Le GABA est le principal neurotransmetteur inhibiteur du SNC et est impliqué dans le développement du cerveau, la plasticité synaptique et la pathogénèse de maladies telles que l’épilepsie, les troubles de l’anxiété et la douleur chronique. Le modèle actuel de fonctionnement du récepteur GABA-B implique l’hétérodimérisation GABA-B1/B2, laquelle est requise au ciblage à la surface membranaire et au couplage des effecteurs. Il y est cependant des régions du cerveau, des types cellulaires et des périodes du développement cérébral où la sous-unité GABA-B1 est exprimée en plus grande quantité que GABA-B2, ce qui suggère qu’elle puisse être fonctionnelle seule ou en association avec des partenaires inconnus, à la surface cellulaire ou sur la membrane réticulaire. Dans le cadre de cette thèse, nous montrons la capacité des récepteurs GABA-B1 endogènes à activer la voie MAPK-ERK1/2 dans la lignée dérivée de la glie DI-TNC1, qui n’exprime pas GABA-B2. Les mécanismes qui sous-tendent ce couplage demeurent mal définis mais dépendent de Gi/o et PKC. L’immunohistochimie de récepteurs endogènes montre par ailleurs que des anticorps GABA-B1 dirigés contre la partie N-terminale reconnaissent des protéines localisées au RE tandis des anticorps C-terminaux (CT) marquent une protéine intranucléaire. Ces données suggèrent que le domaine CT de GABA-B1 pourrait être relâché par protéolyse. L’intensité des fragments potentiels est affectée par le traitement agoniste tant en immunohistochimie qu’en immunobuvardage de type western. Nous avons ensuite examiné la régulation du clivage par le protéasome en traitant les cellules avec l’inhibiteur epoxomicine pendant 12 h. Cela a résulté en l’augmentation du marquage intranucléaire de GABA-B1-CT et d’un interacteur connu, le facteur de transcription pro-survie ATF-4. Dans des cellules surexprimant GABA-B1-CT, l’induction et la translocation nucléaire d’ATF-4, qui suit le traitement epoxomicine, a complètement été abolie. Cette observation est associée à une forte diminution du décompte cellulaire. Étant donné que les trois derniers résidus de GABA-B1-CT (LYK) codent un ligand pseudo-PDZ et que les protéines à domaines PDZ sont impliquées dans la régulation du ciblage nucléaire et de la stabilité de protéines, en complément de leur rôle d’échaffaud à la surface cellulaire, nous avons muté les trois derniers résidus de GABA-B1-CT en alanines. Cette mutation a complètement annulé les effets de GABA-B1-CT sur l’induction d’ATF-4 et le décompte cellulaire. Cette deuxième série d’expériences suggère l’existence possible de fragments GABA-B1 intranucléaires régulés par le traitement agoniste et le protéasome dans les cellules DI-TNC1. Cette régulation d’ATF-4 dépend des résidus LYK de GABA-B1-CT, qui modulent la stabilité de GABA-B1-CT et favorisent peut-être la formation d’un complexe multiprotéique incluant GABA-B1-CT, ATF-4, de même qu’une protéine d’échaffaudage inconnue. En somme, nous démontrons que les sous-unités GABA-B1 localisées au RE, lorsque non-hétérodimérisées avec GABA-B2, demeurent capables de moduler les voies de signalisation de la prolifération, la différentiation et de la survie cellulaire, via le couplage de protéines G et possiblement la protéolyse régulée. Les mécanismes de signalisation proposés pourraient servir de nouvelle plate-forme dans la compréhension des actions retardées résultant de l’activation des récepteurs 7-TMs. / GABA is the principal inhibitory neurotransmitter in the CNS and is implicated in brain development, synaptic plasticity and the pathogenesis of diseases such as epilepsy, anxiety disorders and chronic pain. In the current model of GABA-B function, there is a requirement for GABA-B1/B2 dimerization for targetting to the cell surface and effector coupling. However, there are certain brain regions (putamen), cell types (glial cells) and times during brain development where GABA-B1 is expressed in higher amounts than GABA-B2, suggesting that GABA-B1 might be functional alone or in association with unidentified partners, either at the cell surface or on the ER membranes. In this thesis, we first show the capacity of endogenous GABA-B1 receptors to activate the MAPK-ERK1/2 pathway in the DI-TNC1 glial-derived cell line which does not express GABA-B2. The underlying mechanisms remain incompletely defined but depend on Gi/o and PKC. Immunohistochemistry of endogenous receptors shows that GABA-B1 N-terminal antibodies recognize ER-localized proteins and that C-terminal (CT) antibody shows intranuclear distribution. This data suggests that fragments of the GABA-B1 receptor are generated by proteolysis and indeed we show that agonist treatment affects the intensity of certain C-terminal GABA-B1 fragments both in immunohistochemistry and western blots suggesting that the GABA-B1 receptor is subjected to regulated proteolysis. Since a 13-residue potential PEST sequence was localized immediately distal to the ER retention motif in the GABA-B1 CT, we examined proteasome regulation of the cleavage event. Following a 12h treatment with the proteasome inhibitor, epoxomicin, we detected increases in intranuclear staining for both GABA-B1 and a known interactor, the pro-survival transcription factor ATF-4, using confocal microscopy and by western blotting of nuclear extracts. These increases are due either to proteasome inhibition or activation of the ER stress pathway. In cells overexpressing GABA-B1-CT, ATF-4 induction and nuclear translocation, which normally follows epoxomicin treatment, was completely abolished. This observation was associated to a strong decrease in cell number. Since the last three residues of GABA-B1-CT (LYK) encode a pseudo-PDZ ligand and that PDZ domain protein regulate nuclear targeting and protein stability, in complement to their role in scaffolding at the cell surface, we mutated the last three residues of GABA-B1-CT to alanines. This mutation completely reversed the effect of GABA-B1-CT on ATF-4 induction and on cell number. This second set of data suggests the existence of agonist and proteasome-regulated intranuclear GABA-B1 fragments in DI-TNC1 cells. Further, the GABA-B1-CT pseudo-PDZ ligand appears to be critically important in regulating ATF-4 induction by modulating GABA-B1-CT stability and perhaps by favoring the formation of a multiprotein complex with ATF-4, ATF-4 interactors and an unknown scaffolding protein. Overall, we show that ER-localised GABA-B1 subunits, when not dimerized with GABA-B2, can still modulate proliferation, differentiation and survival pathways, both through G-protein coupling and regulated proteolysis. The signalling mechanisms which we propose could serve as a new platform in understanding the long term effects of 7-TM receptor activation.

