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Opioid receptor modulation of pro-inflammatory cytokine production is vital for host defense and the inflammatory response. Previous results have shown the mu-opioid receptor (MOR) selective agonist, DAMGO, has the capacity to increase the expression of the pro-inflammatory chemokines, CCL2/MCP-1, CCL5/RANTES and CXCL10/IP-10 in peripheral blood mononuclear cells (PBMCs). We have shown that MOR activation is able to induce the expression of TGF-β, and TGF-β appears to be required for induction of CCL5 following MOR activation. This work suggests a novel role for TGF-β in the inflammatory response. NF-κB is a transcription factor that plays a pivotal role in inflammation and the immune response. We have found that NF-kB inhibitors can prevent the MOR-induced activation of CCL2 and CCL5, and that the NF-kB subunit, p65, is phosphorylated at serine residues 311 and 536 in response to μ-opioid receptor activation. In vivo, DAMGO administration can induce binding of p. 65 to the enhancer region of the CCL2 promoter. Furthermore, we demonstrate that PKCζ is phosphorylated following DAMGO-induced MOR activation and, is essential for NF-kB activity as well as CCL2 expression and transcriptional activity. In conclusion, these data suggest a pro-inflammatory role for MOR which involves NF-κB activation and PKCζ as well as a novel role for TGF-β as a regulator of pro-inflammatory chemokines. / Molecular Biology and Genetics

Ces travaux de thèse concernent la synthèse et l’étude des propriétés optiques de fluorophores organiques appliqués à la détection de deux biomarqueurs de la maladie d’Alzheimer : la protéine kinase C (PKC) et les fibrilles de peptides amyloïdes Aβ42. La détection de la PKC est réalisée à l’aide de dyades comprenant un émetteur de type boradiazaindacène (BODIPY) relié de manière covalente à un inhibiteur de cette enzyme. Ces composés obtenus via une synthèse multi-étape présentent des coefficients d’absorption molaires et des rendements quantiques de fluorescence en solution importants. Ces sondes ont ensuite été utilisées en cytométrie de flux par la société Amoneta Diagnostics afin de détecter la PKC en surface des globules rouges et d’évaluer le potentiel de ces sondes dans le diagnostic de la maladie d’Alzheimer par test sanguin. Pour la détection des fibrilles de peptides amyloïdes, deux séries de fluorophores de la famille des 2-(2’-hydroxyphényl)-benzoxazoles ont été étudiées. Ces molécules présentent un phénomène de transfert de proton intramoléculaire à l’état excité (ESIPT) qui leur confère des propriétés optiques remarquables, telles que d’importants déplacements de Stokes et une émission duale. Enfin, un test de détection in vitro a été mis au point. Une interaction entre ces fluorophores et les fibrilles amyloïdes a pu être observée permettant la détection de ces peptides en solution. / This research concerns the synthesis and photophysical studies of organic fluorescent probes applied to the detection of two Alzheimer’s disease biomarkers: the protein kinase C and fibrils of amyloid Aβ42 peptides. PKC detection is achieved by dyads composed of boradiazaindacene dye (BODIPY) covalently linked to an inhibitor of this enzyme. These compounds afforded by a multistep synthesis display high molar absorption coefficients and quantum yields in solution. These probes were then used in flow cytometry by Amoneta Diagnostics to detect PKC on the surface of red blood cells in order to asses the potential of these molecules for the diagnostic of Alzheimer’s disease by blood test. Two series of fluorophores from the 2-(2’-hydroxyphenyl)-benzazole family have been studied for the detection of amyloid fibrils. These molecules display an excited state intramolecular proton transfer (ESIPT) phenomenon which confer them remarkable optical properties such as important Stokes shifts and dual emission. Finally, an in vitro detection test has been developed. Interactions between these dyes and amyloid fibrils has been observed, allowing the detection of these peptides in solution.

Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Development of bovine embryos produced by intracytoplasmic sperm injection (ICSI) is low compared to fertilized embryos. Deficient oocyte activation, inappropriate sperm capacitation, and lack of sperm decondensation are thought to be the main constrains affecting ICSI success in cattle. In the present study, activation compounds were tested to establish an effective protocol for activation of bovine oocytes, and then used for oocyte activation after ICSI. The activation approach consisted of exposing in vitro matured oocytes to Ionomycin (ION) following by a specific CDK1 inhibitor (RO-3306), a specific PKC activator (OAG) or both RO+OAG. In the first experiment, the rate of activation (pronuclear (PN) formation), cleavage, development to the blastocyst stage, and number of cells per blastocyst were evaluated after oocyte treatment. The PN rates were higher (P≤0.01) in the groups activated with ION+RO (48.5 %) and ION+RO+OAG (65.6 %) compared to ION (12.3 %) and ION+OAG (9.2 %). There was no significant effect between the RO concentrations tested (5, 7.5 and 10 μM) on oocyte activation. The PN rate was significantly higher (P≤0.01) when oocytes were exposed to RO for 240 min (84.6 %) compared to 60 (53.6 %) and 120 min (60.0 %). However, there was no difference between groups when treatment with RO started at 0, 30 or 60 min after on ION exposure. Cleavage rate was higher in ION+RO (70.2 %) and ION+RO+OAG (62.4 %) groups compared to ION (11.8 %) and ION+OAG (22.8 %). Blastocyst rate was also higher in the ION+RO+OAG (24.1 %) group, but not statistically different between ION+RO (19.7 %) and ION+OAG (9.5 %) groups. There was no development to the blastocyst stage after treatment with ION alone. The average cell number in blastocysts was not statistically different among treatments. In the second experiment, the effect of activation with ION+RO (10 μM for 240 min) was tested after ICSI using control (ICSI-Cont) or treated by electroporation (ICSI-El) sperm. Most oocytes presented a well-developed female PN (66.4%). Male PN formation was higher (P≤0.05) in the ICSI-El (33.3%) compared to the ICSI-Cont (9.4%) group. In conclusion, this study revealed that the specific inhibition of CDK1 after ION treatment is an effective approach to activate bovine oocytes. Male pronuclear formation after ICSI is increased by sperm electroporation, but is lower than female pronuclear formation. This indicates that deficient sperm decondensation and male PN formation rather than deficient oocyte activation is likely the main problem to develop an effective protocol for bovine ICSI. / O desenvolvimento de embriões bovinos produzidos por injeção intracitoplasmática de espermatozóides (ICSI) é baixo em relação aos embriões fertilizados. Deficiência na ativação de oócitos, capacitação inadequada de espermatozóides e falta de descondensação de espermatozóides são os principais transtornos que afetam o sucesso de ICSI em bovinos. No presente trabalho, foram testados métodos de ativação com o intuito de estabelecer um protocolo efetivo para a ativação de oócitos bovinos após a ICSI. Para isso, um inibidor específico de CDK1 (RO-3306) e um ativador específico de PKC (OAG) foram utilizados após a incubação com Ionomicina (ION) para avaliar a retomada da meiose em oócitos bovinos. Além disso, a incubação em meio Fert durante 6 h e a eletroporação (El) de espermatozoides previamente a ICSI foram testadas para verificar o efeito sobre a descondensação do espermatozoide e formação do pró-núcleo (PN) masculino. Inicialmente oócitos bovinos foram incubados, com diferentes concentrações e períodos de exposição aos tratamentos. Foram avaliados conforme a taxa de ativação oocitária (formação de PN), clivagem, desenvolvimento a blastocisto e número de células nos embriões que se desenvolveram a blastocisto. As taxas de PN foram maiores (P≤0,01) nos grupos ativados com ION+RO (48,5 %) e ION+RO+OAG (65,6 %) comparado com ION (12,3 %) e ION+OAG (9,2 %). Não houve efeito significativo entre as concentrações 5,0, 7,5 e 10,0 μM de RO sobre a taxa de ativação. A taxa de ativação foi significativamente maior (P≤0,01) em oócitos tratados com RO por 240 min (84,6 %) comparado a 60 (53,6 %) e 120 min (60,0%). No entanto, não houve diferença significativa na taxa de ativação quando o tratamento com RO foi iniciado a 0, 30 ou 60 minutos após a incubação com ION. A taxa de clivagem foi inferior nos grupos ION (11,8 %) e ION+OAG (22,8 %) comparada aos grupos ION+RO (70,2 %) e ION+RO+OAG (62,4 %). A taxa de blastocistos também foi maior no grupo ION+RO+OAG (24,1 %), mas não houve diferença estatística entre os grupos ION+RO (19,7 %) e ION+OAG (9,5 %). Não houve desenvolvimento a blastocisto quando os oócitos foram tratados somente com ION. Não foi detectada diferença estatística entre os tratamentos sobre o número médio de células por blastocistos. No segundo experimento, espermatozoides não tratados (ICSI-Cont) ou tratados por electroporação (ICSI-El) foram microinjetados em oócitos, os quais foram ativados com ION+RO por 240 min e fixados cerca de 15 h após a injeção para determinar a taxa de formação de PNs masculino e feminino (2PN). A maioria dos oócitos apresentaram o PN feminino bem desenvolvido (66,4 %). A formação de 2PN (masculino e feminino) foi maior no grupo ICSI-El (33,3 %) comparado ao grupo ICSI-Cont (9,4 %). Em conclusão, esse estudo demonstrou que a inibição específica da CDK1 após o tratamento com ION promove a ativação de oócitos bovinos. O tratamento de espermatozoides com eletroporação melhora a formação do PN masculino após ICSI, mas a taxa é inferior a formação do PN feminino. Esses resultados indicam que a deficiente descondensação do espermatozoide é o principal limitante para estabelecer um protocolo eficaz para ICSI em bovinos.

