2 é outro transportador transmembrânico que tem influência na farmacocinética e farmacodinâmica de certos xenobióticos e substratos endógenos; além disso, acredita-se que este gene contribui para a resistência a várias drogas. Por esses motivos identificamos por sequenciamento ou PCR-RFLP os polimorfismos dos genes ABCC2 (Val417Ile, Ser789Phe e Ala1450Thr) e ABCG2 (Val12Met, Gly126stop códon, Gly141Lys) em 90 pacientes portadores de carcinoma epidermóide de cabeça e pescoço (HNSCC) e tentamos correlacionar a presença do polimorfismo com resposta a tratamento que incluiu cisplatina em todos os pacientes. Não encontramos nenhuma correlação entre a presença de polimorfismo para Val12Met, Gly141Lys e Val417Ile, determinados em 68 pacientes tratados exclusivamente com cisplatina e radioterapia, e a resposta ao tratamento. As curvas de sobrevida, determinadas por Kaplan-Meier, considerando polimórfico os pacientes que continham pelo menos um dos alelos alterados e selvagem os que não tivessem nenhum deles, mostraram que os pacientes selvagens para o polimorfismo Val12Met tiveram tendência a uma pior sobrevida (sobrevida mediana de 18,7 meses) em relação aos pacientes polimórficos (sobrevida mediana não atingida; P=0,089 Teste de log-rank), já os pacientes selvagens para o polimorfismo Gly141Lys tiveram tendência a uma pior sobrevida (sobrevida mediana de 15,8 meses) em relação aos pacientes polimórficos (sobrevida mediana de 25,6 meses; P = 0,16 Teste de logrank). Não observamos correlação entre os outros polimorfismos e sobrevida. Quanto ao GLY126stop, somente um paciente foi identificado como polimórfico. Conclusões: No nosso estudo, a freqüência do polimorfismo Val12Met de 10% está próxima dos 18% descrito na população normal (Kobayashi 2004). Nosso trabalho foi o primeiro a correlacionar estes polimorfismos com resposta ao tratamento em pacientes com carcinoma epidermóide de cabeça e pescoço e indica que Val12Met e Gly141Lys e o gene ABCG2 como um todo são candidatos a um estudo maior / ATP binding cassette (ABC) transporters form one of the largest transmembrane protein families. These proteins use cellular ATP to drive the transport of various substrates across cell membranes including many exogenous and endogenous compounds, which includes drugs used in cancer treatment. ABCC2 is an ATP binding cassette transporter which accepts a diverse range of substrates, including glutathione, glucuronide, and sulfate conjugates of many metabolites and xenobiotics. ABCG2 is a member of the ATP binding cassette (ABC) transporters whose function is to pump out of the cell a wide variety of endogenous and exogenous compounds. Widely expressed in stem cells, ABCG2 is also recognized as a universal marker of stem cells. For these reasons we had identified the following polymorphisms of ABCC2 gene: -Val417Ile, Ser789Phe and Ala1450Thr- and of ABCG2 gene as well: -Val12Met, Gly126stop códon, Gly141Lys in 88 patients with head and neck squamous cell carcinoma (HNSCC). Methodology included PCR - RFLP and direct sequencing. Survival analysis was done using Kaplan-Meier curves and response measured by RECIST criteria. Comparisons were done between polymorphic patients in which at least one polymorphism was present as opposed to the patients without the polymorphism. Correlation with response to treatment was studied for Val12Met, Gly141Lys e Val417Il in 68 patients exclusively treated with concomitant cisplatin and radiotherapy and no correlation was found between these markers and treatment response. Patients without the Val12Met presented a trend towards shorter survival (median survival 18.7 months) as compared to polymorphic patients (median survival not reached, long rank p= 0.089). Although statistical significance was not reached, patients wild type for Gly141Lys polymorphism (median survival 15.8 months) had shorter survival than polymorphic patients (25.6 months, p=0.16). We did not observe any other correlation between other polymorphisms and survival. With respect to Gly126stop, only one patient was identified as polymorphic and survival analysis was not possible. As far as we know this is the first study to try to correlate these polymorphisms with treatment response and survival in HNSCC patients. Although we were unable to draw any definitive conclusions, our results indicate that Val12Met and Gly141Lys deserve to be further studied in the future.