Le réseau des MAPK est composé de pathways de signalisation fermement entrecroisés impliqués dans le cancer. Toutefois, les mécanismes précis qui sous-tendent son influence sur l'équilibre entre la prolifération et la mort cellulaire demeurent insaisissablesDes données publiques ont été intégrés dans une carte de réactions détaillée, représentant l'influence du réseau des MAPKs sur la décision du destin cellulaire. Cette carte a ensuite été utilisée pour des analyses informatiques spécifiquesTout d'abord, les dynamiques du réseau des MAPKs dans les cancers de la vessie ont été analysés.Un modèle Booléen a été construit, représentant la réponse du réseau aux inputs d'intérêt.Les résultats de simulations systématiques ont été trouvés globalement cohérents avec des données publiques, et ont permis de déchiffrer les principaux événements qui sous-tendent les différents comportements observés dans le cancerEnsuite, la carte a été exploitée pour réanalyser des données publiques d'expression de gènes, avec l'objectif d'identifier les principaux acteurs de la transduction des signaux prolifératifs, dans des types cellulaires spécifiques.Des analyses du réseaux et des calculs statistiques ont conduit à l'identification de régions dérégulées dans le réseau des MAPKs, et à la délinéation de points d'intervention optimales dans cinq stades du cancer de la vessie et dans quatre sous-types de lymphome TL'ensemble de ces résultats a conduit à la formulation de nouvelles hypothèses concernant le fonctionnement du réseau des MAPKs dans différents états pathologiques, et à la sélection de composants cibles qui pourraient être envisagées pour le développement de nouveaux traitements / MAPK network consists of tightly interconnected signalling pathways. Although several studies established the involvement of this network in cancer deregulations, the precise mechanisms underlying its influence on the balance between cell proliferation and death remain elusive.Public data were integrated into a detailed reaction map, accounting for the influence of MAPK network on cell fate decision. This map was then used for computational analyses addressing specific cancer-related questions.First, the dynamics of MAPK network in bladder cancers were analysed. A Boolean model was built, accounting for the response of the network to selected inputs. The results of systematic simulations were found globally coherent with published data. Based on in silico experiments, the main events underlying different observed cancer cell behaviours were then deciphered.Next, the MAPK reaction map was exploited to reanalyse public high-throughput gene expression data. The goal was to identify key actors for the transduction of proliferative signals, in specific cell types. Network analyses and statistical computations led to the identification of deregulated MAPK network regions, and to the delineation of optimal intervention points aimed at blocking the proliferative signals transduced from such regions. This approach was used to study five different tumour stages and four different subtypes of T-cell lymphoma.Altogether, these results led to the formulation of novel hypotheses concerning the functioning of MAPK network in different pathological conditions, and to the selection of target components that might be considered for the development of novel treatments.

