Made available in DSpace on 2014-06-11T19:26:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-02-27Bitstream added on 2014-06-13T20:54:00Z : No. of bitstreams: 1 estevao_if_me_sjrp.pdf: 1151592 bytes, checksum: 9e2d3a0a29b1ad6405857d13d891a9f1 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A beta talassemia é um dos mais freqüentes distúrbios genéticos no mundo. Estima-se que 1,5% a 3% da população mundial seja portadora do traço talassêmico. Esses portadores geralmente são oligo ou assintomáticos e têm uma expectativa de vida semelhante à dos não portadores. Entretanto, níveis elevados de ferritina sérica têm sido observados em alguns estudos comparativos entre beta talassemia heterozigota e não portadores e, alguns indivíduos, que nunca foram transfundidos, apresentam sinais clínicos e laboratoriais de sobrecarga de ferro. A fisiopatologia dessa complicação continua em discussão. Vários pesquisadores têm sugerido um efeito modulador da mutação do gene da beta globina e mutações em genes codificadores de proteínas relacionadas ao metabolismo do ferro. Mutações no gene HFE são as mais freqüentemente associadas à hemocromatose hereditária. O objetivo do presente trabalho foi avaliar a freqüência das mutações C282Y e H63D no gene HFE em portadores de beta talassemia heterozigota e analisar sua influência no metabolismo do ferro. Foram estudados 162 portadores de beta talassemia heterozigota, residentes na cidade de São Carlos ou região, caucasóides e, acompanhados no serviço de Hematologia. O diagnóstico de traço talassêmico foi confirmado em todos por meio do eritrograma e da quantificação da Hb A2 e Hb fetal por HPLC. O metabolismo do ferro foi avaliado pelas dosagens de ferro sérico, capacidade total de ligação do ferro, ferritina e saturação da transferrina e, a análise molecular das mutações no gene HFE, pela técnica de PCR-RLFP. Foram realizadas análises de correlação linear de Pearson por idade e gênero entre hemoglobina... / Beta thalassemia is one of the most frequent genetic disorder in the world. It is estimated that 1.5% to 3% of the world population is a thalassemia carrier. These individuals are generally slightly symptomatic or asymptomatic and they have a life expectancy similar to those who are non-carriers. However, high levels of serum ferritin have been observed in some comparative studies between heterozygous for beta thalassemia and non-carriers, and some individuals that were never transfused, present clinic and laboratories signs of iron overload. The physiopathology of this disease continues in discussion. Several researchers have suggested a modulator effect from the mutation of the beta globin gene and mutations in genes related with the iron metabolism. Mutations of the gene HFE are the most frequently associated to the hereditary hemochromatosis. The aim of this study was evaluate the frequency of C282Y and H63D mutations in the HFE gene in beta thalassemia carriers, and analyze its influence in the iron metabolism. 162 beta thalassemia carriers, Caucasoid, residing in the city of Sao Carlos or region and accompanied in the Hematology service were studied. The diagnostic of thalassemia trait was confirmed in every one through a complete erythrogram and quantification of Hb A2 and Hb fetal by HPLC. The iron metabolism was evaluated by serum iron, total iron-binding capacity, serum ferritin and percent saturation of transferring. The molecular analysis of the mutations in the HFE gene was made by PCR-RLFP. There were made analysis of linear Pearson’ correlation, by age and gender, among hemoglobin, Hb A2, VCM and among reticulocytes count and the values of saturation of transferrin and serum ferritin.

