Dissertations / Theses on the topic 'Interacteur'

Les récepteurs des cytokines à structure α-hélicoïdale sont pour la plupart composés de deux sous-unités transmembranaires associées aux protéine tyrosine kinases de la famille Janus (Tyk2, Jak1, Jak2 et Jak3). La fixation de la cytokine à son récepteur induit une dimérisation des sous-unités ce qui entraîne l'activation des Jak par transphosphorylation. Les Jaks ainsi activées phosphorylent les facteurs de transcription STAT (Signal Transducer and Activator of Transcription) qui transloquent dans le noyau. Les tyrosine kinases Jak présentent en position N-terminale un domaine FERM (4.1-ezrin-radixin-moesin), suivi par un domaine dit 'SH2-like' et deux domaines kinases: un domaine dit 'kinase-like' (KL) à fonction régulatrice et un domaine catalytique de type tyrosine kinase en position C-terminale. L'association au récepteur se fait par la région N-terminale. Le laboratoire s'intéresse particulièrement au récepteur de l'interféron de type I (IFNα/β) composé de deux chaînes, IFNAR1 et IFNAR2, et aux kinases Tyk2 et Jak1 auxquels elles s'associent respectivement. La première partie de ma thèse a porté sur l'étude d'un nouveau partenaire de Tyk2, la protéine Pot1 (Partner of Tyk2) et de son rôle potentiel dans la voie de signalisation de l'IFNα. Plusieurs ADNc partiels de Pot1 avaient été isolés dans le laboratoire par criblage double-hybride utilisant comme appât le domaine FERM de Tyk2. Un ADNc complet de Pot1 avait été reconstruit donnant lieu à une protéine de 1003 acides aminés (aa). L'interaction entre cette protéine et les domaines FERM de Tyk2 et de Jak1 avait été confirmée par des expériences in vitro. L'analyse par northern blot de Pot1 montre une expression faible dans plusieurs tissus. L'étude de la localisation de la protéine Pot1 surexprimée avait montré une localisation cytoplasmique au niveau de vésicules non identifiées. Un des aspects de mon travail sur Pot1 a été de déterminer si l'ADNc reconstruit dans le laboratoire, appelé 'lab cDNA', correspond à un vrai transcrit. En effet, les banques de données recensent plusieurs autres transcrits issus du gène Pot1, avec une portion 3' commune et identique au 'lab cDNA', mais une région 5' divergente. Ces transcrits donnent lieu à deux isoformes ne possédant pas les 200 aa ou 250 aa en position N-terminale par rapport à la protéine de référence codée par le 'lab cDNA'. De plus, l'unique protéine murine (mPot1) recensée dans les banques de données ne possède pas les 100 aa en N-terminal par rapport à la protéine humaine de référence. Pour analyser l'expression de différents transcrits, j'ai amplifié l'ADNc de cellules humaines HEK293T avec des primers me permettant de 12 distinguer les différents transcrits. Ces expériences ont permis de confirmer l'expression du transcrit correspondant au 'lab cDNA' et codant pour une isoforme de 1003 aa (112 kDa). Par la suite j'ai étudié la localisation de la protéine Pot1 murine dans les cellules surexprimant mPot1. J'ai observé que mPot1 se trouve dans des vésicules cytoplasmiques, tout comme la protéine humaine, et semble être associée à la membrane de ces vésicules. Toutefois, nous ne pouvons pas écarter la possibilité de localisation inadéquate dû à la forte surexpression de mPot1. Il sera nécessaire de confirmer cette observation par l'analyse de la localisation de la protéine endogène. A ce jour les anticorps disponibles ne permettent pas sa détection. Afin d'évaluer le rôle de Pot1 dans la voie de signalisation de l'IFNα, j'ai mesuré la réponse à l'IFNα de cellules déplétées en Pot1 par interférence à l'ARN. J'ai mesuré la phosphorylation des protéines STAT et l'induction d'un gène rapporteur. Ces expériences ont montré que, dans ce système, la diminution de l'expression de Pot1 n'a pas d'effet sur la signalisation par l'IFNα. Afin d'éclairer la fonction de Pot1, de nouveaux interacteurs de Pot1 ont été recherchés par criblage double-hybride. Parmi les 14 protéines identifiées à haut niveau de confiance, nous nous sommes particulièrement intéressés à GIT1 (G protein-coupled receptor kinase interactor), une protéine adaptatrice impliquée dans de nombreux processus cellulaires, tels que l'internalisation de récepteurs, la signalisation induite par l'EGF (epidermal growth factor) et l'angiotensine II ainsi que la migration cellulaire. Afin d'analyser le rôle éventuel de GIT1 dans la signalisation par l'IFNα, j'ai mesuré plusieurs paramètres (internalisation des sous-unités du récepteur, phosphorylation des STAT, induction d'un gène rapporteur) dans des cellules surexprimant ou déplétées en GIT1. Les résultats obtenus ont écarté l'hypothèse d'une implication de GIT1 dans la réponse transcriptionnelle à l'IFNα. La deuxième partie de mon travail a porté sur l'étude de la mutation V678F introduite dans la protéine Tyk2.Cette substitution est située dans le domaine KL et correspond à la mutation V617F de Jak2, décrite comme étant à l'origine de la Polycythemia vera. La P. vera, ou maladie de Vaquez, est une maladie myéloproliférative caractérisée par une hyperproduction d'érythrocytes et leur hypersensibilité à l'érythropoiétine (Epo). Il a été montré que la mutation V617F induit une augmentation de l'activité kinase de base de Jak2. De même, Jak2V617F induit une prolifération indépendante de facteurs de croissance des cellules murines BaF3, confirmant son potentiel transformant. Toutefois, il a été montré que 13 Jak2V617F nécessite la coexpression d'un récepteur homodimérique, tel que récepteur à l'Epo, pour exercer une activité transformante maximale. Les questions que nous nous sommes posées sont les suivantes: 1) quel est l'effet de la substitution V678F sur l'activité catalytique de Tyk2? 2) quel est l'effet du mutant Tyk2V678F sur la signalisation par l'IFNα? 3) le mutant Tyk2V678F aurait-il un effet plus marquant ou délétère s'il est placé dans un contexte de récepteur homodimérique? A cet effet, nous avons établi, à partir de cellules Tyk2-déficientes, des clones stables exprimant la protéine sauvage ou mutante. Nos résultats montrent que la mutation V678F augmente in vivo et in vitro la capacité de Tyk2 à s'auto-phosphoryler. Par la suite, j'ai mesuré la phosphorylation sur tyrosine des protéines STAT en réponse à l'IFNα. Aucune différence de niveau de phosphorylation de STAT1/2/5 n'a pu être décélée entre les cellules exprimant la protéine sauvage et les cellules exprimant le mutant Tyk2V678F. Par contre, j'ai mis en évidence une phosphorylation basale de STAT3 augmentée en présence de Tyk2V678F. Pour déterminer si cette phosphorylation basale de STAT3 corrèle avec une augmentation de son activité transcriptionnelle, j'ai analysé l'activité transcriptionelle de STAT3 à l'aide d'un gène rapporteur. Les résultats montrent que, dans les cellules exprimant le mutant Tyk2V678F, STAT3 possède une activité transcriptionelle augmentée. Afin d'étudier l'effet du mutant Tyk2V678F placé dans un contexte de récepteur homodimérique, j'ai utilisé des cellules exprimant un récepteur chimérique comprenant l'ectodomaine du récepteur à l'Epo fusionné aux régions transmembranaire et cytoplasmique de la chaîne IFNAR1 (EpoR/R1). A l'aide de ces cellules, j'ai pu confirmer que l'expression du mutant Tyk2V678F engendre une phosphorylation basale de STAT3. De plus, dans ce contexte de récepteur homodimérique, suite à stimulation par le ligand Epo, le mutant Tyk2V678F induit une augmentation ultérieure de la phosphorylation de STAT1, STAT3 et STAT5. Nous avons aussi voulu comparer directement le niveau de phosphorylation de Tyk2V678F et de STAT3 dans différents contextes de récepteurs. Les résultats obtenus montrent que la protéine Tyk2V678F est plus phosphorylée basalement dans les cellules exprimant le récepteur homodimérique EpoR/R1. Ceci est probablement la conséquence d'une transphosphorylation plus efficace de deux kinases mutantes juxtaposées. Par contre, la phosphorylation de STAT3 ne corrèle pas directement avec le niveau d'expression du mutant Tyk2V678F, ce qui suggère une absence de corrélation linéaire entre l'activation de Tyk2 et et de STAT3. 14 En conclusion, nous avons montré que la mutation V678F augmente l'activité catalytique de Tyk2. De plus, le mutant acquiert la capacité de phosphoryler STAT3 et ceci en absence du ligand. Cependant, le mutant Tyk2V678F n'affecte pas la réponse à l'IFNα en terme de phosphorylation de Jak1, STAT1 et STAT2. Ces résultats démontrent une interaction fonctionnelle étroite entre Tyk2 et STAT3. Etant donné que STAT3 constitutivement actif exerce des propriétés oncogéniques et que STAT3 est phosphorylé dans de nombreux cancers, il est possible de prédire aussi un rôle oncogénique pour Tyk2 constitutivement active. Récemment, il a été suggéré qu'un polymorphisme de Tyk2, P1104A, pourrait être associé à la présence de tumeurs. Cette substitution est située dans le domaine tyrosine kinase au sein d'une hélice α présente uniquement dans les membres de la famille Jak. Nous avons introduit cette mutation dans Tyk2 et analysé son effet sur l'activité kinase. Les résultats montrent que le mutant Tyk2P1104A est incapable de s'autophosphoryler in vitro. Toutefois, en réponse à l'IFNα, aucune différence du niveau de phosphorylation de STAT1/2 /3 n'est décélée dans les cellules exprimant Tyk2P1104A par rapport aux cellules exprimant la protéine sauvage. Ces résultats suggèrent que la mutation P1104A abolit la capacité autophosphorylante de l'enzyme, mais n'affecte pas l'activité enzymatique induite par l' l'IFNα envers d'autres substrats in vivo. Ces résultats préliminaires devront être renforcés par des études plus approfondies de l'effet de la mutation P1104A sur la fonction que Tyk2 exerce au sein du récepteur de l'IFNα/β ainsi qu'au sein d'autres récepteurs de cytokines de la réponse immune.

Les tyrosines kinases de la famille Jak (Tyk2, Jak1-3) sont impliquées dans la signalisation par les cytokines hélicoïdales. Une partie de ma thèse a porté sur l’étude de Pot1, un nouvel interacteur de Tyk2. Afin d’évaluer le rôle de Pot1 dans la voie de signalisation de l’IFN, j’ai mesuré la réponse à l’IFN de cellules déplétées en Pot1 en évaluant la phosphorylation des STATs et l’induction d’un gène rapporteur. La diminution de l'expression de Pot1 n’a pas d’effet sur la signalisation par l’IFN. La deuxième partie de mon travail a porté sur l’étude de la substitution V678F dans la Tyk2. Nous avons établi, à partir de cellules Tyk2-déficientes, des clones stables exprimant la protéine sauvage ou mutante. Nous avons aussi analysé le mutant Tyk2V678F associé à un récepteur artificiel de type homodimérique. Nos résultats montrent que la mutation V678F augmente in vivo et in vitro la capacité de Tyk2 à s’auto-phosphoryler, cela plus fortement dans le cas du récepteur homodimérique. De plus, le mutant acquiert la capacité de phosphoryler STAT3 en absence de ligand mais n’affecte pas la réponse à l’IFN en terme de phosphorylation de Jak1 et STAT1/2
Tyk2 is a member of the Jak family of tyrosine kinases (Jak1, Jak2, Jak3 and Tyk2), which are indispensable components of -helical cytokine signaling cascades. Receptors for - helical cytokines are mostly made of two transmembrane subunits that associate with Jaks. Ligand bridging of two receptor chains brings together the associated Jaks, enabling their activation by transphosphorylation. Activated Jaks phosphorylate the STATs (Signal Transducer and Activator of Transcription) which translocate into the nucleus to drive gene expression. The Jaks have an N-terminal FERM (band 4. 1-ezrin-radixin-moesin) domain, followed by an "SH2-like" domain and two kinase domains: a kinase-like (KL) domain and the catalytic tyrosine-kinase domain. The FERM and SH2-like domains are necessary for receptor binding. The KL domain has no catalytic activity, but plays an important regulatory role. The laboratory is particularly interested in the type I interferon (IFN/) receptor, made of two subunits IFNAR1 and IFNAR2, which bind Tyk2 and Jak1, respectively. During the first part of my thesis, I analyzed a new Tyk2 interacting protein, Pot1 (Partner of Tyk2). Pot1 was isolated in a yeast two-hybrid screen using the Tyk2 FERM domain as bait. To assess the role of Pot1 in IFNsignaling, I monitored IFN-induced response in Pot1-depleted cells by measuring STAT phosphorylation and the induction of a reporter gene. These experiments showed that, in this system, Pot1 depletion had no effect on IFN-induced signaling. A two-hybrid screen was performed with Pot1 as bait. Among the 14 proteins found with high interaction confidence, we focused on GIT1 (G protein-coupled receptor kinase interactor 1), an adaptor protein implicated in a number of cellular processes, like cell migration, receptor internalization and EGF and angiotensin II signaling. To analyze the role of GIT1 in IFNsignaling, I monitored IFN-induced receptor internalization, STAT phosphorylation and the induction of a reporter gene in GIT1-depleted cells. The results obtained allow us to exclude a role for GIT1 in type I IFN signaling. During the second part of my thesis, I analyzed the effect of the V678F substitution on Tyk2 function. This mutation, located in the KL domain, corresponds to the V617F mutation of Jak2 found at the origin of Polycythemia vera. To study the effect of the V678F mutation on Tyk2 activity, I reconstituted Tyk2-deficient cells with Tyk2 WT or the V678F mutant and monitored IFN-induced response. Our results show that the V678F mutation augments basal Tyk2 kinase activity measured in vitro. This gain-of-function leads to an increase of the basal STAT3 phosphorylation level, but has no effect on IFN-induced Jak1 and STAT1/2/5 phosphorylation. As opposed to Jak2, Tyk2 has been implicated only in signaling via heterodimeric receptor complexes. Interestingly, it has been shown that Jak2V617F needs the coexpression of a cognate homodimeric receptor to fully exert its transforming activity in the BaF3 cellular model system. Therefore, we analyzed the effect of Tyk2V678F on signaling via an artificial homodimeric receptor. To this end, we used Tyk2-deficient cells that express a chimeric receptor containing the extracellular domain of erythropoietin receptor fused to the intracellular region of IFNAR1. These cells were stably reconstituted with Tyk2WT or the V678F mutant. In this context, Tyk2V678F confers ligand hypersensitivity as seen by STAT1/3/5 phosphorylation. Moreover, the ensemble of these data point to STAT3 as a preferred substrate of Tyk2