A dor é descrita como uma sensação sensorial e emocional desagradável de extrema importância para sobrevivência e integridade do organismo. As dores crônicas de origem neuropática são de difícil tratamento e seus mecanismos fisiopatológicos pouco conhecidos. Este estudo foi realizado após inoculação do vírus HSV-1 na pata traseira esquerda de camundongos machos da linhagem Balb/C, e teve como objetivo investigar comportamentalmente o desenvolvimento de alodínia mecânica e hipernocicepção nas fases herpética e pós herpética, caracterizar a atividade de vias intracelulares de sinalização das proteínas JNK, AKT CREB, P38, ERK, Glutamina Sintetase e Stat 3, através de western blot na coluna dorsal da medula espinal nas fases herpética e pós herpética, além de verificar a presença do vírus HSV-1 na medula espinal e gânglio dos animais por meio da reação em cadeia da polimerase em tempo real (RT-PCR). Os resultados evidenciaram alteração de sensibilidade nos animais a partir do 7º dia que permaneceu até o 28º dia após a inoculação do vírus. Houve uma mudança na expressão das proteínas MAPKs, com aumento na expressão de JNK, AKT e CREB no corno dorsal da medula no 8º e 21º dia após a inoculação do vírus HSV-1, aumento na expressão de P 38 e P ERK no 8º dia após inoculação do vírus e uma diminuição na expressão da proteína Stat 3 no 8º e 21º dia após a inoculação do vírus, sugerindo assim a participação dessas proteínas na alteração de sensibilidade tanto no período herpético quanto pós herpético. Também ocorreu um aumento na amplificação do DNA viral HSV-1 na medula espinal e gânglio espinal esquerdo no período herpético após inoculação do vírus HSV-1 / The pain is described as a sensorial and emotional unpleasant sensation of extreme importance for survival and integrity of the organism. The chronic pains that has neuropathic source are hard to treat and its physiopathologic mechanisms not well known. This study was performed after inoculation of the virus HSV-1 in the left back foot of male Balb/C mouse, and had as main objectives to behaviorally investigate the development of mechanical allodynia and hyper nociception during the herpetic and post-herpetic phase, characterize the activity of the intracellular signalization paths of the proteins JNK, AKT CRB, P38, ERK, glutamine synthetase and Sat 3 during herpetic and post-herpetic phase using Western Blot, besides checking as well for the presence of HSV-1 viral load in the spinal cord and ganglions using RT-PCR. The results evidenced alteration of sensitivity in the animals from the 7th day that remained until the 28th day after inoculation of the virus, a change in the MAPKs proteins expression, with a raise of expression of JNK, AKT e CREB in the dorsal horn of the spinal cord in the 8th and 21st day after the HSV-1 virus inoculation, thus suggesting the participation of these proteins in the alteration of sensitivity both in the herpetic and post herpetic periods. It is also possible to observe the presence of viral load in the spinal cord and left spinal ganglion in the herpetic period after HSV-1 virus inoculation

Unbegrentzte Selbsterneuerungkapazität und Pluripotenz sind charakteristische Merkmale von embryonalen Stammzellen (ES-Zellen). Dennoch sind die molekularen und zellulären Mechanismen, die für das Schicksal der ES-Zellen zuständig sind, noch nicht genau definiert. Um regulierende Faktoren des undifferenzierten Zustands von ES-Zellen zu identifizieren, wurden undifferenzierte ES Zellen, "Embryoid Bodies", spontan differenzierte und mit Retinsäure differenzierte ES Zellen mittels Microarray-Analysen verglichen. Neben bereits etablierten Pluripotenz-Markern, wurde Sprouty4 als eines der am stärksten degerulierten Transkripte unter diesen Bedingungen identifiziert. Sprouty4 ist als Inhibitor des ERK (Extracellular signal-regulated protein kinase)-Signalweges bekannt, aber seine Rolle in ES-Zellen wurde noch nicht definiert. Mittels Genexpression und Western BlotAnalysen konnte gezeigt werden, dass Sprouty4 in undifferenzierten ES-Zellen stark exprimiert ist und im Verlauf der Differenzierung schnell herunterreguliert wird. In vivo war Sprouty4 auf die innere Zellmasse (ICM) der Mausblastozyste beschränkt. Außerdem wurde gezeigt, dass der Sprouty4 Promotor durch direkte Bindung der PluripotenzMarkern Nanog, Klf4 und Stat3 reguliert wird. ES-Zellen, die Sprouty4 konstitutiv exprimieren, waren resistent gegen Differenzierung durch Zugabe von Retinsäure oder Bildung von Embryoid Bodies. Hingegen führte die Expression einer dominant-negativen Mutante von Sprouty4 zu einer erhörten Sensitivierung von ES-Zellen gegenüber der Differenzierung und zur Bildung extraembryonaler Gewebe begleitet von Endoreduplikation. Zusammenfassend konnten unsere Ergebnisse zeigen, dass die enge Regulation des ERK-Signalweges und warscheinlich anderer Signalwege durch Sprouty4 notwendig ist, um die Balance zwichen Pluripotenz und Differenzierung embryonaler Stammzellen zu kontrollieren. / A hallmark feature of embryonic stem (ES) cells is the ability to self-renew indefinitely while maintaining pluripotency. However, the molecular and cellular mechanisms underlying ES cell fate are poorly understood. To identify signaling pathway molecules that maintain the uncommitted state of ES cells, a microarray analysis was performed comparing undifferentiated ES cells, mature embryoid bodies, spontaneously differentiated and retinoic acid-induced differentiated ES cells. Among several well-validated pluripotency markers, Sprouty4 was identified as one of the most highly deregulated transcripts under these conditions. Sprouty4 is known as an inhibitor of the extracellular signal-regulated protein kinase (ERK/MAPK) pathway however its role in ES cells has not yet been defined. Gene expression and western-blot analyses have shown that Sprouty4 is highly expressed in ES cells and strongly downregulated upon differentiation whilst in vivo, Sprouty4 is confined to the founder population of ES cells, the inner cell mass of mouse blastocysts. Moreover, the Sprouty4 promoter was found to be regulated via the direct binding of the intrinsic pluripotency-associated factors Nanog, Klf4 and Stat3. ES cells engineered to constitutively express a wild-type version of Sprouty4 were found to be resistant to differentiation induced by retinoic acid or embryoid bodies formation. Conversely, expression of a dominant negative Sprouty4 mutant activating the ERK/MAPK pathway in a sustained manner sensitized ES cells to differentiation and triggered endoreduplication leading to the formation of extraembryonic tissue. Taken together, these results highlight the essential role of Sprouty4 in the tight regulation of the ERK/MAPK pathway- and probably others- for the balance between pluripotency and lineage commitment in mouse embryonic stem cells.

