Angiotensin II (Ang II) is an important modulator for development of atherosclerosis from early stage foam cell formation to advanced stage plaque rupture. Recently, the importance of locally generated Ang II, especially in macrophages, has become more evident. Generation of Ang II by several enzymes other than ACE and renin has been shown mainly in vitro. Cathepsin G is one these enzymes which is expressed in neutrophils and macrophages. Macrophages are one of the primary and crucial cells in atherosclerotic lesions which become lipid-laden foam cells through lipoprotein uptake. We hypothesized that activation of nuclear factors in foam cells increases Ang II by modulation of the renin angiotensin system (RAS) genes and cathepsin G. We also hypothesized that cathepsin G, through its Ang II generating activity and its other catalytic functions, promotes atherosclerosis. The present study assessed the Ang I and II levels and expression of the RAS genes in THP-1 cells, a human acute monocytic leukemia cell line, and in peritoneal and bone marrow-derived macrophages after exposure to acetylated LDL (ac-LDL). I also evaluated how RAS blockade would affect foam cell formation in THP-1 cells. In parallel, I assessed the role of cathepsin G in Ang II generation and in the progression of atherosclerosis in cathepsin G heterozygous knockout mice on an Apoe-/- background (Ctsg+/-Apoe-/- mice). Ac-LDL treatment increased Ang I and Ang II levels in cell lysates and media from THP-1 cells but not in peritoneal or bone marrow-derived macrophages from wild type C57BL/6 mice. In ac-LDL-treated THP-1 cells, ACE and cathepsin G mRNA levels and activities were elevated. Angiotensinogen mRNA is increased but not the angiotensinogen protein concentration. Renin mRNA level and activity were not altered by ac-LDL treatment. Blocking RAS by an AT1 receptor blocker, ACE inhibitors or a renin inhibitor decreased cholesteryl ester content of THP-1 cells after exposure to ac-LDL. To confirm that the Ang II effect on foam cell formation was not unique to ac-LDL, we treated the THP-1 macrophages with a renin inhibitor or an AT1 receptor inhibitor after exposure to oxidized LDL (ox-LDL). RAS blockade in ox-LDL-treated cells also abolished cholesteryl ester formation. To see how Ang II plays a role in foam cell formation we assessed the effect of RAS inhibitors on SR-A, the principal receptor for mediating ac-LDL entry into the cells and on acyl-CoA:cholesterol acyl transferase (ACAT-1), the enzyme responsible for intracellular cholesterol esterification. RAS blockade in both ac-LDL- and ox-LDL-treated cells decreased SR-A and ACAT-1 protein levels. Cathepsin G partial deficiency on an Apoe-/- background did not change Ang II levels in peritoneal or bone marrow-derived macrophage cell lysates or media. This deficiency also did not affect immunoreactive angiotensin peptide levels in atherosclerotic lesions. After 8 weeks on a high fat diet Ctsg+/-Apoe-/- mice were similar to Ctsg+/+Apoe-/- mice in terms of lesion size and serum cholesterol levels but the Ctsg+/+Apoe-/- mice had more advanced lesions with more collagen and smooth muscle cells and fewer macrophages. Moreover, Ctsg+/+Apoe-/- mice had more apoptotic cells than their Ctsg+/-Apoe-/- littermates. Overall, our findings indicate that Ang II is increased in foam cells and this endogenous Ang II is involved in cholesteryl ester formation, possibly by regulating the levels of ACAT-1 and SR-A. We did not find any role for cathepsin G in generation of Ang II in mice but cathepsin G does, nevertheless, promote the progression of atherosclerotic lesions to a more advanced stage.

