El shock es una situación clínica que se ha venido observando durante cientos de años y cuya presencia ha ido asociada con procesos que culminaban casi siempre con la muerte. El estudio de estos procesos ha permitido llegar a la conclusión de que el shock tiene un componente multifactorial y multietiológico. La cascada de eventos que tienen lugar en un estado de shock parece demasiado compleja como para que la actuación terapéutica sobre uno solo de estos pasos sea suficiente como para bloquear el shock en su conjunto. Sin embargo, desde hace años se vienen estudiando diferentes aproximaciones terapéuticas entre las que se encuentra el antagonismo del factor activador de ls plaquetas (PAF). Los diferentes trabajos desarrollados en esta tesis pretenden ser una aportación al estudio del papel del PAF y de sus antagonistas en el shock. Para ello se han recogido una serie de modelos experimentales, tanto in vitro como in vivo , con el antagonista del PAF UR-12460. Los modelos estudiados son: shock anafiláctico, shock por isquemia-reperfusión esplácnica y coronaria, shock endotóxico y shock hemorrágico. El UR-12460 presenta actividad en modelos de shock anafiláctico activo, tanto en cobayo como en ratón. Además, para una serie de antagonistas del PAF estudiados, se demuestra una buena correlación entre la inhibición de la mortalidad inducida por PAF y la inhibición de la mortalidad inducida por shock anafiláctico activo en ratón, lo cual indica que el PAF está implicado en estos procesos. La ineficacia del UR-12460 en procesos de shock pasivo sugiere una menor importancia relativa del PAF con respecto a otros mediadores como la histamina. Por otra parte, en modelos de shock por isquemia-reperfusión esplácnica en rata, el UR-12460 se ha mostrado parcialmente efectivo, inhibiendo la mortalidad y la trombocitopenia producidas por el shock. Otro modelo de isquemia-reperfusión, pero a nivel miocárdico en este caso, ha sido extensamente estudiado en la rata. La oclusión durante 6 minutos de la arteria coronaria izquierda en la rata anestesiada, y posterior reperfusión durante 10 minutos, produce una serie de alteraciones del ritmo cardíaco que son inhibidas por el UR-12460. Dicho compuesto disminuye la incidencia de taquicardias ventriculares durante la oclusión, y disminuye la aparición de fibrilación ventricular en la reperfusión. Esto apoya el papel del PAF en la arritmogénesis por isquemia-reperfusión. Las pruebas de isquemia-reperfusión en corazón aislado de rata, por el método de Langendorff, combinadas con la incubación posterior del tejido cardíaco con cloruro de trifeniltetrazolio, permiten aunar en un mismo experimento y bajo las mismas condiciones, la valoración macroscópica del área de infarto y la alteración funcional del corazón. En este modelo los corazones se perfunden con sangre diluida en líquido de Krebs y se comparan con corazones que reciben sólo el diluyente. Las marcadas diferencias entre los corazones isquémicos que han recibido sangre diluida y los que no, sugieren que las células sanguíneas son pieza clave en la génesis de los infartos y en el deterioro del funcionalismo cardíaco. Por otra parte, la ausencia de una relación directa entre el tiempo de isquemia-reperfusión y la gravedad del infarto, en ausencia de sangre, sugiere que la situación de anoxia no es un factor determinante en la génesis del infarto. Sí existe relación entre el tiempo de isquemia-reperfusión y las alteraciones de la función mecánica. La presencia de células sanguíneas podría representar un sistema de amplificación del daño causado por la anoxia. Los resultados obtenidos en este modelo con el compuesto UR-12460 y el antioxidante mercaptopropionilglicina sugieren que tanto el antagonismo del PAF como la captación de radicales libres pueden ser útiles en la disminución del infarto y, en general, en la mejoría de la función miocárdica durante la reperfusión y en la disminución de las arritmias. Otro importante modelo es el shock endotóxico, inducido en la rata por la administración de lipopolisacárido (LPS) de E. coli de diversos serotipos. El serotipo 0111:B4 produce un característico efecto bifásico de hipotensión arterial. El UR-12460 no inhibe la profunda e inmediata fase de hipotensión pero sí la segunda fase, más lenta y progresiva. Utilizando el serotipo 0127:B8 se ha estudiado el aumento de permeabilidad vascular en la rata. Se ha comprobado que sólo algunos órganos resultan afectados significativamente y que el aumento de permeabilidad se aprecia a partir de los 15 minutos de administrado el LPS. Por otra parte, hay una buena correlación entre la inhibición de la mortalidad inducida por PAF y la inducida por LPS en ratón, lo que refuerza la participación del PAF como mediador clave en la cascada de acontecimientos que producen la mortalidad por LPS. Este papel queda reforzado por la eficacia mostrada por el UR-12460 al atenuar las alteraciones bioquímicas y enzimáticas inducidas por LPS en rata. La inhibición del aumento del tiempo parcial de tromboplastina activada indica que el PAF puede estar también implicado en los mecanismos que conducen al síndrome de coagulación intravascular diseminada en la rata. El último modelo estudiado es el shock hipovolémico hemorrágico, en el cual la eficacia de los antagonistas probados no se ha demostrado. Es posible que su administración sólo sea beneficiosa en fases iniciales de la hemorragia, cuando el shock no se ha hecho todavía progresivo o no se han liberado otros mediadores.En resumen, puede decirse que el UR-12460 se ha mostrado activo, al menos parcialmente, en varios modelos de shock, reforzando la idea de la participación importante del PAF en estos procesos de tan diversa etiología. Sin embargo, el hecho de que este compuesto no tenga efecto sobre algunos de los marcadores del shock estudiados hace pensar que el futuro de un posible tratamiento del shock pasaría por la administración conjunta de diferentes sustancias, dirigidas cada una a los diferentes mediadores del shock que, además del PAF, intervienen en mayor o menor grado en su etiopatogenia. / Shock is a clinical situation that has been observed for centuries and whose presence has been associated to processes leading usually to death. The study of these processes has allowed to reach the conclusion that shock is a multifactorial and multietiological component syndrome. Since the cascade of events that take place during shock is very complex, it seems unlikely that acting on a single of its steps may be sufficient to block the shock state in its entirety. Nevertheless, in the last years different therapeutic approaches are being studied, among them the antagonism of platelet activating factor (PAF). The different studies carried out in this thesis intend to be a contribution to the study of the role of PAF and of its antagonists in shock. With a view to this we have performed several experimental models, both in vitro and in vivo, using UR-12460, a potent and selective PAF antagonist. The studied models are: anaphylactic shock, splachnic and coronary ischemia-reperfusion-induced shock, endotoxic shock and hemorrhagic shock. UR-12460 was found to be effective in active anaphylactic shock models, both in guinea pigs and mice. Furthermore, for a series of PAF antagonists, we have observed a good correlation between the inhibition of PAF-induced mortality and of active anaphylactic shock -induced mortality in mice, which indicates that PAF is involved in these processes. The ineffectiveness of UR-12460 in passive shock processes suggests a minor relative importance of PAF with respect to other mediators such as histamine. On the other hand, in splachnic ischemia-reperfusion (ischemia-reperfusion) shock models in the rat, UR-12460 has been shown partially effective, inhibiting the mortality and thrombocytopenia induced by shock. Another model of ischemia-reperfusion, but at myocardial level in this case, has been extensively studied in the rat. The occlusion during 6 minutes of the left coronary artery in the anesthetized rat, and subsequent reperfusion during 10 