Fra-1 appartient à la famille des facteurs de transcription AP-1. Son expression est particulièrement élevée dans les cellules de cancer du sein qui n'expriment pas le récepteur aux œstrogènes (RE-), c'est-à-dire les cellules les plus agressives. L'inhibition de Fra-1 dans ces cellules entraîne une diminution de la motilité, de l'invasion et de la prolifération, mais elle entraîne aussi de profonds changements de morphologie. Les cellules RE-, qui présentent un phénotype mésenchymateux s'arrondissent et établissent un plus grand nombre de contacts cellule-cellule après l'inhibition de Fra-1. Dans les cellules RE-, la β-caténine est localisée au noyau ou dans le cytoplasme, ce qui est un marqueur de mauvais pronostic. Au cours de cette thèse, j'ai montré que Fra-1 régule la localisation nucléaire de la β-caténine et ainsi régule son activité transcriptionelle en agissant très tardivement sur la voie Wnt. J'ai également mis en évidence une interaction physique directe entre Fra-1 et la β-caténine qui pourrait être responsable de cet effet. De plus, l'analyse de microarrays par RT-QPCR a révélé la régulation d'autres gènes comme la mœsine, la fibronectine et l'extracellular matrix protein 1, qui pourraient également jouer un rôle dans la régulation de l'agressivité tumorale par Fra-1. Par ailleurs, Fra-1 est une protéine instable et nous avons montré qu'elle est phosphorylée et stabilisée par PKCθ. Fra-1 est d'ailleurs nécessaire à l'effet de la kinase sur la motilité cellulaire. / Fra-1 is a member of the AP-1 transcription factor family. It is aberrantly expressed in breast cancer cells lacking Estrogen Receptor (ER-) expression, which are the most aggressive ones. Fra-1 inhibition in these cells leads to a decreased in motility, invasion and proliferation, but also to deep morphologic changes. ER- cells, which present a mesenchymal phenotype, become rounder and establish a greater number of cell-cell contacts after Fra-1 inhibition. In ER- cells, β-catenin is nuclear or cytoplasmic, which is considering as a poor prognosis marker. During this PhD, I demonstrate that Fra-1, which acts very downstream in the Wnt/β-catenin signaling pathway, regulates the nuclear localization of β-catenin leading to up-regulation transcriptional activity of β-catenin. I also found that Fra-1 directly interacts with β-catenin. In addition, RT-QPCR microarrays analysis has revealed the regulation of other genes such as mœsin, fibronectin and extracellular matrix protein 1, which might also take part in the tumoral aggressiveness regulated by Fra-1. Moreover, we show that Fra-1, which is an unstable protein, is phosphorylated and stabilized by PKCθ. Furthermore, Fra-1 is necessary to mediate the kinase effect on cell motility.