Orientadores: Fernando Ferreira Costa, Dulcinéia Martins de Albuquerque / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-17T10:49:18Z (GMT). No. of bitstreams: 1 Ferreira_RegianeAparecida_M.pdf: 1067582 bytes, checksum: 33631c571ed5b3e047ee71d15d222e81 (MD5) Previous issue date: 2010 / Resumo: As variantes de hemoglobina e as Talassemias estão entre as hemoglobinopatias hereditárias mais frequentes no Brasil. A hemoglobina S é resultante de uma mutação de ponto no gene que codifica a cadeia beta da globina e que em homozigose caracteriza a Anemia Falciforme. Outras variantes neste gene podem ocasionar redução parcial ou completa da síntese de uma ou mais cadeias da globina, originando as Talassemias do tipo beta. Recentemente, alguns estudos relacionaram a interação de fatores de transcrição e microRNAs que mediam, em conjunto, mecanismos regulatórios transcricionais e pós transcricionais e promovem a diferenciação, proliferação e maturação de células eritroides. Dessa maneira, alterações no perfil de expressão de fatores transcricionais e de pequenos RNAs podem ocasionar manifestações clínicas envolvidas na eritropoese de pacientes com Talassemia beta intermediária e com Anemia Falciforme. Este trabalho analisou o perfil de expressão dos fatores transcricionais GATA-1, TAL1, NFE-2, LDB1, SPI-1, LMO2 e EKLF e dos miRNAs miR24, miR144, miR155, miR210, miR221, miR222, miR223 e miR451 em reticulócitos de 9 pacientes com Talassemia beta intermediária e 8 pacientes com Anemia Falciforme, através da PCR quantitativa em tempo real. Os resultados apresentaram alterações significativas no perfil de expressão dos FTs GATA-1, TAL1 e NFE-2 em reticulócitos de pacientes com Talassemia beta intermediária. Nos pacientes com Anemia Falciforme, diferenças significativas foram observadas na expressão dos FTs LMO2 e EKLF e dos microRNAs miR221, miR223 e miR451. Em conjunto, nossos resultados sugerem o envolvimento dos fatores de transcrição em associação aos microRNAs como moduladores da manifestação clínica em pacientes com Talassemia e Anemia Falciforme. Trata-se do primeiro estudo que avalia o perfil de expressão de microRNAs e fatores de transcrição relacionados à eritropoese em reticulócitos de pacientes com Anemia Falciforme e Talassemia beta no Brasil / Abstract: The hemoglobin variants and thalassemias are the most frequent hemoglobinopathies in Brazil. Hemoglobin S results from a point mutation in the gene that encodes the beta globin chain, which is characterized in homozygous sickle cell disease. Other variants of this gene can cause partial or complete reduction of the synthesis of one or more globin chains that result in beta-thalassemias. Recently, some studies have identified the interaction of transcription factors and microRNAs that mediate, together, regulation of transcriptional and post transcriptional mechanisms to promote the differentiation, proliferation and maturation of erythroid cells. Thus, disorders in the transcriptional factors and small RNAs expression can cause clinical manifestations involved in the erythropoiesis of patients with beta thalassemia intermediate and in sickle cell disease. This study analyzed the expression profile of the transcription factors GATA-1, TAL1, NFE2, LDB1, SPI1, LMO2 and EKLF and microRNAs miR24, miR144, miR155, miR210, miR221, miR222, miR223 and miR451, in reticulocytes from 9 patients with beta thalassemia intermediate and 8 patients with sickle cell disease, by real-time quantitative PCR. The results showed significant changes in the expression profiles of the transcription factors, GATA-1, TAL1 and NFE-2, in the reticulocytes of patients with beta thalassemia intermediate. In patients with sickle cell disease, significant differences were observed in the expression of the transcription factors, LMO2 and EKLF, and microRNAs miR221, miR223 and miR451. Together, our results suggest the involvement of transcription factors in association with miRNAs as modulators of the clinical manifestation in patients with beta thalassemia and sickle cell disease. This is the first study that evaluates the gene expression of microRNAs and transcription factors related to erythropoiesis in reticulocytes from patients with sickle cell anemia and beta thalassemia in Brazil / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

Beta-talassemia é uma das doenças genéticas mais comuns no mundo, com graus variados de anemia crônica, tratados por transfusões sanguíneas rotineiras nos casos mais graves. A sobrecarga de ferro acentuada a que esses pacientes são submetidos é a principal responsável pela morbimortalidade, sendo o acúmulo de ferro no miocárdio e a doença cardíaca disso decorrente, a principal causa de morte nessa população. A ressonância magnética cardíaca (RMC) é ferramenta central no acompanhamento desses pacientes, utilizando-se da técnica T2*, capaz de determinar a presença e grau da deposição de ferro no miocárdio, modificando o tratamento da terapia quelante de ferro (TQF). Além disso, outros parâmetros volumétricos e funcionais obtidos no exame de RMC podem estar alterados nesses pacientes. Até a atualidade, inexistem estudos nacionais descrevendo uma população de pacientes beta-talassêmicos pelos parâmetros da RMC. Objetivos: 1. Caracterizar