minutes, produces a number of disturbances in cardiac rhythm that are inhibited by UR-12460. This compound reduces the incidence of ventricular tachycardia during the occlusion phase as well as the onset of ventricular fibrillation in the reperfusion. This finding supports the role of PAF in the arrhythmogenesis by ischemia-reperfusion. The ischemia-reperfusion tests in isolated rat heart using the Langendorff method, combined with the subsequent incubation of the cardiac tissue with triphenyltetrazolium chloride, allow to combine in a single experiment, and under identical conditions, the macroscopic assessment of the infarcted area and the functional alterations of the heart. In this model, hearts are perfused with blood diluted with Krebs solution and are compared with hearts receiving only Krebs. The marked differences observed between the ischemic hearts that have received diluted blood and those that have not suggest that blood cells are a key factor in the genesis of infarction and in the impairment of cardiac function. On the other hand, the absence of a direct relationship between the time of ischemia-reperfusion and the severity of the infarction in the absence of blood suggests that the situation of anoxia is not a relevant factor in the genesis of infarction. There is indeed a relationship between the time of ischemia-reperfusion and the impairment of the mechanical function. The presence of blood cells may represent an amplification system of the damage caused by the anoxia. The results obtained in this model with the compound UR-12460 and the antioxidant mercaptopropionylglycine suggest that both PAF antagonism and free radical scavenging may be useful to reduce infarction and, in general, to improve the myocardial function during reperfusion and to reduce arrhythmias.Another important model is the endotoxic shock, which is induced in the rat by the administration of E. coli lipopolysaccharide (LPS) of different serotypes. Serotype 0111:B4 produces a characteristic biphasic effect of arterial hypotension. UR-12460, although not able to inhibit the steep and immediate phase of hypotension, clearly inhibits the second phase, which is slower and more progressive. The increase in vascular permeability has been studied in the rat using serotype 0127:B8. We have observed that only some organs are significantly affected and that the increase in permeability is noticeable from 15 minutes post-administration of LPS. UR-12460 has been shown to inhibit extravasation in the trachea and seminal vesicle. On the other hand, we have observed a good correlation between the inhibition of the mortality induced by PAF and that induced by LPS in the mouse, which fact reinforces the involvement of PAF as a key mediator in the cascade of events that cause the mortality by LPS. This is additionally supported by the ability shown by UR-12460 to reduce the biochemical and enzymatic alterations induced by LPS in the rat. The inhibition of the increase in the activated partial thromboplastin time shows that PAF may also be involved in the mechanisms leading to disseminated intravascular coagulation syndrome in the rat. The last shock model we have studied is the hemorrhagic hypovolemic shock, where none of the antagonists tested has proved effective. It is possible that their administration may only be beneficial in the initial stages of hemorrhage, when shock has not yet become progressive or other mediators have not yet been released. To sum up, it can be said that UR-12460 has proved effective, at least partially, in several models of shock, reinforcing the theory that PAF has a key role in these processes of such diverse etiology. However, the fact that this compound has no effect on some of the shock markers studied in this thesis brings us to think that the future of a possible treatment for shock requires the administration of different substances, directed to each one of the different mediators of shock that, in addition to PAF, are involved to a greater or lesser extent in its etiopathogenesis.