Les maladies cardiovasculaires sont souvent causées par des arythmies qui proviennent d'une obstruction du système de conduction cardiaque. L'intervenant clé de ce système est le nœud sinu-atrial (SA), qui est responsable de l’initiation de chaque battement cardiaque. L’activation électrique à intervalles réguliers, assurant que le rythme cardiaque est un rythme normal. Le dysfonctionnement du nœud SA entraînerait des instabilités électriques dans le cœur. Une maladie cardiaque acquise, comme la cardiopathie rhumatismale, ou un bloc de conduction ne sont que quelques-uns des nombreux cas qui nécessitent un stimulateur cardiaque électronique pour surveiller la fréquence cardiaque et générer une impulsion lorsqu'elle bat anormalement. Bien que le stimulateur cardiaque électrique soit considéré comme une thérapie fiable, il n'est pas sans limites. Ces limites comprennent les complications chirurgicales, l'infection au plomb ainsi que la durée de vie limitée de la batterie, qui doit être remplacée à intervalles de quelques années, ce qui alourdit le fardeau hospitalier. Plusieurs approches ont été adoptées pour développer une méthode thérapeutique plus adéquate. Une stratégie qui sera étudiée implique l'utilisation d'une greffe de cellules de stimulateur cardiaque, créant fondamentalement un stimulateur biologique. Les approches de thérapie cellulaire utilisent des cellules souches embryonnaires pour évoluer vers les lignées de cellules cardiaques, y compris les cellules stimulatrices cardiaques. Ces cellules de stimulation sont caractérisées par une dépolarisation spontanée qui crée les impulsions rythmiques dans le cœur et contrôle la fréquence cardiaque. Un élément important des cellules du stimulateur cardiaque qui donne lieu à la dépolarisation spontanée sont les canaux « hyperpolarization-activated and cyclic nucleotide-gated » qui sont activés pendant l’hyperpolarisation et conduisent le courant sous le nom de « funny current ». Ce courant augmente la perméabilité intérieure de la cellule aux courants de sodium et de potassium conduisant à la dépolarisation de la cellule. D'autre part, le taux de conduction est déterminé par la connexine 30.2 et la connexine 45, qui sont des protéines transmembranaires qui s’assemblent pour former des jonctions lacunaires. L'expression de HCN et l'expression de la connexine ont toutes deux étés liés au facteur T-box 3 (Tbx3) dans le développement des myocytes auriculaires. Une approche praticable pour moduler l'expression des gènes et par conséquent l'expression des protéines est l'utilisation du conditionnement chimique. Le Phorbol 12- myristate 13-acétate (PMA) est un activateur de Protéine Kinase C (PKC) lié à l'expression de Tbx3, et par conséquent à l'expression de HCN et de connexine, et entraînant une modification de l'activité spontanée. Les cellules souches embryonnaires de souris sont des cellules qui sont isolées de la masse cellulaire interne des embryons. Ces cellules ont la capacité de se différencier en tous les types de cellules somatiques. En combinant les facteurs de croissance, ces cellules peuvent se différencier en cardiomyocytes. Nous émettons l'hypothèse que le conditionnement chronique de cardiomyocytes de souris avec PMA entraîne une régulation à la hausse de l'expression de Tbx3 et par conséquent une régulation à la hausse de l'expression de HCN et de l'expression de connexine, favorisant ainsi le développement des cellules stimulatrices cardiaques dans la population des cardiomyocytes. Afin de vérifier notre hypothèse, nous avons acheté des cellules de la lignée cellulaire E14TG2A de souris. Ces cellules ont été cultivées dans des pétris et différenciées en cardiomyocytes à l'aide d'un protocole en trois étapes (voir la section Méthodes). Les cardiomyocytes sont ensuite exposés à la PMA à des concentrations variables (0.1 µM vs 1 µM) pendant 1h (exposition aiguë) ou 24 h (exposition chronique). Les résultats variaient d'un groupe expérimental à l'autre par rapport au groupe témoin. Dans toutes les conditions expérimentales, il semble y avoir une augmentation initiale de l'activité spontanée, mais elle s'inverse rapidement à la marque des 24 heures, où le rythme diminue. Différentes concentrations jouent un rôle dose-dépendant dans l'effet inhibiteur de longue durée sur la stimulation des cellules. / Cardiovascular diseases are often caused by arrhythmias that originate from an obstruction within the cardiac conduction system. The key player within that system is the sinoatrial (SA) node, which is responsible for initiation the electrical impulses at a regular interval, insuring the heartbeat at a normal pace. Dysfunction of the SA node would lead to electrical instabilities in the heart. An acquired heart disease, such as rheumatic heart disease, or a conduction block are just some of many cases that would require an electronic pacemaker to monitor the heart rate and generate an impulse when it beats abnormally. Although the electric pacemaker is considered as a reliable therapy, it is not without limitations. These limitations include surgery complication, lead infection as well as limited battery lifespan, which requires replacement every few years thus adding to the hospital burden. Several approaches have been taken to develop a more adequate therapeutic method. A strategy that will be investigated involves using a