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O receptor do PAF no microambiente tumoral. / PAF receptor in tumor microenvironment.Silva Júnior, Ildefonso Alves da 30 March 2017 (has links)
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.
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O PAF como regulador endógeno do fenótipo e função das células dendríticas. / PAF as an endogenous modulator of Dendritic Cells phenotype and function.Koga, Marianna Mainardi 16 October 2015 (has links)
Neste trabalho nós mostramos que células dendríticas (DCs) de camundongos BALB/c expressam receptor para o PAF (Fator ativador de Plaquetas) e que sua ativação promove um fenótipo tolerogênico, associado à produção de IL10 e PGE2. O bloqueio do PAF-receptor por antagonistas aumentou a capacidade das DCs induzirem proliferação de linfócitos T. O antagonista WEB2170 potencializou a resposta imune in vivo a concentração de anticorpo IgG2a OVA-específico aumentou 30 vezes no grupo tratado; a concentração de IgG1 foi semelhante nos dois grupos. O bloqueio do PAFR em camundongos imunizados com OVA em adjuvante completo de Freund, aumentou a produção de IgG1 e IgG2a OVA-específicos. Em camundongos imunizados com OVA/alum o antagonista não alterou a produção de IgG1. Estes resultados indicam que a ativação do PAFR em DCs modula a sua função apresentadora de antígenos pela produção de IL10 e PGE2. O bloqueio do PAFR pode ser útil na ativação das DCs em protocolos de vacinação com DCs e/ou como co-adjuvante em protocolos de imunização. / In the present work we show that BALB/c mice dendritic cells (DCs) express the PAF (platelet-activating factor) receptor and that its activation promotes a tolerogenic phenotype via IL10 and PGE2 production. Blocking PAFR by selective antagonists markedly enhanced DCs ability to induce T cell proliferation. The antagonist WEB2170 potentiated the in vivo immune response the IgG2a OVA-specific levels were 30 fold increased in the treated group; IgG1 concentration was similar for both groups. The PAFR blockade in mice immunized with OVA in complete Freunds adjuvant enhanced both IgG1 and IgG2a OVA-specific antibody production. In OVA/alum immunized mice, the antagonist did not change IgG1 production. These results suggest that PAFR activation in DCs modulates their antigen-presenting function through IL10 and PGE2 production. Blocking PAFR may be useful to induce DCs activation in DCs-based vaccination protocols and/or as a co-adjuvant in immunization protocols.
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O PAF como regulador endógeno do fenótipo e função das células dendríticas. / PAF as an endogenous modulator of Dendritic Cells phenotype and function.Marianna Mainardi Koga 16 October 2015 (has links)
Neste trabalho nós mostramos que células dendríticas (DCs) de camundongos BALB/c expressam receptor para o PAF (Fator ativador de Plaquetas) e que sua ativação promove um fenótipo tolerogênico, associado à produção de IL10 e PGE2. O bloqueio do PAF-receptor por antagonistas aumentou a capacidade das DCs induzirem proliferação de linfócitos T. O antagonista WEB2170 potencializou a resposta imune in vivo a concentração de anticorpo IgG2a OVA-específico aumentou 30 vezes no grupo tratado; a concentração de IgG1 foi semelhante nos dois grupos. O bloqueio do PAFR em camundongos imunizados com OVA em adjuvante completo de Freund, aumentou a produção de IgG1 e IgG2a OVA-específicos. Em camundongos imunizados com OVA/alum o antagonista não alterou a produção de IgG1. Estes resultados indicam que a ativação do PAFR em DCs modula a sua função apresentadora de antígenos pela produção de IL10 e PGE2. O bloqueio do PAFR pode ser útil na ativação das DCs em protocolos de vacinação com DCs e/ou como co-adjuvante em protocolos de imunização. / In the present work we show that BALB/c mice dendritic cells (DCs) express the PAF (platelet-activating factor) receptor and that its activation promotes a tolerogenic phenotype via IL10 and PGE2 production. Blocking PAFR by selective antagonists markedly enhanced DCs ability to induce T cell proliferation. The antagonist WEB2170 potentiated the in vivo immune response the IgG2a OVA-specific levels were 30 fold increased in the treated group; IgG1 concentration was similar for both groups. The PAFR blockade in mice immunized with OVA in complete Freunds adjuvant enhanced both IgG1 and IgG2a OVA-specific antibody production. In OVA/alum immunized mice, the antagonist did not change IgG1 production. These results suggest that PAFR activation in DCs modulates their antigen-presenting function through IL10 and PGE2 production. Blocking PAFR may be useful to induce DCs activation in DCs-based vaccination protocols and/or as a co-adjuvant in immunization protocols.
