Segundo estimativas da Organização Mundial de Saúde (OMS) no Brasil nascem 3.500 crianças com anemia falciforme a cada ano e 20% delas não conseguem atingir os cinco anos de idade devido às complicações diretas causadas pela doença. A anemia falciforme é uma doença do grupo das hemoglobinopatias extremamente comum e é causada por uma alteração molecular no cromossoma 11, quando ocorre a substituição do ácido glutâmico pelo aminoácido valina, na posição 6 da cadeia da β- globina. No Brasil acredita-se que a anemia falciforme seja a doença hereditária que mais prevalece, sendo assim, ela é considerada um problema de saúde pública. Alguns estudos apontaram a existência de uma prevalência de aproximadamente 4 a 5% em recém-nascidos portadores da doença e sua maior incidência ocorre no norte e nordeste, regiões brasileiras mais pobres e com maior miscigenação das populações. É urgente a necessidade do desenvolvimento de um diagnóstico viável e acessível a tais áreas, pois existe o reconhecimento pela própria OMS de tal prioridade e que levou o Brasil a criar uma Portaria em 2005 e um Decreto em 2008 que instituem atenção integral aos portadores da doença falciforme. Portanto, foi desenvolvido neste trabalho um método de triagem para a detecção de anemia falciforme, que usou uma tecnologia atual conhecida como point-of-care. Para isso foi utilizado uma gota de sangue em meio tamponado com detergente comercial Limpol® para lisar as hemácias e o hidrossulfito de sódio para reduzir a hemoglobina S e esta foi detectada em papel de cromatografia. O método foi desenvolvido com eficácia, pois foi proposta a substituição da saponina (reagente caro) por detergente comercial Limpol® e do hidrossulfito de sódio (reagente caro) por tiossulfato de sódio, tornando o custo do kit desenvolvido muito baixo. Portanto, sob quaisquer condições adversas e em qualquer região que necessite da triagem diagnóstica populacional, principalmente nas comunidades carentes e de difícil acesso, será possível levar uma solução simples a campo para a execução de um diagnóstico rápido e sem aumento do ônus para o sistema de saúde público brasileiro, consolidando os direitos adquiridos dos indivíduos, mas que não abrangem àqueles que mais necessitam. / According to estimates from the World Health Organization (WHO), every year are born around 3.500 children with sickle cell anemia (SCA) in Brazil and 20% of them wont reach five years old because of complications directly related to the disease. The SCA is a disease of the hemoglobinopathies group extremely common and is caused by a molecular alteration in the chromosome 11, when occurs the replacement from the acid glutamic acid by amino acid valine in the chain of position 6 of the β- globin. In Brazil is believed that the SCA is the hereditary disease that prevails more being considered as a public health problem. Some studies have indicated that exist prevalence in approximately 4-5% of this disease in newborns and the greatest incidence there is mainly in north and northeast, regions with bigger miscegenation from population. Is urgent the necessity in to develop viable accessible diagnostic for those areas whereas it was recognized as a priority by the WHO leading Brazil to create an Ordinance in 2005 and a Decree in 2008 establishing total care to the people with sickle cell disease. Therefore, was developed in this work a screening method to detect the sickle cell anemia, which used a technology known as point-of-care. Was created an alternative method to substitute one international kit which has high cost for the adequation according with of economic Public Health conditions from Brazil. For this a drop of blood in buffered medium with liquid detergent Limpol®, to lyse RBC and the (tiossulfato de sódio) to reduce the RBC S which was detected in simple celulose paper. The method was developed with efficiency, on this account the substitution of saponina (expensive reagent) by liquid detergent Limpol® and (hidrossulfitob de sodio) (expensive reagent) by (tiossulfato de sódio) was successful making the cost of developed kit cheaper. Therefore in any population with adverse conditions or region where there are needs, mainly in poor communities with difficult acces, will be possible to lead a simple solution at field to execution by a quickly diagnostic without increasing of charges to health public brazilian system, consolidating acquired rights to individuals that not are reached by those needs yet.

