Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2008. / Vita. Bibliography: p.116-124

La migration cellulaire collective (MCC) est un processus fondamental qui intervient au cours du développement, de la réparation tissulaire, de l'invasion tumorale et de la formation de métastases. Les cellules qui migrent collectivement possèdent deux types d'interaction avec leur environnement : i) l'un avec leur substrat et ii) l'autre avec les cellules voisines en migration. Deux grandes familles de protéines permettent ces interactions ainsi que la génération de forces mécaniques: i) la famille des intégrines (les récepteurs de la matrice extracellulaire) et ii) la famille des cadhérines (formant les jonctions intercellulaires). Les cadhérines classiques sont impliquées dans la formation des jonctions intercellulaires et sont les principaux acteurs de la MCC au cours du développement normal et tumoral. La transmission de force entre les cellules en migration est nécessaire à leur cohésion et à la communication des cellules entre elles. Des études récentes montrent que les cadhérines sont nécessaires à la transmission des forces au substrat. Néanmoins, les processus par lesquels les cadhérines agissent sur ses forces dans le contexte d'une MCC restent inexplorés. Nous avons identifié l'expression de la P-cadhérine comme étant associée à l'agressivité du rhabdomyosarcome de type alvéolaire (ARMS), sous type ayant le plus mauvais pronostic car très invasif. Nos données, ainsi que de récentes études qui démontrent que la P-cadhérine est impliquée dans l'agressivité des tumeurs du sein, nous ont conduits à étudier le rôle de cette cadhérine dans la migration cellulaire, processus majeur dans le développement tumoral. Nous nous sommes intéressés à l'impact de l'expression de la P-cadhérine sur la migration des myoblastes murins normaux C2C12. Pour ce faire nous utilisons un test de migration in vitro en 2D proche du test de blessure qui consiste à retirer une barrière physique induisant la migration des cellules vers l'espace libre ainsi créé. Nous avons pu monter que l'expression de la P-cadhérine dans les myoblastes C2C12 augmente les paramètres caractéristiques d'une MCC in vitro : la vitesse, la polarité, la persistance et la directionalité de la migration des cellules du front et au sein du feuillet. De plus, à l'aide de techniques microscopiques de mesure des forces nous avons montré une augmentation des forces intercellulaires allant du front vers le feuillet cellulaire au cours de la migration des cellules exprimant la P-cadhérine. Cela suggère une augmentation de la cohésion cellulaire. L'ensemble de ces résultats démontrent clairement que l'expression de la P-cadhérine induit une MCC. Nous avons aussi mesuré et cartographié les forces de traction au substrat connues pour être le moteur de la migration cellulaire. Nos données indiquent que l'expression de la P-cadhérine augmente l'anisotropie de ces forces de traction ainsi que leur intensité, et ce, uniquement au front de migration. L'expression de la P-cadhérine remodèle et stimule la dynamique des plaques focales d'adhérence à cet endroit.Afin de mieux comprendre comment la P-cadhérine modifie la dynamique des adhésions focales et augmente les forces de traction, nous avons étudié l'activité spatiotemporelle des petites protéines G de la famille Rho. Nous montrons que l'expression de la P-cadhérine active Rac1 et Cdc42 au front de migration, entrainant ainsi le remodelage et l'organisation des plaques focales d'adhérence à cet endroit. L'inhibition de Rac1 et Cdc42 bloque la MCC induite par la P-cadhérine. Pour conclure, en combinant la mesure des paramètres de migration cellulaire avec la mesure des forces mécaniques intercellulaires et au substrat, nous avons démontré que la P-cadhérine induit un comportement collectif des cellules et ce dépendamment de l'activité de Rac1 et de Cdc42. De plus nous mettons en avant l'existence de propriétés mécano-transductrices de cette cadhérine au cours de la MCC. / Collective cell migration (CCM), the coordinated movement of multiple cells that are connected by cell-cell adhesion, is a fundamental process in development, tissue repair and tumor invasion and metastasis. Cells part of a moving collective have two different types of interactions, i) one with the substratum, and ii) one with surrounding moving cells. Two protein families allow these interactions and also the generation of mechanical forces: i) typically integrins on the underlying extracellular matrix (ECM) and ii) cadherins at intercellular adhesion