Les œuvres numériques interactives remettent en question le rôle du spectateur. Il est sollicité physiquement et sensoriellement afin de l’inciter à être actif. Dans ce projet de thèse, nous mettons en avant le potentiel créatif enfui dans chaque participant à travers sa gestualité interactive dans la co-création de l’œuvre.À partir de l’exploration des différents gestes performatifs à travers les courants et les disciplines artistiques existants sous forme de performances et d’installations, nous avons identifié et créé le geste performatif recherché dans nos créations artistiques : la gestualité interactive créative et performative. Par ailleurs, dans le cadre de cette recherche-création, la réalisation de performances dansées augmentées et d’installations interactives performatives a été consolidée par l’observation et l’étude des comportements des participants afin de déterminer les possibilités et les limites de la co-création des œuvres réalisées
Interactive digital artworks question the role of the spectator. They solicit him physically and sensually in order to make him active.In this thesis project, we highlight the creative potential embedded in each participant through their interactive gestures in the co-creation of the artwork.Thanks to the exploration of the different performative gestures through existing art movement and disciplines in the form of performances and installations, we identified and created the sought performative gesture in our artistic creations : creative and performative interactive gestures. Moreover, as part of this research-creation, the realization of augmented dance performances and performative interactive installations was consolidated through the observation and the study of the participants’ behavior in order to depict the possibilities and the limits of co-creation in the created artworks

Scania provides a fleet management system for hauliers who want to control their fleets in an efficient way. The system includes an onboard computer with touch screen called Scania Interactor where the driver can access GPS navigation, order support, message service and other functionalities. The purpose of this thesis is to develop a concept for logging the user interaction on the Interactor and analyze the result which enables further development and optimization of the interface. A demonstration application is designed, implemented and tested for the purpose of showing the possibilities and difficulties with logging of interaction. The result shows that logging is quite easy to implement and that the amount of produced data is small and therefore not expensive to transfer via GPRS to a central server where searching and analysis can be done. On the other hand, the biggest challenge lies in the analysis and interpretation of the logged data and to apply it to the development of the interface. Logging of interaction can not answer questions about the user’s opinions but can at least point out potential flaws and areas that can be improved. Another issue related to logging of interaction is the legal aspect. Two laws have been identified and need consideration before logging can be implemented in reality.
Scania tillhandahåller ett system för flottstyrning, kallat Fleet Management System, för åkerier som vill styra sina flottor på ett effektivt sätt. I Scanias system ingår på fordonssidan en fordonsdator med pekskärm kallad Interactor. På den finns ett grafiskt användargränssnitt där chauffören har tillgång till GPS-navigeringsverktyg, orderhantering, meddelandetjänst och andra funktioner för att underlätta i arbetet. Detta examensarbete har gått ut på att ta fram ett koncept för att logga interaktionen som chaufförerna har med Interactorn för att sedan analysera loggen och dra slutsatser som leder till att utvecklingen av Interactorn kan optimeras. En demonstrator har designats, implementerats och testats för att visa på möjligheter och svårigheter med interaktionsloggning. Resultatet visar att det på ett enkelt sätt går att logga interaktion med Interactorn samt att loggen som skapas inte blir särskilt stor och därmed inte så dyr att överföra med GPRS till en central enhet där sökning och analys kan utföras. Däremot ligger det en stor utmaning i att analysera och tolka loggen och sedan applicera det på utvecklingen av Interactorn. Interaktionsloggning kan inte svara på frågor om användarnas uppfattning om Interactorn men kan peka på potentiella brister och områden som kan förbättras. Den lagliga aspekten av interaktionsloggning har också tagits upp där två lagar har identifierats, personuppgiftslagen och lagen om elektronisk kommunikation. Om interaktionsloggning ska implementeras och användas i mer än fältprovsbilar måste dessa lagar tas hänsyn till.

This thesis is about making a support for barcode readers and RFID readers on a computer mounted in heavy vehicles. The computer has several applications designed for heavy vehicles such as an order handling application. The goal was to suggest an architecture to implement a support for the readers. The architecture should be independent of which manufacturer who has made the reader hardware that was connected to the computer. At first a theoretically study was made around RFID technology and standards for the transport and supply sector. Then the architecture was developed and a demo implementation was made. The conclusion of this thesis is to implement support for barcode readers with a wedge application. The wedge application is an application that handles the communication with barcode readers and converts the barcode into key presses from a virtual keyboard. The wedge application is an application that the manufacturer of the barcode reader supplies. The user must install the drivers and wedge application for the reader. On one type of the computers the user is not allowed to install own applications. This is solved by Scania by installing drivers and wedge applications from the largest actors on the barcode reader market. The implementation of RFID readers is not as urgent as for barcode reader. RFID is mostly used in shape of smart labels. Smart labels are a combination of an RFID tag and a barcode and therefore the barcode can be used on smart labels. When and if the implementation of RFID will be made the EPC standard should be used. This is probably he standard that will be used in supply chains. The EPC standard specifies a protocol between the RFID reader and the host computer.
Det här examensarbetet handlar om att göra ett stöd för streckkodsläsare och RFID-läsare till en dator monterad i en lastbil. Datorn har flera applikationer gjorda för lastbilar till exempel en orderhanteringsapplikation. Målet var att föreslå en arkitektur för att implementera stöd för läsare. Arkitekturen ska vara oberoende av vilken tillverkare som har gjort hårdvaran som kopplas till datorn. Först gjordes en teoristudie av RFID-teknologin och standarder för transport- och distributionsnätet. Sedan utvecklades arkitekturen och en demo gjordes. Slutsatsen av det här examensarbetet är att implementera stöd för streckkodsläsare med en så kallad wedge-applikation. Wedge-applikationen hanterar kommunikationen med streckkodsläsare och konverterar streckkoden till tangentbordstryckningar från ett virtuellt tangentbord. Wedge-applikationen tillhandahåller tillverkaren av streckkodsläsaren. Användaren måste installera drivrutiner och wedge-aplikationen för sin läsare. På en av datorerna är det inte tillåtet att installera egna applikationer. Detta löses genom att Scania installerar drivrutiner och wedge-applikationer från de största tillverkarna inom streckodsläsarområdet. Implementeringen av RFID-läsare är inte lika brådskande som för streckkodsläsare. RFID används oftast i form av smart labels. Smart labels är en kombination av en RFID-tag och en streckkod och därför kan streckkoden användas på en smart label. Om och när implementeringen av RFID sker så bör EPC-standarden användas. Det är troligvis denna standard som kommer att användas inom distribution. EPC-standarden specificerar ett protokoll som används mellan RFID-läsaren och värddatorn.

La protéine kinase activée par AMP (AMPK) est un senseur et régulateur central de l'état énergétique cellulaire, mais ces voies de signalisation ne sont pour le moment que partiellement comprises. Deux criblages non-biaisés pour la recherche de partenaires d'interaction et de substrats d'AMPK ont précédemment été réalisés dans le laboratoire. Ces derniers ont permis l'identification de plusieurs candidats (protéines), mais leur rôle fonctionnel et physiologique n'était pas encore établi. Ici nous avons caractérisé la fonction de la relation entre AMPK et quatre partenaires d'interaction : gluthation S-transferases (GSTP1 and GSTM1), fumarate hydratase (FH), l'E3 ubiquitine-ligase (NRDP1), et les protéines associées à la membrane (VAMP2 and VAMP3). Chacune de ces interactions parait avoir un rôle différent dans la signalisation AMPK, agissant en amont ou en aval de la protéine AMPK. GSTP1 et GSTM1 contribueraient à l'activation d'AMPK en facilitant la S-glutathionylation d'AMPK en conditions oxydatives moyennes. Cette régulation non-canonique suggère que l'AMPK peut être un senseur de l'état redox cellulaire. FH mitochondrial est l'unique substrat AMPK clairement identifié. Etonnamment le site de phosphorylation se trouve dans le peptide signal mitochondrial, ce qui pourrait affecter l'import mitochondrial. NRDP1, protéine pour laquelle nous avons pour la première fois développé un protocole de production de la protéine soluble, est faiblement phosphorylée par l'AMPK. L'interaction ne sert pas à l'ubiquitination d'AMPK, mais affecte le renouvellement de NRDP1. Finalement, l'interaction de VAMP2/3 avec AMPK n'implique pas d'évènement de phosphorylation ou d'activation d'un des partenaires. Nous proposons un mécanisme de recrutement d'AMPK par VAMP2/3 (" scaffold ") au niveau des vésicules en exocytose. Ce recrutement favoriserait la phosphorylation de substrats de l'AMPK à la surface des vésicules en exocytoses. Une fois mis en commun, nos résultats enrichissent les connaissances sur les voies de signalisation AMPK, et suggèrent une grande complexité de ces dernières. Plus que les kinases en amont et des substrats en aval, la régulation de la signalisation d'AMPK se fait via des modifications secondaires autres que la phosphorylation, via des effets sur le renouvellement de protéines, et probablement via un recrutement spécifique de l'AMPK dans certains compartiments cellulaires.

Le Syndrome de l'X fragile (FXS) est la forme la plus fréquente de retard mental héréditaire. Il est causé par l’inactivation du gène FMR1 codant une RNA Binding Protein (FMRP) impliquée dans le contrôle de la traduction. Afin de mieux comprendre la fonction de FMRP, nous nous somme intéressé à ses interacteurs et mon travail s’est organisé en deux parties: la caractérisation de l'interaction FMRP/ARNm GRK4 et la caractérisation de la fonction neuronale de CYFIP1/2, deux protéines interacteurs de FMRP. Nous avons confirmé l'interaction FMRP/ARNm GRK4 et identifié une portion contenant deux motifs ACUK / WGGA, connu pour être de nouvelles cibles pour FMRP. FMRP se lie à GRK4 via son domaine RGG-box et régule négativement sa traduction. Dans les cellules granulaires du cervelet, GRK4 se lie au récepteur GABABR(GBR), induisant sa désensibilisation. Sachant l’importance de la signalisation GBR du cervelet dans la coordination motrice, un niveau élevé de GRK4 peut contribuer au déficit de l'apprentissage moteur et la coordination des mouvements dans FXS. Nous avons également caractérisé la fonction neuronale de CYFIP1/2 en induisant leur knockdown (KD). Ces protéines appartiennent au complexe WAVE (WRC) qui est impliqué dans la régulation de l’établissement de la polarité axonale et dendritique. Nous avons identifié un mécanisme de co-régulation de la transcription des ARNm codant les membres du WRC lors de l’altération de l'expression de CYFIP1/2. Le KD CYFIP1/2 modifie également neuronale ramification et connectivités. L'interaction de FMRP/CYFIP1/2 permettrait de comprendre les mécanismes impliqués dans le développement des anomalies des épines dendritiques dans FXS
Fragile X syndrome (FXS) is the most common form of inherited mental retardation. It is caused by the silencing of the FMR1 gene, which encodes for an RNA-binding protein (FMRP) involved in translational control. To better understand the function of FMRP, we are interested in its interactors and so my work was organized into two main parts: the characterization of the interaction between FMRP and GRK4 mRNA and the characterization of the neuronal function of CYFIP1/2, two FMRP interacting proteins. We confirmed in vivo and in vitro the FMRP/ GRK4 mRNA interaction and identified a portion containing two ACUK/WGGA motifs, known to be a novel targets for FMRP. FMRP binds this target via its RGG box domain and negatively modulates the expression of GRK4 at the translational level only in cerebellum. In cerebellar granule cells, GRK4 interacts directly with the GABAB receptor (GBR), promoting its desensitization. Since in cerebellum GBR signaling has a relevant role in motor coordination, an elevated level of GRK4 can contribute to deficits of motor learning and movement coordination in FXS. Next, we characterized the function of CYFIP1/2 in neurons by inducing their knockdown (KD). CYFIP1/2 are components of the canonical WAVE regulatory complex (WRC), important in the spatiotemporal regulation of actin dynamics to get correct axonal and dendrites polarity and branching. We identified a co-regulation of transcription of mRNA coding the WRC members when the expression of CYFIP1/2 is disturbed. KD CYFIP1/2 also alters neuronal branching. The FMRP/CYFIP1/2 interaction would allow us to understand the mechanisms involved in the development of dendritic spines abnormalities in FXS