Angiotensin II (Ang II) is an important modulator for development of atherosclerosis from early stage foam cell formation to advanced stage plaque rupture. Recently, the importance of locally generated Ang II, especially in macrophages, has become more evident. Generation of Ang II by several enzymes other than ACE and renin has been shown mainly in vitro. Cathepsin G is one these enzymes which is expressed in neutrophils and macrophages. Macrophages are one of the primary and crucial cells in atherosclerotic lesions which become lipid-laden foam cells through lipoprotein uptake. We hypothesized that activation of nuclear factors in foam cells increases Ang II by modulation of the renin angiotensin system (RAS) genes and cathepsin G. We also hypothesized that cathepsin G, through its Ang II generating activity and its other catalytic functions, promotes atherosclerosis. The present study assessed the Ang I and II levels and expression of the RAS genes in THP-1 cells, a human acute monocytic leukemia cell line, and in peritoneal and bone marrow-derived macrophages after exposure to acetylated LDL (ac-LDL). I also evaluated how RAS blockade would affect foam cell formation in THP-1 cells. In parallel, I assessed the role of cathepsin G in Ang II generation and in the progression of atherosclerosis in cathepsin G heterozygous knockout mice on an Apoe-/- background (Ctsg+/-Apoe-/- mice). Ac-LDL treatment increased Ang I and Ang II levels in cell lysates and media from THP-1 cells but not in peritoneal or bone marrow-derived macrophages from wild type C57BL/6 mice. In ac-LDL-treated THP-1 cells, ACE and cathepsin G mRNA levels and activities were elevated. Angiotensinogen mRNA is increased but not the angiotensinogen protein concentration. Renin mRNA level and activity were not altered by ac-LDL treatment. Blocking RAS by an AT1 receptor blocker, ACE inhibitors or a renin inhibitor decreased cholesteryl ester content of THP-1 cells after exposure to ac-LDL. To confirm that the Ang II effect on foam cell formation was not unique to ac-LDL, we treated the THP-1 macrophages with a renin inhibitor or an AT1 receptor inhibitor after exposure to oxidized LDL (ox-LDL). RAS blockade in ox-LDL-treated cells also abolished cholesteryl ester formation. To see how Ang II plays a role in foam cell formation we assessed the effect of RAS inhibitors on SR-A, the principal receptor for mediating ac-LDL entry into the cells and on acyl-CoA:cholesterol acyl transferase (ACAT-1), the enzyme responsible for intracellular cholesterol esterification. RAS blockade in both ac-LDL- and ox-LDL-treated cells decreased SR-A and ACAT-1 protein levels. Cathepsin G partial deficiency on an Apoe-/- background did not change Ang II levels in peritoneal or bone marrow-derived macrophage cell lysates or media. This deficiency also did not affect immunoreactive angiotensin peptide levels in atherosclerotic lesions. After 8 weeks on a high fat diet Ctsg+/-Apoe-/- mice were similar to Ctsg+/+Apoe-/- mice in terms of lesion size and serum cholesterol levels but the Ctsg+/+Apoe-/- mice had more advanced lesions with more collagen and smooth muscle cells and fewer macrophages. Moreover, Ctsg+/+Apoe-/- mice had more apoptotic cells than their Ctsg+/-Apoe-/- littermates. Overall, our findings indicate that Ang II is increased in foam cells and this endogenous Ang II is involved in cholesteryl ester formation, possibly by regulating the levels of ACAT-1 and SR-A. We did not find any role for cathepsin G in generation of Ang II in mice but cathepsin G does, nevertheless, promote the progression of atherosclerotic lesions to a more advanced stage.