Buscando sintetizar análogos de PAF imobilizados em resina para isolamento dos correspondentes receptores T. cruzi, foi adotada uma estratégia de síntese convergente que implicava no acoplamento à resina de uma cadeia hidrofóbica análoga à presente no fosfolipídio. Através de modificação sequencial do reagente comercial D-Manitol para sua ligação à resina, os análogos seriam sintetizados em um total de 12 etapas. Após a obtenção com sucesso do primeiro intermediário e de dificuldades na obtenção do segundo intermediário, a preparação de derivados de PAF a partir de D-Manitol foi abandonada já que revisão da literatura demonstrou ser possível a síntese destes mesmos análogos em apenas cinco etapas. A nova rota sintética adotada teve como ponto de partida a fosfatidilcolina extraída de gemas de ovos e, através desta nova abordagem, foram sintetizados com sucesso três intermediários. Devido a dificuldades na adaptação de protocolos sintéticos clássicos a compostos carregados como os fosfolipídios, não foi possível a obtenção dos derivados de PAF inicialmente propostos. Os estudos de bioinformática, os quais visaram localizar a sequencia deste provável receptor de PAF no proteoma do parasita, sugerem a existência de proteínas com características de receptores de membrana acoplados à proteína G (GPCRs) e entre estas, possivelmente receptores de PAF a serem explorados na terapia da doença de Chagas. / In order to synthesize resin-immobilized PAF analogs to further promote the isolation of the T.cruzi correlative receptor, we adopted a convergent synthesis strategy. The strategy implied the coupling between the resin and a hydrophobic chain resembling PAF, in which resin-immobilized PAF analogs would be synthesized by means of sequencial 12 step modification of commercially available reagent D-manitol. Despite successful achievement of the first intermediary, the synthesis of the second intermediary was accompanied by difficulties which led to an extensive literature review and subsequent change of synthetic route. The preffered route was a 5 step approach with egg phosphatidilcholine as the starting material, which rendered the successful synthesis of three intermediates. However, due to difficulties to adapt classical synthetic protocols to charged compounds as phospholipids, we could not obtain the initially proposed PAF analogs. Bioinformatic studies were conducted in order to locate the aminoacid sequence of the probable PAF receptor in T.cruzi proteome. Our data suggest the existence of a receptor resembling G protein coupled membrane receptors (GPCRs), which probably include PAF receptors, which, in turn, might be a potential target to the treatment of Chagas disease.

Neste trabalho investigamos o papel do receptor do Fator ativador de Plaquetas (PAF) em diferentes tumores. Observamos que animais PAFR KO são mais resistentes ao crescimento do melanoma B16F10 e do carcinoma TC-1 e apresentaram maior infiltrado de linfócitos CD4+, neutrófilos e de macrófagos M1 do que animais WT. Células de carcinoma humano (C33/SiHa/HeLa/SSC78/ SSC90) expressam PAFR e tiveram sua proliferação in vitro reduzida por um antagonista de PAFR e aumentada pela adição de PAF. A irradiação gama induziu ligantes PAFR. O bloqueio do PAFR durante a radioterapia aumentou a morte induzida pela irradiação. Em modelo de repopulação tumoral observamos que tumores PAFR+ (KBP) tiveram um crescimento acelerado em relação à tumores PAFR- (KBM). Nossos dados sugerem que durante a irradiação ocorre ativação de PAFR nas células tumorais aumentando sua sobrevivência e proliferação. Ao mesmo tempo que ativa PAFR nos macrófagos reprogramando-os para um perfil pró-tumoral. A associação da radioterapia com antagonistas de PAFR pode ser uma estratégia terapêutica promissora. / We investigate the role of the platelet activating factor receptor (PAFR) in tumors. We observed that PAFR KO animals are more resistant to the growth of B16F10 melanoma and TC-1 carcinoma than WT animals. PAFR KO had more infiltration of CD4+ cells, neutrophil and M1 macrophages than WT animals. Human carcinoma cells (C33 / SiHa / HeLa / SSC78 / SSC90) express PAFR and had their in vitro proliferation reduced by a PAFR antagonist and increased by the addition of PAF. Gamma irradiation induced PAFR ligands. Blocking PAFR during radiotherapy increased radiation-induced cell death. In tumor repopulation model, PAFR+ tumors (KBP) had an accelerated growth compared to PAFR- (KBM) tumors. Our data suggest that during irradiation occurs PAFR activation in tumor cells increasing their survival and proliferation. Also, irradiation promote activation of PAFR in macrophages by reprogramming them to a pro-tumor profile. We propose that PAFR represents a possible target for improving the efficacy of radiotherapy through inhibition of tumor repopulation.