sites. Classical cadherins are involved in cell-cell adhesion and are major drivers of collective cell migration in embryonic development and tumorigenesis.Mechanical coupling between migratory cells may result in the production of force-dependent signals by which the cells influence each other. Moreover, whereas recent data showed that cadherin-dependent cell-cell adhesions are important for the force transmission to the ECM, how intercellular adhesion impacts on cell-ECM forces in the context of collective cell migration is totally unexplored. We identified P-cadherin expression to be associated with alveolar rhabdomyosarcoma (ARMS) aggressiveness, tumors with a bad prognosis due to the propensity for early and wide dissemination. Our data and recent findings showing that P-cadherin is associated with breast tumor invasiveness and aggressiveness, led us to investigate the role of P-cadherin in cell migration. We analyzed cell migration of normal mouse C2C12 myoblasts that express P-cadherin using a “wound-healing like assay” in which migration is analyzed after removal of a physical barrier. We observed that P-cadherin expression in C2C12 myoblasts increased the speed, polarity, persistence and directionality of migration toward the free space of both cells at the border and cells into the sheet. Using monolayer stress microscopy we showed that P-cadherin increases inter-cellular stresses and force transmission across the cell sheet. According to those observations we concluded that P-cadherin induces CCM.Traction forces exerted by the cells on the substrate are important for cell migration. Using traction force microscopy, we demonstrated that P-cadherin expression increases the traction forces anisotropy specifically at the multicellular leading row. To better understand how these mechanical signals induce CCM, we studied both the organization of the focal adhesions and the spatio-temporal activity of Rho GTPase. We showed that P-cadherin expression activates Rac1 and Cdc42 which induces extensive focal adhesions remodeling at the leading edge of cells at the leading row. Rac1 and Cdc42 inhibition impaired P-cadherin-induced CCM, focal adhesion remodeling and forces generation. In conclusion, combining a detailed measurement of the parameters of cell migration with physical measure of the intercellular stresses and traction forces, we have shown that P-cadherin promotes collective behavior of cells during migration through Rac1 and Cd42 activity. Also, those results provide evidence for mechano-transmission properties of P-cadherin during collective cell migration.

En plus des anomalies génétiques, plusieurs études ont rapporté des altérations des protéines chez les patients atteintes de Syndromes Myéloprolifératifs (SMP) Ph- qui pourraient participer à leurs phénotypes cliniques. Néanmoins, les altérations protéiques dans ces pathologies ne sont pas bien connues. Dans ce contexte, nous avons utilisé une approche protéomique par spectrométrie de masse pour nous aider à déchiffrer le paysage des anomalies des protéomes érythrocytaire et granulocytaire qui pourraient être liées à des altérations cellulaires fonctionnelles et à la physiopathologie des SMP. Nous avons ainsi pu identifier des dérégulations importantes de protéines qui varient selon le statut génétique des patients, [JAK2(+), JAK2(-) ou CALR(+)], selon la charge allélique de JAK2V617F, mais aussi selon le type de SMP JAK2(+) ou selon la. Ces dérégulations protéiques perturbent des voies de signalisation comme la voie IQGAP1/Rho GTPase qui pourrait être liée aux thromboses par des modifications de l'intégrité membranaire via la dérégulation de PAK1 ou des protéines du cytosquelette d'actine. Des modifications des voies de signalisation des ROS ou mTOR ont été également identifiées dans les granulocytes. En outre, nous avons montré que la protéine CALR pourrait avoir un double rôle oncogénique grâce à son expression élevée dans les SMP JAK2V617F, différent de l'altération de sa fonction décrites dans les patients CALR mutés. En conclusion, nous avons montré que les dérégulations protéomiques pourraient jouer un rôle oncogénique important dans la physiopathologie des SMP Ph- et qu’elles pourraient être impliquées dans certaines complications de ces pathologies telles que les accidents thrombotiques. / Besides genetic abnormalities, several studies have reported protein alterations in Ph-Myeloproliferative neoplasms (MPN) which could participate to the clinical phenotype of patients. Nevertheless, little is known about protein alterations in these