Angiotensin II (Ang II) is an important modulator for development of atherosclerosis from early stage foam cell formation to advanced stage plaque rupture. Recently, the importance of locally generated Ang II, especially in macrophages, has become more evident. Generation of Ang II by several enzymes other than ACE and renin has been shown mainly in vitro. Cathepsin G is one these enzymes which is expressed in neutrophils and macrophages. Macrophages are one of the primary and crucial cells in atherosclerotic lesions which become lipid-laden foam cells through lipoprotein uptake. We hypothesized that activation of nuclear factors in foam cells increases Ang II by modulation of the renin angiotensin system (RAS) genes and cathepsin G. We also hypothesized that cathepsin G, through its Ang II generating activity and its other catalytic functions, promotes atherosclerosis. The present study assessed the Ang I and II levels and expression of the RAS genes in THP-1 cells, a human acute monocytic leukemia cell line, and in peritoneal and bone marrow-derived macrophages after exposure to acetylated LDL (ac-LDL). I also evaluated how RAS blockade would affect foam cell formation in THP-1 cells. In parallel, I assessed the role of cathepsin G in Ang II generation and in the progression of atherosclerosis in cathepsin G heterozygous knockout mice on an Apoe-/- background (Ctsg+/-Apoe-/- mice). Ac-LDL treatment increased Ang I and Ang II levels in cell lysates and media from THP-1 cells but not in peritoneal or bone marrow-derived macrophages from wild type C57BL/6 mice. In ac-LDL-treated THP-1 cells, ACE and cathepsin G mRNA levels and activities were elevated. Angiotensinogen mRNA is increased but not the angiotensinogen protein concentration. Renin mRNA level and activity were not altered by ac-LDL treatment. Blocking RAS by an AT1 receptor blocker, ACE inhibitors or a renin inhibitor decreased cholesteryl ester content of THP-1 cells after exposure to ac-LDL. To confirm that the Ang II effect on foam cell formation was not unique to ac-LDL, we treated the THP-1 macrophages with a renin inhibitor or an AT1 receptor inhibitor after exposure to oxidized LDL (ox-LDL). RAS blockade in ox-LDL-treated cells also abolished cholesteryl ester formation. To see how Ang II plays a role in foam cell formation we assessed the effect of RAS inhibitors on SR-A, the principal receptor for mediating ac-LDL entry into the cells and on acyl-CoA:cholesterol acyl transferase (ACAT-1), the enzyme responsible for intracellular cholesterol esterification. RAS blockade in both ac-LDL- and ox-LDL-treated cells decreased SR-A and ACAT-1 protein levels. Cathepsin G partial deficiency on an Apoe-/- background did not change Ang II levels in peritoneal or bone marrow-derived macrophage cell lysates or media. This deficiency also did not affect immunoreactive angiotensin peptide levels in atherosclerotic lesions. After 8 weeks on a high fat diet Ctsg+/-Apoe-/- mice were similar to Ctsg+/+Apoe-/- mice in terms of lesion size and serum cholesterol levels but the Ctsg+/+Apoe-/- mice had more advanced lesions with more collagen and smooth muscle cells and fewer macrophages. Moreover, Ctsg+/+Apoe-/- mice had more apoptotic cells than their Ctsg+/-Apoe-/- littermates. Overall, our findings indicate that Ang II is increased in foam cells and this endogenous Ang II is involved in cholesteryl ester formation, possibly by regulating the levels of ACAT-1 and SR-A. We did not find any role for cathepsin G in generation of Ang II in mice but cathepsin G does, nevertheless, promote the progression of atherosclerotic lesions to a more advanced stage.

<p> Mammalian sialidases are hydrolytic enzymes that initiate the removal of terminal a2-3, a2-6 and a2-8 sialic acid residues from various sialylated glycoconjugates. Sialidases are reportedly involved in numerous cellular functions involving proliferation, differentiation, antigenic expression, inflammation and the tumorigenicity of malignant cells. Recently, sialidase has been implicated in various immune signaling pathways, involving immune effector cells, such as activated lymphocytes and macrophages. The human lysosomal sialidase gene encodes a 46 kD glycoprotein which exists in a multienzyme complex with β-galactosidase and PPCA. Neurodegenerative diseases such as Tay-Sachs and Sandhoff are characterized by the progressive storage of glycoproteins and sialylated oligosaccharides in the nervous system. The induction of inflammatory mediators is a critical step in the pathogenesis of neurodegeneration that remains largely undefined. As such, an in vitro model of Tay-Sachs disease was used to identify potential mediators involved in disease progression. In addition, we have used the THP-1 monocytic cell line as a model of human macrophages