graft of pacemaker cells, fundamentally creating a biological pacemaker. Cell therapy approaches use embryonic stem cells to evolve into the cardiac cell lines, including pacemaker cells. These pacing cells are characterized by spontaneous depolarization that create the rhythmic impulses in the heart and control the heart rate. An important element of the pacemaker cells that give rise to the spontaneous depolarization are the hyperpolarization- activated and cyclic nucleotide-gated (HCN) channels that are activated during hyperpolarization and conduct the funny current by increasing the cell’s inward permeability to sodium-potassium currents. On the other hand, the conduction rate is determined by connexin 30.2 and connexin 45, which are transmembrane proteins that assemble to form gap junctions. Both HCN expression and connexin expression has been linked to T-box factor 3 (Tbx3) in the development of atrial myocytes. A practicable approach to modulate gene expression and consequently protein expression is using chemical conditioning. Phorbol 12-myristate 13-acetate (PMA) is a Protein Kinase C (PKC) activator that has linked to Tbx3 expression, and consequently HCN and connexin expression, and lead to a modification in spontaneous activity. Mouse embryonic stem cells (ESCs) are cells that are isolated from the inner cell mass of early embryos. These cells can differentiate into all somatic cell types. Given the proper combination of growth factors, these cells can differentiate into cardiomyocytes. We hypothesize that chronic conditioning of mice cardiomyocytes with PMA lead to an upregulation of Tbx3 expression and consequently an upregulation of HCN expression and connexin expression, therefore promoting the development of pacemaker cells within the cardiomyocyte population. In order to test our hypothesis, we purchased cells from the mouse E14TG2A cell line. These cells were cultured in glass bottom petri dishes and differentiated into cardiomyocytes using a three-step protocol (shown in Methods section). The cardiomyocytes are then exposed to PMA in varying concentration (0.1 µM vs 1 µM) for either 1h (acute exposure) or 24 h (chronic exposure). The results varied between the experimental groups compared to the control. In all experimental conditions there seems to be an initial increase in spontaneous activity, but this is quickly reversed at the 24 h mark, where pacing decreased. Different concentration plays a dose-dependent role in long-lasting inhibitory effect on the pacing of the cells

Sob o modelo de criptografia de chave pública baseada em identidades (ID-PKC), a própria identidade dos usuários é usada como chave pública, de modo a dispensar a necessidade de uma infra-estrutura de chaves públicas (ICP), na qual o gerenciamento de certificados digitais é complexo. Por outro lado, sistemas nesse modelo requerem uma entidade capaz de gerar chaves secretas. Essa entidade é conhecida por PKG (Private Key Generator); ela possui uma chave-mestra e mantém custódia das chaves secretas geradas a partir dessa chave-mestra. Naturalmente, a custódia de chaves é indesejável em muitas aplicações. O conceito de Criptografia de Chave Pública sem Certificado, ou Certificateless Public Key Cryptography (CL-PKC), foi proposto para que a custódia de chaves fosse eliminada, mantendo, porém, as características de interesse: a não necessidade de uma ICP e a eliminação de certificados digitais. CL-PKC deixa de ser um sistema baseado em identidades, pois é introduzida uma chave pública, gerada a partir de uma informação secreta do usuário. Nesta dissertação, apresentamos a construção de dois esquemas, um CL-PKE e um CL-PKS, baseados em emparelhamentos bilineares sobre curvas elípticas. Ambas propostas: (1) eliminam custódia de chaves; (2) dispensam certificados digitais; (3) são mais eficientes, sob certos aspectos, que esquemas anteriormente publicados; (4) e são seguros contra ataques adaptativos de texto cifrado escolhido (em CL-PKE) e contra ataques adaptativos de mensagem escolhida (em CL-PKS), sob o modelo de oráculos aleatórios. / Under the model of Identity Based Cryptography (ID-PKC), the public key can be the user's identity, therefore it does not require a Public Key Infrastructure (PKI) with its complex management of Digital Certificates. On the other hand, this system requires a Private Key Generator (PKG), a trusted authority who is in possession of a master key and can generate any of the private keys. In this way, PKG can exercise the so-called key escrow, which is undesirable in many applications. The concept of Certificateless Public Key Cryptography (CL-PKC) was proposed in order to remove the key escrow characteristic of IBC, while it does not require PKI neither Digital Certificates to certify the public keys. CL-PKC is no more an IBC because public keys are introduced, to bind the identities with its secret keys. In this thesis we construct two schemes, one CL-PKE and one CL-PKS, based on bilinear pairing functions which: (1) does not allow key escrow by the PKG; (2) does not require Digital Certificates; (3) is more efficient, in some aspects, than previously published CL-PKE and CL-PKS schemes; (4) and is secure in the sense that it is strong against adaptive chosen ciphertext attacks (in CL-PKE) and adaptive chosen message attacks (in CL-PKS), under Random Oracle Model.