Mestrado em Biotecnologia - Biotecnologia Molecular
Neste trabalho avaliou-se o efeito de mutações em tRNA mitocondrial humano usando a levedura Saccharomyces cerevisiae como modelo. Em termos genéricos S. cerevisiae é um microrganismo muito estudado e conhecido, sendo possível modificar o seu genoma de forma rápida e precisa. Como bom modelo permite estudar este tipo de doenças através da mutação dos seus genes que codificam para tRNAs através de um procedimento biobalístico. Com este procedimento é possível introduzir mutações nos genes que codificam para tRNAs mitocondriais da levedura em qualquer posição, incluindo mutações equivalentes às que são patogénicas em humanos. S. cerevisiae é também um bom modelo pois os seus mutantes que exibem deficiências respiratórias, complexas ou muitos graves, têm a capacidade de crescer pois continuam a realizar fermentação em glucose, permitindo assim efectuar estudos funcionais moleculares. Para além do referido, o uso de diferentes estirpes de S. cerevisiae com diferentes mutações permite estudar genes nucleares isolados capazes de suprimir o fenótipo anormal dos mutantes, neste caso o crescimento num meio respirável. Na primeira parte deste projecto caracterizaram-se três estirpes diferentes de S. cerevisiae wild-type, MCC123, D273-10B/1A e W303-1B e subsequentemente o efeito de substituições nos tRNAs mitocondriais com o propósito de definir uma relação entre a eficiência mitocondrial e a presença de diferentes contextos nucleares. Concluiu-se que as estirpes MCC123 e W303-1B são mais apropriadas para o estudo de mutações severas, em contraste a D273-10B/1A é mais adequada para estudar mutações mais leves. Na segunda parte estudou-se a actividade supressora da sobre-expressão dos genes que codificam para leucil-tRNA sintetase, o seu domínio C-terminal e algumas pequenas sequências em diferentes mutantes nos tRNAs incapazes de crescer em meios contendo glicerol (GlnC6T, AspC61T, GlyG30A e HisG51A). A tRNA sintetase inteira (genes de levedura e de humano) e o Cterminal têm alguma capacidade de suprimir o fenótipo anormal, mas o melhor resultado foi obtido sobre-expressando as sequências mais pequenas que codificam para péptidos de quinze aminoácidos do domínio C-terminal (β30-31 e β32-33). Outras sequências testadas não tiveram qualquer efeito supressivo. Também foi caracterizada a capacidade supressora dos factores de elongação da síntese proteica mitocondrial de levedura e humana em células carregando a mutação AspC61T. Os nossos resultados, sobre-expressando tRNA sintetases e factor EF-Tu, mostram que a capacidade catalítica não é necessária para a supressão e sugerimos que esteja envolvida uma catividade tipo chaperone.
In this work we evaluate the effect of mitochondrial tRNA mutations using the yeast Saccharomyces cerevisiae as model. This simple organism is good model since it is possible to transform the mitochondria by a biolistic procedure. This makes possible to introduce mutations at any desired position in yeast mt tRNA genes, including mutations equivalent to those that are pathogenic in humans. Yeast offers a unique possibility in that mutants exhibiting complete or very severe respiratory deficiencies can grow by fermentative metabolism on glucose and are therefore amenable to molecular functional studies. Moreover using the yeast strains with the different mutations is possible to isolate nuclear genes able to suppress the defective phenotype of the mutants, in this case to allow the growth on respirable medium (containing glycerol as unique carbon source). In the first part of this project we characterized three different S. cerevisiae wild-type strains, MCC123, D273-10B/A1 and W303-1B and subsequently the effect of mt tRNA substitutions in order to define a relationship between the mitochondrial efficiency and the presence of different nuclear backgrounds. We concluded that MCC123 and W303-1B are more appropriate to study severe mutations whereas the D273-10B/A1 is more suitable to study milder mutations. In the second part of ours experiments we studied the suppression activity overexpressing the mt leucyl-tRNA synthetases genes, it C-terminal domain and some shorter sequences in different tRNA mutants unable to grow in glycerol containing media (GlnC6T, AspC61T, GlyG30A and HisG51A). The entire tRNA synthetases (yeast and human genes) and the C-terminal have some capability of suppress the defective phenotype, but the best result was obtained overexpressing two shorter sequences coding for fifteen aminoacids from its C-terminal domain (β30-31 and β32-33). Other sequences tested did not have any supress effect. I also report a further characterization of the functionality of mt yeast and human mt protein synthesis elongation factors in their suppressing activities in cells bearing the AspC61T mutation. Our results, overexpressing either the tRNA synthetases or the EF-Tu factor, show that the catalytic function is not necessary for suppression and we suggest that a chaperone like activity is involved.

2016 - 2017
The Vector Autoregressive (VAR) Models can be considered as a dynamic multivariate extension of the univariate autoregressive models. This family of models has become very popular in macroeconomics analysis after the work of Sims(1980) and they are widely used in time series literature thanks to their flexibility. As a matter of fact, by setting appropriately a VAR model, we can describe efficiently the dynamics of the economy and provide quite accurate forecasts. During recent years, researchers developed different VAR models with the purpose to represent better the data generating process. Among these, the nonlinear VAR models have gained a central role in macroeconometric analysis in testing the theory, due to their capacity to capture a richer set of dynamics regarding current macroeconomic phenomenons. Depending on the specific model, they can allow, for example, different states (regimes) of the world, to allow the coefficients of the model to vary over time in each time unit, allowing for interactions between variables potentially revealing important information. The first paper included in this thesis is a survey which have the purpose to examine linear and nonlinear VAR models. The second and third papers present two empirical applications of the Interacted Panel VAR Model, which is a new nonlinear methodology we illustrated over the first paper. Specifically, we analyze in both papers the behavior of government spending multiplier when the interest rate is at the Zero Lower Bound (ZLB). This is a highly topical question since the outbreak of Great Recession, given that many policy makers have wondered whether fiscal stimulus would be able to help the economy to recover from recession. In particular, there exist two different and opposite theoretical predictions. New Keynesian DSGE models show that, when the interest rate is at the ZLB, a raise in government spending has a strong and positive impact on the economy. On the other side, theoretical prediction indicate very low multipliers, showing that an increase in government spending does not stimulate private activity. Although there exist many theoretical predictions about the size of government spending multiplier at the ZLB, very few empirical evidences are provided. These two paper aim to shed light on the size of the government spending multiplier at the ZLB. Among the nonlinear VAR models, we choose the Interacted (Panel) VAR Model because it offers an important advantage compared to others nonlinear approaches. Thanks to the interaction term, we are able to investigate among the entire sample. This can be done also within a time varying framework, but it implies a larger number of estimates which requires informative priors. In order to be as more agnostic as possible, we also use a Bayesian approach for inference but with uninformative priors. In the first paper we develop an Interacted VAR Model and conduct our analysis on the United States sample. In order to identify government spending shocks we use the sign restrictions approach, furthermore we use the forecast series of government spending to account for the potential effects of anticipation that can pose serious problems for the identification of government spending shocks. We find that the government spending multiplier ranges between 3.4 and 3.7 at the ZLB, while it ranges from 1.5 to 2.7 away from the ZLB. Then, we develop a Factor-Augmented IVAR (FAIVAR) model with the purpose to address another limited information problem. It confirms our results from a qualitatively point of view. As a matter of fact, the government spending multiplier ranges between 2.0 and 2.1 at the ZLB and between 1.5 and 1.8 away from the ZLB. These results are also in line with some recent studies which predict higher multipliers at the ZLB than in normal times... [edited by author]
XVI n.s.

La monoubiquitinación de la histona H2A cumple un rol esencial en la represión génica. Diversas enzimas desubiquitinantes (DUBs) han sido identificadas, que remueven específicamente la ubiquitina de H2A en distintos contextos celulares. Sin embargo poco se sabe sobre la regulación de esta actividad enzimática y de la interrelación con la maquinaria de ubiquitinación. En el presente trabajo hemos estudiado USP21, una DUB específica de H2A, que modula la iniciación de la transcripción. Hemos identificado BEND3 como interactor del USP21 nuclear. BEND3 se distingue por la presencia de cuatro dominios BEN, está localizado en la heterocromatina y actúa como represor de la transcripción. Hemos validado y delimitado la interacción entre USP21 y BEND3 y hemos observado que BEND3 está polyubiquitinado. La estabilidad proteica de BEND3 y su localización subcelular fueron independientes de la actividad catalítica de USP21 y los niveles de H2Aub no fueron influenciados por BEND3. Finalmente, el análisis de la expresión génica en células madre embrionarias de ratón revela la expresión diferencial de genes involucrados en crecimiento, proliferación celular y ciclo celular.
Monoubiquitination of histone H2A fulfills an essential role in gene repression. Several deubiquitinating enzymes (DUBs) have been identified that specifically remove ubiquitin in different cellular contexts. However, the regulation of their enzymatic activity as well as their interplay with the ubiquitination machinery are not well understood. We studied the H2A specific deubiquitinase USP21, which modulates transcriptional initiation. We identified BEND3 as interactor of nuclear USP21. BEND3 is characterized by a quadruple repeat of BEN domains, is localized to heterochromatin, and acts as a transcriptional repressor. We validated and mapped the interaction between USP21 and BEND3 and found that BEND3 is polyubiquitinated. BEND3 protein stability and subcellular localization were independent of the catalytic activity of USP21 protein, and H2Aub levels were not influenced by BEND3. Finally microarray gene expression analysis in mouse embryonic stem cells depleted for BEND3 revealed the differential expression of genes involved in cellular growth proliferation and cell cycle amongst others.

Title from PDF of title page (University of Missouri--Columbia, viewed on March 10, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Thesis advisor: Dr. Steve Osterlind. Includes bibliographical references.

This study developed an advance understanding of the mechanisms of hydraulic fracture initiation, propagation, and natural fractures interaction based-on thorough investigation of effects of various parameters associated with hydraulic and natural fractures through numerical modelling, simulation and laboratory experiments. Thesis progressively demonstrates different steps of the development of numerical modelling, simulation and experimental validation. Factors influencing the initiation and propagation of hydraulic fracture, and natural fractures interactions are thoroughly discussed based upon comprehensive sensitivity studies.

El nostre interès pels temes relacionats amb la lectura, i concretament per la manera com es porta a terme l’ensenyament de la comprensió lectora, és fruit de la nostra activitat professional a l’àrea de Psicologia de l'Educació i en particular en l’àmbit de la intervenció psicoeducativa. Precisament en aquest àmbit hem pogut constatar personalment que de l’elevat nombre d'alumnes que no assoleixen l’èxit acadèmic, una bona part presenten problemes a l’hora d’entendre el contingut d’un text, i més amplament, dificultats relacionades amb l’aprenentatge de la lectura. A aquesta constatació succeïa una altra, relativa a la manca de recursos i dificultats que experimenten els ensenyants, en general, per a establir seqüencies d'instrucció susceptibles d'ajudar aquells alumnes. La nostra manera d'entendre els objectius de la intervenció psicoeducativa ens portava a interrogar-nos sobre els processos d'ensenyament/aprenentatge que calia dissenyar per assegurar que els alumnes poguessin comprendre, interpretar i utilitzar els textos als que amb tanta freqüència es veuen confrontats dins i fora del marc escolar. Hem d'admetre que no disposàvem de resposta, més enllà d'algunes intuïcions, per a l'interrogant que ens havíem plantejat: com s’havia de fer? En les pàgines que segueixen hem intentat abordar des de la nostra pròpia perspectiva aquesta ampla problemàtica, conscients que el nostre treball constitueix nomes una passa en el camí que condueix al coneixement dels factors i variables que intervenen en l'ensenyament. El primer capítol constitueix una aproximació als models teòrics que és possible identificar en el camp de la lectura; els dos capítols següents, ja a partir d'aquells models, analitzen respectivament la manera d’entenendre l'aprenentatge i l'ensenyament de la comprensió lectora. La separació entre ensenyament i aprenentatge s'ha produït únicament a efectes d'assolir una major claredat en l'exposició; en el capítol quart s'ofereix una breu revisió de treballs d'investigació que s'han ocupat del mateix fenomen que ens preocupa a nosaltres. El cinquè vol desembocar en la proposta d’un model susceptible de donar compte dels factors que intervenen en l'ensenyament de la comprensió lectora. En el capítol sisè es descriuen les línies generals del disseny d'investigació elaborat per tal de contrastar el model formulat i el capítol setè es dedica a l'anàlisi de les dades obtingudes. Les conclusions i la discussió s'aborden en el vuitè i últim capítol, en el qual s'expliciten també les implicacions del nostre treball envers la recerca i la formació del professorat.