which play a key role in potentiating a variety of immune responses. </p> <p> Translocation of neul from lysosomes to the cell surface and the resulting interaction with signaling molecules suggests neul is involved in the regulation of immune activities. We have investigated the role of sialidase on CD44 expression, an inflammation-associated glycoprotein found on the cell surface. Our data indicate that sialidase interacts with CD44 on the cell surface which may contribute to disease progression in Tay-Sachs disease. We illustrate that overexpression of sialidase stimulates interleukin-6 (IL-6) secretion in both human Tay-Sachs neuroglia and THP-1 derived macrophages. Moreover, the sialidase inhibitor 2-deoxy-2, 3-dehydro-N-acetylneuraminic acid (DANA) was found to attenuate IL-6 secretion and sialidase expression in THP-1 derived macrophages. </p> / Thesis / Master of Science (MSc)

Orientador: Alexandrina Sartori / Resumo: A leishmaniose visceral (LV) é uma doença negligenciada causada por Leishmania donovani na Índia e África ou Leishmania infantum na Europa e América Latina. O desenvolvimento de resposta imune eficaz e subsequente eliminação destes patógenos, requer o reconhecimento inicial da Leishmania, o qual é intermediado por receptores de reconhecimento padrão expressos por células da imunidade inata, entre eles os receptores de ligação a nucleotídeo (NLRs). Alguns NLRs ativam uma plataforma de proteínas, os inflamassomas, responsáveis pela ativação da caspase-1 e maturação de IL-1β e IL-18 e outra classe de NLRs, chamada NODs, ativam vias que culminam na ativação de NF-κB e produção de mediadores inflamatórios. O envolvimento desses receptores na LV ainda é pouco elucidado. Mesmo diante dos mecanismos de defesa do hospedeiro, esses parasitas conseguem sobreviver dentro dos macrófagos utilizando várias estratégias para escapar da resposta imune. Para um melhor entendimento dos mecanismos imunes envolvidos na LV, caracterizamos o perfil transcricional e a formação de inflamassomas e NODsomas de células THP-1 infectadas com L. infantum. Os resultados mostram que a L. infantum não induziu produção de TNF-α, IL-6 e IL-1β e nem ativação de caspase-1 após 8, 24 e 48 horas de infecção. Além disso, a infecção resultou em padrão de expressão gênica similar às células sem estímulo e distinto de células estimuladas com LPS, indicando que os parasitas entram nas células de forma mais silenciosa. Ap... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Visceral leishmaniasis (VL) is a neglected infectious disease caused by Leishmania donovani in India and Africa or Leishmania infantum in Europe and Latin America. The development of an effective immune response and subsequent elimination of these pathogens requires the initial recognition of the Leishmania that is mediated by pattern recognition receptors expressed in innate immunity cells, such as nucleotide-binding receptors (NLRs). Some NLRs activate a multiprotein platform named inflammasomes, responsible for the activation of caspase-1 and consequent maturation of IL-1β and IL-18; and another class of NLRs, the NODs, activate pathways that trigger NF-κB activation and production of inflammatory mediators. The involvement of NLRs in LV is poorly elucidated. Even in the presence of host defense mechanisms, these parasites can survive within the macrophages by employing successful strategies to escape from immune response. For a better understanding of the immune mechanisms involved in LV, we characterized the transcriptomic profiling and assembly of inflammasomes and NODsomas during infection with L. infantum in THP-1 cells. The results show that L. infantum did not induce the production of TNF-α, IL-6 and IL-1β nor activation of caspase-1 after 8, 24 and 48 hours of infection. In addition, the infection resulted in a pattern of gene expression similar to the non-stimulated cells and distinct from LPS-stimulated cells, indicating that the parasites enter inside cells in a... (Complete abstract click electronic access below) / Doutor

Submitted by Mariana Gatto (marianagatto11@hotmail.com) on 2018-05-21T17:47:50Z No. of bitstreams: 1 Tese Mariana Gatto.pdf: 3235524 bytes, checksum: b7c9938cd744aff0ff8a8ee5f858831e (MD5) / Approved for entry into archive by Sulamita Selma C Colnago null (sulamita@btu.unesp.br) on 2018-05-22T14:28:44Z (GMT) No. of bitstreams: 1 gatto_m_dr_bot.pdf: 3235524 bytes, checksum: b7c9938cd744aff0ff8a8ee5f858831e (MD5) / Made available in DSpace on 2018-05-22T14:28:44Z (GMT). No. of bitstreams: 1 gatto_m_dr_bot.pdf: 3235524 bytes, checksum: b7c9938cd744aff0ff8a8ee5f858831e (MD5) Previous issue date: 2018-04-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A leishmaniose visceral (LV) é uma doença negligenciada causada por Leishmania donovani na Índia e