Sob o modelo de criptografia de chave pública baseada em identidades (ID-PKC), a própria identidade dos usuários é usada como chave pública, de modo a dispensar a necessidade de uma infra-estrutura de chaves públicas (ICP), na qual o gerenciamento de certificados digitais é complexo. Por outro lado, sistemas nesse modelo requerem uma entidade capaz de gerar chaves secretas. Essa entidade é conhecida por PKG (Private Key Generator); ela possui uma chave-mestra e mantém custódia das chaves secretas geradas a partir dessa chave-mestra. Naturalmente, a custódia de chaves é indesejável em muitas aplicações. O conceito de Criptografia de Chave Pública sem Certificado, ou Certificateless Public Key Cryptography (CL-PKC), foi proposto para que a custódia de chaves fosse eliminada, mantendo, porém, as características de interesse: a não necessidade de uma ICP e a eliminação de certificados digitais. CL-PKC deixa de ser um sistema baseado em identidades, pois é introduzida uma chave pública, gerada a partir de uma informação secreta do usuário. Nesta dissertação, apresentamos a construção de dois esquemas, um CL-PKE e um CL-PKS, baseados em emparelhamentos bilineares sobre curvas elípticas. Ambas propostas: (1) eliminam custódia de chaves; (2) dispensam certificados digitais; (3) são mais eficientes, sob certos aspectos, que esquemas anteriormente publicados; (4) e são seguros contra ataques adaptativos de texto cifrado escolhido (em CL-PKE) e contra ataques adaptativos de mensagem escolhida (em CL-PKS), sob o modelo de oráculos aleatórios. / Under the model of Identity Based Cryptography (ID-PKC), the public key can be the user's identity, therefore it does not require a Public Key Infrastructure (PKI) with its complex management of Digital Certificates. On the other hand, this system requires a Private Key Generator (PKG), a trusted authority who is in possession of a master key and can generate any of the private keys. In this way, PKG can exercise the so-called key escrow, which is undesirable in many applications. The concept of Certificateless Public Key Cryptography (CL-PKC) was proposed in order to remove the key escrow characteristic of IBC, while it does not require PKI neither Digital Certificates to certify the public keys. CL-PKC is no more an IBC because public keys are introduced, to bind the identities with its secret keys. In this thesis we construct two schemes, one CL-PKE and one CL-PKS, based on bilinear pairing functions which: (1) does not allow key escrow by the PKG; (2) does not require Digital Certificates; (3) is more efficient, in some aspects, than previously published CL-PKE and CL-PKS schemes; (4) and is secure in the sense that it is strong against adaptive chosen ciphertext attacks (in CL-PKE) and adaptive chosen message attacks (in CL-PKS), under Random Oracle Model.

La scoliose idiopathique de l’adolescent (SIA) est une maladie dont la cause est encore inconnue, et qui génère des déformations complexes du rachis, du thorax et du bassin. La prévalence est de 4% dans la population adolescente au Québec. Cette pathologie affecte surtout les filles durant leur poussée de croissance pubertaire. Parmi plusieurs hypothèses émises, l’hypothèse neuroendocrinienne, impliquant une déficience en mélatonine comme agent étiologique de la SIA a suscité beaucoup d’intérêt. Cette hypothèse découle du fait que l’ablation de la glande pinéale chez le poulet produit une scoliose ressemblant sous plusieurs aspects à la pathologie humaine. La pertinence biologique de la mélatonine dans la scoliose est controversée, étant donné que la majorité des études chez l’homme n’ont pu mettre en évidence une diminution significative des niveaux de mélatonine circulante chez les patients scoliotiques. Nous avons démontré un dysfonctionnement dans la signalisation de la mélatonine au niveau des tissus musculo-squelettiques chez une série de patients atteints de SIA (Moreau & coll. 2004). Nous avons confirmé ce défaut chez un plus grand nombre de patients ainsi qu’en utilisant une nouvelle technologie (spectroscopie cellulaire diélectrique) n’ayant pas recours à un prétraitement des cellules donnant ainsi des résultats plus précis. Cette technique a montré la présence des mêmes groupes fonctionnels identifiés auparavant par la technique d’AMPc. Le dysfonctionnement de la signalisation de la mélatonine est dû à une phosphorylation accrue des protéines G inhibitrices. Ce défaut pourrait être causé par un déséquilibre de l’activité des kinases et phosphatases capables de réguler la phosphorylation des protéines Gi. Parmi ces kinases, PKCd a suscité initialement notre intérêt vu qu’elle peut phosphoryler les protéines Gi. Nous avons démontré que cette kinase interagit avec le récepteur de la mélatonine MT2 et que cette interaction varie selon le groupe fonctionnel auquel un patient SIA