La drépanocytose est une maladie génétique du globule rouge, due à une mutation ponctuelle du gène β de la chaine de l’hémoglobine. Néanmoins, la physiopathologie de la maladie va bien au-delà des anomalies érythrocytaires, avec notamment un dysfonctionnement vasculaire et leucocytaire qui font toute la complexité de la maladie. Au cours de ce travail, nous avons étudié le rôle de l’endothéline-1, peptide vaso-constricteur impliqué de nombreuses pathologies notamment vasculaires, sur l’activation et l’adhérence des polynucléaires neutrophiles dans la drépanocytose. Nous avons travaillé à la fois sur un modèle murin de drépanocytose (les souris SAD) et sur des échantillons sanguins de patients. Sur le modèle animal, nous avons réalisé des expériences de microscopie intravitale, permettant de tester in vivo l’effet des antagonistes des récepteurs ETA et ETB à l’endothéline sur le recrutement des polynucléaires neutrophiles. Sur les échantillons de sang de patients drépanocytaires, nous avons testé in vitro l’adhérence en flux des neutrophiles à l’endothélium vasculaire en réponse au blocage des récepteurs ETA et ETB. Nous avons enfin étudié l’expression des récepteurs ETA et ETB à la surface des neutrophiles et les voies de signalisations découlant de leur activation.Sur le modèle de souris drépanocytaire, l’inhibition des récepteurs ETA, mais surtout ETB permet de limiter le recrutement leucocytaire important provoqué par un stimulus inflammatoire. Ces résultats confirment le rôle d’ETA et celui, plus inattendu, d’ETB dans toutes les étapes d’adhérence des polynucléaires neutrophiles et dans leur transmigration tissulaire en contexte drépanocytaire.Sur des échantillons humains, nous avons confirmé le rôle crucial d’ETB dans l’adhérence des polynucléaires neutrophiles. Nous avons également confirmé la présence de récepteurs ETA et ETB à la surface des neutrophiles. Le récepteur ETB active une voie de signalisation responsable d’une mobilisation du calcium intra cytoplasmique, mais indépendante de l’activation de la phospho-inositide 3-kinase. Enfin, nous avons montré la capacité de ces cellules à synthétiser et excréter elles-mêmes de l’endothéline-1, pérennisant ainsi probablement la réponse inflammatoire et le recrutement leucocytaire. En conclusion, notre travail a permis de mettre en évidence le rôle important du récepteur ETB dans le recrutement des polynucléaires neutrophiles dans la drépanocytose. Ces données suggèrent que les antagonistes des récepteurs à l’endothéline pourraient être bénéfiques en prévention des phénomènes vaso-occlusifs chez les patients drépanocytaires. / Sickle cell disease is a genetic disorder affecting red blood cells, due to a point mutation in the β chain of the hemoglobin gene. However, the pathophysiology of the disease goes well beyond the erythrocyte abnormalities, including vascular and white blood cell dysfunctions that contribute to the complexity of the disease. In this project, we investigated the role of endothelin-1, a powerful vasoconstrictor peptide involved in many vascular diseases, on activation and adhesion of neutrophil in sickle cell disease.We worked on both a mouse model of sickle cell disease (SAD mice) and blood samples from patients. In mice, we performed intravital microscopy experiments, to test the in vivo effect of endothelin receptor antagonists ETA and ETB on neutrophils recruitment. On blood samples from patients with sickle cell disease, we tested in vitro adhesion of neutrophils to vascular endothelium in response to the blocking of ETA and ETB receptors. Finally, we studied the expression of ETA and ETB receptors on neutrophils and the signaling pathways resulting in their activation.In our mouse model of sickle cell disease, the inhibition of both endothelin receptors ETA and ETB limits the major leukocyte recruitment caused by an inflammatory stimulus. These results confirm the role of ETA and the more unexpected important role of ETB in all stages of neutrophil adhesion and transmigration in sickle cell context.On human samples, we demonstrated the crucial role of ETB in neutrophils adhesion. We also confirmed the expression of ETA and ETB receptors on neutrophils. ETB receptor activates a signaling pathway responsible for intracytoplasmic calcium mobilization, but not involving the activation of phosphoinositide 3-kinase. Finally, we have shown the ability of neutrophils to synthesize and secrete endothelin-1, which can contribute to sustain the inflammatory stimulation and increased leukocyte recruitment.In conclusion, our work has highlighted the important role of ETB receptor in the recruitment of neutrophils in sickle cell disease. These data suggest that the antagonists of endothelin could be beneficial in prevention of vaso-occlusive events in sickle cell patients.