a partir dos diversos parâmetros dos exames de RMC, a população dos pacientes talassêmicos acompanhados no Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto. 2. Avaliar o impacto dos diversos parâmetros da RMC com informações clinicas como mudança terapêutica, ocorrência de insuficiência cardíaca e óbito. Métodos: foram avaliados 44 pacientes com diagnóstico de beta-talassemia em seguimento no serviço de hematologia, para os quais foram solicitados exames de RMC na rotina clínica. Os exames incluíram sequências cine SSFP (steady state free precession) nos diversos planos cardíacos, incluindo uma sequência do eixo curto de ambos os ventrículos, esta utilizada para as medidas de volumes e função ventriculares. A sequência T2* utilizada foi gradiente eco com pré-pulso de saturação do sangue, com aquisição de 9 ecos consecutivos em apenas uma apneia. Sequência semelhante foi obtida para cálculo do T2* hepático. Foi feita revisão dos prontuários dos pacientes para avaliação dos dados clínicos. Resultados: 44 pacientes foram avaliados (25 do sexo masculino 56,8 %), com idade de 23,1±10,3 anos e diagnóstico de beta-talassemia maior em 30 casos (68,2 %) e talassemia intermedia em 14 casos (31,8 %). Foram descritos os volumes, massa e função para ambos os ventrículos, bem como a distribuição da medida do T2* miocárdico. Não foram observadas diferenças desses parâmetros entre os diferentes grupos de impregnação miocárdica pelo T2*. O seguimento médio do estudo foi de 4,0 (±1,1) anos com 4,7 (±1,6) exames por paciente. Foram identificados cinco pacientes com doença cardíaca e dois óbitos. Esses pacientes apresentaram redução significativa do T2* miocárdico, da fração de ejeção do ventrículo direito (VD), massa ventricular direita e esquerda e do volume sistólico final do VD, quando comparados aos indivíduos sem acometimento cardíaco reportado. Conclusão: Caracterizou-se a população de pacientes beta-talassêmicos acompanhados nesta instituição, em relação aos diversos parâmetros da RMC, salientando-se o impacto da introdução do método na mudança terapêutica. A medida do T2* miocárdico está correlacionada ao desenvolvimento de cardiopatia e pior evolução clínica, incluindo óbito. Acometimento do VD pode ser indicador precoce da impregnação miocárdica. / Beta-thalassemia is one of the most common genetic disorders worldwide, with different degrees of chronic anemia, treated with routine blood transfusions in severe cases. Morbidity and mortality is mainly related to the chronic iron overload and iron burden these patients endure. Myocardial iron overload and the development of cardiac disease remain the main cause of death for this population. Cardiac magnetic resonance (CMR) plays a central role in patient management, utilizing T2* sequences, which allows for detection and quantification of myocardial iron deposits, thus prompting changes in iron chelation therapy (ICT). Besides, there are other significant CMR volumetric and functional parameters to present abnormalities in these patients. To the present date, no national data has been published, describing a beta-thalassemia patient population from the standpoint of CMR parameters. Objectives: 1. To characterize, from the different CMR parameters, the beta-thalassemia patient population followed at a Brazilian tertiary hospital. 2. To evaluate the impact of these different CMR parameters, in relation to clinical data, such as therapeutic change, cardiac disease and death. Methods: 44 beta-thalassemia patients followed by the hematology service of this institution were evaluated, to whom CMR exams were performed in routine clinical practice. CMR exams included SSFP (steady state free precession) cine images through the different cardiac planes, including a short axis stack through both ventricles, used to calculate ventricular volumes and function. A gradient echo sequence with a dark blood pre-pulse saturation was used to calculate myocardial T2* values, with 9 consecutive echo times acquisition in one breath hold. A similar sequence was used to determine liver T2* values. Medical records were reviewed for clinical data. Results: 44 patients were evaluated (25 males, 56.8 %), with a mean age of 23,1 (±10,3) years, and diagnosis of betathalassemia major in 30 patients (68,2 %) and beta-thalassemia intermedia in 14 (31,8 %). Ventricular volumes, mass and function were described, as well as myocardial T2* distribution. There were no statistical difference observed among the different CMR parameters and the myocardial T2* degrees. Mean follow up was 4,0 (±1,1) years, with 4,7 (±1,6) exams per patient. Five patients with cardiac disease were identified with two deaths during observation. These patients showed a significantly reduced myocardial T2* and RVEF, and an elevated ventricular mass, for both ventricles, and final systolic right ventricular volume, when compared to patients without cardiac disease. Conclusion: A betathalassemia patient population was characterized through the different CMR parameters, highlighting the impact of CMR introduction to treatment decision. Myocardial T2* is related to cardiac disease development, clinical worsening and death. Right ventricular functional worsening could be an early sign of myocardial iron involvement.