Dysregulation of mitochondrial dynamics is an emerging theme in neurodegenerative disorders, including Parkinson disease (PD). Autosomal recessive PD (ARPD) can be caused by mutations in the mitochondrial kinase PINK1 or the cytosolic E3 ubiquitin ligase Parkin. PINK1 and Parkin act in a common genetic pathway to control mitochondrial dynamics. To gain insights into the regulation of mitochondrial dynamics, binding partners of PINK1 were identified using different approaches: yeast two-hybrid screening of cytosolic and membrane bound forms of PINK1 and affinity purification of epitope-tagged PINK1 from a stable cell line under basal conditions and during mitochondrial stress. Many potential substrates of PINK1 were identified, including proteins with roles in mitochondrial dynamics. CluH was persistently identified in the PINK1 co-precipitation studies. CluH is a large tetratricopeptide protein that is highly conserved. CluH has previously been implicated in the regulation of mitochondrial dynamics in yeast, slime mold, plants and the fruit fly, but remains uncharacterized in mammals. I therefore investigated the expression profile of CluH in mouse tissues, and the subcellular localization in human cells using confocal microscopy. I also examined whether CluH could regulate mitochondrial morphology and function. I demonstrated that siRNA knock-down of CluH led to hyperfused mitochondria while over-expression of CluH led to fragmented mitochondria. Knock-down of CluH also resulted in decreased complex I activity, suggesting that CluH is required for mitochondrial integrity. The domains of CluH required for altering mitochondrial dynamics were also determined. Finally, I examined the interplay of CluH with the fission/fusion machinery and found that CluH can interact with fission proteins MiD49 and Drp1, consistent with a role for CluH in promoting mitochondrial fission. Taken together, these studies have identified CluH as a new player in mitochondrial dynamics, which interacts with the ARPD protein PINK1.

ΔNp63α è un fattore trascrizionale ampiamente studiato nel cancro alla mammella. Studi recenti indicano che esso è implicato nella tumorigenesi e nell’auto-rinnovamento delle cellule staminali di suddetto cancro. Anche se il profilo trascrizionale di p63 è stato ampiamente caratterizzato, la nostra conoscenza sui suoi possibili interattori implicati nella regolazione del cancro alla mammella è limitata. In questo lavoro abbiamo inizialmente effettuato un saggio di doppio ibrido per cercare dei putativi interattori della proteina che potessero avere un ruolo nella tumorigenicità del cancro al seno. Fra questi, abbiamo identificato Setdb1, una istone lisina metiltrasferasi, che interagisce specificamente con l’isoforma alfa troncata della proteina, ΔNp63α, interazione che contribuisce alla stabilità di p63. Setdb1 risulta essere amplificato nei tumori primari della mammella e la sua assenza rende le cellule tumorali meno prone alla crescita. Abbiamo inoltre trovato trenta geni che risultano aumentare dopo il silenziamento di Setdb1 o p63 (quindi geni che sono repressi da entrambe le proteine) e l’espressione di quattro di essi risulta essere positivamente correlata alla sopravvivenza dei pazienti di cancro al seno. Questi risultati suggeriscono che p63 e Setdb1 potrebbero agire come oncogeni in questo tipo di cancro andando a reprimere dei geni che si comportano da soppressori dei tumori.
ΔNp63α has been widely studied in breast cancer, and recent studies indicate that it is involved in both breast tumorigenesis and self-renewal potential of breast cancer stem cells. Although the p63 transcriptional profile has been extensively characterized, our knowledge about p63 binding partners, potentially involved in the regulation of breast tumor progression, is limited. Here, we performed the yeast-twohybrid approach to identify p63α interactors involved in breast tumorigenesis. We found that Setdb1, a histone lysine methyltransferase, interacts with ΔNp63α and that this interaction contributes to p63 protein stability. Setdb1 is often amplified in primary breast tumours and its depletion confers growth disadvantage to breast cancer cells. Also, we identified a list of thirty genes repressed by ΔNp63 in a Setdb1dependent manner and the expression of four of them is positively correlated to survival of breast cancer patients. These results suggest that p63 and Setdb1 may have diagnostic and prognostic potential via the repression of common target genes.

Aquesta tesi proposa el seguiment en profunditat d'un procés de disseny i d'implementació d'un artefacte interactiu. Explora l'agència de dissenyadors, usuaris i artefactes interactuant junts i configurant processos que van més enllà de la producció d'un objecte acabat. També mostrar com el disseny d'interactius no només és un disseny de tecnologia, sinó també de context d'ús i on els processos socials -entre humans i no humans, també- juguen un paper determinant. La visió del codisseny que es proposa obre una mirada crítica sobre com entenem actualment els processos de disseny i els agents implicats, qüestionant que el centre d'atenció es posi en l'experiència d'usuari sense tenir en compte l'experiència dels propis dissenyadors i el context d'ús. Metodològicament, aquesta tesi revela com quan el dissenyador experimenta amb diferents mètodes etnogràfics la seva forma de comprendre el món requereix no només observar i descriure, sinó també fer i donar forma a la pròpia tecnologia.
Esta tesis propone el seguimiento en profundidad de un proceso de diseño y de implementación de un artefacto interactivo. Explora la agencia de diseñadores, usuarios y artefactos interactuando juntos y configurando procesos que van más allá de la producción de un objeto terminado. También muesra cómo el diseño de interactivos no sólo es un diseño de tecnología, sino también de contexto de uso y donde los procesos sociales -entre humanos y no humanos, también- juegan un papel determinante. La visión del codiseño que se propone abre una mirada crítica sobre cómo entendemos actualmente los procesos de diseño y los agentes implicados, cuestionando que el centro de atención se ponga en la experiencia de usuario sin tener en cuenta la experiencia de los propios diseñadores y el contexto de uso. Metodológicamente, esta tesis revela que cuando el diseñador experimenta con diferentes métodos etnográficos su forma de comprender el mundo requiere no sólo observar y describir, sino también hacer y dar forma a la propia tecnología.
This Ph.D. thesis proposes to follow a design and implementation process of an interactive artifact. It explores the agency of designers, users and devices interacting together and forming processes that go beyond the production of a finished object. It also shows how the design of interactive artifacts is not only the development of a technology but also the configuration of a context of use t where social processes among humans and non-humans, play a decisive role. The proposed vision of co-design opens a critical view at how we currently understand design processes and stakeholders. I argue that in interactive design the experience of users include the experience of the designers and that the non-human components play also a significative role in how thee artifact becomes. Methodologically, this PhD dissertation reveals how the designer needs to make and shape technology, not only observe and describe, when he experiments with different ethnographic methods to understand the world.

Aquest estudi mostra els efectes d’una intervenció psicològica amb entrenadors de vela infantil- classe Optimist, que combina un programa d’assessorament en grup per millorar l’estil de comunicació i el clima motivacional (MAC) amb un programa d’assessorament personalitzat a cada entrenador (PAPE). Prèviament es realitza un estudi pilot per adaptar el Coaching Behaviour Assessment System (CBAS) com a instrument per avaluar els efectes del programa d’assessorament amb entrenadors d’Optimist. Els principals resultats indiquen que el disseny plantejat en aquest estudi permet analitzar de forma independent els canvis produïts per cada programa i permeten constatar que el programa de grup MAC és eficaç per millorar algunes conductes, especialment les respostes davant els errors i, en menor mesura, davant els encerts. Els resultats també indiquen que els efectes del programa personalitzat PAPE són superiors als induïts per MAC, i que el disseny permet ajustar la intervenció PAPE a les necessitats de cada entrenador en funció dels canvis induïts ja per MAC. Globalment, els resultats indiquen una clara millora de l’estil de comunicació de quatre dels cinc entrenadors d’Optimist participants i també mostren efectes favorables en les variables psicològiques dels regatistes, analitzades de forma complementària: motivació intrínseca, compromís, clima motivacional percebut pels regatistes, percepció de l’estil de comunicació del seu entrenador, i també mostren efectes en l’autopercepció conductual dels entrenadors.
This study shows the effects of a psychological intervention to children’s sailing coaches of optimist class. The intervention combines a group assessment programme to improve the communication style and the motivational climate (MAC) with a personalised coach intervention (PAPE). Previously, a pilot study has been carried out in order to adapt the Coaching Behaviour Assessment System (CBAS) as the tool to evaluate the effects of personal intervention programmes with children’s sailing coaches of the optimist class. The results indicate that the design of this this study allows an analysis of the effects of each programme independently (MAC and PAPE). It also enables us to confirm the efficacy of MAC on the coach communication style, especially taking into consideration adequate responses after mistakes and, to a lesser extent, after successes. Results also reveal that the effectiveness of the personalised intervention (PAPE) was more salient than MAC and indicate that this design allows suiting the personalised programme to the needs of each coach after the changes in the group intervention (MAC). Globally, the study results indicate that the effects of the psychological intervention were positive in four of the five coaches participating. Furthermore, in complementary data, it reflects positive effects on the psychological variables of the athletes: intrinsic motivation, commitment, motivational climate, perception of their coach behaviour, and also on the self-behavioural perception of the coaches.

The conserved human protein KANK1 has been identified as a tumour suppressor and its expression is down-regulated in several tumour types. Roles for this protein in actin regulation, cell migration and cell polarity have been documented in cultured mammalian cells. In C. elegans the KANK1 orthologue, VAB-19, is required for normal development as it helps stabilise attachment structures between muscle and epidermal cells. Despite these studies, the precise cellular role of KANK remains elusive. It was found that the Drosophila KANK orthologue binds directly to EB1, a crucial regulator of microtubule plus-end dynamics. I aimed to determine the role of KANK with respect to this indirect microtubule interaction using Drosophila. I identified residues which mediate the interaction between KANK and EB1, and showed they are essential for localisation of KANK to microtubule plus-ends in Drosophila culture cells. I found that KANK expression increases during embryogenesis and peaks in the late embryonic development when KANK is shown to localise to sites of attachment between muscle and epidermal cells. This suggests a role for the protein in stabilisation of muscle attachment during embryonic development, a process previously shown to require EB1. I generated a KANK deletion mutant and found they are viable and fertile but show a mild neuronal phenotype, specifically early branching of the neurons and less organised neuron bundles. My results suggest previously unknown roles for KANK in myogenesis and neurogenesis in Drosophila embryogenesis.

Résumé : Les maladies cardiovasculaires représentent la principale cause de mortalité mondiale, soit le tiers des décès annuels selon l’Organisation mondiale de la Santé. L’hypercholestérolémie, caractérisée par une élévation des niveaux plasmatiques de lipoprotéines de faible densité (LDL), est l’un des facteurs de risque majeur pour les maladies cardiovasculaires. La proprotéine convertase subtilisine/kexine type 9 (PCSK9) joue un rôle essentiel dans l’homéostasie du cholestérol sanguin par la régulation des niveaux protéiques du récepteur LDL (LDLR). PCSK9 est capable de se lier au LDLR et favorise l’internalisation et la dégradation du récepteur dans les lysosomes. L’inhibition de PCSK9 s’avère une cible thérapeutique validée pour le traitement de l’hypercholestérolémie et la prévention des maladies cardiovasculaires. Par contre, plusieurs mécanismes responsables de la régulation et la dégradation du complexe PCSK9-LDLR n’ont pas encore été complètement caractérisés comme la régulation par la protéine annexin A2 (AnxA2), un inhibiteur endogène de PCSK9. De plus, plusieurs évidences suggèrent la présence d’une ou plusieurs protéines, encore inconnues, impliquées dans le mécanisme d’action de PCSK9. Celles-ci pourraient réguler l’internalisation et le transport du complexe PCSK9-LDLR vers les lysosomes. Les objectifs de cette thèse sont de mieux définir le rôle et l’impact de l’AnxA2 sur la protéine PCSK9 en plus d’identifier de nouveaux partenaires d’interactions de PCSK9 pour mieux caractériser son mécanisme d’action sur la régulation des niveaux de LDLR. Nous avons démontré que l’inhibition de PCSK9 par l’AnxA2 extracellulaire s’effectue via sa liaison aux domaines M1+M2 de la région C-terminale de PCSK9 et nous avons mis en évidence les premières preuves d’un contrôle intracellulaire de l’AnxA2 sur la traduction de l’ARNm de PCSK9. Nos résultats révèlent une liaison de l’AnxA2 à l’ARN messager de PCSK9 qui cause une répression traductionnelle. Nous avons également identifié la protéine glypican-3 (GPC3) comme un nouveau partenaire d’interaction extracellulaire avec le PCSK9 et intracellulaire avec le complexe PCSK9-LDLR dans le réticulum endoplasmique des cellules HepG2 et Huh7. Nos études démontrent que GPC3 réduit l’activité extracellulaire de PCSK9 en agissant comme un compétiteur du LDLR pour la liaison avec PCSK9. Une meilleure compréhension des mécanismes de régulation et de dégradation du complexe PCKS9-LDLR permettra de mieux évaluer l’impact et l’efficacité des inhibiteurs de la protéine PCSK9.
Abstract : Cardiovascular disease is the leading cause of global mortality, responsible for one third of global deaths, according to the latest statistics from World Health Organization. Hypercholesterolemia, characterized by increased plasma low-density lipoprotein (LDL) cholesterol, is a major determinant of cardiovascular disease risk. Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a critical role in cholesterol homeostasis by regulating LDL receptor (LDLR) protein levels. PCSK9 binds to the LDLR and promotes its internalization and degradation in late endosomal/lysosomal compartments. Inhibition of PCSK9 action on LDLR has emerged as a novel therapeutic target for hypercholesterolemia and the prevention of cardiovascular disease. Annexin A2 (AnxA2) was reported as an endogenous extracellular inhibitor of PCSK9 activity upon cell-surface LDLR degradation and mechanisms of PCSK9’s regulation by AnxA2. However, its role on PCSK9 regulation still need better characterization in hepatocellular carcinoma cell lines. Moreover, many evidences suggest the presence of additional unknown interaction partners involve in the LDLR regulation and degradation mediated by PCSK9. These unknown partners could regulate the internalization and trafficking of the PCSK9-LDLR complex to lysosomes. The objectives of this thesis are to better define the role and impact of AnxA2 on PCSK9 and to identify novel PCSK9 interacting partners that participate and regulate the PCSK9-LDLR complex formation and degradation. We demonstrated that PCSK9 inhibition by extracellular AnxA2 occurs via its interaction with the M1+M2 modules of PCSK9’s C-terminal region. Most importantly, we revealed a new role of intracellular AnxA2 in the reduction of PCSK9 protein levels via a translational mechanism. Our results suggest a translational repression from the binding of AnxA2 to PCSK9’s mRNA. Also, we successfully identified a novel and functional interaction between glypican-3 (GPC3) and PCSK9. We demonstrated the extracellular GPC3 interaction with PCSK9 and the intracellular GPC3 with both PCSK9 and LDLR in human hepatocellular carcinoma cell lines HepG2 and Huh7. Our studies revealed that extracellular GPC3 can act as an endogenous competitive binding partner of PCSK9 to the LDLR, and hence reducing its activity towards LDLR degradation. The continued understanding of PCSK9 interactions is critical, from a mechanistic point of view as well as from the optimization of therapeutic interventions.