Chlamydia trachomatis ist ein obligat intrazellulär lebendes Bakterium. Es wird mit einer Vielzahl von Krankheiten in Verbindung gebracht. Das Überleben von Chlamydia trachomatis hängt in nahezu allen Aspekten von der Wirtszelle ab, angefangen bei der Anheftung an die Wirtszelle, über die Invasion, bis zur intrazellulären Replikation. Für ein vollständiges Bild der Pathogenese sind sowohl das Verstehen der dazu beitragenden bakteriellen wie auch Wirtszellfaktoren essenziell. Die vorliegende Arbeit konzentriert sich dabei auf die an der Infektion beteiligten Wirtsfaktoren. Mit Hilfe eines RNA-Interferenz-vermittelten Funktionsverlustscreens wurden 59 Wirtszellgene identifiziert, die einen Einfluss auf die Infektion und Vermehrung von Chlamydia trachomatis hatten. Unter Zuhilfenahme von bioinformatischen Signaltransduktionsweganalyse-Programmen konnten einige der Hits bekannten zellulären Signalnetzwerken, unter anderem dem Ras/Raf/Mek/Erk-Signalweg, zugeordnet werden. Insbesondere der Funktionsverlust zweier validierter Targets, Ras und Raf1, erhöhte das Chlamydien-Wachstum und deren Vermehrung. Bisher wurde angenommen, dass die bei der Chlamydien-Infektion beobachtete Aktivierung der Kinase Erk, die mit der Aktivierung der Phospholipase cPLA2, der Induktion des Interleukins 8 sowie der Stabilisierung des antiapoptotischen Faktors Mcl-1 in Verbindung steht, über den Ras/Raf/Mek-Signalweg vermittelt wird. Ich konnte jedoch zeigen, dass die Chlamydien-induzierte Erk-Aktivierung unabhängig von Ras und Raf1 stattfindet. Vielmehr wird während der Chlamydien-Infektion, Raf1, abhängig von der Kinase Akt, durch Phosphorylierung an Serin259 inaktiviert. Zudem wird das inaktivierte Raf1 zur bakteriellen Inklusion rekrutiert. Dies lässt vermuten, dass das Überleben derChlamydien und deren Wachstum nicht nur von der Erk-Aktivierung und dessen Substrate, sondern auch von der Inaktivierung von Raf1 und dessen Rekrutierung zur Inklusion abhängt. / Chlamydia trachomatis is a Gram-negative obligate intracellular bacterial pathogen with a major impact on human health. As an obligate intracellular pathogen, Chlamydia rely on host cell for all aspects of their survival. Here we present RNAi screen that identified 59 host cell genes influencing C. trachomatis infection and infectivity. Network analysis of hits revealed several prominent signaling networks, including Ras-Raf-MEK-ERK pathway. Knockdown of Ras and Raf1 components of the aforesaid pathway led to increased Chlamydial growth and survival. In Chlamydia infections, ERK activation is believed to be activated through upstream kinases Ras-Raf-MEK and involved in activation of cPLA2, induction of IL8 and stabilization of the anti-apoptotic Mcl-1. However, I could show that ERK activation after Chlamydia infection is independent of Ras and Raf1. Moreover, I also showed that Raf1 is inactivated by phosphorylation at Ser259 in an Akt dependent manner. Consequently, the Ser259 phosphorylated Raf1 was recruited to the Chlamydia inclusion in an Akt and 14-3-3β dependent manner. This strongly suggests that Chlamydia survival and replication in the host cell depends not only on the activation of ERK and its downstream targets such as cPLA2, but also on the inactivation of Raf1 by phosphorylation and recruitment to the inclusion.