África ou Leishmania infantum na Europa e América Latina. O desenvolvimento de resposta imune eficaz e subsequente eliminação destes patógenos, requer o reconhecimento inicial da Leishmania, o qual é intermediado por receptores de reconhecimento padrão expressos por células da imunidade inata, entre eles os receptores de ligação a nucleotídeo (NLRs). Alguns NLRs ativam uma plataforma de proteínas, os inflamassomas, responsáveis pela ativação da caspase-1 e maturação de IL-1β e IL-18 e outra classe de NLRs, chamada NODs, ativam vias que culminam na ativação de NF-κB e produção de mediadores inflamatórios. O envolvimento desses receptores na LV ainda é pouco elucidado. Mesmo diante dos mecanismos de defesa do hospedeiro, esses parasitas conseguem sobreviver dentro dos macrófagos utilizando várias estratégias para escapar da resposta imune. Para um melhor entendimento dos mecanismos imunes envolvidos na LV, caracterizamos o perfil transcricional e a formação de inflamassomas e NODsomas de células THP-1 infectadas com L. infantum. Os resultados mostram que a L. infantum não induziu produção de TNF-α, IL-6 e IL-1β e nem ativação de caspase-1 após 8, 24 e 48 horas de infecção. Além disso, a infecção resultou em padrão de expressão gênica similar às células sem estímulo e distinto de células estimuladas com LPS, indicando que os parasitas entram nas células de forma mais silenciosa. Após a infecção houve aumento da expressão de alguns genes como ACTG1, ACTB, CD36 e DUSPs relacionados com vias de motilidade celular e regulação de MAPKs. Os genes CSF1 e CDC20 foram dois dos 30 mais expressos após infecção e estão relacionados com ciclo celular e diferenciação de macrófagos para um perfil anti-inflamatório. O gene GBP1, associado com ativação de inflamassomas, foi sub expresso após a infecção. Além disso, infecção com L. infantum resultou na expressão de poucos genes relacionados com a via dos NLRs, destacando-se entre esses o TNFAIP3 e IL1RN, referentes à modulação negativa dessa via. Os resultados obtidos indicam que a L. infantum entra nas células THP-1 de forma mais silenciosa, desativa vias inflamatórias, entre essas a via de receptores NLRs e evita a montagem de uma resposta imunológica efetora. Provavelmente o parasita usa esses recursos como mecanismos adicionais de escape para garantir sua sobrevivência dentro das células. / Visceral leishmaniasis (VL) is a neglected infectious disease caused by Leishmania donovani in India and Africa or Leishmania infantum in Europe and Latin America. The development of an effective immune response and subsequent elimination of these pathogens requires the initial recognition of the Leishmania that is mediated by pattern recognition receptors expressed in innate immunity cells, such as nucleotide-binding receptors (NLRs). Some NLRs activate a multiprotein platform named inflammasomes, responsible for the activation of caspase-1 and consequent maturation of IL-1β and IL-18; and another class of NLRs, the NODs, activate pathways that trigger NF-κB activation and production of inflammatory mediators. The involvement of NLRs in LV is poorly elucidated. Even in the presence of host defense mechanisms, these parasites can survive within the macrophages by employing successful strategies to escape from immune response. For a better understanding of the immune mechanisms involved in LV, we characterized the transcriptomic profiling and assembly of inflammasomes and NODsomas during infection with L. infantum in THP-1 cells. The results show that L. infantum did not induce the production of TNF-α, IL-6 and IL-1β nor activation of caspase-1 after 8, 24 and 48 hours of infection. In addition, the infection resulted in a pattern of gene expression similar to the non-stimulated cells and distinct from LPS-stimulated cells, indicating that the parasites enter inside cells in a more silent way. After infection, there was increased expression of some genes, such as ACTG1, ACTB, CD36 and DUSPs related to cellular motility and regulation of MAPKs pathways. The CSF1 and CDC20 genes were two of the 30 most expressed after infection and were related to cell cycle pathway and macrophage differentiation to an anti-inflammatory profile. The GBP1 gene, associated with inflammasome activation, was downregulated after infection. In addition, infection with L. infantum resulted in the expression of few genes related to the NLRs pathway, such as TNFAIP3 and IL1RN that are related to down modulation of this pathway. The results indicate that L. infantum enters inside the THP-1 cells more quietly, deactivates inflammatory pathways, including the NLR receptor pathway, and avoids the assembly of an effector immune response. Probably the parasite uses these strategies as additional escape mechanism to ensure its survival within host cells.

