Le facteur de transcription NF-κB régule l’expression d’une pléthore de gènes impliqués dans divers processus physiologiques notamment la prolifération et la survie cellulaires, l’inflammation ainsi que les réponses immunes. Il intervient également dans de nombreux processus pathologiques à l’exemple de certains cancers et maladies auto-immunes.L’activation de NF-κB suite à l’engagement de différents immunorécepteurs requiert la mise en place de larges signalosomes formés suite au recrutement de différents adaptateurs au niveau des immunorécepteurs engagés. Ces adaptateurs subissent des ubiquitinations non-dégradatives nécessaires pour la transduction du signal. Nous avons démontré dans un précédent travail que ces protéines ubiquitinylées s’accumulent au niveau de la membrane du réticulum endoplasmique (RE) via la protéine réticulaire Métadhérine (MTDH). De plus, nos résultats suggèrent que l’ubiquitination est un prérequis nécessaire à l’adressage de ces protéines au RE. Afin d’évaluer la contribution des E3 ubiquitine ligases en charge de relayer NF-κB au niveau des organites, nous avons effectué le crible d’une banque de siRNAs dirigés contre les 46 E3 ligases transmembranaires en utilisant comme modèle la signalisation du TNF récepteur. Ce crible nous a permis d’identifier l’E3 ligase RNF121 comme régulateur positif de NF-κB. Bien que le mécanisme d’action de RNF121 ne soit pas complètement élucidé, nos données suggèrent qu’il agirait au niveau de la régulation de l’inhibiteur de NF-κB « IκBα ».Durant la deuxième partie de cette thèse, je me suis intéressée à la dynamique mitochondriale. Les mitochondries sont des organites dynamiques, dont la forme est maintenue grâce à une balance entre deux processus antagonistes appelés : fission et fusion. Il a été rapporté qu’en présence de certains stress modérés, les mitochondries hyperfusent, ce processus est appelé SIMH (Stress-Induced Mitochondrial Hyperfusion). Nous avons pu démontrer que la SIMH s’accompagne de l’activation de la voie canonique du facteur de transcription NF-κB via l’E3 ubiquitine ligase mitochondriale MULAN. Nos résultats suggèrent que durant le SIMH, MULAN forme un complexe avec TRAF2 et module son ubiquitination. Ces résultats suggèrent que la mitochondrie, de part sa dynamique, convertie un signal de stress en un signal de survie via l’activation de NF-κB. Pris dans leur ensemble, nos résultats illustrent la complexité de la régulation de NF-κB par ubiquitination et attribuent aux organites un nouveau rôle dans la transduction de la signalisation. / The transcription factor NF-κB regulates the expression of several genes implicated in various physiological processes such as cell proliferation and survival, inflammation and immune responses. Dysregulation of its activation is involved in diverse pathologies such as cancer and auto-immune diseases. Following the engagement of immunoreceptors, NF-κB signaling requires a large signalosome assembly containing different adaptor proteins. These adaptors undergo poly-ubiquitinylation in a non-degradative manner, which is essential for signal transduction. We demonstrated in previous work that these ubiquitinylated proteins accumulate at the surface of the endoplasmic reticulum (ER) via a reticular protein called “Methaderin” (MTDH). Furthermore, our results suggest that ubiquitinylation is a necessary prerequisite for protein addressing to the ER. To evaluate the contribution of E3 ubiquitin ligases in this process we performed a screen of a siRNA bank, targeting the 46 human transmembrane E3 ligases, using the TNF receptor signaling as a model. This screen enabled the identification of RNF121, a Golgi E3 ligase, as a positive regulator of NF-κB. Although the mechanism by witch RNF121 acts is not yet elucidated, our data suggest that it acts on the regulation of NF-κB inhibitor (IκBα). In the second part of this thesis, we investigated mitochondrial dynamics. Mitochondria are dynamic organelles; their shape is maintained due to a balance between two antagonist processes called: fusion and fission. It is known that moderate stress triggers mitochondrial hyperfusion, this process is called: SIMH (Stress-Induced Mitochondrial Hyperfusion). During this thesis, we demonstrated that SIMH is accompanied by NF-κB activation through the mitochondrial E3 ubiquitin ligase MULAN. Our results suggest that during SIMH, MULAN forms a complex with the protein TRAF2 and modulates its ubiquitinylation to allow NF-κB signaling transduction. This work shows that, through their dynamics, mitochondria convert stress signals into a prosurvival response via NF-κB activation.In summary, our results illustrate the complexity of the ubiquitin-dependant regulation of NF-κB and attribute a new role in signaling transduction to the organelles.