appartient. Par la suite nos travaux se sont dirigés vers la découverte d’effecteurs cellulaires régulés par la mélatonine et plus spécifiquement l’ostéopontine (OPN), compte tenu de son rôle présumé comme mécanorécepteur et dans certaines structures jouant un rôle dans la proprioception, le contrôle postural et la fonction vestibulaire. L’OPN a été identifiée initialement par sa surexpression au niveau protéique et de l’ARNm dans la musculature paraspinale uniquement chez les poulets scoliotiques. Nous avons également utilisé un autre modèle animal, la souris C57Bl/6 naturellement déficiente en mélatonine. Nous avons généré des souris bipèdes en amputant les membres antérieurs de souris OPN KO, des souris CD44 KO ainsi que des souris contrôles C57Bl/6. Nos résultats ont montré qu’aucune souris OPN KO (n=50) ou CD44 KO (n=60) ne développe la maladie, contrairement aux souris contrôles C57Bl/6 (n=50) dont 45% deviennent scoliotiques. Ces résultats nous ont poussés à investiguer le rôle de cette protéine dans l’étiopathogenèse de la maladie chez l’humain. Nos résultats ont montré une augmentation des niveaux circulants d’OPN chez les patients atteints de la SIA et que l’élevation en OPN corrélait avec la sévérité de la maladie. Nos études chez les enfants asymptomatiques nés de parents scoliotiques et qui sont plus à risque de développer la maladie ont aussi démontré des différences significatives au niveau des concentrations en OPN en comparaison avec les sujets sains. En effet, plusieurs enfants à risque présentaient des niveaux d’OPN supérieurs à 800ng/ml suggérant un plus grand risque de développer une scoliose indiquant aussi que l’augmentation des niveaux en OPN précède le début de la maladie. / Adolescent idiopathic scoliosis (AIS) is the most common form of scoliosis that affects a significant number of young teenagers, mainly females. Historically, several hypotheses were postulated to explain the aetiology of AIS. The neuroendocrine hypothesis involving a melatonin deficiency as the source for AIS has generated great interest. This hypothesis stems from the fact that experimental pinealectomy in chickens, and more recently in rats maintained in a bipedal mode, produces scoliosis. The biological relevance of melatonin in idiopathic scoliosis is controversial since no significant decrease in circulating melatonin level has been observed in a majority of studies. Analysis of melatonin signal transduction in musculoskeletal tissues of AIS patients demonstrated for the first time a defect occurring in a cell autonomous manner in different cell types isolated from AIS patients suffering of the most severe form of that disease. We confirmed this defect by analysing more AIS patients and by using a new technology (cellular dielectric spectroscopy) which gives more precise results because it allows the measurement and analysis of receptor activation without the need to pretreat cells. This technique showed the same functional classification into three functional groups as identified by cAMP technique. Melatonin signalling dysfunction is caused by phosphorylation of serine residues affecting the activity of G inhibitory (Gi) proteins normally associated with melatonin receptors present at the cell surface. This defect could be caused by an imbalance in the activity of kinases or phosphatases that can regulate Gi proteins phosphorylation. Among these kinases PKCd was initially of interest because it has been shown that it can phosphorylate Gi proteins. We showed that this kinase interacts with melatonin receptor MT2 and that this interaction varies from one functional group to another. Thereafter, we moved one step further to characterise downstream effector regulated by melatonin. This work has led to the identification of osteopontin (OPN) which is a relevant candidate because it can act as a mecanosensor and it is involved in proprioception, postural and vestibular control. OPN was initially identified in pinealectomized chickens where it was shown to be upregulated at protein and mRNA levels only in scoliotic ones. We also used another animal model, C57Bl/6 mice which are naturally deficient in melatonin. We generated bipedal mice by amputating forelimbs of OPN knock-out mice, CD44 knock-out mice as well as C57Bl/6 wild type mice. Our results showed that all bipedal mice OPN Knock-out or CD44 Knock-out did not develop scoliosis contrasting with C57Bl/6 wt mice where 45% develop scoliosis. These results prompted us to investigate the role of this protein in scoliosis etiopathogenesis in humans. We showed an increase in the OPN circulating levels in AIS patients and this elevation correlates with disease severity. Elevated plasma OPN levels were also found in the asymptomatic at-risk group (offspring of scoliotic patients), suggesting that these changes precede scoliosis onset.