Buscando sintetizar análogos de PAF imobilizados em resina para isolamento dos correspondentes receptores T. cruzi, foi adotada uma estratégia de síntese convergente que implicava no acoplamento à resina de uma cadeia hidrofóbica análoga à presente no fosfolipídio. Através de modificação sequencial do reagente comercial D-Manitol para sua ligação à resina, os análogos seriam sintetizados em um total de 12 etapas. Após a obtenção com sucesso do primeiro intermediário e de dificuldades na obtenção do segundo intermediário, a preparação de derivados de PAF a partir de D-Manitol foi abandonada já que revisão da literatura demonstrou ser possível a síntese destes mesmos análogos em apenas cinco etapas. A nova rota sintética adotada teve como ponto de partida a fosfatidilcolina extraída de gemas de ovos e, através desta nova abordagem, foram sintetizados com sucesso três intermediários. Devido a dificuldades na adaptação de protocolos sintéticos clássicos a compostos carregados como os fosfolipídios, não foi possível a obtenção dos derivados de PAF inicialmente propostos. Os estudos de bioinformática, os quais visaram localizar a sequencia deste provável receptor de PAF no proteoma do parasita, sugerem a existência de proteínas com características de receptores de membrana acoplados à proteína G (GPCRs) e entre estas, possivelmente receptores de PAF a serem explorados na terapia da doença de Chagas. / In order to synthesize resin-immobilized PAF analogs to further promote the isolation of the T.cruzi correlative receptor, we adopted a convergent synthesis strategy. The strategy implied the coupling between the resin and a hydrophobic chain resembling PAF, in which resin-immobilized PAF analogs would be synthesized by means of sequencial 12 step modification of commercially available reagent D-manitol. Despite successful achievement of the first intermediary, the synthesis of the second intermediary was accompanied by difficulties which led to an extensive literature review and subsequent change of synthetic route. The preffered route was a 5 step approach with egg phosphatidilcholine as the starting material, which rendered the successful synthesis of three intermediates. However, due to difficulties to adapt classical synthetic protocols to charged compounds as phospholipids, we could not obtain the initially proposed PAF analogs. Bioinformatic studies were conducted in order to locate the aminoacid sequence of the probable PAF receptor in T.cruzi proteome. Our data suggest the existence of a receptor resembling G protein coupled membrane receptors (GPCRs), which probably include PAF receptors, which, in turn, might be a potential target to the treatment of Chagas disease.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-02-10T16:31:37Z No. of bitstreams: 1 Vinicius Costa Souza Ferreira, Papel do fator... 2014.pdf: 10087481 bytes, checksum: 8c34a10bbc4c46304eaf9166412e6f0f (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-02-10T16:31:51Z (GMT) No. of bitstreams: 1 Vinicius Costa Souza Ferreira, Papel do fator... 2014.pdf: 10087481 bytes, checksum: 8c34a10bbc4c46304eaf9166412e6f0f (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-02-10T16:32:18Z (GMT) No. of bitstreams: 1 Vinicius Costa Souza Ferreira, Papel do fator... 2014.pdf: 10087481 bytes, checksum: 8c34a10bbc4c46304eaf9166412e6f0f (MD5) / Made available in DSpace on 2015-02-10T16:32:18Z (GMT). No. of bitstreams: 1 Vinicius Costa Souza Ferreira, Papel do fator... 2014.pdf: 10087481 bytes, checksum: 8c34a10bbc4c46304eaf9166412e6f0f (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / A leishmaniose visceral é causada pelo parasita Leishmania infantum. A infecção ocorre quando flebótomos infectados se alimentam na derme do hospedeiro vertebrado, inoculando o parasita. A infecção produz uma resposta com diversas moléculas inflamatórias, como os mediadores lipídicos. O fator de ativação de plaquetas (PAF) é um potente mediador lipídico derivado de um lisofosfolipídio. PAF participa da fisiologia normal da célula e possui um perfil pró-inflamatório. A participação de mediadores lipídicos, como eicosanóides e PAF, já foi identificada na imunopatogênese das leishmanioses. PAF gerado pelo hospedeiro tem efeito leishmanicida e de controle da infecção por L. amazonensis. PAF-acetilhidrolases (PAF-AH) são fosfolipases A2 que hidrolisam PAF e foi demonstrado que PAF-AH podem ser um fator de virulência devido a essa habilidade. O objetivo desse estudo foi avaliar o papel do PAF e de uma PAF-AH na infecção de macrófagos por L. infantum. Foi observado que PAF 1μM, quando adicionado durante e após a infecção, foi capaz de diminuir 50% da infecção após 72 horas, bem como a viabilidade dos parasitas dentro