Hematopoietic tumors are the most common neoplastic disorders in felines. Many of these tumors are associated with infection by feline leukemia virus (FeLV) and one of the described mechanisms is the integration of viral material into the feline genome, which can lead to genomic instability and consequent alteration of genes related to proliferation control and cell death. When larger DNA fragments are affected, such changes can be observed by cytogenetic analysis. The present study aimed to observe cytogenetic changes in felines with hematopoietic neoplasms. The study was performed in eight felines, seven of them with a diagnosis of leukemia or lymphoma. The control consisted of a healthy feline FeLV negative with normal karyotype. At least 10 metaphases of each animal were analyzed. Additionally, 1000 lymphocytes of these two patients, were analyzed and classified cytologically by their viability (necrosis, apoptosis), mitotic state (mononuclear, binucleate (BN), mitotic multinucleate) and their chromosomal damage or instability state (presence of micronucleus (MN) in mononuclear, binucleate, as well as nuclear buds (NBUD). The results of this observation were statistically analyzed by the Wilcoxon paired test. In this chromosomal analysis two of these animals presented monosomy of the E3 chromosome, one with diagnosis of acute myeloid leukemia of the M6a (LMA-M6a) FAB subtype and another with multicenter lymphoma (LM). There were significant differences in LMA-M6a scores (N = 7, Z = 2.36, p = 0.01) and LM scores (N = 8, Z = 2.52, p = 0.01) when group control. However, there was no difference between AML and lymphoma (N = 8, Z = 0.07, p = 0.94). The E3 chromosome has 1401 genes, several related to cell cycle control (Plk1, Sun, Mad1l1, Mcm7), DNA repair (Pms2, Usp42), tumor suppression (Bcl7b). The loss of DNA fragments, such as the loss of the E3 chromosome in the two patients described, led to the haploinsufficiency of important genes for the cell cycle, and this could be a cause of genomic instability and consequently susceptibility to the development of cancer. The observation of cytogenetic alterations, in this way, allows a better understanding of the cancer in the feline species and translational research. / Os tumores hematopoiéticos são os distúrbios neoplásicos mais comuns em felinos. Muitos desses tumores estão associados à infecção pelo vírus da leucemia felina (FeLV) e um dos mecanismos descritos é pela integração de material viral no genoma felino, que pode levar à instabilidade genômica e consequente alteração de genes relacionados com o controle da proliferação e morte celular. Quando fragmentos maiores de DNA são afetados, tais alterações podem ser observadas pela análise citogenética. O presente estudo objetivou observar as alterações citogenéticas em felinos com neoplasias hematopoiéticas. O estudo foi realizado em oito felinos, sete destes com diagnóstico de leucemia ou linfoma. O controle foi constituído por um felino saudável FeLV negativo com cariótipo normal. Foram analisadas no mínimo 10 metáfases de cada animal. Adicionalmente, destes dois pacientes foram analisados 1.000 linfócitos e classificados citologicamente pelo seu estado de viabilidade (necrose, apoptose), seu estado mitótico (mononucleado, binucleado (BN), multinucleado, mitótico) e seu dano cromossômico ou estado de instabilidade (presença de micronúcleo (MN) em célula mononucleada, binucleada, bem como brotos nucleares (NBUD). Os resultados dessa observação foram analisados estatisticamente pelo teste pareado de Wilcoxon. Nesta análise cromossômica dois destes animais apresentaram monossomia do cromossomo E3, um com diagnóstico de leucemia mieloide aguda do subtipo FAB M6a (LMA-M6a) e outro com linfoma multicêntrico (LM). Houve diferença significativa nas contagens de LMA-M6a (N=7; Z=2,36; p=0,01) e de LM (N=8; Z=2,52; p=0,01) quando comparados com o grupo controle. No entanto, não houve diferença entre LMA e linfoma (N=8; Z=0,07; p=0,94). No cromossomo E3 são descritos 1401 genes, sendo vários relacionados com controle de ciclo celular, (Plk1, Sun, Mad1l1, Mcm7), reparo de DNA (Pms2, Usp42) e supressão tumoral (Bcl7b).A perda de fragmentos de DNA, como o cromossomo E3 nos dois pacientes descritos, que leva a haploinsuficiência de genes importantes para o ciclo celular, poderia ser a causa de instabilidade genômica e, consequentemente suscetibilidade ao desenvolvimento do câncer. A observação de alterações citogenéticas, dessa forma, possibilita o melhor entendimento do câncer na espécie felina e serve como subsídio para a pesquisa translacional.