Orientador: Fernando Ferreira Costa / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-19T02:13:16Z (GMT). No. of bitstreams: 1 Fertrin_KleberYotsumoto_D.pdf: 1904934 bytes, checksum: a74beda8b565fcdc3f59ad37d66ca23e (MD5) Previous issue date: 2011 / Resumo: As hemoglobinopatias são distúrbios hereditários em que uma mutação genética leva a alteração da produção normal de hemoglobina, tal como na anemia falciforme e nas talassemias ß. Na maioria dessas doenças, ocorre anemia com necessidade transfusional variável, o que pode acarretar sobrecarga corporal de ferro. Na talassemia ß intermediária, ocorre aumento espontâneo e desproporcional da absorção do ferro, com consequente excesso desse metal mesmo na ausência de transfusões. Com a evolução da terapia transfusional e o aumento da expectativa de vida desses pacientes, o conhecimento sobre a regulação do metabolismo do ferro tornou-se fundamental para melhor controle da sobrecarga de ferro. O principal regulador desse metabolismo é a hepcidina, um polipeptídeo produzido majoritariamente pelo fígado, porém também sintetizado por células do sistema fagocítico-mononuclear, em que seu papel é pouco conhecido. Uma citocina capaz de suprimir a produção de hepcidina é o GDF-15 (fator de crescimento e diferenciação 15). Neste estudo, com a avaliação de amostras de sangue de 103 pacientes com anemia falciforme, talassemia ß intermediária, anemia por deficiência de cobalamina ou outros tipos de anemia, constatou-se que o aumento dos níveis desse fator ocorre tanto em quadros de hemólise crônica quanto na presença de eritropoese ineficaz, constituindo um sinal da medula óssea modulador da absorção de ferro nos estados de aumento da eritropoese. Entretanto, evidenciou-se que a associação de supressão da hepcidina com altos níveis de GDF-15 ocorre nas hemoglobinopatias, mas não nas demais causas de anemia. Na anemia megaloblástica, a ausência de sobrecarga de ferro com níveis normais de hepcidina ao diagnóstico e sua queda durante o tratamento sugerem regulação da hepcidina independente de GDF-15 neste tipo de anemia. A análise da razão hepcidina/ferritina mostrou-se mais fidedigna que os níveis de hepcidina circulante na identificação dos estados em que há propensão a absorção aumentada de ferro por alta atividade eritropoética, e sugerem que o estado inflamatório crônico da anemia falciforme poderia exercer um fator protetor contra sobrecarga de ferro, quando comparados a talassemia intermediária, pela elevação relativa da produção de hepcidina. Além disso, observou-se uma correlação negativa entre a expressão gênica de hepcidina (gene HAMP) em monócitos humanos e os níveis de GDF-15, denotando um provável efeito regulatório semelhante ao descrito em hepatócitos. Não se identificou correlação entre essa expressão nos monócitos e marcadores de sobrecarga de ferro, corroborando a hipótese de a hepcidina ter outra função nessas células, não relacionada diretamente à absorção de ferro. Pacientes com anemia falciforme em uso de hidroxiureia apresentaram maiores níveis de expressão de hepcidina monocítica e obteve-se evidência in vitro de uma ação estimuladora dessa expressão por esse fármaco, caracterizando a hidroxiureia com potencial atividade agonista de hepcidina, de futuro interesse em estudos de sua aplicação clínica nos estados em que exista deficiência monocítica dessa proteína. Trata-se do primeiro estudo avaliando comparativamente hemoglobinopatias e outros tipos de anemia com e sem componente eritropoético ineficaz do ponto de vista dos reguladores da absorção de ferro, além de caracterizar, pela primeira vez, a expressão de hepcidina extra-hepática nos distúrbios da síntese de hemoglobina / Abstract: Hemoglobinopathies are inherited diseases in which a genetic mutation leads to abnormal production of hemoglobin, such as in sickle cell anemia or in the ß-thalassemias. In the majority of these disorders, anemia causes variable degrees of transfusion dependency, which may lead to iron overload. In ß-thalassemia