No description available.

The system of mononuclear phagocytes includes macrophages that modulate their phenotype based on microenvironmental signals. Their properties vary considerably in a differentiated stage. M1 macrophages, which are classically activated (typically by IFN-γ), are involved in phagocytosis and produce some pro-inflammatory cytokines, that can stimulate other immune cells. A phenotypically different cell population are M2 macrophages, which are alternatively activated by exposure by Th2 cytokines. M2 macrophages produce preferentially anti-inflammatory cytokines IL-10, TGF-β and participate in repair and tissue healing. The main aim of this study was to standardize a model of differentiation of THP-1 cells and human monocytes towards the M2 phenotyp using an in vitro model. This is represented in particular by increased expression of CD163 and CD206 molecules. The second aim was to assess the dynamics of expression (and co-expression) of CD163 and CD206 molecules in monocytes of patients after kidney transplantation. Expression of surface markers was determined by flow cytometry. Both THP-1 cells and human monocytes, isolated from buffy coat fraction, were stimulated by IL-4, TNF-α, TGF-β and IL-10. Changes in CD163 and CD206 expression were measured after day 1, day 3 and day 6 of stimulation. The most...

En dehors de son rôle de stockage de l’énergie cellulaire, l’adénosine-triphosphate (ATP) est également une molécule de signalisation extracellulaire qui agit sur deux familles de récepteurs, les récepteurs métabotropiques P2Y et les récepteurs ionotropiques P2X. Dans le système nerveux central, les récepteurs P2X et la signalisation purinergique sont impliqués dans de nombreuses fonctions physiologiques comme la modulation de la transmission synaptique et la communication neurone-glie ainsi que dans diverses pathologies telles que les douleurs chroniques, l’épilepsie ou les maladies neurodégénératives. Enregistrer l’activité des récepteurs P2X in situ reste aujourd’hui encore difficile de par la paucité des outils pharmacologiques et de par les propriétés biophysiques particulières de ces récepteurs canaux. De surcroit, les mécanismes de libération de l’ATP sont encore mal caractérisés et la détection de cette molécule dans l’espace extracellulaire est limitée par la faible résolution spatio-temporelle des techniques disponibles. A ce jour, il n’existe aucune méthode satisfaisante permettant de suivre l’activité des récepteurs P2X ou détecter la libération d’ATP en temps réel. Pour pallier à ces manques, nous avons développé de nouveaux outils fluorescents basés sur la fusion du rapporteur calcique GCaMP6s aux récepteurs P2X.Notre étude montre d’abord que ces biosenseurs P2X-GCaMP6s sont capables de rapporter spécifiquement et dynamiquement, en fluorescence, l’activité des récepteurs P2X dans différentes lignées cellulaires (HEK, astrocytes, macrophages), ainsi que dans des neurones hippocampiques en culture. Dans un deuxième temps, l’outil P2X2-GCaMP6s a été modifié afin de créer un biosenseur de haute affinité pour l’ATP extracellulaire. Deux mutants, dont l’affinité apparente est de l’ordre de la centaine de nanomolaire, ont permis de détecter et de quantifier une libération d’ATP endogène. En combinant l’utilisation de ces biosenseurs avec des approches pharmacologiques et génétiques, nous avons montré que lors d’un choc hypotonique l’activation du canal VRAC LRRC8 contribue à la libération d’ATP par les cellules HEK et par des monocytes humains différenciés en macrophages. Enfin, nous avons montré que la libération d’ATP lors du gonflement des cellules déclenchait le phénomène de régulation du volume cellulaire, permettant aux cellules de retrouver leur volume initial.Ces biosenseurs fluorescents permettent donc de visualiser de façon dynamique l’activité des récepteurs P2X et la libération d’ATP. Ces outils étant compatibles avec des approches in vivo, ils devraient permettre une meilleure caractérisation des mécanismes moléculaires de la communication purinergique. / Adenosine 5'-triphosphate (ATP) is an extracellular signaling molecule acting on two