Les modifications post-traductionnelles des protéines constituent un niveau crucial de régulation de l’expression des gènes. Parmi elles, la conjugaison peptidique impliquant l’ubiquitine intervient entre autre dans la régulation de la stabilité protéique. La fixation de ce peptide de 76 acides aminés, extrêmement conservé, sous forme de chaîne de polyubiquitine, nécessite l’intervention de trois enzymes (E1, E2 et E3) et constitue un signal de dégradation de la protéine ainsi modifiée. Cette voie de régulation intervient dans de très nombreux processus biologiques. Les complexes SCF sont impliqués dans la voie de protéolyse ciblée. Ils représentent l' une des classes les plus fréquentes d'ubiquitine ligase E3 et ils sont composés de quatre sous-unités (Rbx, Cullin, SKP1, et F-box). La structure et la fonction des complexes SCF, ont été étudiées chez la levure, l’Homme et la plante modèle A. thaliana. Cependant, peu de travaux ont été réalisés chez des plantes cultivées, en particulier les céréales, telles que le blé. Cinq gènes codant pour la sous-unité Skp1 (TSK1, TSK3, TSK6, TSK11 et TSK16), cinq gènes codant pour la sous-unité F-box (ZTL, ATFBL5, EBF, TIR1 et ABA-T), un gène codant pour la sous-unité Cullin1 et un gène codant pour la protéine RBX du complexe SCF du blé, ont été isolés et clonés. Les différents tests d’interaction entre les quatre sous-unités du complexe SCF ont été réalisés par la méthode du double-hybride dans la levure en utilisant la technologie Gateway. Ces études ont montré que les deux protéines, TSK1 et TSK3, fixent spécifiquement différentes sous-unités F-box. Parallèlement, nous avons montré que la protéine TSK11 représente une structure particulière. Des études d’insertion/délétion sur la protéine TSK11 ont permis d’identifier un nouveau domaine indispensable à l’interaction. Les analyses par PCR semi-quantitative des différents gènes codant pour la sous-unité Skp1, dans trois tissus différents (feuille tige et racine), ont mis en évidence une expression constitutive des gènes TSK3, TSK6 et TSK11. Tandis que les gènes TSK1 et TSK16 sont exprimés préférentiellement dans les racines. Les analyses par PCR semi-quantitative sur des plantules de blé à différents stades de développement, ont mis en évidence une surexpression du gène TSK11 au moment de la floraison. Ce qui suggère que TSK11 est probablement un équivalent fonctionnel d’ASK1 chez Arabidopsis thaliana. / The selective degradation of proteins is an important means of regulating gene expression and plays crucial roles in the control of various cellular processes. The Ubiquitin (Ub)–Proteasome System (UPS) is the principal non-lysosomal proteolytic pathway in eukaryotic cells and is required for the degradation of key regulatory proteins. Ubiquitin is a 76-residue protein that can be attached covalently to target proteins through an enzymatic conjugation cascade involving three enzymes denoted, E1, E2 and E3.The SCF complex is a type of ubiquitin-protein ligase (E3) that acts as the specific factor responsible for substrate recognition and ubiquitination. Some polyubiquitinated proteins are then targeted to the 26S proteasome for degradation. The SCF complex consists of four components including SKP1, Cullin1, Rbx1 and a large gene family of F-box proteins. Twenty one SKP1-related genes have been described in the Arabidopsis genome and some of these genes have been analyzed genetically. By contrast, little is known about the function and structure of SKP1 homologues in wheat. Some of the Triticum SKP1-related protein (TSKs) have been characterized in this study. Five complete sequences of SKP1 (TSK1, TSK3, TSK6, TSK11 and TSK16), five F-box (ZTL, ATFBL5, EBF, TIR1 and ABA-T), one Cullin1 and one Rbx, were successfully cloned and biochemically characterized. Yeast two-hybrid analysis showed that TSK1 and TSK3 are capable of interacting with different F-box proteins. Furthermore, TSK11 contains an additional domain that changed its interaction capabilities. In vitro analysis using a chimeric protein showed that this additional domain could modify the interaction between a SKP-like protein and two F-box proteins. Expression analyses revealed that TSK1 and TSK16 were expressed predominantly in roots. While, TSK3, TSK6 and TSK11 were expressed in several wheat organs. In addition, the TSK11 was up-regulated in the leaves at the flowering stage.