pathologies. In this context, we used an integrative proteomic approach to decipher the landscape of the erythrocyte and granulocyte proteome abnormalities that could be related to functional cell alterations and to MPN physiopathology. We could identify significant protein deregulations that varied not only according to genetic status JAK2(+), JAK2(-) or CALR(+) but also among JAK2(+) MPNs or depending on the JAK2V617F allele burden. These protein deregulations involved pathway alterations such as the IQGAP1/Rho GTPase signaling that could be related to thrombosis via alterations on the membrane integrity by deregulation of PAK1 or the actin cytoskeleton signaling. ROS or mTOR signaling alterations were also identified in granulocytes. Finally, we stress out that CALR protein could have a dual oncogenic role through its up-regulation in JAK2V617F MPNs different from its altered function described in CALR mutated patients. Altogether, we showed that proteomic deregulations might play an important oncogenic role in MPN physiopathology and could be implicated in complication as thrombotic accidents

Orientador: Edson Antunes / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T11:05:17Z (GMT). No. of bitstreams: 1 Calmasini_FabianoBeraldi_M.pdf: 937196 bytes, checksum: e7b4d280e06537641c107fb45b2ceb67 (MD5) Previous issue date: 2013 / Resumo: A próstata é densamente inervada pelo sistema nervoso autônomo simpático, parassimpático e não-adrenérgico não-colinérgico (NANC) os quais atuam sinergicamente para o correto funcionamento deste órgão. O principal representante da inervação NANC é o óxido nítrico (NO), porém sua função na próstata ainda não está bem definida. Sendo assim, o presente trabalho utilizou o modelo de inibição crônica da NO sintase (NOS) com L-NAME para avaliar possíveis alterações funcionais, morfológicas e bioquímicas da musculatura lisa prostática (MLP) em ratos adultos Wistar (200-250 g). Os ratos foram tratados por 4 semanas com L-NAME na dose de 20 mg/kg/dia através de ingesta hídrica. Especificamente, realizamos os seguintes experimentos na próstata dos ratos controles e tratados com L-NAME: 1) curvas concentração-efeito in vitro à fenilefrina (agonista ?1-adrenérgico) e ao carbacol (agonista muscarínico não seletivo), na presença e ausência do inibidor seletivo da rho-quinase Y27632 (1 uM), e curvas concentração-efeito ao agonista purinérgico, ?,?-metileno ATP, e à estimulação elétrica (contração neurogênica); 2) curvas concentração-efeito a agentes relaxantes como nitroprussiato de sódio (doador de NO), Y27632 (inibidor da rho-quinase) e isoproterenol (agonista ?-adrenérgico); 3) determinação dos níveis de AMPc e GMPc; e 4) análise histomorfométrica. A próstata dos animais tratados cronicamente com L-NAME apresentou aumento em seu peso relativo, alterações histomorfométricas caracterizadas como aumento na área epitelial e na muscular lisa e diminuição no índice de contorno dos ácinos. A contração da MLP induzida pela fenilefrina, carbacol, ?,?-metileno ATP e estímulo elétrico foi maior nos animais tratados cronicamente com L-NAME em relação aos animais controle. A incubação in vitro com o inibidor da rho-quinase, Y27632, restaurou ao nível controle as contrações aumentadas induzidas pela fenilefrina e carbacol no grupo L-NAME. O relaxamento da MLP nos animais do grupo L-NAME produzido pelo isoproterenol foi significativamente menor comparado ao grupo controle. Não houve diferença para o relaxamento induzido pelo SNP e Y27632 entre os grupos. Os níveis de AMPc e GMPc na próstata dos animais tratados cronicamente com L-NAME foram significativamente menores em relação aos animais do grupo controle. O tratamento com L-NAME não alterou os níveis das espécies reativas de oxigênio. Em suma, nossos dados mostram que a inibição crônica de NO leva a alterações funcionais in vitro da MLP caracterizadas por aumento na resposta contrátil à fenilefrina, carbacol, ?,?-metileno ATP e ao estímulo elétrico, além de redução no relaxamento ?