Segundo estimativas da Organização Mundial de Saúde (OMS) no Brasil nascem 3.500 crianças com anemia falciforme a cada ano e 20% delas não conseguem atingir os cinco anos de idade devido às complicações diretas causadas pela doença. A anemia falciforme é uma doença do grupo das hemoglobinopatias extremamente comum e é causada por uma alteração molecular no cromossoma 11, quando ocorre a substituição do ácido glutâmico pelo aminoácido valina, na posição 6 da cadeia da β- globina. No Brasil acredita-se que a anemia falciforme seja a doença hereditária que mais prevalece, sendo assim, ela é considerada um problema de saúde pública. Alguns estudos apontaram a existência de uma prevalência de aproximadamente 4 a 5% em recém-nascidos portadores da doença e sua maior incidência ocorre no norte e nordeste, regiões brasileiras mais pobres e com maior miscigenação das populações. É urgente a necessidade do desenvolvimento de um diagnóstico viável e acessível a tais áreas, pois existe o reconhecimento pela própria OMS de tal prioridade e que levou o Brasil a criar uma Portaria em 2005 e um Decreto em 2008 que instituem atenção integral aos portadores da doença falciforme. Portanto, foi desenvolvido neste trabalho um método de triagem para a detecção de anemia falciforme, que usou uma tecnologia atual conhecida como point-of-care. Para isso foi utilizado uma gota de sangue em meio tamponado com detergente comercial Limpol® para lisar as hemácias e o hidrossulfito de sódio para reduzir a hemoglobina S e esta foi detectada em papel de cromatografia. O método foi desenvolvido com eficácia, pois foi proposta a substituição da saponina (reagente caro) por detergente comercial Limpol® e do hidrossulfito de sódio (reagente caro) por tiossulfato de sódio, tornando o custo do kit desenvolvido muito baixo. Portanto, sob quaisquer condições adversas e em qualquer região que necessite da triagem diagnóstica populacional, principalmente nas comunidades carentes e de difícil acesso, será possível levar uma solução simples a campo para a execução de um diagnóstico rápido e sem aumento do ônus para o sistema de saúde público brasileiro, consolidando os direitos adquiridos dos indivíduos, mas que não abrangem àqueles que mais necessitam. / According to estimates from the World Health Organization (WHO), every year are born around 3.500 children with sickle cell anemia (SCA) in Brazil and 20% of them wont reach five years old because of complications directly related to the disease. The SCA is a disease of the hemoglobinopathies group extremely common and is caused by a molecular alteration in the chromosome 11, when occurs the replacement from the acid glutamic acid by amino acid valine in the chain of position 6 of the β- globin. In Brazil is believed that the SCA is the hereditary disease that prevails more being considered as a public health problem. Some studies have indicated that exist prevalence in approximately 4-5% of this disease in newborns and the greatest incidence there is mainly in north and northeast, regions with bigger miscegenation from population. Is urgent the necessity in to develop viable accessible diagnostic for those areas whereas it was recognized as a priority by the WHO leading Brazil to create an Ordinance in 2005 and a Decree in 2008 establishing total care to the people with sickle cell disease. Therefore, was developed in this work a screening method to detect the sickle cell anemia, which used a technology known as point-of-care. Was created an alternative method to substitute one international kit which has high cost for the adequation according with of economic Public Health conditions from Brazil. For this a drop of blood in buffered medium with liquid detergent Limpol®, to lyse RBC and the (tiossulfato de sódio) to reduce the RBC S which was detected in simple celulose paper. The method was developed with efficiency, on this account the substitution of saponina (expensive reagent) by liquid detergent Limpol® and (hidrossulfitob de sodio) (expensive reagent) by (tiossulfato de sódio) was successful making the cost of developed kit cheaper. Therefore in any population with adverse conditions or region where there are needs, mainly in poor communities with difficult acces, will be possible to lead a simple solution at field to execution by a quickly diagnostic without increasing of charges to health public brazilian system, consolidating acquired rights to individuals that not are reached by those needs yet.

La drépanocytose est une hémoglobinopathie essentiellement connue pour ses répercussions hématologiques, hémodynamiques et vasculaires chez les patients homozygotes (SCA). Bien que ces sujets présentent une intolérance à l'effort, la littérature est très pauvre concernant les répercussions musculaires de la maladie. Ce travail doctoral nous a permis de caractériser pour la première fois les répercussions structurales, énergétiques et microvasculaires du muscle d’hommes (étude 1) et de femmes (étude 2) drépanocytaires par rapport à des sujets SCT et contrôles (CON). Nos analyses histologiques et biochimiques ont mis en évidence chez les sujets SCA masculins une amyotrophie qui explique au moins en partie la diminution de l’IMC des SCA au dépend de la masse maigre. Nous avons également observé l’altération de plusieurs indices du métabolisme oxydatif (CS, β-HAD et COx) qui pourrait relever de la restriction d’approvisionnement et d’utilisation tissulaire en O2 et expliquer en partie l’intolérance à l’effort. Enfin, un remodelage microvasculaire caractérisé par une raréfaction, une moindre tortuosité et une fragilisation des microvaisseaux a également été démontré. Ce remodelage microvasculaire pourrait contribuer à limiter le risque d’enclavement des hématies falciformées et ainsi réduire les risques de vaso-occlusions. La recherche de ces différents stigmates dans le muscle de la population féminine homologue a rapporté un remodelage musculaire similaire à celui observé chez les hommes SCA, mais ce dernier semble atténué, suggérant un effet