Le blé tendre, Triticum aestivum, est une des céréales les plus cultivées au monde et est d’une importance considérable pour l’alimentation humaine, fournissant environ un cinquième des calories consommées par l’Homme. Le rendement en grain chez les céréales dépend majoritairement du nombre et de la taille des grains. Chez le riz (Oryza sativa), le gène GW2 a été isolé dans un locus à effet quantitatif majeur pour la taille et le poids du grain. Ce gène code pour une enzyme E3 ligase de type RING, qui régule négativement la taille et le poids du grain de riz. L’homologue de GW2 chez le blé tendre, le gène TaGW2, est exprimé par trois copies TaGW2-A,TaGW2-B et TaGW2-D, portées par chacun des génomes homéologues A, B et D. Les trois copies présentent des profils d’expression distincts au cours du développement du grain. TaGW2-A a été cartographié dans une région de QTLs pour le rendement, sur le chromosome 6AS ; et du polymorphisme dans sa séquence promotrice et intronique a été retrouvé associé au poids de 1000-grains dans une core collection mondiale de blé tendre. Afin de rechercher la fonction de TaGW2, l’extinction stable des trois copies TaGW2 a été entreprise par ARN interférence. De manière surprenante, les plantes transgéniques montrent des réductions significatives des dimensions et du poids du grain de blé (- 22,5 et - 30% du volume et de la masse du grain, respectivement), ainsi que du nombre de cellules de l’albumen (- 25%), comparé aux plantes témoins dans nos conditions ; suggérant que TaGW2 est un régulateur positif de la taille finale du grain chez le blé tendre. La protéine TaGW2-A a été caractérisée aux niveaux moléculaire et biochimique : elle est une E3 ubiquitine ligase fonctionnelle in vitro, et s’accumule dans la cellule au niveau du nucléole, du nucléoplasme et du cytoplasme. Sa fonction E3 ligase semble notamment influencer sa localisation subcellulaire. Afin de déterminer la ou les voie(s) de signalisation dans la(es)quelle(s) intervient TaGW2, une banque ADNc de grains de blé a été construite et criblée par double-hybride avec 320 acides aminés de la protéine TaGW2-A. Les premiers interacteurs potentiels identifiés suggèrent d’une part un rôle de TaGW2 dans la régulation de la division cellulaire, et d’autre part une fonction E3 Nedd8 ligase, en plus de son activité E3 ligase. / Wheat, Triticum aestivum, is one of the world’s major cereal crops and is of considerable importance to human nutrition, supplying one-fifth of the calories consumed by humans. For important food crops such as wheat, rice and maize, grain yield mainly depends on grain number and size. In rice (Oryza sativa), GW2 was isolated from a major quantitative trait locus for grain size and weight, and encodes an E3 RING ligase that negatively regulates these yield components. Wheat has TaGW2 homologs in A, B and D genomes; and copies show distinct expression pattern during whole grain development in wheat. TaGW2-A was mapped in a genomic region on 6AS, encompassing previous reported QTLs for yield; and polymorphisms in TaGW2-A (promoter and intron 7) were associated with thousand-grain weight, in a worldwide wheat core collection. To investigate TaGW2 function, RNA interference was used to down-regulate TaGW2 transcripts levels. Surprisingly, transgenic wheat lines significantly showed decreased grain weight and size-related dimensions, and endosperm cell number compared to controls. The present study thus suggests that TaGW2 is a positive regulator of the final grain size in wheat, conversely to GW2 in rice. Biochemical and molecular analyses of the protein TaGW2-A revealed that 1) TaGW2-A is a functional E3 ubiquitine ligase in vitro, 2) TaGW2-A accumulates in the nucleolus, the nucleoplasm, and the cytosol, 3) E3 ubiquitine ligase activity seems to impact TaGW2-A subcellular localization. To investigate the TaGW2 signalling pathway(s), cDNA library from whole wheat grains was built and screened with the bait protein TaGW2(1-320). Preliminary results from the interactomic study suggest that TaGW2 may regulate cell division. Moreover, TaGW2 may also function as an E3 Nedd8 ligase, besides its E3 ubiquitin ligase function.