Este trabalho apresenta um conjunto de métodos para a proteção e o monitoramento de transformadores de potência baseado em sistemas inteligentes e na aplicação das transformadas de Clarke e Wavelet. A abordagem inteligente utilizada permite analisar a condição operativa dos transformadores de potência e detectar a ocorrência de defeito interno, distinguindo-a de outras situações de operação, como, a energização, a energização solidária, a saturação dos transformadores de corrente e a sobreexcitação. As tomadas de decisão das técnicas desenvolvidas são realizadas pela lógica fuzzy após o pré-processamento dos sinais de entrada por meio de diversos métodos, os quais variam de acordo com o algoritmo que esta sendo executado. Os algoritmos propostos foram testados por meio de simulações realizadas através do software Alternative Transients Program (ATP). É importante salientar que nas simulações do ATP foram modelados diversos equipamentos que constituem o sistema elétrico de potência, incluindo um gerador síncrono com regulação de velocidade, linhas de transmissão com variação em frequência, transformadores de potência com suas respectivas curvas de saturação, transformadores de potencial e de corrente. Estas modelagens tiveram por objetivo gerar dados das distintas situações de operação para a verificação e análise da metodologia proposta. Os resultados da pesquisa mostram a aplicabilidade dos algoritmos propostos na proteção e no monitoramento dos transformadores de potência, mesmo nas condições mais adversas, como na ocorrência da saturação dos transformadores de corrente, uma vez que os sinais de entrada distorcidos pela saturação são corrigidos por uma rede neural artificial. Os resultados apresentados comparam as respostas obtidas pelas técnicas propostas em relação às saídas de um relé comercial, habilitado à proteção diferencial percentual. / This work presents a set of methods for protecting and monitoring power transformers based on intelligent systems and the application of Clarke and Wavelet transforms. The intelligent approach allowed us to analyze the operating condition of power transformers and it discriminates between an internal fault and different operating conditions, as energization, sympathetic inrush, saturation of current transformers and overexcitation. Decision making is performed by fuzzy logic after the preprocessing of the input signals through various methods, varying according to which algorithm is running. It is important to point out that in the simulations using ATP many different power system equipment had been modeled, including a synchronous generator with speed regulation, transmission lines with variation in frequency, power transformers with their saturation curves, potential transformers and current transformers. The objective of these tests was to generate data for distinct situations for the verification and the analysis of the proposed methodologies. The results of the research show the applicability of the algorithms considered in protection and monitoring of power transformers, even in adverse conditions, such as saturation of current transformers, since the input signals are distorted by CT saturation corrected by artificial neural network. The results are compared to the ones presented by a commercial percentage differential relay.

Esta pesquisa apresenta um estudo da resposta dinâmica dos motores de indução trifásicos submetidos a afundamentos de tensão. As simulações computacionais sobre um sistema de distribuição, assim como a modelagem dos motores de indução trifásicos baseada em dados reais, foram realizadas utilizando-se do software ATP (Alternative Transients Program). Estas tiveram por objetivo gerar situações representativas da operação do sistema elétrico de potência (SEP), caracterizando afundamentos de tensão, fenômenos estes pertencentes à classe de variações de tensão de curta duração (VTCD), no contexto da qualidade da energia elétrica (QEE). Da observação destes afundamentos, verificaram-se as situações que vieram ou não, a comprometer a alimentação do equipamento analisado e, conseqüentemente, seu desempenho. Pelos resultados observados, evidencia-se que a metodologia de análise aplicada é satisfatória e condizente com o encontrado em situações reais de operação dos motores de indução trifásicos, denotando certas características intrínsecas no contexto das VTCDs. / This research shows a study on the dynamic response of three-phase induction motors submitted to voltage sags. Computer simulations about a distribution system, as well as the modeling of three-phase induction motors based on actual data, were made using ATP (Alternative Transients Program) software. These simulations intended to generate representative situations of the electrical power system (EPS) operation, characterizing voltage sags, which is a phenomenon belonging to the class of short duration voltage variations (SDVV), in the electrical power quality context (EPQ). From the observation of these sags, we verified the situations that jeopardized or not the supply of the analyzed equipment and, consequently, its performance. From the observed results, it becomes evident that the methodology of the applied analysis is satisfactory and in agreement with the one found in actual situations of three-phase induction motors operations, indicating certain characteristics intrinsic to the SDVV context.