genre / Sickle cell anemia (SCA) is a hemoglobinopathy particularly known for its hematologic, hemodynamics and vascular repercussions. Although SCA patients are exercise intolerant, no studies have looked at muscle repercussions. We assessed repercussions of sickle cell anemia on skeletal muscle and its microvasculature in men (study 1) and women (study 2) for the first time. Our results showed that men with SCA displayed an amyotrophy which can at least partly explain the decrease of BMI at the expense of lean muscle mass. We also pointed out a decrease in muscle oxidative capacities (CS, β-HAD and COx) which could result from O2 supply and utilization disorders and partly explain their exercise intolerance. Finally, a microvascular remodeling characterized by a rarefaction, a decrease in microvessel tortuosity and a microvessel weakening was also highlighted. This remodeling could contribute to maintain local blood flow and reduce risks of entrapment of sickle red blood cells in the microvasculature, hence reducing vaso-occlusive risks. Muscle repercussions on a similar female population testified of the same muscle remodeling as the one observed in men with SCA, but this latter seemed to happen at a lesser extent in women with SCA, suggesting a gender effect

La drépanocytose, ou anémie falciforme, présente une variabilité interindividuelle considérable, conditionnée par de multiples facteurs, dynamiques et interactifs, depuis le niveau moléculaire jusqu’au niveau du patient. L’hémoglobine drépanocytaire, ou hémoglobine S (HbS, tétramère a2bS 2), est un mutant de l’hémoglobine A (a2b2) : elle possède à sa surface une valine (hydrophobe) substituant un acide glutamique natif (négativement chargé). Cette mutation entraîne l’agrégation de l’HbS désoxygénée en polymères, ainsi que l’altération des propriétés de l’érythrocyte -dont sa rhéologie et ses interactions avec les différentes cellules vasculaires. C’est pourquoi la polymérisation de l’HbS constitue un facteur étiologique clef, sinon le primum movens, de la drépanocytose, et une hypothèse thérapeutique (étayée par l’observation) postule que la réduction des fibres intra-érythrocytaires de HbS pourrait améliorer le statut clinique des patients en abaissant la fréquence et la sévérité des crises vasoocclusives. Dans l’optique de mieux comprendre et de mieux gérer la variabilité individuelle drépanocytaire, il apparaît donc indispensable de disposer, en premier lieu, d’une description réaliste de la polymérisation de l’HbS. L’objectif de ce travail de thèse est la mise en place et la validation d’un modèle mathématique de la polymérisation de l’HbS désoxygénée, en tant que processus cinétiquethermodynamique, sous l’influence de la concentration et de la température –les deux facteurs modulateurs les plus importants. A partir d’un modèle existant, mais linéaire et incomplet (Ferrone et al., 1985), nous avons procédé à son implémentation, à sa correction et à sa mise à jour, ainsi qu’à l’évaluation quantitative de ses performances dynamiques, par intégration complète et simulation numérique (Simulink©). Ceci nous a permis de réaliser un diagnostic et d’effectuer un certain nombre de raffinements, concernant en particulier (i) la voie de nucléation hétérogène (formation de néo-fibres sur les fibres préexistantes), (ii) la non-idéalité de la solution protéique de HbS, induite par le volume exclus des fibres polymères (coefficients d’activité calculé à partir de la « théorie des particules convexes »), ainsi que (iii) la structuration spatiale des polymères en domaines. Le modèle développé dans ce travail servira de base pour une description (i) de l’influence dynamique de l’oxygénation et des hémoglobines non-polymérisantes sur la polymérisation de HbS, puis (ii) des polymères de HbS sur les propriétés membranaires et rhéologiques de l’érythrocyte drépanocytaire. / Sickle cell disease pathology exhibits a strong interindividual variability, which depends upon multiple, dynamic and interacting factors, from the molecular to the patient level. Sickle hemoglobin, hemoglobin S (HbS, a2bS 2 tetramer), is a mutant of HbA (a2b2), with a surface valine (hydrophobic) substituting a native glutamic acid (negatively charged). Such a mutation endows deoxygenated HbS with the propensity to agregate into polymers, altering erythrocyte properties –including its rheology and its interactions with vascular and circulatory cells. Thus HbS polymerization is a key etiological factor of sickle cell disease, if not the primum movens. Indeed, one therapeutical hypothesis (supported by observation) postulates that the reduction of intra-erythrocytic HbS fibers could improve patients clinical status by lowering the frequency and the severity of vasooclusive crisis. In order to better understand and manage sickle cell disease variability, it is essential to have a realistic description of HbS polymerization. This work aims at developing and validating a mathematical model of deoxygenated HbS polymerization, as a kinetic and thermodynamic process under the influence of concentration and temperature –the two most important modulators. Building on an existing, but linearized and uncomplete (Ferrone et al., 1985) model, we have implemented, corrected and updated, and quantitatively evaluated its dynamical performances: this was done by full numerical integration using Simulink©. This allowed us to make several improvements, related in particular to : (i) the heterogeneous nucleation pathway (seeding and formation of new fibers from pre-existing ones), (ii) the non-ideality of the HbS protein solution, caused by polymer fibers excluded volume (activity coefficients were calculated with the CPT, Convex Particle Theory), and (iii) the spatial organization of polymers into domains. The model developped in this work will ground the description of the dynamic influence (i) oxygenation and non-polymerizing hemoglobins, (ii) HbS polymers interactions with membrane and consequences upon rheological properties of sickle cell erythrocyte.