Adenovirus humanos são responsáveis por infecção respiratória aguda (IRA) em crianças e adultos, sendo os das espécies B e C mais frequêntes. Com o objetivo de estudar a variabilidade genética de HAdVs, 3087 amostras de aspirado de nasofaringe foram colhidas de crianças, em São Paulo, de 1995 à 2006. A PCR direcionada ao gene VA-RNA detectou 677 adenovírus (22%). O sequenciamento dos genes hexon, fibra e região E3 foram utilizados para determinar os sorotipos e estudar sua variabilidade genética. Dos 677 adenovírus, 69% são da espécie B, 23% da C e 0,7% da E. Variabilidades genéticas foram observadas em todas as regiões estudadas, por meio de mutações, evidenciadas por substituições, recombinações e deleções. Os genes que apresentaram maior variabilidade foram VA-RNA e E3 ORF7.7. Genomas virais de DNA, como dos adenovírus, podem se manter estáveis em condições diversas, contudo, a pressão sofrida por esses genomas, através da resposta imunológica do hospedeiro, fazem com que seu mecanismos evolutivos entrem em operação e variabilidades genéticas sejam observadas. / Human adenoviruses (HAdV) cause acute respiratory disease (ARD) in children and adults, being the adenovirus from species B and C the most frequently detected. With the aim of study the genetic variability of HAdV, 3087 nasopharyngeal aspirate were collected from children in São Paulo, from 1995 to 2006. PCR assay directed to adenovirus VA-RNA gene detected 677 HAdV (22%). Sequencing of the hexon and fiber genes and the E3 region were done to determine the serotypes and study genetic variability. Among the 677 adenoviruses detected 69 % were classified as species B, 23% as species C and 0,7 % as species E. Genetic variability was observed in all studied region, specially at the 7.7Orf of the E3 region and the VA-RNA gene. Genetic modifications were observed as recombination, substitutions and deletions. It is known that viral DNA genomes, as adenovirus, remain genetically stable under a variety of conditions, however, the pressure of the host on these viruses make that their evolutionary mechanisms come to operation and genetic variability are observed.

Given the relative paucity of treatments relating to both the 1532 and 1538 Bern Gespräche, alongside a growing historiography which has offered a clearer understanding of the backdrop around which these two debates were held, the focus of this research project will be to provide a comparative analysis of the recorded dialogues from the debates at Bern. This ecclesiologically focused comparison aims to discern whether the debate relating to the nature of the church at the 1538 session was merely a redundant exercise and continuation of the earlier 1532 disputation or whether the latter debate offered anything substantively new to the ongoing religious dialogue between these two groups. Furthermore, all of the respective views on the nature of the church manifest in these debates will be examined in light of the preceding Anabaptist/Reformed dialogue of the period to determine their place contextually. Having embarked upon the aforementioned goals several conclusions may be definitively drawn. First, the major ecclesiological suppositions expressed by both the Anabaptist and Reformed participants at the 1538 debate were, in fact, retained using the same core theological elements employed by their predecessors at the 1532 debate. Yet, despite this striking similarity, the independent nature of these debates must also be acknowledged. This may primarily be found in that both groups expressed their retained ecclesiologies with notable variation in things such as language, argumentative content, biblical corroboration, and illustrative evidence. Finally, both the similar and independent nature of these events will be shown to have been largely derived from the Anabaptist/Reformed dialogue already begun as the Swiss Brethren movement emerged from under Zwingli’s reform efforts in Zürich. Each of these conclusions should help to paint a more accurate portrait of not only what was accomplished through these debates, but where each stands contextually during the period.