OmiHtrA2 is a highly conserved mammalian serine protease that belongs to the HtrA family of proteins. Omi shares homology with the bacterially expressed heat shock protease HtrA, which functions as a protease at higher temperatures and a chaperone at lower temperatures. Additionally, Omi shares sequence similarity with the mammalian homologs L56/HtrA1 and PRSP/HtrA3. Omi was first isolated as an interacting protein of Mxi2, an alternatively spliced form of the p38 stress-activated kinase, using a modified yeast two-hybrid system. Omi localizes in the mitochondria and in response to apoptotic stimuli the mature form of this protein translocates to the cytoplasm. In the cytoplasm Omi participates in both the caspase-dependent as well as caspase-independent apoptosis. Additionally, recent studies suggest that Omi may have another unique function, maintaining homeostasis within the mitochondria. In an effort to further elucidate the function of Omi, a yeast two-hybrid screening was performed to isolate novel interacting proteins. This screening identified a novel protein (HOPS), as a specific interactor of Omi. The predicted amino acid sequence of this protein does not provide any information about its potential function in mammalian cells. However, experiments show that HOPS is cleaved in vitro by Omi. Furthermore, in response to apoptotic stimuli, HOPS is also degraded in vivo. This study suggests that HOPS could be a physiological substrate of Omi that is cleaved and removed during apoptosis.
M.S.
Department of Molecular Biology and Microbiology
Health and Public Affairs
Molecular Biology and Microbiology

Les papillomavirus humains (PVH) sont des virus dont certains génotypes sont responsables de cancers dans la population générale. Leur pathogénicité et leur oncogénicité reposent principalement sur les propriétés biologiques des protéines virales E6 et E7. L’objectif de notre thèse a été de rechercher les partenaires cellulaires de la protéine E7 de 11 PVH à tropisme cutané ou muqueux, oncogène ou non-oncogène par criblage double hybride chez la levure et de corréler la fonction de ces partenaires avec la pathogénicité et l’oncogénicité des PVH. Par comparaison des profils d’interactions, une analyse des oncoprotéines de ces génotypes nous a permis d’identifier des protéines cellulaires spécifiquement ciblées par les génotypes muqueux ou cutanés. Nous avons montré une corrélation étroite entre la phylogénie et l’interactomique. Ce travail a permis de discriminer les PVH grâce à trois équations booléennes basées sur sept marqueurs. Ces données pourraient avoir une valeur prédictive pour déterminer la pathogénie et l’oncogénicité d’un PVH dont on ignore les propriétés biologiques. Enfin, nos études ont permis d’identifier des interactions qui pourraient être des cibles pour des drogues capables d’éradiquer les infections par les PVH.

The progressive appearance of affordable tabletop technology and devices urges humancomputer interaction researchers to provide the necessary methods to make this kind of devices the most useful to their users. Studies show that tabletops have distinctive characteristics that can be specially useful to solve some types of problems, but this potential is arguably not yet translated into real-world applications. We theorize that the important components that can transform those systems into useful tools are application frameworks that take into account the devices affordances, a third party application ecosystem, and multi-application systems supporting concurrent multitasking. In this dissertation we approach these key components: First, we explore the distinctive affordances of tabletops, with two cases: TurTan, a tangible programming language in the education context, and SongExplorer, a music collection browser for large databases. Next, in order to address the difficulty of building such applications in a way that they can exploit these affordances, we focus on software frameworks to support the tabletop application making process, with two different approaches: ofxTableGestures, targeting programmers, and MTCF, designed for music and sound artists. Finally, recognizing that making useful applications is just one part of the problem, we focus on a fundamental issue of multi-application tabletop systems: the difficulty to support multi-user concurrent multitasking with third-party applications. After analyzing the possible approaches, we present GestureAgents, a content-based distributed application-centric disambiguation mechanism and its implementation, which solves this problem in a generic fashion, being also useful to other shareable interfaces, including uncoupled ones.
L’aparició progressiva de tecnologia i dispositius tabletop barats urgeix a la comunitat de recerca en interacció persona-ordinador a proveir els mètodes necessaris per transformar aquests dispositius en eines realment útils pels usuaris. Diversos estudis indiquen que els tabletops tenen algunes característiques peculiars que poden ser especialment útils per solucionar algun tipus de problemes, però tanmateix sembla que el seu potencial encara no arriba a transformar-se en aplicacions reals. Creiem que els components importants per a transformar aquests sistemes en eines útils són frameworks d’aplicació que tinguin en compte les capacitats dels dispositius, un ecosistema d’aplicacions fetes per desenvolupadors independents, i sistemes multi-aplicació amb suport per multitasca concurrent. En aquesta tesi doctoral ens aproximem a aquests components clau: En primer lloc, explorem les capacitats dels tabletops, usant dos casos: TurTan, un llenguatge de programació tangible en el context educatiu, i SongExplorer, un navegador de col·leccions musicals per a grans bases de dades. A continuació, amb ànim d’abordar la complexitat a l’hora de crear aquest tipus d’aplicacions de tal manera que usin aquestes capacitats, ens centrem en els frameworks de programari per donar suport en el procés de creació d’aplicacions tabletop, amb dues aproximacions diferents: ofxTableGestures, dirigit a programadors, i MTCF, dissenyat per a artistes musicals i del so. Finalment, reconeixent que crear aplicacions útils és només part del problema, ens centrem en una questió fonamental dels sistemes multi-aplicació: la dificultat d’acceptar la interacció multitasca de forma concurrent i multi-usuari amb aplicacions externes. Després d’analitzar-ne les possibles aproximacions, presentem GestureAgents, un mecanisme de desambiguació (i la seva implementació corresponent) basat en el contingut, distribuït i centrat en les aplicacions, que soluciona aquest problema d’una forma genèrica, esdevenint útil també per altres interfícies compartibles, incloses les desacoblades.

The cytokine TRAIL is a promising cancer therapeutic candidate as it induces apoptosis selectively in transformed cells. TRAIL-induced clustering of its receptors (DR) is essential for the DISC complex formation, which induces cell death. The mechanism for TRAIL's tumour selective effect is largely unknown. We identified the cytoskeleton proteins non-muscle myosin heavy chain IIa, IIb (NMHCIIa, NMHCIIb), myosin regulatory light chain (MLC2) and ß-actin as novel DR-interactors. An initially weak and TRAIL-induced abrogation of NMHCII/DR interaction correlated with efficient DISC formation in tumour cells. In contrast, a robust NMHCII/DR interaction that was sustained upon TRAIL stimulus was accompanied by incomplete DISC arrangement. Weakening the NMHCII/DR interaction in normal cells using chemical inhibitors enhanced TRAIL-induced apoptosis. Intriguingly, siRNA-mediated NMHCIIa- but not NMHCIIb depletion potently released TRAIL resistance in normal cells and influenced DISC composition. Reduced NMHCII/DR interaction in transformed cells was characterised by diminished MLC2 phosphorylation and altered protein expression of upstream regulatory kinases. Our results suggest that normal cell resistance to TRAIL-apoptosis is based on the interaction of cytoskeleton components with DR that is impaired upon transformation. Since NMHCII function in cell adhesion and migration, it will be interesting to study possible roles of the interaction in cell detachment and altered TRAIL sensitivity; moreover this link may provide clues as to the cause of TRAIL resistance in some cancers.

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CNPQ – Conselho Nacional de Desenvolvimento Científico e Tecnológico
Os ambientes de Ensino a Distancia (EAD) estao cada vez mais presentes no dia-a-dia de professores e alunos, seja no formato exclusivamente a distancia (e-learning) ou como uma ferramenta de apoio ao ensino presencial (b-learning). Apesar do aumento de sua utilizacao, o uso dessas ferramentas nao deve representar somente um meio digital de armazenamento e compartilhamento de dados. Elas tambem devem ser uteis tanto para o aluno quanto para o professor no processo de mediacao do aprendizado. Neste contexto, o monitoramento das atividades nessas ferramentas e de grande importancia para tornar o seu uso mais eficiente. Apenas coletar e sumarizar os registros das atividades dos alunos de forma quantitativa – numero de acesso, por exemplo – para posteriormente condensa-los em relatorios, pode nao fornecer informacoes suficientes para justificar o uso desses ambientes. E importante tambem avaliar questoes qualitativas, identificando caracteristicas que possibilitem personalizar o ensino. Outro problema encontrado e que para cada um desses ambientes existentes, os registros das atividades sao armazenados numa estrutura e num formato proprios de cada software. Isso, por sua vez, acaba por dificultar a criacao de ferramentas padronizadas de analise. Nos trabalhos relacionados apresentados no capitulo 3 e possivel verificar que, apesar de utilizar a mesma base teorica (teoria socio-historica) para fundamentar as interações educacionais, todos os trabalhos analisam de forma qualitativa somente ferramentas de comunicacao, tais como forum chat, mensagens, e-mail, ignorando o restante dos recursos existentes nos AVAs. Entretanto, as interacoes educacionais tambem ocorrem atraves de outras tecnologias, por exemplo: ao disponibilizar um arquivo pdf ou uma pagina com conteudo para o aluno, o professor estara interagindo com esse estudante, mesmo que de forma assincrona. Outro exemplo pode ser representado por uma atividade, que ao ser entregue pelo aluno, podera receber um feedback do professor. Ainda com relacao ao processo de mediacao online, outro ponto que foi considerado, e que em ambientes online alguns alunos talvez nao utilizem as ferramentas de comunicacao, ou as utilizem em pouca frequencia, mas ao mesmo tempo esse mesmo apresenta boas notas e acessa os recursos disponibilizados pelo professor. Neste caso, o acompanhamento do processo de mediacao por parte do professor fica comprometida se ele visualizar somente registros de ferramentas de comunicacao. No entanto, ao visualizar as interacoes do aluno no restante dos recursos o professor podera ter um panorama mais completo sobre o estado do aluno. Com base no que foi citado acima, para o presente trabalho serao utilizadas como base teorica a Abordagem Socio-historica e a Teoria dos Atos da Fala. Uma vez fundamentado o processo de interacao com base na primeira teoria, sera apresentado um modelo de classificacao dos atos ilocucionarios para as interacoes dos usuarios em ambientes de EAD, ou seja, sera identifiada a intencionalidade de uma acao realizada pelo usuario. Para isso, o modelo fara o uso de tecnlogias da Web Semantica, padroes de registros educacionais e rede bayesiana para classificacao dos atos da fala. Sendo que ao final do trabalhos são apresentados os resultados dos experimentos realizados para tal modelo. Alem disso, para trabalhos futuros projeta-se a utilizacao do presente modelo na identificacao da categoria de mediacao que o aluno encontra-se, isso sera realizado partindo-se do pressuposto de que os atos da fala classificados no presente trabalho sirvam de base para tal proposta (o que e justificado no capitulo 4.1 “Analise comparativa entre a TAF e a TSH”). O texto inicialmente aborda a Teoria Socio-historica, a Teoria dos Atos da Fala, Ambientes Virtuais de Aprendizagem e Registros Padronizados de Interacao, assim como a Web Semantica e Redes Bayesianas. No capitulo seguinte sao vistos os trabalho que fazem uso da Teoria Socio-historica e sua relacao ao processo de mediacao nos ambientes virtuais de ensino. No capitulo 4 e apresentado o Modelo de Interacoes Pedagogicas assim como informacoes sobre o prototipo desenvolvido. O capitulo 5 descreve os experimentos e os resultados alcancados. O trabalho e finalizado com a apresentacao das consideracoes finais a proposta de trabalhos futuros.
The Distance Learning environments (EAD) are becoming more present in the daily lives of teachers and students, either solely in the distance format (e-learning) or as a support tool to presencial teaching (b-learning). Despite the increase in its use, the use of these tools should not only represent a digital storage mean and data sharing. They should also be useful for both the student and the teacher in the learning mediation process. In this context, monitoring of activities in these tools is very important to make its use more efficient. Just collecting and summarizing the logs of the student's activities in a quantitative manner - e.g access number - to further condense them in reports, may not provide enough information to justify the use of these environments. It is also important to assess qualitative issues, by identifying characteristics that enable personalized learning. Another problem found is that for each of these existing environments, the activities logs are stored in their own format and structure of each software. This, in turn, makes it difficult to create standardized analysis tools. In the papers related described in Chapter 3 it is possible to check that, despite using the same theoretical base (socio-historical theory) to support educational interactions, all works analyze qualitatively only communication tools such as forum chats, messages, e-mail, bypassing the rest of the existing resources in AVAs. However, educational interactions also occur through other technologies; for example by providing a PDF file or page content to the student, the teacher will be interacting with this student, even asynchronously. Another example can be represented by an activity which when delivered by the student he may receive feedback from the teacher. Still regarding the online mediation process, another point that was considered is that in online environments, some students may not use the communication tools, or use it in low frequency, but at the same time, it presents good grades and access features made available by the teacher. In this case, monitoring of the mediation process by the teacher is compromised if he sees only records of communication tools. However, by seeing the interactions of students in the rest of the resources the teacher can have a more complete overview on the state of the student. Based on what was mentioned above, the present study will use as a theoretical basis the socio-historical approach and the Theory of Speech Acts. Once the interaction process is grounded based on the first theory, it will be presented a classification model of the illocutionary acts to the interactions of the users in EAD environments, in other words, it will be identified the intentionality of an action performed by the user. For this, the model will make use of Web Semantic technologies, educational standards of records and Bayesian network for classification of speech acts. At the end of the study, it will be presented the results of the experiments performed to such model. Also, for future work it is foreseen the use of this model to identify the category of mediation that the student is found; this will be done starting from the assumption that speech acts classified in this study is used as a basis for such proposal (which is explained in Chapter 4.1 "Comparative analysis between TAF and the TSH"). The text initially addresses the socio-historical theory, the Theory of Speech Acts, Virtual Learning Environments and Interaction of Standardized Records, as well as, the Web Semantic and Bayesian networks. In the next chapter are seen the work that makes use of socio-historical theory and its relation to the mediation process in virtual learning environments. In Chapter 4, it is presented the Pedagogical Interaction Model as well as information on the developed prototype. The paper ends with the presentation of the final considerations on the proposal for future work.