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Participação do PAF-R na fagocitose de células apoptóticas, no fenótipo de macrófagos e na imunossupressão causada por terapia fotodinâmica. / Participation of PAF-R in the phagocytosis of apoptotic cells, in macrophage phenotype and in the immunosuppression caused by photodynamic therapy.Ferracini, Matheus 18 September 2014 (has links)
Macrófagos (Mf) produzem PAF e PAF-R e eliminam partículas alteradas via CD36. Uptake de oxLDL requer associação CD36/PAF-R. Avaliamos isto na eferocitose. Bloqueio do PAF-R e de lipid rafts (LR) inibiu eferocitose. Esta induziu associação PAF-R/CD36 e destes com flotilina-1 (marca LR). Eferocitose induziu IL-10 e IL-12p40. Bloqueio do PAF-R inibiu mais IL-10 e inibição da COX-2 teve efeito similar, sugerindo que eferocitose depende da interação PAF-R/CD36 em LR e que isto induz prostanoides e perfil regulador. Mf adquirirem diferentes fenótipos. Estudamos a participação do PAF-R. Bloqueio do PAF-R antes dos estímulos (IFN-g/LPS, IL-4 ou IgG-SRBC/LPS) inibiu marcadores MCP-1, TNF-a, iNOS, receptor manose, arginase-1 e IL-10, mas não IL-12p40, sugerindo que PAF-R modula fenótipo de Mf. PAF e PAF-like são gerados por estressores oxidativos. Ativação do PAF-R induz imunossupressão sistêmica (IS). Mostramos que terapia fotodinâmica (PDT) in vitro gerou ligantes do PAF-R e in vivo inibiu reação de CHS em WT, mas não em PAF-R KO, sugerindo que PDT induz IS via PAF-R. / Macrophages (Mp) produce PAF and PAF-R and scavenge altered particles via CD36. oxLDL uptake requires association CD36/PAF-R. We analyzed that on efferocytosis. PAF-R and lipid rafts (LR) blockage inhibited efferocytosis. Efferocytosis induced association CD36/PAF-R and both with LR marker protein, and induced IL-10 and IL-12p40. PAF-R and COX-2 blockage inhibited more IL-10, suggesting that efferocytosis depends on PAF-R/CD36 interaction in LR and that this induces prostanoids and regulatory profile. Mp acquire different phenotypes. PAF-R participation in that was analyzed. PAF-R blockage before stimuli (IFN-g/LPS, IL-4 or IgG-SRBC/LPS) inhibited markers MCP-1, TNF-a, iNOS, mannose receptor, arginase-1 and IL-10, but not IL-12p40, suggesting that PAF-R modulates Mp phenotype. PAF and PAF-like are generated by oxidative stressors. PAF-R activation induces systemic immunosuppression (SI). We showed that photodynamic therapy (PDT) in vitro generated PAF-R ligands and in vivo inhibited CHS reaction in WT, but not PAF-R KO, suggesting that PDT induces SI via PAF-R.