Phosphorylierung und Dephosphorylierung gehören zu den zentralen Regulationsmechanismen jeder Zelle. Während einer der bekanntesten Signalwege, der Mitogen-aktivierte Protein-Kinase(MAPK)-Signalweg bereits intensiv untersucht wurde, ist über die MAPK-Phosphatasen als wesentliche Inaktivatoren dieser Signalwegfamilie bisher nur wenig bekannt. Die MAPK-Signalwege sind in Tumoren häufig aktiv. Dies ließe sich durch aktivierende Mutationen der Kinasen oder ihrer übergeordneten Rezeptoren erklären. Ein anderer Ansatz geht von der stromalen Entzündungsreaktion aus, die viele solide Tumoren begleitet. Zytokine, die durch die Entzündungszellen gebildet werden, sind physiologische Aktivatoren der MAPK und eine fehlende Gegenregulation durch inaktivierende Phosphatasen würde ebenso zu hoher Aktivität der MAPK führen. In der vorliegenden Arbeit wurde ein solches Entzündungsgeschehen in Ovarialkarzinomzellinien simuliert und die Reaktion der Phosphatasen MKP-1 und MKP-3 auf proinflammatorische Zytokine untersucht. MKP-1 und MKP-3 reagierten mit erheblichen Unterschieden auf die Zugabe proinflammatorischer Zytokine, das Reaktionsmuster reichte von starker Aktivierung (MKP-1 in SKOV-3 und OVCAR-3) bis hin zu verringerter Aktivität der Phosphatasen (MKP-1 in OAW42 und CAOV-3). Zur Ergänzung der Zellkulturstudien wurde die Expression der Phosphatase MKP-1 in primären Ovarialkarzinomen, Zystadenomen sowie gesunden Ovarien immunhistochemisch untersucht. Der Proteinnachweis in insgesamt 101 Gewebeproben ergab eine signifikant geringere Expression der Phosphatase MKP-1 in Karzinomen im Vergleich zu normalem Ovarialepithel oder Zystadenomen. Innerhalb der Gruppe der Karzinome zeigte die MKP-1-Expression dennoch eine hohe Varianz, hierbei waren Malignome mit deutlicher MKP-1-Expression mit einer wesentlich schlechteren Prognose der Erkrankung verbunden. Die Phosphatase war in der multivariaten Analyse des rezidivfreien Überlebens ein unabhängiger Prognoseparameter (RR=4,03; 95%CI=1,72-9,48; p=0,001). Ein kürzeres rezidivfreies Überleben ist häufig mit der frühen Entwicklung von Chemoresistenzen verbunden. Ein möglicher Zusammenhang zwischen der Phosphatase MKP-1 und Chemoresistenz wurde in Zellkulturversuchen unter Verwendung von Cisplatin, einem wesentlichen Bestandteil der Standardchemotherapie bei Ovarialkarzinomen, untersucht. Die Expression der MKP-1 konnte durch Zugabe von Cisplatin deutlich induziert werden. Bemerkenswerterweise zeigten resistente Zellinien dabei eine frühe Reaktion, sensible Zellen reagierten deutlich verzögert. Diese frühe Induktion der MKP-1 könnte die therapeutisch induzierte Apoptose blockieren. Weitere Erkenntnisse über die daran beteiligten Signalwege sowie pharmakologische Inhibitoren der Phosphatasen sind daher vielversprechende Ansätze zur Optimierung der Chemotherapie. / Protein phosphorylation and dephosphorylation is a central regulatory system of cells. The mitogen-activated-protein-kinase(MAPK)-pathway as a typical example is one of the most investigated signalling pathways in cancers. In contrast, much less is known about MAPK-phosphatases, their physiological inactivators. MAPK pathways are frequently up-regulated in cancers. This might be explained by activating mutations of kinases or of up-stream receptors. Another view is based on the inflammatory stroma infiltrate that accompanies most solid carcinomas. Cytokines produced by inflammatory cells are physiological activators of MAPK pathways and missing balance of inactivating phosphatases would also result in up-regulated MAPK pathways. In this study, such an inflammatory situation was simulated in cell culture models and expression patterns of MAPK-phosphatases MKP-1 and MKP-3 were investigated after addition of proinflammatory cytokines. The expression of MKP-1 and MKP-3 after cytokine addition differed widely between the ovarian cancer cell lines investigated, ranging from strong induction in SKOV-3 and OVCAR-3 to down-regulation of phosphatases in OAW42 and CAOV-3. In addition to cell culture experiments, expression of MKP-1 was examined immunohistochemically in primary ovarian cancers, adenomas and normal ovaries (total of 101 samples). There was a lower expression of phosphatase MKP-1 in ovarian cancers compared to surface epithelium of normal ovaries and cystadenomas. However, MKP-1 expression in the group of carcinomas showed a high variation, including also a number of negative cases. Among all investigated cancer samples, those with a higher expression of MKP-1 were associated with poorer prognosis. Multivariate survival analysis revealed this phosphatase as an independant prognostic factor for progression-free survival (RR=4,03; 95%CI=1,72-9,48; p=0,001). Short progression-free survival is usually associated with early development of chemoresistance. Consequently, poor prognosis might result from different efficiencies of initial adjuvant chemotherapeutical treatments. Based on this presumption, the effects of cisplatin, a typically used drug against ovarian cancer, were investigated in cell culture. The phosphatase MKP-1 was highly inducable by cisplatin, remarkably as early reaction in cisplatin-resistant cell lines and with distinct delay in sensitive cells. This early induction of MKP-1 in resistant cells might block drug-induced apoptosis. Further studies about influencing pathways and pharmacological inhibitors of phosphatase MKP-1 might be promising efforts to optimize chemotherapy.