dos macrófagos num mecanismos independente do seu receptor PAFR e da produção de óxido nítrico. PAF 10μM interrompeu o crescimento de promastigotas de L. infantum em cultura axênica. Uma PAFAH, com elevada identidade e semelhança com PLA2/PAF-AH de outros tripanossomatídeos, foi identificada no genoma de L. infantum. A clonagem e expressão recombinante produziu uma proteína de cerca de 69kDa, com atividade PAF-AH. Frações celulares do parasita, enriquecidas com estruturas de membrana também apresentaram atividade PAF-AH. Os resultados indicam que PAF é capaz de diminuir a infecção de macrófagos por L. infantum e que o parasita possui uma PAF-AH funcional possivelmente envolvida com sua virulência. / Visceral leishmaniasis is caused by Leishmania infantum parasites. Infection occurs when infected sandflies feed on vertebrate host skin delivering the parasite which survive, multiply and spread on the parasitophorous vacuoles of macrophages. The inflammatory response during the infection leads to the production of diverse bioactive molecules, as lipid mediators. The platelet activating factor (PAF) is a lipid mediator derived from a lysophospholipid. PAF has a role in normal cellular physiology, acting as proinflamatory molecule. The participation of some lipid mediators, as eicosanoids and PAF has been identified in leishmaniasis. PAF produced by the host is able to kill the parasite and control the infection by L. amazonensis. PAF-acetylhydrolases (PAF-AH) are phospholipases A2 (PLA2) that hydrolyse PAF, and possibly involved in pathogen virulence. The aim of this study was to evaluate the role of PAF on macrophages infection by L. infantum and identify a PAF-AH expressed by the parasite. PAF 1μM, added during and after the infection, was able to reduce approximately 50% of infection, as well as, the viability of parasites inside macrophages. Apparently this reduction occurs by an classical PAF receptor and nitric oxide production independent mechanism. PAF 10μM inhibited L. infantum promastigotes growing in axenic culture. A PAF-AH with high identity to PLA2/PAF-AH of others trypanosomatids was identified in L. infantum genome. The cloning and recombinant expression produced a 69kDa protein with PAF-AH activity. Cellular fractions from parasites, with membrane structures also presented PAF-AH activity. The results suggest that PAF is able to decrease machophage infection by L. infantum witch has a functional PAFAH possibly related to its virulence

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Abstract Neutrophils are the major cellular component of acute inflammatory response. The mechanism by which fMLP or PAF activates neutrophils is not fully elucidated. Stimulation of MAPKs and activation of NF-kappa B in neutrophils regulate various cell functions, including superoxide production. Neutrophils isolated from blood taken from healthy donors, were incubated with specific inhibitors, GF109203X (PKC inhibitor), calphostin C (PKC-gamma isoform inhibitor), wortmannin (PI3K inhibitor), U73122 (PLC inhibitor), aristolochic acid (PLA2 inhibitor), SKF96365 (SOC channel inhibitor), EGTA (extracellular calcium chelator), SB203580 (p38 MAPK inhibitor), and PD98059 (MEK inhibitor), followed by fMLP or PAF treatment. MAPK activation by fMLP or PAF is based on immunoblot analysis. NF-kappa B activation is detected by EMSA, and superoxide production is measured by flow cytometry. The data indicate that neutrophil MAPK signaling pathways mediated by fMLP and PAF are different. PAF-induced ERK MAPK phosphorylation was involved PI3K, PKC, PLA2, PLC, and extracellular calcium, wheres fMLP-induced phosphorylation doesn¡¦t involve PKC

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Comunicação e Expressão. Programa de Pós-Graduação em Jornalismo / Made available in DSpace on 2013-07-16T04:21:08Z (GMT). No. of bitstreams: 1 297913.pdf: 9045831 bytes, checksum: 36103336678ce2f6381cf50cbcff0dab (MD5) / A presente pesquisa analisa a revista Pif Paf, publicada por Millôr Fernandes em 1964, e o espaço ocupado por essa publicação no campo jornalístico brasileiro. A partir da constatação de que a linguagem humorística da revista entra em choque com a padronização do texto jornalístico # baseada no ideal da objetividade # implementada pela grande imprensa brasileira na década de 50, busca-se compreender a trajetória histórica da formatação da linguagem jornalística. Para isso, a pesquisa se baseia nas análises de Pierre Bourdieu sobre o mercado de bens simbólicos, o campo de produção erudita e o campo da indústria cultural (onde se insere o jornalismo) e nos trabalhos de John Hartley e Michael Shudson sobre a formação do campo jornalístico, sua função social e linguagem.