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Participação do PAF-R na fagocitose de células apoptóticas, no fenótipo de macrófagos e na imunossupressão causada por terapia fotodinâmica. / Participation of PAF-R in the phagocytosis of apoptotic cells, in macrophage phenotype and in the immunosuppression caused by photodynamic therapy.Matheus Ferracini 18 September 2014 (has links)
Macrófagos (Mf) produzem PAF e PAF-R e eliminam partículas alteradas via CD36. Uptake de oxLDL requer associação CD36/PAF-R. Avaliamos isto na eferocitose. Bloqueio do PAF-R e de lipid rafts (LR) inibiu eferocitose. Esta induziu associação PAF-R/CD36 e destes com flotilina-1 (marca LR). Eferocitose induziu IL-10 e IL-12p40. Bloqueio do PAF-R inibiu mais IL-10 e inibição da COX-2 teve efeito similar, sugerindo que eferocitose depende da interação PAF-R/CD36 em LR e que isto induz prostanoides e perfil regulador. Mf adquirirem diferentes fenótipos. Estudamos a participação do PAF-R. Bloqueio do PAF-R antes dos estímulos (IFN-g/LPS, IL-4 ou IgG-SRBC/LPS) inibiu marcadores MCP-1, TNF-a, iNOS, receptor manose, arginase-1 e IL-10, mas não IL-12p40, sugerindo que PAF-R modula fenótipo de Mf. PAF e PAF-like são gerados por estressores oxidativos. Ativação do PAF-R induz imunossupressão sistêmica (IS). Mostramos que terapia fotodinâmica (PDT) in vitro gerou ligantes do PAF-R e in vivo inibiu reação de CHS em WT, mas não em PAF-R KO, sugerindo que PDT induz IS via PAF-R. / Macrophages (Mp) produce PAF and PAF-R and scavenge altered particles via CD36. oxLDL uptake requires association CD36/PAF-R. We analyzed that on efferocytosis. PAF-R and lipid rafts (LR) blockage inhibited efferocytosis. Efferocytosis induced association CD36/PAF-R and both with LR marker protein, and induced IL-10 and IL-12p40. PAF-R and COX-2 blockage inhibited more IL-10, suggesting that efferocytosis depends on PAF-R/CD36 interaction in LR and that this induces prostanoids and regulatory profile. Mp acquire different phenotypes. PAF-R participation in that was analyzed. PAF-R blockage before stimuli (IFN-g/LPS, IL-4 or IgG-SRBC/LPS) inhibited markers MCP-1, TNF-a, iNOS, mannose receptor, arginase-1 and IL-10, but not IL-12p40, suggesting that PAF-R modulates Mp phenotype. PAF and PAF-like are generated by oxidative stressors. PAF-R activation induces systemic immunosuppression (SI). We showed that photodynamic therapy (PDT) in vitro generated PAF-R ligands and in vivo inhibited CHS reaction in WT, but not PAF-R KO, suggesting that PDT induces SI via PAF-R.
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Modulation allostérique de la fonction des récepteurs FP et PAFBivona, Dario Antonio 07 1900 (has links)
Les récepteurs couplés aux protéines-G (RCPGs) constituent la première étape d’une série de cascades signalétiques menant à la régulation d’une multitude de processus physiologiques. Dans le modèle classique connu, la liaison du ligand induit un changement de conformation du récepteur qui mène à sa forme active. Une fois activés, les RCPGs vont réguler l’activité d’une protéine membranaire cible qui peut être tant une enzyme qu’un canal ionique. L’interaction entre le récepteur et la cible nécessite l’intermédiaire d’une protéine hétérotrimérique appelée « protéine G », qui est activée pour favoriser l’échange du GDP (guanosine diphosphate) pour un GTP (guanosine triphosphate) et assurer la transduction du signal du récepteur à l’effecteur. Les mécanismes moléculaires menant à l’activation des effecteurs spécifiques via l’activation des RCPGs par les protéines G hétérotrimériques sont encore plutôt méconnus.
Dans notre étude nous nous sommes intéressés aux récepteurs FP et PAF, à leurs ligands naturels, la PGF2α et le Carbamyl-PAF respectivement, et à des ligands à action antagoniste sur ces récepteurs. Des ligands considérés comme agonistes, sont des molécules qui interagissent avec le récepteur et induisent les mêmes effets que le ligand naturel. Les antagonistes, par contre, sont des molécules qui interagissent avec le récepteur et bloquent l’action du ligand naturel en prévenant le changement conformationnel du complexe, et ils peuvent avoir une action compétitive ou non-compétitive.
Nous avons étudié aussi des ligands orthostériques et allostériques du récepteur FP des prostaglandines et du récepteur PAF. Un ligand orthostérique peut se comporter comme agoniste ou antagoniste en se fixant au site de liaison du ligand (agoniste) naturel. Un ligand allostérique est un agoniste ou antagoniste se fixant à un site autre que celui du ligand naturel entraînant un changement de conformation ayant pour conséquence soit une augmentation (effecteur positif), soit une diminution (effecteur négatif) de l'activité du ligand naturel. / G protein coupled receptors (GPCRs) are involved in the first step of most signalling pathways that regulate a variety of physiological events. The classical view of GPCR activation suggests that ligand binding to the inactive receptor will trigger a conformational change leading to an active conformation of the receptor. The GPCRs activated regulate the activity of a target membrane protein which can then activate other signalling proteins such as enzymes and ionic channels. The interaction between the receptor and the target requires an intermediary, in this case an heterotrimeric protein named « G protein », which is activated in order to facilitate the exchange of GDP (guanosine diphospate) for a GTP (guanosine triphosphate) and allow the transduction of the signal from the receptor to the effector. The molecular mechanisms leading to the activation of signalling effectors via the activation of GPCRs by its heterotrimeric G protein have not yet been well characterized.