Orientador: Claudia Regina Bonini- Domingos / Banca: Flávio Augusto Naoum / Banca: Sônia Maria Oliani / Resumo: A Doença Falciforme é caracterizada por anemia hemolítica e eventos vaso-oclusivos que resultam em crises de dor aguda, com danos crônicos e progressivos nos tecidos e órgãos. Pacientes com Doença Falciforme mostram ativação da coagulação sanguínea e sistema fibrinolítico, bem como aumento da atividade plaquetária e consumo de inibidores da coagulação, especialmente durante as crises vaso-oclusivas. A hiper-homocisteinemia, resultante de mutações no gene da metilenotetrahidrofolato redutase (MTHFR) e no gene da cistationinabeta- sintase (CBS), constituem fatores de risco isolados para doenças vasculares. Diante da importância em se identificar fatores de risco para doenças tromboembólicas, a complexidade da fisiopatologia vascular na da Doença Falciforme e a associação de polimorfismo como potenciais modificadores genéticos, objetivamos avaliar a frequência de polimorfismos nos genes CBS e MTHFR em Doentes Falciformes. Foram pesquisados 300 pacientes de várias localidades do país, com hemoglobinopatia S (Hb SS, Hb SC, Hb S/Beta talassemia) para a presença da mutação C677T no gene da MTHFR e 844ins68, no gene da CBS. Foram coletados 5 mL de sangue venoso em EDTA, após consentimento informado. As metodologias clássicas para o diagnóstico de hemoglobinopatias foram realizadas para a confirmação do fenótipo de cada portador. O DNA foi extraído dos leucócitos pelo método fenol/clorofórmio. As detecções das mutações referentes aos genótipos de Hemoglobina e dos polimorfismos pesquisados foram realizadas por amplificação de um segmento gênico, com iniciadores que flanqueiam a região de inserção e análise por RFLP e AE. Posteriormente foi feita a revelação em gel de agarose, corado com ...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Sickle Cell Disease is an inflammatory condition with dependent pathophysiology of vaso-occlusive episodes. Mutations in methylenetetrahydrofolate reductase gene (MTHFR) and cystathionine beta-synthase (CBS) gene are risk factors for vascular disease. Given the importance of identifying risk factors for vasoocclusive events in sickle cell patients, aimed to evaluate the frequency of mutations C677T in the MTHFR gene and 844ins68 in the CBS gene in these patients.We evaluated 300 sickle cell patients, HbSS, HBSC, HbS/Beta thalassemia, from Brasília, Goiânia, Rio de Janeiro, São Jose do Rio Preto and São Paulo. Were collected 5mL of venous blood in EDTA after informed consent. The classical methods were performed to confirm the phenotype of hemoglobin. The mutations to the genotypes of hemoglobin and polymorphisms studied were evaluated by Restriction Fragment Length Polymorphism and Allele Specific. The results showed that 93 patients (31.00%) were heterozygous and 13 (4.33%) homozygous for the C677T mutation and 90 were heterozygotes (30.00%) and 8 homozygous (2.66%) for the 844ins68 mutation, both with significant difference for genotypic frequency between the locatities. The allelic frequencies are in Hardy-Weinberg equilibrium for both mutations. Analyzing punctually the patients of the city of Goiania The frequence of mutations were significant and the presence of related vaso-occlusive events was more incident in patients with HbSS (p=0007). The 844ins68 mutation was approximately three times higher in patients with the presence of vaso-occlusive complications (p=0011). The C677T mutation didn't show association with the risk for vaso-occlusive manifestations (p=0,193) in patients. The C677T/844ins68 interaction occurred in 12.08% of patients and showed an increase of twice the risk of vaso-occlusive manifestations (RR:2.16). The frequencies of mutations... (Complete abstract click electronic access below) / Mestre

Les vésicules sont des bicouches lipidiques refermées sur elles même, séparant ainsi un volume intérieur du milieu extérieur. Elles sont donc utilisées dans de nombreuses applications nécessitant l'encapsulation d'une substance mais également pour la construction d'objets biomimétiques. Une nouvelle méthode simple, appelée continuous Droplet Interface Crossing Encapsulation (cDICE), a été développée pour la fabrication de vésicules de taille et de contenu finement contrôlés. Ainsi, des vésicules dans la gamme de taille 5-70 μm de diamètre et encapsulant des solutions aussi variées que des colloïdes micrométriques, protéines, cellules, solutions visqueuses (40 mPas) ou solutions salines (>300 mosm), sont produites de façon continue et à fréquence élevée (∼ 150 Hz). Des vésicules " drépanocytaires " ont également pu être produites grace à cette méthode. La drépanocytose ou anémie falciforme est une maladie génétique dont la principale conséquence est la vaso-occlusion de la circulation sanguine. La conception de canaux microfluidiques se rapprochant au mieux des conditions physiologiques (vitesse d'écoulement, concentration d'oxygène, hématocrite...) de la microcirculation a permis une approche biomimétique de la vaso-occlusion, mettant en évidence l'importance de paramètres physiques tels que la géométrie de l'écoulement, le taux d'oxygène, la présence de globules blancs et l'hémoglobine libre en solution sur la formation d'agrégats cellulaires qui pourraient jouer un rôle dans la vaso-occlusion.