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Pós-Graduação em Biologia Molecular, 2015. / Submitted by Fernanda Percia França (fernandafranca@bce.unb.br) on 2015-11-23T19:17:46Z No. of bitstreams: 1 2015_AnneCarolineDiasNeves.pdf: 2726609 bytes, checksum: 9b96c0c43e123e5240c0572c1d0bb7d6 (MD5) / Approved for entry into archive by Marília Freitas(marilia@bce.unb.br) on 2016-05-26T20:23:23Z (GMT) No. of bitstreams: 1 2015_AnneCarolineDiasNeves.pdf: 2726609 bytes, checksum: 9b96c0c43e123e5240c0572c1d0bb7d6 (MD5) / Made available in DSpace on 2016-05-26T20:23:23Z (GMT). No. of bitstreams: 1 2015_AnneCarolineDiasNeves.pdf: 2726609 bytes, checksum: 9b96c0c43e123e5240c0572c1d0bb7d6 (MD5) / Durante o processo inflamatório são ativadas uma quantidade grande de células, entre elas se destacam os leucócitos polimorfonucleares neutrófilos por serem os mais abundantes do sangue periférico (40% a 70% das células brancas) e por chegarem primeiro ao local da injúria tecidual ou local da infecção. O conhecimento dos mecanismos de ativação desse tipo celular torna-se importante para se compreender a regulação imunológica em diversas situações patológicas, sendo algumas destas consequentes à falha na regulação de ativação de polimorfonucleares. Os casos mais comuns encontrados são nas complicações da doença granulomatosa crônica, doença pulmonar obstrutiva crônica, síndrome da angústia respiratória aguda, artrite reumatóide e síndrome da resposta inflamatória sistêmica. Pesquisas relataram que o Fator de Agregação Plaquetária (PAF) está relacionado à ativação dos neutrófilos nessas condições, onde o estudo das proteínas e das suas modificações pós traducionais tornam-se pontos centrais para entender a sinalização celular dos neutrófilos nas doenças citadas anteriormente. No presente trabalho foi feito o estudo proteômico de neutrófilos quiescentes com a identificação de mais de 3000 proteínas e de neutrófilos estimulados pelo PAF que revelou um perfil protéico que caracteriza essa estimulação, onde proteínas que fazem parte de processos essenciais tiveram a abundância aumentada como reorganização do citoesqueleto, sinalização intracelular mediada por RAS e Proteina G, subunidades da ATPases vacuolar, e alguns produtos característico do processo inflamatório como NFkβ e prostaglandina E. Por outro lado, como os neutrófilos estão em seu estado estimulado outras proteínas tiveram abundância diminuída, entre elas proteínas responsáveis pelo processo de diferenciação e amadurecimento dos neutrófilos e proteínas que fazem parte do sistema de controle de processos essenciais da célula como síntese de proteínas, metabolismo energético e motilidade celular. A identificação de proteínas acetiladas nos indicou as possíveis proteínas alvos para regulação de importantes vias de estimulação dos neutrófilos como tradução, formação estrutural do núcleo multilobulado dos neutrófilos, transporte de vitaminda B12, assim como em pontos de regulação na síntese de ATP e de subunidades de ATPases. A identificação dessas proteínas com abundância diferencial e acetiladas é de fundamental importância para indicar possíveis alvos terapêuticos de doenças auto-imunes e inflamatórias causadas pelo PAF. ____________________________________________________________________________________ ABSTRACT / During the inflammatory process is activated a large number of cells, these include polymorphonuclear neutrophil leukocytes to be the most abundant in the peripheral blood (40% to 70% of white cells) and reaching the first site of tissue injury or local infection. Knowledge of this type of cellular activation mechanisms becomes important to understand the immune regulation in various pathological situations, some of these failed in the subsequent activation of polymorphonuclear regulation. The most common cases are found