Nesta pesquisa, apresentam-se os principais eventos relacionados com a proteção de transformadores e sua correlação com os distúrbios de qualidade da energia elétrica (QEE). Neste sentido, foi desenvolvido um algoritmo que utiliza a transformada Wavelet (TW) e a lógica Fuzzy (LF) para classificar os eventos transitórios associados à proteção de transformadores. Estes eventos foram observados em um sistema elétrico de potência (SEP) simulado com a utilização do software Alternative Transients Program (ATP). Importa ressaltar que o sistema modelado apresenta transformadores ligados em paralelo, possibilitando o estudo de eventos decorrentes desta situação, como a energização solidária (Sympathetic Inrush). Por este SEP, modelado sobre parâmetros reais, foram simuladas várias situações transitórias, que provocam o aparecimento de correntes diferenciais, sendo estas direcionadas para análise do algoritmo desenvolvido. Afirma-se que, nos testes realizados, o algoritmo proposto apresentou um desempenho satisfatório perante as mais variadas situações a que foi submetido, identificando as causas das correntes diferenciais, sejam proporcionadas por defeitos ou por outras condições de operação aplicadas. / In this research, the main events related to the transformer protection and its correlation with the power quality disturbances (PQ) are presented. In this context, an algorithm based on Wavelet transform (WT) and Fuzzy logic (FL) was developed to classify the transient events associated with the transformer protection. These events were observed in an electrical power system (EPS) simulated using the Alternative Transients Program (ATP) software. It should be emphasized that the modeled system presents transformers connected in parallel, allowing the study of events of this situation, such as sympathetic inrush. For the simulated EPS, modeled based on real parameters, various transients situationswere simulated, causing the appearance of differentials currents which were directed to the analysis. The proposed algorithm showed a satisfactory performance tomany situations, identifying the causes of the differentials currents, either provided by faults or other operation conditions.

TRPM4, un canal cationique non-sélective activé par le Ca2+ intracellulaire, est un acteur moléculaire important impliqué de la régulation du signal calcique et l’activation des lymphocytes T conventionnels mais son rôle dans la fonction des lymphocytes T régulateurs (Tregs Foxp3+) reste inconnu. Dans un modèle de souris transgéniques dans lequel le gène Trpm4 a été sélectivement invalidé dans la population des Tregs Foxp3+ (souris Foxp3(YFP)Cre+Trpm4flox/flox), nous avons démontré dans différents modèles in vivo d’inflammation aiguë et chronique que TRPM4 contrôle la fonction suppressive et la mort de ces cellules. Dans le modèle de fibrosarcome induit par le méthylcholanthrène (3-MCA) ou implanté (modèle MCA205), dans lequel le rôle des Tregs est documenté, l’absence de fonction de TRPM4 induit une diminution significative de l’incidence et de la croissance tumorale. Dans l’environnent inflammatoire chronique et hypoxique de ces tumeurs, l’expression de TRPM4 protège les Tregs infiltrant la tumeur de la mort cellulaire induit par l’ATP extracellulaire et stimule ainsi le développent et la progression tumorale. L’absence d’expression de TRPM4 dans les Tregs stimule la réponse anti-tumorale médiée par l’IFNg et induit la régression des tumeurs. En conclusion, en inhibant l’entrée de Ca2+ extracellulaire, TRPM4 régule négativement les fonctions suppressives des Tregs et protège ces cellules de la mort cellulaire induite par l’activation. / TRPM4, a Ca2+-activated non-selective cation ion channel is an important regulator of Ca2+ signaling and cell activation in conventional T cells, but its role in Foxp3+ Tregs function remains unknown. Using a model in which Trpm4 gene was selectively invalidated in Foxp3+ Tregs population (Foxp3(YFP)Cre+Trpm4flox/flox mice) we have shown in different in vivo models of acute and chronic inflammation that TRPM4 is an important regulator of Tregs functions and survival. In a model of primary carcinogenesis induced by methylcholantrene (3-MCA) or implanted fibrosarcoma (MCA205 model), in which Tregs role has been documented, lack of TRPM4 expression and function induced significantly decreased incidence and tumor growth. We found that within chronic inflammatory and hypoxic tumor microenvironment, TRPM4 protected Tregs from ATP-induced cell death and therefore promoted tumor initiation and progression. In contrast, TRPM4 deficiency in Tregs favored IFN-g-mediated spontaneous anti-tumor immune response. Thus, through inhibiting Ca2+ influx, TRPM4 acts as a negative modulator of Tregs suppressive functions and protects Tregs from activation-induced cell death.