major classes of membrane receptors, metabotropic P2Y receptors and ionotropic P2X receptors. In the central nervous system, P2X receptors are involved in diverse functions such as modulation of synaptic transmission or neuron-glia communication and are implicated in different pathologies including chronic pain, epilepsy or neurodegenerative diseases. Recording P2X receptor activity is difficult because of the paucity of pharmacological tools and because P2X receptors are prone to desensitization. In addition, measuring extracellular ATP concentration is challenging since the mechanisms and the source of ATP are still poorly characterized. In addition, classical ATP detecting approaches have clear spatial and temporal limitations. As a consequence, following P2X activity and visualizing or quantifying ATP release in real time remains challenging. To overcome these issues, we developed new fluorescent biosensors based on the fusion of the fluorescent calcium reporter GCaMP6s to P2X receptors.We first determined that fluorescence specifically reports on the activity of the P2X2 channel in different cell line (HEK, astrocytes, macrophages) and in primary culture of hippocampal neurons. We next engineered P2X2 receptor to create high affinity ATP biosensors. We identified two mutants with EC50s for ATP in the 100 nanomolar range that allow for the detection and the quantification of endogenous ATP release evoked by cell swelling. Using pharmacological approaches and knock-out cells, we demonstrated the implication in ATP release of the recently identify volume-regulated anion channel, LRRC8A in HEK cells and differentiated human macrophages. Finally, we provided evidence that the LRRC8-dependant ATP release is necessary for the cellular regulation of volume decrease after swelling.Our results show that these fluorescent ATP biosensors can be used to dynamically track P2X channel activity and can be used in vivo to decipher the molecular mechanisms involved in purinergic signaling.

A diferenciação das células THP-1 em macrófagos, induzida por PMA, é associada ao aumento da expressão de PPAR. A UTR 5` de PPAR regula negativamente sua síntese, porém, o mecanismo molecular envolvido não foi esclarecido. Neste estudo, o estado traducional das células THP-1 diferenciadas por PMA foi investigado em associação à superprodução de PPAR. A presença de uORFs no transcrito de PPAR, contendo códons de iniciação compatíveis com seqüências de Kosak, poderia ser a causa do efeito inibitório da UTR 5`. A incorporação reduzida de L-[U-14C]leucina revelou que a superprodução de PPAR ocorre durante inibição global da tradução, confirmada pela redução dos polissomos. Além disso, desfosforilação de 4E-BP1 foi observada após tratamento com PMA e é associada a inibição da iniciação da tradução e estimulação da tradução dependente de IRES. De fato, a estrutura da UTR 5` de PPAR apresenta características de transcritos que formam IRES. Assim, a produção de PPAR pode ser regulada por IRES e ocorre concomitantemente com a inibição da tradução dependente de cap / The differentiation of THP-1 cells in macrophages, induced by PMA, is associated to overexpression of PPARb. Previous studies have shown that the PPARb 5\' UTR negatively regulates its expression. In our study the translational status of PMA-differentiated THP-1 cells was investigated in association to PPARb overexpression. Putative compatible Kosak initiation codons were identified in the PPARb uORFs and could be involved in the inhibitory effect of 5\' UTR. Decreased incorporation of L-[U-14C]leucine in proteins revealed that the overproduction of PPARb in PMA-differentiated THP-1 cells coincides with a global decrease in the protein synthesis process. Translation impairment was confirmed by polysome profile assay. An intense dephosphorylation of 4E-BP by PMA treatment was observed. Dephosphorylated 4E-BP causes inhibition of eIF4E cap-dependent translation initiation and favors IRES-dependent translation. The PPARb 5\' UTR structure has some characteristics that resemble the one described for IRES. Therefore, the PPARb production may be controlled by IRES