in the complications of chronic granulomatous disease, chronic obstructive pulmonary disease, acute respiratory distress syndrome, rheumatoid arthritis and systemic inflammatory response syndrome. Research reported that platelet aggregation factor (PAF) is related to the activation of neutrophils such conditions, where the study of proteins and their post-translational modifications become key points to understand cell signaling in neutrophils in the above-mentioned diseases. In the present work was done the proteomic study of quiescent neutrophils with the identification of over 3,000 proteins and neutrophils stimulated by PAF which revealed a protein profile that characterizes this stimulation, in which proteins that are part of key processes had increased abundance as reorganization cytoskeleton, intracellular signaling mediated by Ras and G-protein subunits of vacuolar ATPases, and some typical products of the inflammatory process as NFkβ and prostaglandin E. On the other hand, since neutrophils are stimulated in their state had decreased abundance other proteins, including proteins responsible for the process of differentiation and maturation of neutrophils and proteins that are part of the key process control system of the cell as protein synthesis, metabolism energy and cell motility. The identification of acetylated proteins indicated the possible targets proteins for regulation of important pathways stimulation of neutrophils such as translation, formation of the structural core of the multi-lobed neutrophil, vitaminda B12 transportation, as well as set points in the synthesis of ATP and of subunit ATPases. Identifying these proteins with differential abundance and acetylated is fundamental to indicate possible therapeutic targets for autoimmune and inflammatory diseases caused by PAF.

Platelet activating factor (PAF) was administered to female mice in order to investigate its effect on ovulation rate and on oocyte quality including their in-vitro embryonic development, implantation and uterine receptivity. In experiment 1, 4-week-old female mice were assigned to receive PAF or phosphate buffered saline for 4 consecutive days. On the second day of this treatment, pregnant mares' serum gonadotrophin was administered and human chorionic gonadotrophin (HCG) 48 h later, after which copulation occurred. Oocytes were collected on the following day and evaluated. The mean number of oocytes and zygotes (two pronuclear stage embryos) recovered from the PAF-treated group was not diffferent from the control group (31 versus 27), but the proportion of zygotes was higher in PAF-treated group than in controls (83 versus 68%, P < 0.05, PAF versus controls). Although the rate of in-vitro first cleavage was not different in the two groups (82 versus 69% respectively), hatching was higher in the PAF-treated group than control mice (99 versus 83%, P < 0.01). In experiment 2, the in-vitro developed blastocysts from experiment 1 were transferred into the uterus of day 3 pseudopregnant PAF-treated or control recipients. Three different combinations of intrauterine transfer were performed; PAF embryo to control recipient (PAF → C: n = 19), control embryo to PAF recipient (C → PAF: n = 19), and control embryo to control recipient (C → C: n = 22). Implantation and abortion were assessed on day 19 post-transfer. The implantation rate of C → PAF (23.7%) was lower than C → C (31.1%, P < 0.05), but was not different from PAF → C (31.2%). Further, C → PAF showed a higher abortion rate per embryo (29.6%) than PAF → C (12.7%, P < 0.05), but was not different from C → C (24.4%). In the present study, PAF administration enables females to produce oocytes with a higher potential for fertilization, in vitro development and implantation, but has a detrimental effect on uterine receptivity to embryos.