-adrenérgico. A inibição da via da rho-quinase in vitro restaurou os padrões contráteis para os agonistas adrenérgicos e colinérgicos. A deficiência crônica de NO gera alterações funcionais, bioquímicas e morfológicas em próstata de ratos com participação da via da rho-quinase nesse processo / Abstract: The prostate is densely innervated by the sympathetic and parasympathetic as well as non-adrenergic non-cholinergic (NANC) autonomic innervation that act synergistically for its correct functioning. The main NANC mediator is nitric oxide (NO), but the role of this mediator in the regulation of prostate smooth muscle (PSM) is not well established. Thus, the present study used the model of chronic NOS inhibition with L-NAME to evaluate the functional, morphological and biochemical alterations in PSM of Wistar adult male rats (200-250 g). Rats were treated with L-NAME (20 mg/kg/day) for 4 weeks in the drinking water. Specifically, we performed the following experiments in prostates from control and L-NAME-treated rats: 1) in vitro concentration-response curves to phenylephrine (?1-adrenergic agonist) and carbachol (non-selective muscarinic agonist) in the presence and absence of the selective rho-kinase inhibitor Y27632 (1 ?M) and concentration-response curves to the purinergic agonist ?-?-methylene-ATP, as well as electrical stimulation (neurogenic contraction); 2) in vitro concentration-response curves to the relaxing agents sodium nitroprusside (NO donor), Y27632 (rho-kinase inhibitor) and isoproterenol (non-selective ?-adrenergic receptor agonist); 3) determination of cAMP and cGMP levels, and 4) histomorphometric analysis. The relative weight of the prostate from L-NAME-treated rats was greater than control animals. Histomorphometric changes characterized by an increase in epithelial acini and smooth muscle area, as well as by a decrease in luminal contour índex were found in L-NAME treated rats. The phenylephrine and carbachol-induced prostate contractions were higher in L-NAME compared with control group. Prior incubation of PSM with the rho-kinase inhibitor Y27632 (1 ?M) significantly inhibited the enhanced carbachol- and phenylephrine-induced PSM contractions in L-NAME group, restoring the Emax to control levels. The PSM contractions induced by ?,?-methylene ATP and electrical field stimulation were also greater in L-NAME compared with control group. The PSM-induced relaxations in response to SNP remained unaltered, whereas isoproterenol-induced relaxations were lower in L-NAME compared with control group. The cAMP and cGMP levels in prostate homogenate were lower in L-NAME compared with control group. No difference was found in reactive oxygen species levels. In summary, our data showed that chronic NO inhibition led to in vitro functional changes in PSM characterized by increased contractile response to phenylephrine, carbachol, ?-?-methylene-ATP and electrical-field stimulation, accompanied by reduction of ?-adrenergic-induced relaxation. The in vitro inhibition of rho-kinase pathway restored the contractile pathway for the adrenergic and cholinergic agonists. The chronic NO-deficiency generates functional, biochemical and morphological changes in rat prostate with involvement of the rho-kinase pathway in this process / Mestrado / Farmacologia / Mestre em Farmacologia

La sécrétion d'hormones et de neuropeptides par les cellules neuroendocrines est assurée par un processus d'exocytose, contrôlé notamment par les GTPases Rho. La compréhension des mécanismes moléculaires régulant la sécrétion neuroendocrine est primordiale. En effet, la majorité des cancers neuroendocrines tels que les phéochromocytomes, sont associés à une perturbation du processus de sécrétion. Actuellement, les mécanismes moléculaires qui induisent de telles perturbations de la sécrétion ainsi que l’impact de l’activité sécrétrice sur le développement des tumeurs neuroendocrines ne sont pas élucidés. Mes travaux de thèse proposent pour la première fois un lien fonctionnel direct entre l'activité sécrétrice des cellules et la vitesse de développement des phéochromocytomes ainsi qu’une altération des voies moléculaires impliquant certaines protéines Rho, en démontrant un lien entre la baisse de l’activité de Rac1 et Cdc42 observée dans les phéochromocytomes et la diminution de l’expression de leurs régulateurs ARHGEF1 et FARP1. / Neuroendocrine cells secrete hormones and neuropeptides through calcium-regulated exocytosis, controlled especially by Rho GTPases. Neuroendocrine tumours, such pheochromocytomas, are generally associated with a dysfunction of secretion. Although this aspect is well known by clinicians, it has never been explored at the molecular level. Moreover, the potential link between secretion and tumour development remains uninvestigated. Altogether, our results demonstrate for the first time the importance of secretion in tumor development of pheochromocytomas and an alteration of the Rho GTPase pathway, by demonstrating a link between the inhibition of Rac1 and Cdc42 activity observed in pheochromocytomas and the decrease of their activators ARHGEF1 and FARP1 expression.