Using the dynamic conditional correlation (DCC) model due to Engle (2002), we estimate time varying correlations of quarterly real GDP growth among the G7 countries. In general, we find that rather heterogeneous patterns of international synchronization exist during US recessions. During the 2007-2009 recession, however, international co-movement increased substantially. (authors' abstract)

This paper provides a comprehensive empirical assessment of the relation between the cyclicality of fiscal expenditure policy, output volatility, and economic growth, using a large cross-section of 88 countries over the period 1960 to 2004. Identification of the effects of (endogenous) cyclical expenditure policy is achieved by exploiting the exogeneity of countries political and institutional characteristics, which we find to be relevant determinants of the cyclicality of expenditures. There are three main results: First, both pro- and countercyclical expenditure policy amplify output volatility, much in a way like pure fiscal shocks that are unrelated to the cycle. Second, output volatility, due to variations in cyclical and discretionary fiscal policy, is negatively associated with economic growth. Third, there is no direct effect of cyclicality on economic growth other than through output volatility. These findings advocate the introduction of fiscal rules that limit the use of (discretionary and) cyclical fiscal (expenditure) policy to improve growth performance by reducing volatility. (author's abstract)

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Afin d'améliorer l'efficacité des immunothérapies anti-tumorales, il est crucial de mieux comprendre les régulations cellulaires et moléculaires contrôlant l'initiation et le maintien d'une réponse immunitaire adaptative anti-tumorale. Dans ce contexte, l'équipe a identifié l'E3 ubiquitine ligase ASB2a (Ankyrin repeat-containing protein with a SOCS Box 2 alpha) comme un régulateur de la motilité des cellules hématopoïétiques via la polyubiquitination et la dégradation par le protéasome de la Filamine A. Mon projet de thèse visait à étudier si ASB2a joue un rôle dans la mise en place et/ou le maintien de la réponse immunitaire anti-tumorale. L'analyse des données de séquençage d'ARN issues des biopsies d'une cohorte de patients atteints de cancer colorectaux révèle que l'expression d'ASB2 est plus élevée dans les sous-types de mauvais pronostic et est inversement corrélée avec la survie des patients. Dans le but de déterminer s'il existe un lien fonctionnel entre l'expression d'ASB2 et la progression du cancer du côlon, nous avons utilisé un modèle de tumeurs coliques chez des souris invalidées de façon conditionnelle et inductible pour le gène ASB2 dans les cellules hématopoïétiques. Dans un premier temps, nous avons validé la pertinence de ce modèle d'invalidation en montrant qu'ASB2a était responsable des différents niveaux de Filamine A observés entre les différentes populations de cellules dendritiques conventionnelles de la rate. J'ai ensuite démontré que l'invalidation d'ASB2 réduit le développement des tumeurs coliques, résultats en accord avec la pathologie humaine. Ce phénotype est causé par l'activation d'une réponse immunitaire anti-tumorale adaptative de type 1 plus forte chez les souris invalidées pour ASB2. L'ensemble de ces travaux mettent en évidence le potentiel d'ASB2a comme cible thérapeutique innovante dans le traitement du cancer colorectal. / In order to enhance the efficiency of anti-tumor immunotherapies, it is crucial to better understand the cellular and molecular regulations sustaining the initiation and maintenance of an adaptive anti-tumor immune response. In this context, the team identified the E3 ubiquitin ligase ASB2a (Ankyrin repeat-containing protein with a SOCS Box 2 alpha) as a regulator of hematopoietic cell mobility through the polybiquitination and proteasomal degradation of the Filamin A. My thesis project aimed to study if ASB2a plays a role in the establishment and/or maintenance of the anti-tumor immune response. The analysis of RNA sequencing derived from the biopsy of colorectal cancer patients revealed that ASB2a expression is higher in the subtypes associated with poor prognosis and is inversely correlated with patient relapse-free survival. In order to determine whether there is a functional link between ASB2 expression and colon cancer progression, we used a colorectal cancer model applied to ASB2 knockout mice in hematopoietic cells. First, we validate the relevance of this knockout model by showing that ASB2a was responsible for the different levels of Filamin A observed between the different populations of spleen conventional dendritic cells. Then, I have shown that ASB2 invalidation reduces the development of colonic tumors, in accordance with human pathology. This phenotype is due to the activation of a stronger type 1 adaptive anti-tumor immune response in ASB2 knockout mice. This study highlight the potential of ASB2a as an innovative therapeutic target in the treatment of colorectal cancer.