A dor neuropática pode ser causada por lesões e/ou disfunções no sistema somatossensorial. Nestes tipos de dores, alterações plásticas ao longo de todo o sistema sensorial nociceptivo estão associadas à cronificação do processo doloroso. A plasticidade observada pode ser resultante da indução e/ou repressão de genes, os quais geralmente são modulados por fatores de transcrição. Um dos principais fatores de transcrição até então conhecido é a proteína ativadora-1 (AP-1), que pode ser estruturalmente formado principalmente por proteínas das famílias Jun e Fos. Entretanto, na dor neuropática, a participação e o papel do AP-1 não estão bem elucidados. Dessa forma, a hipótese deste trabalho é que a ativação do AP-1 contribua para a indução e/ou manutenção da dor neuropática, através da ativação de células gliais e de proteinocinases ativadas por mitógenos (MAPK) e por indução da produção e liberação de mediadores pró-inflamatórios, bem como de metaloproteinases da matriz extracelular (MMP) na medula espinal. Esses fatores contribuem para sensibilização central causada pela SNI, facilitando a transmissão dolorosa. Assim, a inibição do AP-1 seria uma potencial estratégia terapêutica no tratamento da dor neuropática. Foi utilizado o modelo experimental de dor neuropática de lesão limitada do nervo isquiático (SNI, Spared Nerve Injury) em camundongos, os quais receberam injeção intratecal (i.t.) do inibidor de AP-1, SR11302, ou seu veículo (DMSO, tween® 20 e salina). O tratamento com o inibidor de AP-1 reduziu a hipernocicepção mecânica causada pela SNI, e o perfil de redução sugeriu que esse fator de transcrição esteja relacionado com a manutenção da dor neuropática. No sétimo dia após a SNI, observou-se na medula espinal dos camundongos, ativação da microglia, dos astrócitos e das MAPK, além de aumento na expressão de TNF-, interleucina (IL)-6, IL-1, IL-17A, quimiocina derivada de queratinócito (KC), proteína quimiotáxica de monócitos (MCP-1), óxido nítrico (NO), NO sintase induzível e das MMP-2 e -9. Todos esses eventos estão associados à sensibilização central, portanto, contribuem para a facilitação da transmissão nociceptiva. O tratamento com o inibidor de AP-1 SR11302 impediu, pelo menos parcialmente, a ativação das células gliais e das MAPK e bloqueou o aumento na expressão de todos esses mediadores pró-inflamatórios e das MMPs na medula espinal. Assim, o fator de transcrição AP-1 e, consequentemente, suas vias a jusante (downstream) são potenciais alvos farmacológicos no tratamento da dor neuropática. / Neuropathic pain results from nerve damage or dysfunction, which is associated to the painful process of chronification. This process may include participation of the inducible genes, which may be modulated by transcription factors, including the activator protein-1 (AP-1), which can structurally be formed by proteins from Jun and Fos families. However, the participation and the role of AP-1 neuropathic pain remain unclear. Our hypothesis is that the activation of AP-1 would contribute for the induction and/or maintenance of neuropathic pain, by inducing the glial cells activation and mitogen-activated protein kinases, and by inducing the production/release of pro-inflammatory mediators and extracellular matrix metalloproteinase (MMP) in mices spinal cord. All these factors are contributing to SNI-evoked central sensitization, facilitating pain transmission. Thus, inhibition of AP-1 would be a potential drug target in the treatment of neuropathic pain. The animals received inhalatory anesthesia (2% isoflurane) and were submitted to an experimental model of neuropathic pain Spared Nerve Injury (SNI). The animals were treated intrathecally (i.t.) with AP-1 inhibitor SR11302 or vehicle (DMSO, tween®20 and saline). Treatment with AP-1 inhibitor reduced the SNI-induced mechanical hypernociception, suggesting that this transcription factor is related to the maintenance of neuropathic pain. On the seventh day after SNI, there was in the spinal cord of mice microglia, astrocytes and MAPK activation, increased of expression of TNF-, interleukin (IL)-6, IL-1, IL-17A, keratinocyte-derived chemokine (KC), monocyte chemoattractant protein-1 (MCP-1), nitric oxide (NO) and inducible NO synthase, and increased the expression of MMP-2 and -9. All of these effects are related with central sensitization, thus facilitating nociceptive transmission. However, treatment with SR11302 prevented, at least partially, activation of MAPK and glial cells, as well as prevented the increase in expression of all these pro-inflammatory mediators and MMPs in the spinal cord. Thus, inhibition of AP-1 and hence its way downstream is a potential pharmacological target in the treatment of neuropathic pain.