L'épithélium intestinal se renouvelle tous les 4 à 6 jours chez les mammifères grâce aux cellules souches localisées au fond des cryptes. Le renouvellement dépend des signaux émis par le microenvironnement et requiert une phase de prolifération des cellules souches, de différenciation et d'apoptose/desquamation des cellules épithéliales. La signalisation Wnt, joue un rôle majeur dans l'homéostasie intestinale par l'action de deux gradients inversés le long de l'axe crypte/lumière ; la signalisation Wnt canonique, active au fond des cryptes, contrôle la prolifération alors que la signalisation non-canonique, active vers le haut des cryptes contrôle la différenciation. Il a été montré que ces deux voies contrôlent l'activité de la GTPase atypique RhoU/Wrch1. RhoU fait partie des GTPases qui s'activent spontanément, son activité est donc directement proportionnelle à son niveau d'expression dans la cellule. Enfin, cette GTPase atypique est sous exprimée dans de nombreuses tumeurs gastriques et colorectales.Compte tenu de ces données, nos objectifs étaient donc de caractériser les changements morphologiques induits par l'invalidation conditionnelle de RhoU dans l'épithélium intestinal murin et d'en déterminer les mécanismes d'action. Nos résultats montrent que la déplétion de RhoU n'est pas létale, cependant elle a induit une augmentation de 20% de la densité cellulaire et une désorganisation de la structure de l'épithélium dans le haut des cryptes du colon. Cette augmentation concerne aussi bien les lignages sécrétoires et absorptifs, cependant, l'absence de RhoU a induit une sur-représentation du lignage sécrétoire. Dans la lignée de tumeur colorectale DLD-1, nous avons montré que l'absence de RhoU mime le phénotype d'augmentation de la densité cellulaire observé chez la souris. L'invalidation de RhoU ne modifie pas la distribution des phases du cycle cellulaire ni de celle de la mitose, cependant, elle réduit le nombre des cellules en apoptose dans le colon des souris et dans les cellules DLD-1. L'invalidation de RhoU a réduit la signalisation Hippo et a altéré la contractilité cellulaire via une augmentation de la phosphorylation de la protéine MLC2. Des travaux récents ont montré que la diminution du niveau MLC2 phosphorylée est nécessaire pour l'activation des caspases par un stimulus apoptotique. Ceci suggère que cette perturbation de la contractilité peut être à l'origine de cette diminution de l'apoptose qui est la cause majeure responsable de ce phénotype. En conclusion, RhoU est un régulateur de l'homéostasie intestinale chez la souris via son rôle modérateur de la mort cellulaire. / In Mammals, the intestinal epithelium is renewed every 4-6 days through the stem cells located at the bottom of crypts. The renewal depends on signals from the micro-environment and requires a proliferation phase of stem cells, then a differentiation and apoptosis/desquamation phases of epithelial cells. Wnt signaling plays a major role in intestinal homeostasis by the action of two reversed gradients along the axis crypt/ lumen: canonical Wnt signaling, active in the bottom of crypts, control proliferation while non canonical signaling, active in the top of the crypts control cell differentiation. It was shown that these two pathways are regulator of the atypical GTPase RhoU/Wrch1. The RhoU protein activates spontaneously, its activity is directly proportional to its expression level in the cell and is expressed as in gastric and colorectal tumors. In view of these informations, our objectives were therefore to characterizethe morphological changes induced by conditional invalidation of RhoU in the intestinal epithelium of mice and to determine the mechanisms of action. Our results show that RhoU depletion is not lethal. However, it induces an increase of cell density (+20%) and a disruption of the epithelium structure in the top of the colonic crypts. This increase affects both absorptive and secretory lineages. However, the absence of RhoU induced over-representation of secretory lineage. In colorectal tumor cell line DLD-1, we have shown that the absence of RhoU mimics the phenotype of cell density increase observed in mice. RhoU invalidationdid not change the distribution of cell cycle phases and mitosis, however, it reduces the number of apoptotic cells in the colon of mice and in the DLD-1 cells. RhoU invalidation reduced Hippo signaling and altered cell contractility via the increase of the protein MLC2 phosphorylation. Recent work has shown that the reduction of MLC2-P level is necessary for the caspase protein activation by an apoptotic stimulus. Suggesting that the perturbation of contractility may be the cause of this apoptosis decrease which is the main cause responsible of this phenotype. Finally, RhoU is a regulator of the intestinal homeostasis in micevia its moderating role of cell death.

and keywords: During metastasis, cancer cells can invade the extracellular matrix using various strategies. When invading individually, they employ either the amoeboid invasion mode, during which the cell body dynamically deforms by enhanced contractility to squeeze through pores within the matrix, or protease dependent mesenchymal migration that takes advantage of the possibility to digest the surrounding matrix. Cells migrating in one mode can actively switch to the other by mesenchymal-amoeboid (MAT) or amoeboid-mesenchymal transitions (AMT). This enables escape mechanisms and considerably complicates anti-metastatic treatment. It is well known that Rho GTPases are master regulators of cytoskeleton re-arrangements and thus, unsurprisingly, play a major role in both invasion modes and can directly drive the transitions. However, upstream activation of these pathways is still largely unclear. This thesis aimed to optimize 3D conditions suitable for studying plasticity of cell invasion in vitro, establish AMT and MAT in melanoma cells based on manipulation of Rho GTPases and verify novel candidates regulating cell invasion plasticity based on previous RNA sequencing of cells before and after MAT. Last, by synthesis of published data, results from sequencing and new findings presented in this...