Made available in DSpace on 2014-06-11T19:26:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-26Bitstream added on 2014-06-13T18:29:22Z : No. of bitstreams: 1 jacob_ma_me_sjrp.pdf: 1262459 bytes, checksum: 7bde47cc7e532096f30ddf3b74ef0606 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Ministério da Saúde / A Doença Falciforme é caracterizada por anemia hemolítica e eventos vaso-oclusivos que resultam em crises de dor aguda, com danos crônicos e progressivos nos tecidos e órgãos. Pacientes com Doença Falciforme mostram ativação da coagulação sanguínea e sistema fibrinolítico, bem como aumento da atividade plaquetária e consumo de inibidores da coagulação, especialmente durante as crises vaso-oclusivas. A hiper-homocisteinemia, resultante de mutações no gene da metilenotetrahidrofolato redutase (MTHFR) e no gene da cistationinabeta- sintase (CBS), constituem fatores de risco isolados para doenças vasculares. Diante da importância em se identificar fatores de risco para doenças tromboembólicas, a complexidade da fisiopatologia vascular na da Doença Falciforme e a associação de polimorfismo como potenciais modificadores genéticos, objetivamos avaliar a frequência de polimorfismos nos genes CBS e MTHFR em Doentes Falciformes. Foram pesquisados 300 pacientes de várias localidades do país, com hemoglobinopatia S (Hb SS, Hb SC, Hb S/Beta talassemia) para a presença da mutação C677T no gene da MTHFR e 844ins68, no gene da CBS. Foram coletados 5 mL de sangue venoso em EDTA, após consentimento informado. As metodologias clássicas para o diagnóstico de hemoglobinopatias foram realizadas para a confirmação do fenótipo de cada portador. O DNA foi extraído dos leucócitos pelo método fenol/clorofórmio. As detecções das mutações referentes aos genótipos de Hemoglobina e dos polimorfismos pesquisados foram realizadas por amplificação de um segmento gênico, com iniciadores que flanqueiam a região de inserção e análise por RFLP e AE. Posteriormente foi feita a revelação em gel de agarose, corado com... / Sickle Cell Disease is an inflammatory condition with dependent pathophysiology of vaso-occlusive episodes. Mutations in methylenetetrahydrofolate reductase gene (MTHFR) and cystathionine beta-synthase (CBS) gene are risk factors for vascular disease. Given the importance of identifying risk factors for vasoocclusive events in sickle cell patients, aimed to evaluate the frequency of mutations C677T in the MTHFR gene and 844ins68 in the CBS gene in these patients.We evaluated 300 sickle cell patients, HbSS, HBSC, HbS/Beta thalassemia, from Brasília, Goiânia, Rio de Janeiro, São Jose do Rio Preto and São Paulo. Were collected 5mL of venous blood in EDTA after informed consent. The classical methods were performed to confirm the phenotype of hemoglobin. The mutations to the genotypes of hemoglobin and polymorphisms studied were evaluated by Restriction Fragment Length Polymorphism and Allele Specific. The results showed that 93 patients (31.00%) were heterozygous and 13 (4.33%) homozygous for the C677T mutation and 90 were heterozygotes (30.00%) and 8 homozygous (2.66%) for the 844ins68 mutation, both with significant difference for genotypic frequency between the locatities. The allelic frequencies are in Hardy-Weinberg equilibrium for both mutations. Analyzing punctually the patients of the city of Goiania The frequence of mutations were significant and the presence of related vaso-occlusive events was more incident in patients with HbSS (p=0007). The 844ins68 mutation was approximately three times higher in patients with the presence of vaso-occlusive complications (p=0011). The C677T mutation didn´t show association with the risk for vaso-occlusive manifestations (p=0,193) in patients. The C677T/844ins68 interaction occurred in 12.08% of patients and showed an increase of twice the risk of vaso-occlusive manifestations (RR:2.16). The frequencies of mutations... (Complete abstract click electronic access below)