A musculatura esquelética tem um papel fundamental para a manutenção da homeostase do organismo. A perda de massa muscular está relacionada a prejuízos na qualidade de vida de indivíduos saudáveis, além de piorar o prognóstico de pacientes com doenças sistêmicas, como o câncer, o diabetes e a insuficiência cardíaca. Em quadros mais graves de insuficiência cardíaca, a perda excessiva de massa muscular associada a um reduzido consumo de oxigênio de pico, são considerados como preditores independentes de mortalidade. O aumento do estresse oxidativo tem sido apontado como um dos principais desencadeadores do aumento da degradação de proteínas na atrofia muscular. Na presente tese, investigamos a contribuição do estresse oxidativo para a ativação das vias de sinalização NF-kB, FOXO e MAPK na atrofia muscular desencadeada pela insuficiência cardíaca. Para compreender melhor os mecanismos envolvidos na ativação dessas vias pelo estresse oxidativo, utilizamos a linhagem de células musculares C2C12. Observamos que o tratamento com peróxido de hidrogênio (1,2mM, 12h) induziu um aumento do estresse oxidativo, o qual foi capaz de aumentar a atividade do proteassoma, desencadeando a atrofia dos miotúbulos. Verificamos também um aumento da expressão proteica de alguns componentes dessas vias de sinalização, como p-p38 e NF-kB; apontando para uma ativação diferenciada dessas vias pelo estresse oxidativo. Para verificar se essas vias de sinalização relacionadas ao estresse oxidativo estavam também relacionadas à atrofia desencadeada pela insuficiência cardíaca, avaliamos um modelo experimental de ratos com insuficiência cardíaca induzida pelo infartado do miocárdio. Observamos uma redução da área de secção transversa do músculo plantar, acompanhada de um aumento da inflamação sistêmica, de p38 e das atividades de NF-kB e do proteassoma. Como o treinamento físico aeróbico tem se apresentado como uma estratégia terapêutica não farmacológica eficaz na redução do estresse oxidativo e no restabelecimento da atividade do sistema ubiquitina proteassoma, submetemos os ratos infartados ao treinamento físico aeróbico em esteira rolante. O treinamento físico aeróbico preveniu a perda de massa muscular, reduzindo a inflamação sistêmica e as atividades de NF-kB e do proteassoma. Em conjunto, os resultados apontam para o estresse oxidativo como um fator preponderante para o aumento da degradação de proteínas relacionada à atrofia muscular, seja por indução de inflamação (TNF-α) ou por sua ação direta. Além disso, observamos que as vias de sinalização são ativadas de forma diferenciada nos dois modelos, sugerindo que a degradação de proteínas nos miotúbulos está relacionada ao controle de qualidade de proteínas e, nos ratos infartados, às alterações do metabolismo, servindo como fonte de energia. Já o treinamento físico aeróbico comprovou sua eficácia no restabelecimento da atividade do proteassoma, reduzindo a inflamação e a atividade de NF-kB, prevenindo assim, a perda de massa muscular / About 40% of human body mass consists of skeletal muscles, which are involved in all aspects of movement including breathing, eating, posture, walking and reflexes. Skeletal muscle is also important as a source of heat generation and as a regulator of intermediary metabolism. Loss of skeletal muscle mass and function (skeletal muscle atrophy) leads to several functional impairments, affecting health and quality of life. It occurs in several chronic diseases such as cancer, diabetes and heart failure. In heart failure, atrophy is considered an independent predictor of poor prognosis. Oxidative stress has a crucial role in atrophy, activating different signaling pathways capable of stimulating the ubiquitin proteasome system to degrade proteins. In this study, we investigated the oxidative stress contribution to NF-kB, FOXO and MAPK signaling pathway activation in heart failure-induced atrophy. To better understand the mechanisms involved with oxidative stress and signaling pathways activation in atrophy, we have used C2C12 skeletal muscle cells. We observed that, even in high hydrogen peroxide concentrations, oxidative stress increased proteasome activity, phosphorylated p38 and NF-kB protein expression, causing myotubes atrophy. In an experimental heart failure model of infarcted rats, we evaluated plantaris muscle and verified a reduced cross sectional area, accompanied by increased systemic inflammation, p-38 protein expression and increased both NF-kB and proteasome activities. As aerobic exercise training causes a lot of beneficial effects on skeletal muscle structure and function in chronic diseases, we submitted infarcted rats to 8 weeks of aerobic exercise training on a treadmill. Aerobic exercise training prevented atrophy by reducing inflammation and both NF-kB and proteasome activities. Collectively, our data suggest a differentiated activation by oxidative stress in muscle cells and animal models. In the first case, protein degradation was involved with protein quality control; and, in the other, oxidative stress is a second messenger, stimulating protein degradation to provide substrates to metabolism. Aerobic exercise training re-established proteasome activity by reducing inflammation and NF-kB activity, preventing muscle atrophy

Carcinomas papilíferos (CP) estão relacionados à ativação da via de sinalização MAPK por diversos oncogenes. Estudos têm relacionado a incidência de CP com a disponibilidade de iodo, um dos mais importante reguladores da função tiroidiana. Recentemente, nosso grupo descreveu que a inibição da via de sinalização TGF<font face=\"symbol\">b pode estar vinculada ao desenvolvimento de CP. Nosso objetivo foi avaliar os efeitos do excesso de iodo na transformação de células tiroidianas. Utilizamos células PCCL3 com indução do oncogene RET/PTC3, tratadas com excesso de iodo. Observamos que o iodo reduziu a proliferação, sem alterar a morte das células e recuperou a expressão de genes específicos tiroidianos, evento geralmente vinculado à transformação maligna tiroidiana. O tratamento com excesso de iodo, ainda, reduziu a expressão de proteínas envolvidas na via MAPK e promoveu o restabelecimento da expressão da via TGF<font face=\"symbol\">b, uma importante via inibitória da proliferação de células tiroidianas. Concluímos que o excesso iodo tenha uma ação antioncogênica durante a transformação maligna tiroidiana. / Papillary Carcinomas (PC) are frequently related to MAPK signaling pathway, activation by several oncogenes. Recent studies had related PC incidence and iodine availability, one of the most important thyroid function regulators. Our group has recently showed that TGF<font face=\"symbol\">b pathway inhibition is directly related to PC development. Our aim was to evaluate iodine excess effects during thyroid cells transformation. We used the PCCL3 cells with RET/PTC3 oncogene induction, treated with iodine excess. We observed that iodine reduced the cell proliferation, not altering cell death and recovered thyroid specific genes expression, event frequently liked to thyroid cells malignant transformation. Iodine excess treatment also reduced MAPK proteins expression and reestablished TGF<font face=\"symbol\">b pathway components expression, an important inhibitory pathway of epithelial cells. We can conclude that iodine excess treatment plays an antioncogenic role during thyroid malignant transformation.