Orientadores: Carla Fernanda Franco Penteado, Fernando Ferreira Costa / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T18:17:52Z (GMT). No. of bitstreams: 1 Pallis_FlaviaRubia_D.pdf: 2236341 bytes, checksum: 3d0b519ba21fcfbc645749dccc82a06a (MD5) Previous issue date: 2015 / Resumo: A vaso-oclusão compreende um processo complexo e multicelular iniciado pela adesão de hemácias e leucócitos ao endotélio ativado, causando a obstrução vascular, ativação de células endoteliais vasculares e lesões que podem induzir uma resposta inflamatória contínua na anemia falciforme (AF). Estudos preliminares mostraram que os eosinófilos de pacientes com AF encontram-se ativados no sangue periférico, porém o envolvimento dessa célula no processo de vaso-oclusão ainda não está bem caracterizado. Pouco se sabe sobre o papel das proteínas da via das Rho GTPases na adesão dos eosinófilos ao endotélio vascular, bem como nas complicações pulmonares da doença. O objetivo desse trabalho foi avaliar in vitro o papel dos eosinófilos e das proteínas pertencentes à família das Rho GTPases na fisiopatologia da AF, e avaliar in vivo o papel dessa via na resposta inflamatória pulmonar induzida pela OVA em camundongos com AF. Sangue periférico de indivíduos saudáveis (controles) e pacientes com AF, em terapia com hidroxiureia (SSHU) ou não (SS), foi coletado para avaliar a adesão estática e em fluxo. A adesão dos eosinófilos de pacientes com AF à HUVEC estimulada com TNF-'alfa' foi significativamente maior quando comparado a adesão dos eosinófilos á HUVEC de indivíduos controle, em condições de fluxo e estática. No entanto, a adesão dessas células à HUVEC estimulada com TNF-? foi menor nos pacientes SSHU, quando comparada com o com aos SS. O pré-tratamento das HUVEC com o inibidor de Rac1, reduziu a adesão dos eosinófilos de pacientes SS ou SSHU. Em condições de fluxo, o número de eosinófilos aderidos à HUVEC reduziu significativamente quando estas foram tratadas com Y-27632 ou NSC23766 nos três grupos avaliados, porém essa inibição foi maior no grupo SS. In vivo a OVA induziu inflamação pulmonar caracterizada pelo aumento na contagem de leucócitos, principalmente eosinófilos, no lavado broncoalveolar dos camundongos. Essa inflamação foi potencializada nos camundongos com AF (Berkeley e Transplantados) quando comparados aos controles (C57BL6). Os camundongos Transplantados apresentaram níveis elevados de mediadores pró-inflamatórios, tais como, IL-4, IL-5, IL-6, IL-13, RANTES, Eotaxina, MCP-1, MMP-9 e TIMP-1 quando comparados aos camundongos do grupo não sensibilizado (NS). Nenhuma diferença foi observada nos níves de RNAm pulmonar das metaloproteinases e seus inibidores quando comparados os camundongos Transplantados do grupo OVA com os NS, porém a expressão de IL-6 é significativamente maior no pulmão dos animais falciformes desafiados com OVA. Na avaliação funcional dos brônquios, os dados mostraram que a potência para metacolina foi maior nos camundongos Berkeley, quando comparados aos Transplantados e ainda maior quando comparados com os C57BL6. Os animais que foram tratados com os inibidores da via RhoA/ROCK, Y-27632 ou Fasudil, apresentaram menor contagem total e diferencial das células que migraram para os pulmões e níveis reduzidos dos mediadores pró-inflamatórios avaliados. O pré-tratamento com o Fasudil reduziu a potência e a resposta máxima para metacolina dos brônquios da linhagem Berkeley. Não foi observada diferença na avaliação funcional da reatividade da traqueia no modelo de asma experimental nos grupos avaliados. Tomados em conjunto, os resultados indicam que a via RhoA/ROCK tem papel importante na adesão dos eosinófilos ao endotélio e que a inibição dessa via pode atenuar a asma associada a doença falciforme. Deste modo, sugerimos que os inibidores dessa via podem ser novos agentes terapêuticos para o tratamento das manifestações clínicas da AF / Abstract: Vaso-occlusion, comprising a complex and multicellular process, initiated by the adhesion of erythrocytes and leukocytes to the activated endothelium, leading to vascular obstruction, activation of vascular endothelial cells and continuous lesions in patients with sickle cell anemia (SCA). Preliminary results demonstrated that eosinophils from SCA patients exist in an activated state, however the participation of this cell in