L’apoptose est une forme de mort cellulaire essentielle au développement et au maintien de l’homéostase chez les animaux multicellulaires. La machinerie apoptotiq ue requiert la participation des caspases, des protéases conservées dans l’évolution et celle des organelles cytoplasmiques. Les lysosomes subissent des ruptures partielles, labilisation de la membrane lysosomale (LML), qui entraînent l’activation des cathepsines dans le cytoplasme de cellules cancéreuses humaines en apoptose induite par la camptothecin (CPT), incluant les histiocytes humains U-937. Ces modifications lysosomales se manifestent tôt durant l’activation de l’apoptose, concomitamment avec la perméabilisation de la mitochondrie et l’activation des caspases. Une étude protéomique quantitative et comparative a permis d’identifier des changements précoces dans l’expression/localisation de protéines lysosomales de cellules U-937 en apoptose. Lors de deux expériences indépendantes, sur plus de 538 protéines lysosomales identifiées et quantifiées grâce au marquage isobarique iTRAQ et LC-ESIMS/ MS, 18 protéines augmentent et 9 diminuent dans les lysosomes purifiés de cellules en cours d’apoptose comparativement aux cellules contrôles. Les candidats validés par immuno-buvardage et microscopie confocale incluent le stérol-4-alpha-carboxylate 3- déhydrogénase, le prosaposin et la protéine kinase C delta (PKC-d). Des expériences fonctionnelles ont démontrées que la translocation de PKC-d aux lysosomes est requise pour la LML puisque la réduction de son expression par ARN interférents ou l’inhibition de son activité à l’aide du rottlerin empêche la LML lors de l’apoptose induite par la CPT. La translocation de PKC-d aux lysosomes conduit à la phosphorylation et l’activation de la sphingomyelinase acide lysosomale (ASM), et à l’accroissement subséquent du contenu en céramide (CER) à la membrane lysosomale. Cette accumulation de CER endogène aux lysosomes est un évènement critique pour la LML induite par la CPT car l’inhibition de l’activité de PKC-d ou de ASM diminue la formation de CER et la LML.Ces résultats révèlent un nouveau mécanisme par lequel la PKC-d active l’ASM qui conduit à son tour à l’accumulation de CER à la membrane lysosomale et déclenche la LML et l’activation de la voie lysosomale de l’apoptose induite par la CPT. En somme, ce mécanisme confirme l’importance du métabolisme des sphingolipides dans l’activation de la voie lysosomale de l’apoptose. / Apoptosis is a distinct form of regulated cell death which is essential for the development and homeostasis maintenance of multicellular animals. Apoptosis is an evolutionary conserved process involving a specific molecular pathway, known as the caspase cascade, and the different cytoplasmic organelles. A lysosomal pathway, characterized by partial rupture, labilization of lysosomal membranes (LML), and cathepsin activation in the cytoplasm, is evoked during camptothecin-induced apoptosis in human cancer cells, including human histiocytic lymphoma U-937 cells. These lysosomal events begin rapidly and simultaneously with mitochondrial permeabilization and caspase activation within 3 h after drug treatment. Comparative and quantitative proteome analyses were performed to identify early changes in lysosomal protein expression/localization from U-937 cells undergoing apoptosis. In two independent experiments, among a total of more than 538 proteins putatively identified and quantitated by iTRAQ isobaric labelling and LC-ESI-MS/MS, 18 proteins were found to be upregulated and 9 downregulated in lysosomes purified from early apoptotic compared to control cells. Protein expression was validated by Western blotting on enriched lysosome fractions, and protein localization confirmed by fluorescence confocal microscopy of representative protein candidates, whose functions are associated with lysosomal membrane fluidity and dynamics. These include sterol-4-alpha-carboxylate 3-dehydrogenase (NSDHL), prosaposin (PSAP) and protein kinase C delta (PKC-d). Functional experiments demonstrate that PKC-d translocation to lysosomes is required for LML, as silencing its expression with RNA interference or suppressing its activity with the inhibitor rottlerin prevents CPT-induced LLM. PKC-d translocation to lysosomes is associated with lysosomal acidic sphingomyelinase (ASM) phosphorylation and activation, which in turn leads to an increase of ceramide (CER) content at lysosomes. The accumulation of endogenous CER at lysosomes is a critical event for CPT-induced LLM as suppressing PKC-d or ASM activity reduces both CPT-mediated CER generation at lysosomes and CPT-induced LLM.These findings reveal a novel mechanism by which PKC-d mediates ASM phosphorylation/activation and CER accumulation at lysosomes in CPT-induced LLM, rapidly activating the lysosomal pathway of apoptosis after CPT treatment. Taken together, these results confirm the importance of sphingolipid metabolism in the activation of the lysosomal pathway of apoptosis.