We focused our study on two GPCRs, the FP and PAF receptors, their natural ligands, PGF2α and Carbamyl-PAF respectively, and their antagonist ligands. Agonists are ligands that bind to the target receptor and trigger the same effects as the natural ligand of the GPCR. In contrast with agonists, antagonist ligands are molecules that prevent the effects of the natural ligand by keeping the GPCR from changing to its active conformation and can be competitive or non-competitive.
We have also studied orthosteric and allosteric ligands of the FP and PAF receptors. An orthosteric ligand binds the same site as the natural ligand of the receptor and can act as an agonist or an antagonist. In the contrary, an allosteric ligand will rather have a different binding site then the natural ligand (agonist) and can positively or negatively modulate the effects of the natural ligand.
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Modulation allostérique de la fonction des récepteurs FP et PAFBivona, Dario Antonio 07 1900 (has links)
Les récepteurs couplés aux protéines-G (RCPGs) constituent la première étape d’une série de cascades signalétiques menant à la régulation d’une multitude de processus physiologiques. Dans le modèle classique connu, la liaison du ligand induit un changement de conformation du récepteur qui mène à sa forme active. Une fois activés, les RCPGs vont réguler l’activité d’une protéine membranaire cible qui peut être tant une enzyme qu’un canal ionique. L’interaction entre le récepteur et la cible nécessite l’intermédiaire d’une protéine hétérotrimérique appelée « protéine G », qui est activée pour favoriser l’échange du GDP (guanosine diphosphate) pour un GTP (guanosine triphosphate) et assurer la transduction du signal du récepteur à l’effecteur. Les mécanismes moléculaires menant à l’activation des effecteurs spécifiques via l’activation des RCPGs par les protéines G hétérotrimériques sont encore plutôt méconnus.
Dans notre étude nous nous sommes intéressés aux récepteurs FP et PAF, à leurs ligands naturels, la PGF2α et le Carbamyl-PAF respectivement, et à des ligands à action antagoniste sur ces récepteurs. Des ligands considérés comme agonistes, sont des molécules qui interagissent avec le récepteur et induisent les mêmes effets que le ligand naturel. Les antagonistes, par contre, sont des molécules qui interagissent avec le récepteur et bloquent l’action du ligand naturel en prévenant le changement conformationnel du complexe, et ils peuvent avoir une action compétitive ou non-compétitive.
Nous avons étudié aussi des ligands orthostériques et allostériques du récepteur FP des prostaglandines et du récepteur PAF. Un ligand orthostérique peut se comporter comme agoniste ou antagoniste en se fixant au site de liaison du ligand (agoniste) naturel. Un ligand allostérique est un agoniste ou antagoniste se fixant à un site autre que celui du ligand naturel entraînant un changement de conformation ayant pour conséquence soit une augmentation (effecteur positif), soit une diminution (effecteur négatif) de l'activité du ligand naturel. / G protein coupled receptors (GPCRs) are involved in the first step of most signalling pathways that regulate a variety of physiological events. The classical view of GPCR activation suggests that ligand binding to the inactive receptor will trigger a conformational change leading to an active conformation of the receptor. The GPCRs activated regulate the activity of a target membrane protein which can then activate other signalling proteins such as enzymes and ionic channels. The interaction between the receptor and the target requires an intermediary, in this case an heterotrimeric protein named « G protein », which is activated in order to facilitate the exchange of GDP (guanosine diphospate) for a GTP (guanosine triphosphate) and allow the transduction of the signal from the receptor to the effector. The molecular mechanisms leading to the activation of signalling effectors via the activation of GPCRs by its heterotrimeric G protein have not yet been well characterized.
We focused our study on two GPCRs, the FP and PAF receptors, their natural ligands, PGF2α and Carbamyl-PAF respectively, and their antagonist ligands. Agonists are ligands that bind to the target receptor and trigger the same effects as the natural ligand of the GPCR. In contrast with agonists, antagonist ligands are molecules that prevent the effects of the natural ligand by keeping the GPCR from changing to its active conformation and can be competitive or non-competitive.
We have also studied orthosteric and allosteric ligands of the FP and PAF receptors. An orthosteric ligand binds the same site as the natural ligand of the receptor and can act as an agonist or an antagonist. In the contrary, an allosteric ligand will rather have a different binding site then the natural ligand (agonist) and can positively or negatively modulate the effects of the natural ligand.
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