Women of all backgrounds have contributed to the environmental history of the United States, but most of the environmental historical scholarship places such women alongside men and by doing so clouds their involvement as well as their achievements. This discussion introduces readers to pieces of environmental history that engage gender as a framework, while also acknowledging that there is not an individual women’s environmental experience by covering specific yet contrasting geographical spaces. The American West and the New York Adirondacks offer diverse perspectives and experiences of pioneering women who interacted with the environment, including Diné women, park rangers, Adirondack guides and residents, nature lovers, conservationists, and more. This research unearths the stories and experiences of these women, creating a more balanced and fuller image of the ways in which humans interacted with nature, while shining a light on the undervalued narratives of the frequently uplifting and consistently complex history of American women in relation to the environment.

The transcription/DNA repair factor IIH (TFIIH) is made of two distinct sub-complexes, the core-TFIIH and the CDK-activating kinase (CAK), bridged together by the XPD subunit. The CDK7 kinase subunit of the CAK sub-complex targets different substrates according to the protein complex it belongs to: as free-CAK it phosphorylates specific cyclin-dependent kinases (Cdks) and promotes cell cycle progression; as subunit of the entire TFIIH (holo-TFIIH), CDK7 phosphorylates and activates RNA polymerase II and specific transcription factors. In this context, the bridging factor XPD plays a key role in modulating the association/dissociation between the CAK and the core-TFIIH, thus linking transcription to cell cycle control and DNA repair. Mutations in the ERCC2/XPD gene are responsible for distinct clinical entities, including the cancer-prone xeroderma pigmentosum (XP) and the multisystemic cancer-free trichothiodystrophy (TTD). To understand how mutations in the same gene give rise to hereditary disorders with opposite cancer proneness, we investigated whether XPD mutations affect the cellular composition of TFIIH complex and, in turn, the CAK-mediated signaling pathways. Therefore, by native chromatin immunoprecipitation studies, we investigated the association/dissociation dynamic of the two TFIIH sub-complexes throughout the cell cycle and found that in XPD mutated fibroblasts, isolated from either XP or TTD patients, most of the CAK sub-complex dissociates from the chromatin and from the core-TFIIH. Considering that the CAK substrate specificity depends on it free or core-bound state, we looked at the protein interaction profile of CDK7 kinase subunit both in physiological and pathological conditions. The CDK7 immunoprecipitation followed by mass spectrometry analysis allowed the identification of new CDK7 interactors either associated or detached from the chromatin. Some of the identified proteins display a different interaction profile in TTD and/or XP cells, compared to control fibroblasts. We focused our attention on DDX1, an ATP-dependent RNA helicase that is involved in the resolution of RNA/DNA hybrids accumulated at DNA double strand breaks. We found that the interaction between DDX1 and the chromatin-bound CDK7 is stronger in TTD primary and immortalized fibroblasts than in XP or control fibroblasts. To gain knowledge on the functionality of the identified interaction we performed various in vitro assays. Upon purification of DDX1 from E. coli and XPD, CAK and TFIIH complex from Baculovirus and in vitro co-immunoprecipitation experiments, we established that DDX1 binds not only the CAK sub-complex but also the XPD subunit and the core-TFIIH. Moreover, the presence of XPD stabilizes the DDX1 interaction with the CAK but not with the core-TFIIH. We found that the C-terminal region of XPD is essential for DDX1 binding and that TTD- and XP-specific point mutations mapping in the C-terminus of XPD do not affect their interaction in vitro. Finally, by in vitro kinase assay we assessed that DDX1 is a substrate of the free-CAK but not of the holo-TFIIH complex. Future studies will clarify the functional relevance of the CAK-XPD interaction with DDX1. Overall, our results point to an altered association of the CAK to the chromatin as consequence of XPD mutations and identify DDX1 as a novel interactor of CDK7, showing a different binding profile in TTD compared to control and XP fibroblasts.

The glutamatergic synapse is involved in the modulation of higher brain functions, such as learning and memory processes. The activation of specific subtypes of N-methyl-D-aspartate-type glutamate receptors (NMDARs) located at synapses accounts for different electrophysiological properties of neurotransmission and consequent synaptic plasticity mechanisms. NMDAR subunit composition is relevant for physiological neuronal functions and is often altered in many neurological disorders. In particular, augmented levels of synaptic NMDARs containing the GluN2A subunit can be observed after potentiation of neurotransmission during Long-Term Potentiation (LTP). Several studies revealed that LTP at hippocampal synapses underlies encoding and consolidation of memory. Rabphilin-3A (Rph3A) was recently characterized as a specific GluN2A intracellular binding partner promoting stabilization of GluN2A containing NMDARs at postsynapses through a trimeric complex with the main postsynaptic scaffolding protein PSD-95. Silencing of Rph3A or disruption of Rph3A/GluN2A/PSD-95 interactions leads to reduction in GluN2A-containing NMDARs due to receptor endocytosis. The modulation of this complex in the striatum of parkinsonian animals showing a dyskinetic behavior was able to restore normal locomotive behavior, indicating Rph3A could represent a new pharmacological target in brain diseases. However, the role of Rph3A in hippocampal functions such as synaptic plasticity and learning and memory has not yet been elucidated. In the present PhD project we aimed to investigate the role of Rph3A/GluN2A/PSD-95 complex in these events applying different stimulation protocols both in vitro and in vivo. In addition, we investigated the possible interplay between these synaptic complexes and the synapse-to-nucleus messenger Ring Finger Protein 10 (RNF10). We discovered that Rph3A is present in almost 50% of hippocampal dendritic spines at resting conditions. However, after potentiation of neurotransmission the number of Rph3A positive spines increases, paralleled by augmented formation of Rph3A/GluN2A/PSD-95 trimeric complex. Interference with Rph3A/GluN2A interaction through different experimental approaches leads to failure in LTP induction both at molecular and morphological levels, impairing also hippocampal dependent spatial learning. Dendritic spines displaying Rph3A show higher maturation degree in morphological parameters and recruit more newly-synthetized proteins compared to Rph3A negative ones, suggesting that Rph3A positive spines represent more stable and mature neuronal connections. Furthermore, the molecular motor transporter Myosin-VA was previously described as a binding partner of Rph3A and involved in GluA1-containing AMPA receptors delivery to synapses during synaptic potentiation. Interestingly, a trimeric complex composed by Rph3A/MyoVA/GluA1 is detected in hippocampus and their interaction is increased after cLTP application. Finally, our results show that impairing the synapse-to-nucleus transport of RNF10 during chemical-LTP is detrimental for Rph3A synaptic localization, indicating the integration of synapse-to-nucleus signals during synaptic plasticity probably impacts on Rph3A postsynaptic functions. In conclusion, our results suggest that Rph3A postsynaptic role is not only given by the stabilization of GluN2A containing NMDARs accounting for adequate LTP induction and hippocampal learning process, but also for modulation of synaptic responsiveness through the delivery of GluA1-containing AMPA receptors during synaptic potentiation.