the vasooclusive process in not well establish. The role of proteins via the Rho GTPases in the adhesion of eosinophils to the vascular endothelium and in the pulmonary complications of SCA disease it is unknown. The aim of this study was to evaluate in vitro the role of the eosinophils and proteins belonging to the Rho GTPases family in SCA pathophysiology, and evaluate in vivo the role of this pathway in pulmonary inflammatory responses induced by OVA in SCA mice. Peripheral blood of healthy individuals (controls) and SCA patients in therapy or not with hydroxyurea (SCAHU) was collected for static and flow adhesion experiments. For static adhesion assays, eosinophils were isolated from control subjects or SCA patients on or off HU therapy. Adhesion of SCA eosinophils to HUVEC (Human Umbilical Vein Endothelial Cells) under TNF-'alfa'-stimulated conditions was higher when compared with control eosinophils, in flow conditions and static assay. Furthermore, SCAHU eosinophils demonstrated significantly lower adhesive properties, compared to SCA eosinophils. The adhesion of eosinophils from SCA or SCAHU patients were reduced when HUVEC were pretreated with NSC23766 inhibitor, compared to non-treated HUVEC. Under flow conditions, the number of eosinophils adhered to HUVEC cells was reduced when they were treated with Y-27632 or NSC23766 in the three groups investigated, however this inhibition was higher in the SCA patients. In vivo OVA induced lung inflammation, characterized by increased leukocyte particularly eosinophils, counts in the mice bronchoalveolar fuid (BALF). This inflammation was enhanced in SCA mice (Berkeley and Transplanted) when compared to controls (C57BL6). Transplanted mice showed high levels of pro-inflammatory mediators such as IL-4, IL-5, IL-6, IL-13, RANTES, Eotaxin, MCP-1, MMP-9 and TIMP-1 as compared to the control mice group (NS). Furthermore, the SCA mice induced with OVA showed higher lung gene expression of IL-6; however, there was no difference in the expression of MMP-2, MMP-8, MMP-9, MMP-12, TIMP-1 and TIMP-2, compared to NS. In the functional assessment of the bronchi reactivy, data showed that the potency to methacholine in asthmatic model was was greater in the SCA mice (Berkeley), compared to the Transplanted SCA mice, and even greater when compared to C57BL6. The animals treated with the inhibitors of the RhoA/ROCK pathway, Y-27632 or Fasudil, showed lower total and differential cells counts due to migration to the lungs. Pretreatment with the RhoA/ROCK pathway inhibitor significantly reduced the proinflammatory mediator levels evaluated. Treatment with Fasudil reduced maximal response for methacholine in bronchi from Berkeley model. No significant difference was observed in the tracheal reactivity after OVA challenge in all groups investigated. Taken together, our data indicated that RhoA/ROCK pathway play an important role in the eosinophil adhesion to the endothelium and, the inhibition of this pathway could alleviates asthma in SCA patients. Thus, we suggest that RhoA/ROCK inhibitors represents novel therapeutic agents for the treatment of SCA clinical manifestations / Doutorado

O Corpo Ciliar (CC) é uma fonte de células tronco da retina de animais adultos, mas sua ativação permanece desconhecida. GTPases Rho são proteínas que reorganizam do citoesqueleto de actina, regulam vias de sinalização e transcrição gênica, sobrevivência celular e proliferação. Neste trabalho, investigamos a expressão das GTPases Rho nas células do CC e seu efeito na regulação do ciclo celular. As GTPases RhoA, RhoB e Rac1 foram expressas nas células do CC e sua ativação pelo ácido lisofosfatidico (LPA) aumentou a expressão dos genes progenitores retinianos Pax6 e Chx10. A inibição das proteínas por Toxina A de Clostridium difficile aumentou a proliferação no CC e potencializou o efeito proliferativo dos fatores de crescimento. A inibição especifica destas proteínas diminuiu a expressão dos transcritos de p21cip, p27kip, p16INK4a e p19INK4d e aumentou de Ki67, CiclinaA e D1. O estudo da via de Wnt indicou que Rac1 regulou os genes de componentes da degradação de -catenina e Lef1. Concluímos que a inativação das GTPases Rho induziu a proliferação das células progenitoras retinianas localizadas no CC e regulou a via de Wnt. Sua ativação induziu o perfil de célula