Le virus de la fièvre catarrhale ovine (Bluetongue virus, BTV) est l’agent étiologique de la maladie du même nom, une arbovirose non contagieuse transmise aux ruminants domestiques et sauvages par l’intermédiaire de morsures de moucherons hématophages du genre Culicoides. Il existe actuellement 27 sérotypes décrits de BTV à travers le monde qui se distinguent par les pathologies qu’ils induisent et leur capacité à infecter et se propager chez leur(s) hôte(s) mammifère(s). Le premier objectif de mon projet de thèse visait à identifier les interactions cellulaires spécifiques des sérotypes 8 et 27 pour identifier des facteurs de pathogénicité/virulence et/ou de franchissement de barrière d’espèces. Pour atteindre cet objectif, l’ensemble des protéines virales du BTV a été criblé par la méthode du double-hybride en levure contre deux banques d’ADN complémentaires, l’une d’origine bovine et l’autre d’origine culicoïde. A l’issue de 70 cribles, une centaine de nouvelles interactions virus-hôtes a été mise en évidence et révèle un enrichissement pour quatre processus cellulaires : l’épissage des ARNm, les ribosomes, la SUMOylation et l’apoptose. Cette étude nous a ainsi permis de réaliser le premier interactome pour le BTV qui se poursuit au travers de multiples validations biochimiques et fonctionnelles des interactions identifiées. En parallèle de ce travail de protéomique, le second objectif de mon projet de thèse a été de déterminer l’impact du BTV sur la voie MAPK/ERK, une voie cellulaire essentielle à la prolifération et différenciation cellulaire et classiquement modulée lors d’infections virales. En plus de son rôle antagoniste sur la voie des interférons de type I, nous avons démontré la capacité de la protéine NS3 de BTV à activer la voie MAPK/ERK. En effet, nous avons démontré que NS3 a la capacité d’augmenter le niveau de phosphorylation des protéines kinases ERK1/2 mais également du facteur de traduction eIF4E. Cette fonction, qui semble être spécifique au BTV par rapport aux autres orbivirus, implique l’interaction de NS3 avec la protéine cellulaire BRAF, une protéine MAP3 kinase jouant un rôle majeur dans l’activation de la voie MAPK/ERK. L’activation cette voie par NS3 pourrait être un mécanisme de détournement de la traduction cellulaire au profit de celle du virus mais aussi constituer un élément de réponse pour expliquer l’hyper-inflammation observée dans le cas d’une infection par ce virus / Bluetongue virus (BTV) is the etiological agent of the bluetongue (BT) disease, a non-contagious arbovirus that affects a wide range of wild and domestic ruminants. It is transmitted by blood-feeding midges of the genus Culicoides. There are currently 27 serotypes described of BTV in the world that are distinguished by their differences in term of pathology/virulence and their capacity to infect and disseminate in their mammalian host(s). The first objective of my thesis project was to identify specific cellular interactions of serotype 8 and 27 to reveal new factors of pathogenicity/virulence and/or cross species barrier. To reach this goal, all the proteins encoded by BTV were used as baits to screen, by a high-throughput yeast two-hybrid (Y2H) approach, two complementary DNA libraries originating from hosts naturally infected by BTV : Culicoides and cattle. Therefore, 70 screens were performed to identify a hundred of new virus-host interactions and reveal an enrichment for four cellular processes : mRNA splicing, ribosomes, SUMOylation and apoptosis. This study allowed us to build the first interactome of BTV which continues through multiple biochemical and functional validations of the identified interactions. In parallel to this proteomics work, my second objective was to determine the impact of BTV on the MAPK/ERK pathway, a cellular pathway essential for cell proliferation and differentiation usually modulated during viral infections. In addition to its antagonist role on the type I interferon pathway, we have demonstrated the ability of BTV-NS3 to activate the MAPK/ERK pathway. Indeed, we have demonstrated that NS3 has the ability to increase the level of phosphorylation of ERK1/2 protein and the eIF4E translation factor. This function, which seems to be specific to BTV compared to other orbiviruses, involves the interaction of NS3 with BRAF cellular protein, a MAP3 kinase protein that plays a major role in the regulation of the MAPK/ERK pathway. These results could provide a better understanding of the molecular basis underlying the hijacking of the translation machinery to support virus replication but also constitute a hypothesis to explain the hyperinflammation observed in the BTV infection context