Els sistemes de conversió de text en parla (CTP-SU) s'encarreguen de produir veu sintètica a partir d'un text d'entrada. Els CTP basats en selecció d'unitats (CTP-SU) recuperen la millor seqüència d'unitats de veu enregistrades prèviament en una base de dades (corpus). La recuperació es realitza mitjançant algorismes de programació dinàmica i una funció de cost ponderada. La ponderació de la funció de cost es realitza típicament de forma manual per part d'un expert. No obstant, l'ajust manual resulta costós des d'un punt de vista de coneixement prèvi, i imprecís en la seva execució. Per tal d'ajustar els pesos de la funció de cost, aquesta tesi parteix de la prova de viabilitat d'ajust perceptiu presentada per Alías (2006) que empra algorismes genètics interactius actius (active interactive Genetic Algorithm - aiGA). Aquesta tesi doctoral investiga les diferents problemàtiques que es presenten en aplicar els aiGAs en l'ajust de pesos d'un CTP-SU en un context real de selecció d'unitats. Primerament la tesi realitza un estudi de l'estat de l'art en l'ajust de pesos. Tot seguit, repassa la idoneïtat de la computació evolutiva interactiva per realitzar l'ajust revisant amb profunditat el treball previ. Llavors es presenten i es validen les propostes de millora. Les quatre línies mestres que guien les contribucions d'aquesta tesi són: la precisió en l'ajust dels pesos, la robustesa dels pesos obtinguts, l'aplicabilitat de la metodologia per qualsevol funció de cost i el consens dels pesos obtinguts incorporant el criteri de diferents usuaris. En termes de precisió la tesi proposa realitzar l'ajust perceptiu per diferents tipus (clústers) d'unitats respectant les seves peculiaritats fonètiques i contextuals. En termes de robustesa la tesi incorpora diferents mètriques evolutives (indicadors) que avaluen aspectes com l'ambigüitat en la cerca, la convergència d'un usuari o el nivell de consens entre diferents usuaris. Posteriorment, per estudiar l'aplicabilitat de la metodologia proposada s'ajusten perceptivament diferents pesos que combinen informació lingüística i simbòlica. La última contribució d'aquesta tesi estudia l'idoneïtat dels models latents per modelar les preferències dels diferents usuaris i obtenir una solució de consens. Paral•lelament, per fer el pas d'una prova de viabilitat a un entorn real de selecció d'unitats es treballa amb un corpus d'extensió mitjana (1.9h) etiquetat automàticament. La tesi permet concloure que l'aiGA a nivell de clúster és una metodologia altament competitiva respecte les altres tècniques d'ajust presents en l'estat de l'art.
Los sistemas de conversión texto-habla (CTH-SU) se encargan de producir voz sintética a partir de un texto de entrada. Los CTH basados en selección de unidades (CTH-SU) recuperan la mejor secuencia de unidades de voz grabadas previamente en una base de datos (corpus). La recuperación se realitza mediante algoritmos de programación dinámica y una función de coste ponderada. La ponderación de la función de coste se realiza típicamente de forma manual por parte de un experto. Sin embargo, el ajuste manual resulta costoso desde un punto de vista de conocimiento previo e impreciso en su ejecución. Para ajustar los pesos de la función de coste, esta tesis parte de la prueba de viabilidad de ajuste perceptivo presentada por Alías (2006) que emplea algoritmos genéticos interactivos activos (active interactive Genetic Algorithm - aiGA). Esta tesis doctoral investiga las diferentes problemáticas que se presentan al aplicar los aiGAs en el ajuste de pesos de un CTH-SU en un contexto real de selección de unidades. Primeramente la tesis realiza un estudio del estado del arte en el ajuste de pesos, posteriormente repasa la idoneidad de la computación evolutiva interactiva para realizar el ajuste revisando en profundidad el trabajo previo. Entonces se presentan y se validan las propuestas de mejora. Las cuatro líneas maestras que guían las contribuciones de esta tesis son: la precisión en el ajuste de los pesos, la robustez de los pesos obtenidos, la aplicabilidad de la metodología para cualquier función de coste y el consenso de los pesos obtenidos incorporando el criterio de diferentes usuarios. En términos de precisión la tesis propone realizar el ajuste perceptivo por diferentes tipos (clusters) de unidades respetando sus peculiaridades fonéticas y contextuales. En términos de robustez la tesis incorpora diferentes métricas evolutivas (indicadores) que evalúan aspectos como la ambigüedad en la búsqueda, la convergencia de un usuario o el nivel de consenso entre diferentes usuarios. Posteriormente, para estudiar la aplicabilidad de la metodología propuesta se ajustan perceptivamente diferentes pesos que combinan información lingüística y simbólica. La última contribución de esta tesis estudia la idoneidad de los modelos latentes para modelar las preferencias de los diferentes usuarios y obtener una solución de consenso. Paralelamente, para dar el paso de una prueba de viabilidad a un entorno real de selección de unidades se trabaja con un corpus de extensión media (1.9h) etiquetado automáticamente. La tesis permite concluir que el aiGA a nivel de cluster es una metodología altamente competitiva respecto a las otras técnicas de ajuste presentes en el estado del arte.
Text-to-Speech Systems (TTS) produce synthetic speech from an input text. Unit Selection TTS (US-TTS) systems are based on the retrieval of the best sequence of recorded speech units previously recorded into a database (corpus). The retrieval is done by means of dynamic programming algorithm and a weighted cost function. An expert typically performs the weighting of the cost function by hand. However, hand tuning is costly from a standpoint of previous training and inaccurate in terms of methodology. In order to properly tune the weights of the cost function, this thesis continues the perceptual tuning proposal submitted by Alías(2006) which uses active interactive Genetic Algorithms (aiGAs). This thesis conducts an investigation to the various problems that arise in applying aiGAs to the weight tuning of the cost function. Firstly, the thesis makes a deep revision to the state-of-the-art in weight tuning. Afterwards, the thesis outlines the suitability of Interactive Evolutionary Computation (IEC) to perform the weight tuning making a thorough review of previous work. Then, the proposals of improvement are presented. The four major guidelines pursued by this thesis are: accuracy in adjusting the weights, robustness of the weights obtained, the applicability of the methodology to any subcost distance and the consensus of weights obtained by different users. In terms of precision cluster-level perceptual tuning is proposed in order to obtain weights for different types (clusters) of units considering their phonetic and contextual properties. In terms of robustness of the evolutionary process, the thesis presents different metrics (indicators) to assess aspects such as the ambiguity within the evolutionary search, the convergence of one user or the level of consensus among different users. Subsequently, to study the applicability of the proposed methodology different weights are perceptually tuned combining linguistic and symbolic information. The last contribution of this thesis examines the suitability of latent models for modeling the preferences of different users and obtains a consensus solution. In addition, the experimentation is carried out through a medium size corpus (1.9h) automatically labelled in order fill the gap between the proof-of-principle and a real unit selection scenario. The thesis concludes that aiGAs are highly competitive in comparison to other weight tuning techniques from the state-of-the-art.

Hhex transcription factor is expressed in multiple endoderm-derived tissues, like the liver, where it is essential for proper development. The pleiotropic effect of Hhex in the embryo and its dual role as a transcriptional repressor/activator suggest the presence of different interaction partners capable of modulating its activity and function. In the current study we identified two new Hhex protein interactors: SOX13 and c-Myc. We show that Hhex interacts directly with SOX13. By doing so, Hhex sequesters SOX13 from the SOX13•TCF1 complex, overturning SOX13-dependent repression of the Wnt pathway. On the other hand, Hhex induces proliferation of non-tumorigenic human fibroblast through a Myc-dependent mechanism. Hhex and c-Myc interact directly upregulating Cyclin D1, a c-Myc target gene involved in cell cycle progression and proliferation. Elevation of Cyclin D1 might be the final effector of Hhex capacity to regulate cell proliferation.

Les modifications post-traductionelles des histones contribuent à l’expression génique et à la stabilité du génome. La méthylation de la lysine 4 de l’histone H3 (H3K4me), une marque associée aux promoteurs de gènes transcrits, est déposé par les methyltransferases hautement conservées de la famille SET1, dans le contexte du complexe COMPASS. SET-2, l’homologue de SET1 chez Caenorhabditis elegans, est responsable de la déposition de H3K4me dans la lignée germinale, et son inactivation provoque une perte progressive de la fertilité. Le but de mon travail de thèse a été d’étudier comment SET-2 et la méthylation de H3K4 contribuent au maintien de la lignée germinale. J’ai montré que l’absence de SET-2 provoque une sensibilité accrue aux dommages à l’ADN. Cependant, les voies de signalisation et de réparation de ces dommages sont fonctionnelles dans le mutant set-2. Par séquençage de l’ADN, j’ai par ailleurs montré que la stérilité progressive observée en l’absence de set-2 n’est pas due à une capacité de réparation réduite. L’ensemble de mes résultats suggère que H3K4me pourrait agir en aval de la signalisation de dommages à l’ADN, en influençant l’organisation de la chromatine aux sites des cassures double brin. J’ai d’autre part mis en évidence une nouvelle fonction pour la méthylation de H3K4 dans l’organisation de la chromatine en montrant que set-2 interagit génétiquement avec le complexe Condensine II et la Topoisomérase II, facteurs clefs de l’organisation mitotique des chromosomes. Des expériences de microscopie par FLIM-FRET ont d’ailleurs validé une fonction de H3K4 méthylée dans l’organisation de la chromatine dans la lignée germinale. Enfin, j’ai montré par analyses transcriptomiques que la protéine CFP-1 du complexe COMPASS est impliquée dans la régulation du programme transcriptionnel de la lignée germinale et que cette fonction est indépendante de SET-2. L’ensemble de mes résultats montre comment la régulation chromatinienne impacte le maintien d’une lignée germinale fonctionnelle à plusieurs niveaux
Post-translational modifications of histones contribute to gene expression and genome stability. Methylation of lysine 4 of histone H3 (H3K4me), a mark associated with actively transcribed genes, is deposited by the highly conserved SET1 family methyltransferases acting in COMPASS related complexes. SET-2, the SET1 homologue in Caenorhabditis elegans, is responsible for the deposition of H3K4me in the germ line, and its inactivation causes progressive loss of fertility. The purpose of my PhD work was to study how SET-2 and the methylation of H3K4 contribute to the maintenance of the germ line. I have shown that the absence of SET-2 causes increased sensitivity to DNA damage. However, the DNA damage-induced signaling and repair pathways are functional in the set-2 mutant. By DNA sequencing, I have also shown that the progressive sterility observed in the absence of set-2 is not due to a reduced repair capacity. Together, my results suggest that H3K4 methylation may act downstream of DNA damage signaling, potentially by influencing the organization of chromatin at the sites of double-strand breaks. I have also described a new function for H3K4 methylation in the organization of chromatin by showing that set-2 genetically interacts with the Condensitin II complex and Topoisomerase II, key factors in mitotic chromosome organization. Moreover, FLIM-FRET microscopy experiments have validated a role for H3K4 methylation in germline chromatin organization. Finally, using transcriptomic analyses, I have described a function for CFP-1, a component of the COMPASS complex, in the regulation of the germline transcriptional program independent of SET-2. Altogether, my results show how chromatin regulation affects the maintenance of a functional germline through multiple mechanisms

Spinal Muscular Atrophy (SMA) is a neuronal degenerative disease caused by the mutation or loss of the Survival Motor Neuron (SMN) gene. The cause for the specific motor neuron susceptibility in SMA has not been identified. The high axonal transport/localization demand on motor neurons may be one potentially disrupted function, more specific to these cells. We therefore used a large-scale immunoprecipitation (IP) experiment, to identify potential interactors of SMN involved in neuronal transport and localization of mRNA targets. We identified KH-type splicing regulatory protein (KSRP), a multifunctional RNA-binding protein that has been implicated in transcriptional regulation, neuro-specific alternative splicing, and mRNA decay. KSRP is closely related to chick zipcode-binding protein 2 and rat MARTA1, proteins involved in neuronal transport/localization of beta-actin and microtubule-associated protein 2 mRNAs, respectively. We demonstrated that KSRP is arginine methylated, a novel SMN interactor (specifically with the SMN Tudor domain; and not with SMA causing mutants). We also found this protein to be misregulated in the absence of SMN, resulting in increased mRNA stability of KSRP mRNA target, p21cip/waf1. A role for SMN as an axonal chaperone of methylated RBPs could thus be key in SMA pathophysiology.

This dissertation constitutes a quest for an approach to describing language use capable of integrating the findings of disciplines such as linguistics, anthropology, sociology and philosophy within a pedagogical framework based on a modular concept of communicative competence which involves four areas of knowledge and skills: linguistic, sociolinguistic, discourse and strategic.

La recherche des marqueurs biologiques de la tendreté a fait l’objet de nombreux travaux chez les animaux producteurs de viande et en particulier les bovins. A l’issue de ces études, une expression différentielle de la protéine Hsp27 entre des groupes de tendreté extrême a été mise en évidence. Cette protéine est présente à un « carrefour » biologique de l’interactome lié à la tendreté. Comprendre les mécanismes d’action de la protéine Hsp27 dans la tendreté de la viande bovine est l'un des défis de recherche dans le domaine de la production de viande. Dans cette optique, mon travail de thèse (2010-2013) avait pour objectif d’analyser le rôle de Hsp27 dans le développement du muscle et son implication dans le déterminisme des caractéristiques des tissus liés à la qualité de la viande. La première étape de ce travail a consisté à produire un modèle de souris présentant une inactivation du gène de la protéine Hsp27 (KO HspB1) et d’analyser leur phénotype comparativement à des témoins. Les souris KO HspB1 sont viables, fertiles et ne présentent aucune anomalie majeure, mais ont un format plus petit que celui de leurs témoins. L’analyse de leurs caractéristiques musculaires par une technique immunohistoligique mise au point spécifiquement (Publication 1) n’a pas révélé de différences. Au niveau ultrastructural, l'observation du muscle des souris par microscopie électronique à transmission a révélé des différences ultrastructurales entre les deux génotypes à T0 post-mortem avec des écarts entre les myofibrilles très espacées chez les souris KO HspB1 et un appareil contractile musculaire moins organisé. Ces différences sont encore plus marquées à T72 heures post-mortem. Ainsi le phénotype musculaire fin des souris KO HspB1 est plus altéré (Publication 2). Une analyse bio-informatique a été réalisée dans l'objectif de compléter la liste des interacteurs de la protéine Hsp27 et des gènes cibles de l’invalidation d’HspB1 susceptibles de participer à des différences de structure du muscle et de la tendreté. Les partenaires ou cibles prédits de Hsp27 sont des protéines impliquées dans différentes fonctions, comme des Heat shock proteines, des régulateurs de l'apoptose, des facteurs de traduction, des protéines du cytosquelette et des antioxydants. Les abondances de 15 protéines ont été quantifiées par Western-bloting dans deux muscles (m. Soleus, m. Tibialis). Elles sont modifiées chez les souris dépourvues de Hsp27 principalement dans le muscle le plus oxydatif. Cette étude démontre l'existence de liens fonctionnels entre Hsp27 et ses cibles prédites qui pourraient participer au phénotype fin des souris (Publication 3). Pour compléter cette étude, une analyse protéomique du muscle Tibialis anterior a été menée en utilisant la technique d’électrophorèse bidimensionnelle couplée à la spectrométrie de masse. La comparaison des protéomes spécifiques de ces deux génotypes a permis de mettre en évidence des profils d’expression différents pour plusieurs protéines. Elle confirme l’effet muscle spécifique du KO et révèle un lien avec le métabolisme du calcium et des Hsps différentes de celles mises en évidence dans le muscle oxydatif (Publication 4). L'ensemble des données issues de cette étude réalisée dans une espèce modèle apporte des connaissances nouvelles susceptibles d’éclairer sur les mécanismes moléculaires impliqués dans l’établissement de la tendreté de la viande bovine. Elle suggère que le statut en Hsp, les processus apoptotiques et la protection contre le stress oxydatif contribuent à l'évolution de l'ultrastructure post-mortem des muscles et à la tendreté de la viande. Ces nouvelles connaissances seront validées ultérieurement sur muscle bovin
Thanks to genomics, we have previously identified markers of beef tenderness, and computed a bioinformatic analysis that enabled us to build an interactome in which we found Hsp27 at a crucial node. Understanding the role of Hsp27 in the development of muscle and in the determinism of beef tenderness is one of the research challenges in meat production. In this context, my pHDthesis (2010-2013) aimed to analyze the role of Hsp27 in muscle development and its involvement in the determination of the characteristics related to the quality of the meat tissue. In this study, we generated mice devoid of Hsp27 protein by homologous recombination of the HspB1 gene as an animal model. The HspB1-/ - mice were viable and fertile, showing no apparent abnormality but a smaller than their control format. The muscle structure of animals was examined by optical microscopy and transmission electron microscopy. The first approach, made by a developed immunohistochemical classification (Publication 1), did not reveal any differences in the characteristics of muscle fibers (contractile and metabolic type, shape, perimeter, cross-sectional area) but a trend for a higher proportion of small fibers. Different myosin heavy chains electrophoretic profiles were also observed in HspB1-/- mice. At the ultrastructural level, examination of the myofibrillar material showed destructured myofibrils and higher gaps between myofibrils in HspB1-/-, and a greater disintegration of myofibrils at 72h postmortem (Publication 2). We have used a network-based approach for understanding the contribution of Hsp27 to tenderness through the prediction of its interactors related to tenderness. We have revealed the direct interactors of Hsp27. The predicted partners of Hsp27 included proteins involved in different functions e.g. members of Hsp families, regulators of apoptosis, translation factors, cytoskeletal proteins and antioxidants. The abundances of 15 proteins were quantified by Western blotting in two muscles of HspB1-null mice and their controls. We observed changes in the amount of most of the Hsp27 predicted targets in mice devoid of Hsp27 mainly in the most oxidative muscle (Soleus. Our study demonstrates the functional links between Hsp27 and its predicted targets. It suggests that Hsp status, apoptotic processes and protection against oxidative stress are crucial for post-mortem muscle metabolism, subsequent proteolysis, and therefore for beef tenderness (Publication 3). To complete this study, we performed a proteomic analysis of m. Tibialis anterior (glycolytic muscle), using 2D gel electrophoresis, to detect changes in protein abundance subsequent to the invalidation of HspB1 gene. This study confirms the muscle specific effect of HspB1 invalidation and reveals a new list of Heat shock proteins different from those highlighted in oxidative muscle and relationships with calcium (Publication 4). All together, these results provided from a model species showed the very important role of Hsp27 for muscle ultrastructure and revealed its implication in different muscle biological pathways. This provided new elements for understanding the crucial role for Hsp27 in the modulation of the tenderizing process of muscle during meat ageing that will be further examined in beef