ATP synthase est une protéine essentielle associée à la membrane interne mitochondriale, qui synthétise l'ATP par couplage d’un transport de protons au travers de la membrane, en dissipant un gradient électrochimique de protons créé par la chaîne respiratoire. Cette réaction assure l’alimentation en énergie des processus biologiques cellulaires. Si la membrane mitochondriale se dépolarise, la réaction inverse d’hydrolyse d’ATP est rapidement bloquée par un inhibiteur soluble naturel de l’ATPase mitochondriale, IF1. Cette régulation efficace et réversible évite le gaspillage de l’énergie par la cellule. Chez la levure, IF1 est une petite protéine de 63 amino-acides. Elle se fixe sur l'une des trois interfaces catalytiques de l’ATP synthase et inhibe l’hydrolyse d’ATP. Bien que les structures cristallographiques des complexes F1-ATPase inhibés par IF1 aient été résolus, l'étape initiale de reconnaissance et celle du verrouillage d’IF1 restent peu claires au niveau moléculaire.Pendant ma thèse, nous nous sommes intéressés au mécanisme d’inhibition de l’ATPase par IF1. Par des analyses des structures disponibles et des alignements de séquence, nous avons sélectionné de nombreux résidus localisés dans différentes régions des sous-unités α et β de l'ATP synthase de Saccharomyces cerevisiae et susceptibles de participer au processus de fixation d'IF1. En utilisant le mutagenèse dirigée combinée à des experiences cinétiques, nous avons étudié les effects des mutations sur l’inhibition de l’ATP synthase par IF1 chez Saccharomyces cerevisiae. Dans ce travail, nous avons identifié des résidus ou motifs des sous-unités α et β de l’ATP synthase impliqués dans les étapes de reconaissance et/ou verrouillage d’IF1, ce qui nous permet de compléter les études structurales et d'esquisser un mécanisme de fixation d'IF1. / ATP synthase is an essential protein complex located in the mitochondrial inner membrane, which synthesize ATP by coupling to a rotary proton transport across the membrane at the expense of the electrochemical proton gradient created by the electron transport chain. This reaction guarantees the supply of energy to biological processes in a cell. When mitochondria get deenergized, i.e. the protomotive force across the mitochondrial inner membrane collapses, the ATP synthase switches from ATP synthesis to hydrolysis. This hydrolytic activity is then immediately prevented by a natural soluble mitochondrial ATPase inhibitor, IF1. This efficient reversible inhibition system protects cells from wasting energy. In yeast, IF1 is a small protein consisting of 63 amino acids. It binds to one of the three (αβ) catalytic interfaces of ATP synthase and thereby blocks the rotary catalysis. Although the crystal structure of the dead-end IF1 inhibited F1-ATPase complex has been resolved, IF1 initial binding and locking to ATPase still remain unclear events at the molecular level.During my thesis, we have been interested in the dynamic mechanism of ATPase inhibition by IF1. By means of analyses of published structures and protein sequence alignment, we selected numerous residues located in different regions of Saccharomyces cerevisiae ATP synthase α, β subunits, which might potentially paticipate in IF1 binding process. Using site-directed mutagenesis combined with kinetic experiments, we studied the effect of mutations of the selected candidates on the rate and extent of ATPase inhibition by IF1. In this way we identified residues or motifs in ATP synthase α, β subunits involved in IF1 recognition and/or locking steps, which allows complementing structural studies and drawing an outline of IF1 binding.