progenitora, sugerindo uma nova ferramenta para o mecanismo de reparo retiniano. / Ciliary Body (CB) is a potential source of stem cells in the adult retina, but its activation is still unknown. Rho GTPases play a role in actin-based cytoskeleton reorganization, regulate signaling pathways and gene transcription, cell survival and cell proliferation. In this study we investigated the expression of Rho GTPases in CB cells and their role on cell cycle regulation. The GTPases RhoA, RhoB and Rac1 were present in CB cells and the activation by lysophosphatidic acid (LPA) increased the expression of the progenitor genes Pax6 and Chx10. The inhibition by Clostridium difficile Toxin A increased the proliferation of CB cells and potentiated the proliferative effect of growth factors. The specific inhibition decreased the expression of p21cip, p27kip, p16INK4a and p19INK4d as well as increased Ki67, cyclinA and D1 transcripts. The Wnt pathway study indicated that Rac1 regulated -catenin degradation genes components and Lef1. Taken together, the inactivation of Rho GTPases stimulated the proliferation of progenitor cells located in CB as well as regulate the Wnt signaling pathway. The proteins activation was correlated to progenitor profile induction. These different mechanisms may provide a potential new approach on retinal repair.

Notre système immunitaire détecte les microorganismes via des molécules absentes de l’hôte appelées MAMPs. Mais étant donné que les MAMPs sont exprimés par tous les microorganismes indépendamment de leur potentiel pathogène, ce mécanisme n’explique pas comment le système immunitaire distingue les microorganismes pathogènes des non-pathogènes. De récents travaux ont mis en évidence un mécanisme de détection de l’activité des facteurs de virulence bactériens. En utilisant la drosophile, notre laboratoire a précédemment démontré que l’activation de la Rho GTPase Rac2 par la toxine CNF1 d’Escherichia coli induisait une réponse immunitaire innée conservée au cours de l’évolution chez le mammifère et similaire à l’immunité induite par les effecteurs chez la plante. Par la suite, nous avons évalué l’importance de cette réponse immunitaire au cours de la bactériémie chez la souris et démontré le rôle central de la cytokine IL-1β dans l’élimination des bactéries en réponse à la détection de CNF1. Des expériences in vitro nous ont permis d’identifier les mécanismes moléculaires mis en jeu et l’inflammasome responsable de l’activation de la caspase-1 et du clivage de l’IL-1β. De manière intéressante, CNF1 est toujours co-exprimée avec la toxine hémolysine-α (HlyA) dans les souches pathogènes d’E. coli. En outre, nous avons découvert que l’HlyA bloquait l’élimination des bactéries induite par CNF1 au cours de la bactériémie et inhibait la sécrétion de l’IL-1β. Ici, nous avons rapporté le premier exemple d’immunité induite par une toxine (CNF1) et contrecarrée par une autre (HlyA). / Our immune system detects microorganisms via molecules absent from the host called MAMPs. Since MAMPs are shared by all microorganisms regardless of their pathogenic potential, this mechanism does not explain how the immune system distinguishes between pathogenic and non pathogenic microorganisms. The detection of the activities of pathogen-encoded virulence factors has emerged as a new paradigm of pathogen recognition. Using Drosophila we previously demonstrated that the Escherichia coli CNF1 toxin-induced activation of the Rho GTPase Rac2 is sufficient to initiate a defense signal evolutionarily conserved from flies to mammals and similar to Effector-Triggered Immunity in plants. We further addressed the importance of this innate immune mechanism during bacteremia in mice and demonstrated the central role of the IL-1β cytokine in the clearance of bacteria in response to the detection of CNF1. In vitro experiments allowed us to identify the involved molecular mechanisms and the inflammasome responsible of caspase-1 activation and IL-1β maturation. Interestingly, CNF1 is always co-expressed with α-hemolysin toxin in pathogenic E. coli. In addition, we found that HlyA blocked the elimination of bacteria induced by CNF1 during bacteremia and inhibited the secretion of IL-1β. Here, we have reported the first example of immunity induced by a toxin (CNF1) and counteracted by another (HlyA).