Les courts ARN non codants tels que les microARN, les piARN et les siARN sont de petites molécules d’ARN de 20 à 30 nucléotides de long qui sont très bien conservées au cours de l’évolution. Elles s’associent à des protéines Argonautes afin de former un complexe effecteur appelé RISC (RNA induced silencing complex). Ces courtes séquences, ne codant pour aucune protéine, agissent comme de puissants régulateurs de l’expression des gènes. De nombreuses évidences supportent qu’une dérégulation du niveau d’expression de ces courts ARN non codants contribue au développement et au maintien de nombreuses pathologies telles que le cancer. De ce fait, il est essentiel pour la cellule de contrôler la stabilité des courts ARN non codants. Le contrôle de la maturation et de la stabilité de ces courts ARN non codants sont des mécanismes peu connus. L’objectif principal de mon doctorat a donc été de mieux comprendre comment le niveau des courts ARN non codants est contrôlé. Afin d’étudier plus en détail comment le niveau des microARN est régulé, nous avons identifié la phosphatase PPM-2 (PP2Cα chez l’humain) et l’E3 ubiquitine ligase HECD-1 (HectD1 chez l’humain) comme étant de nouveaux interacteurs du complexe de dégradation des microARN. Nous avons utilisé des approches de génétique et de biologie moléculaire chez le nématode C. elegans, pour étudier le rôle de la perte de fonction de ppm-2 et d’hecd1 dans la voie des courts ARN non codants. Nos travaux ont montré que la perte de fonction de ppm-2 induit des défauts développementaux qui sont associés à des défauts de la voie des microARN. De plus, l’absence de ppm-2 exacerbe les phénotypes développementaux observés dans des animaux où la voie des microARN est altérée. De manière intéressante, chez le mutant ppm-2, nous avons constaté que d’autres voies de courts ARN non codants, telles que la voie des piARN et celle de l’endosiARN nucléaire, sont affectées. Du point de vue moléculaire, nous avons observé une déstabilisation du niveau d’expression de plusieurs protéines Argonautes dans le mutant ppm-2. En effet, ces dernières sont envoyées à la dégradation par la voie du protéasome seulement chez des animaux mutés pour ppm-2. Concernant l’étude de HECD1, nous avons remarqué que la perte de fonction de cette ubiquitine ligase entrainait une diminution de la progéniture et une létalité embryonnaire attribuable à des défauts dans la gamétogénèse. De plus, nous avons observé une accumulation de miARN fonctionnels chez des animaux mutés pour hecd-1. L’ubiquitine ligase HECD-1 pourrait être impliquée dans la transcription ou la dégradation des miARN. En conclusion, nos résultats suggèrent que PPM-2 permet de contrôler la stabilité des protéines Argonautes en les dirigeant dans une voie alternative de dégradation et que l’ubiquitine ligase HECD-1 pourrait être impliquée dans la régulation des miARN en modulant leur transcription ou leur dégradation. Mes travaux de doctorat nous ont permis de mettre en lumière un nouveau modulateur des courts ARN non codants, PPM-2, qui agit via le contrôle de la régulation des Argonautes. Les avancées de la recherche dans le domaine des courts ARN non codants pourra permettre le développement de nouvelles thérapies.
Les courts ARN non codants tels que les microARN, les piARN et les siARN sont de petites molécules d’ARN de 20 à 30 nucléotides de long qui sont très bien conservées au cours de l’évolution. Elles s’associent à des protéines Argonautes afin de former un complexe effecteur appelé RISC (RNA induced silencing complex). Ces courtes séquences, ne codant pour aucune protéine, agissent comme de puissants régulateurs de l’expression des gènes. De nombreuses évidences supportent qu’une dérégulation du niveau d’expression de ces courts ARN non codants contribue au développement et au maintien de nombreuses pathologies telles que le cancer. De ce fait, il est essentiel pour la cellule de contrôler la stabilité des courts ARN non codants. Le contrôle de la maturation et de la stabilité de ces courts ARN non codants sont des mécanismes peu connus. L’objectif principal de mon doctorat a donc été de mieux comprendre comment le niveau des courts ARN non codants est contrôlé. Afin d’étudier plus en détail comment le niveau des microARN est régulé, nous avons identifié la phosphatase PPM-2 (PP2Cα chez l’humain) et l’E3 ubiquitine ligase HECD-1 (HectD1 chez l’humain) comme étant de nouveaux interacteurs du complexe de dégradation des microARN. Nous avons utilisé des approches de génétique et de biologie moléculaire chez le nématode C. elegans, pour étudier le rôle de la perte de fonction de ppm-2 et d’hecd1 dans la voie des courts ARN non codants. Nos travaux ont montré que la perte de fonction de ppm-2 induit des défauts développementaux qui sont associés à des défauts de la voie des microARN. De plus, l’absence de ppm-2 exacerbe les phénotypes développementaux observés dans des animaux où la voie des microARN est altérée. De manière intéressante, chez le mutant ppm-2, nous avons constaté que d’autres voies de courts ARN non codants, telles que la voie des piARN et celle de l’endosiARN nucléaire, sont affectées. Du point de vue moléculaire, nous avons observé une déstabilisation du niveau d’expression de plusieurs protéines Argonautes dans le mutant ppm-2. En effet, ces dernières sont envoyées à la dégradation par la voie du protéasome seulement chez des animaux mutés pour ppm-2. Concernant l’étude de HECD1, nous avons remarqué que la perte de fonction de cette ubiquitine ligase entrainait une diminution de la progéniture et une létalité embryonnaire attribuable à des défauts dans la gamétogénèse. De plus, nous avons observé une accumulation de miARN fonctionnels chez des animaux mutés pour hecd-1. L’ubiquitine ligase HECD-1 pourrait être impliquée dans la transcription ou la dégradation des miARN. En conclusion, nos résultats suggèrent que PPM-2 permet de contrôler la stabilité des protéines Argonautes en les dirigeant dans une voie alternative de dégradation et que l’ubiquitine ligase HECD-1 pourrait être impliquée dans la régulation des miARN en modulant leur transcription ou leur dégradation. Mes travaux de doctorat nous ont permis de mettre en lumière un nouveau modulateur des courts ARN non codants, PPM-2, qui agit via le contrôle de la régulation des Argonautes. Les avancées de la recherche dans le domaine des courts ARN non codants pourra permettre le développement de nouvelles thérapies.
Small non-coding RNAs, like microRNAs, piRNAs or siRNAs, are small RNA molecules, 20 to 30 nucleotides long that are conserved during evolution. They form an induced silencing complex (RISC) in association with Argonaute proteins to regulate gene expression. Small non-coding RNAs are involved in the regulation of genes implicated in cell proliferation, differentiation and development. Many evidences support that deregulation of the expression level of those small non-coding RNAs contribute to the development of pathologies such as cancer. It is therefore essential for cells to control small non-coding RNA stability. The control of maturation and stability of those small molecules are poorly understood. The main objective of my doctorate was to better understand how the stability of small non-coding RNAs is controlled. In order to study in more detail how miRNAs are regulated, we identified two factors involved in miRNA turnover in C. elegans. We found that the phosphatase PPM-2 (PP2Cα in human) and the E3 ubiquitin ligase HECD-1 (HectD1 in human) are new components of the miRNA degradation complex. Using the power of the nematode C. elegans and molecular biology, we characterized the role of the loss of function of PPM-2 and HECD-1 in small non-coding RNA pathways. Loss of this phosphatase induces developmental defects which are associated with a defect in the miRNA pathway. Genetically, the phosphatase mutant exacerbates the phenotypes that are observed in animals where the miRNA pathway is affected. Interestingly, we further observed that the loss of the phosphatase affects other small non-coding RNA pathways like the piRNA and the siRNA pathways. At the molecular level, we observed a decrease in the expression level of many Argonaute proteins in phosphatase mutant animals. Upon blocking proteasomal degradation with MG132, we noticed that Argonaute proteins are sent to proteasomal degradation in phosphatase mutant animals. Concerning HECD-1, we noticed that the loss of function of the E3 ubiquitin ligase leads to the decrease of progeny and embryonic lethality due to defects in gametogenesis. Moreover, we observed an accumulation of functional miRNAs. This protein can be implicated in transcription or turnover of miRNAs. VIIn conclusion, our data suggest that PPM-2 controls the stability of Argonaute proteins by sending them through an alternative degradation pathway and that HECD-1 could be implicated in miRNA regulation by modulating their transcription or degradation. My doctoral work helped to highlight a new modulator of small non-coding RNAs, PPM-2, which acts through the regulation of Argonaute protein. A better understanding of the mechanisms controlling the stability and the function of these strong regulators will be useful to develop new therapies.
Small non-coding RNAs, like microRNAs, piRNAs or siRNAs, are small RNA molecules, 20 to 30 nucleotides long that are conserved during evolution. They form an induced silencing complex (RISC) in association with Argonaute proteins to regulate gene expression. Small non-coding RNAs are involved in the regulation of genes implicated in cell proliferation, differentiation and development. Many evidences support that deregulation of the expression level of those small non-coding RNAs contribute to the development of pathologies such as cancer. It is therefore essential for cells to control small non-coding RNA stability. The control of maturation and stability of those small molecules are poorly understood. The main objective of my doctorate was to better understand how the stability of small non-coding RNAs is controlled. In order to study in more detail how miRNAs are regulated, we identified two factors involved in miRNA turnover in C. elegans. We found that the phosphatase PPM-2 (PP2Cα in human) and the E3 ubiquitin ligase HECD-1 (HectD1 in human) are new components of the miRNA degradation complex. Using the power of the nematode C. elegans and molecular biology, we characterized the role of the loss of function of PPM-2 and HECD-1 in small non-coding RNA pathways. Loss of this phosphatase induces developmental defects which are associated with a defect in the miRNA pathway. Genetically, the phosphatase mutant exacerbates the phenotypes that are observed in animals where the miRNA pathway is affected. Interestingly, we further observed that the loss of the phosphatase affects other small non-coding RNA pathways like the piRNA and the siRNA pathways. At the molecular level, we observed a decrease in the expression level of many Argonaute proteins in phosphatase mutant animals. Upon blocking proteasomal degradation with MG132, we noticed that Argonaute proteins are sent to proteasomal degradation in phosphatase mutant animals. Concerning HECD-1, we noticed that the loss of function of the E3 ubiquitin ligase leads to the decrease of progeny and embryonic lethality due to defects in gametogenesis. Moreover, we observed an accumulation of functional miRNAs. This protein can be implicated in transcription or turnover of miRNAs. VIIn conclusion, our data suggest that PPM-2 controls the stability of Argonaute proteins by sending them through an alternative degradation pathway and that HECD-1 could be implicated in miRNA regulation by modulating their transcription or degradation. My doctoral work helped to highlight a new modulator of small non-coding RNAs, PPM-2, which acts through the regulation of Argonaute protein. A better understanding of the mechanisms controlling the stability and the function of these strong regulators will be useful to develop new therapies.