intermedia, an increase in iron absorption occurs spontaneously and regardless from the total body iron stores, generating iron overload even in the absence of repeated transfusions. Owing to advances in transfusion medicine and to the improvement in the overall life expectancy of patients with hemoglobin disorders, further knowledge on the regulation of iron metabolism has become increasingly important for appropriate management of iron overload. The main regulator of iron metabolism is hepcidin, a polypeptide mainly produced by the liver, although its synthesis also occurs in phagocytic-mononuclear cells, in which its role is less known. Growth differentiation factor 15 (GDF-15) is a cytokine capable of downregulating hepcidin production. This study analyzed 103 blood samples from patients with sickle cell anemia, ß-thalassemia intermedia, cobalamin deficiency anemia and other types of anemia, showing elevation of GDF-15 plasmatic levels both in chronic hemolytic states and ineffective erythropoiesis, thus characterizing it as a signalling molecule produced by the bone marrow to stimulate iron absorption in the presence of increased erythropoietic activity. Nevertheless, hepcidin suppression was only associated with high levels of GDF- 15 in the hemoglobinopathies. In megaloblastic anemia, absence of iron overload with normal hepcidin levels, associated with their reduction during treatment, suggest that hepcidin regulation occurs independently from GDF-15 in thie type of anemia. Analysis of hepcidin/ferritin ratio proved to be more reliable to identify patients prone to increased iron absorption due to erythropoietic hyperactivity than hepcidin levels themselves and suggests that the chronic inflammatory state in sickle cell anemia may protect from iron overload by relatively increasing hepcidin levels in comparison to levels found in thalassemia intermedia. Moreover, we found a negative correlation between GDF-15 levels and HAMP monocytic expression, a regulatory mechanism similar to what has been observed in hepatic cell lines. In further analyses of the present study, no correlation between hepcidin expression and iron overload markers was observed in monocytes from patients with hemoglobinopathies, corroborating the hypothesis that the monocytic counterpart of hepcidin could have a different function, unrelated to iron regulation. Patients with sickle cell anemia under hydroxyurea treatment have been shown to present with higher levels of hepcidin expression in monocytes, and a cell culture model managed to demonstrate the upregulating effect of hydroxyurea in vitro, thus highlighting the possibility of exploring this drug in the future as a potential hepcidin agonist and, therefore, as a therapeutic intervention in diseases with impaired monocytic hepcidin production. This is the first study of molecules involved in iron metabolism regulation comparing hemoglobinopathies and other anemia types with and without ineffective erythropoiesis. Furthermore, this is the first characterization of extra-hepatic hepcidin expression in hemoglobin disorders / Doutorado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Doutor em Fisiopatologia Medica

L’érythropoïèse est le processus de formation des globules rouges. L’anémie demeure à l’heure actuelle un problème de santé publique majeur. Par conséquent, une meilleure compréhension des mécanismes impliqués dans le contrôle de ce processus dans des conditions physiologiques et pathologiques, ainsi que l’établissement de stratégies thérapeutiques ciblées constituent un enjeu de recherche majeur. Le récepteur de la transferrine 1 (CD71/RTf1) est un élément essentiel de l'érythropoïèse, la majorité des travaux de recherche étant focalisés sur son rôle indéniable dans le métabolisme du fer. Cependant, de nouveaux ligands du RTf1 ont été découverts ouvrant de nouvelles perspectives relatives aux fonctionnalités de ce récepteur. Ayant démontré que le RTf1 fixait les immunoglobulines A1 (IgA1), nous nous sommes intéressés au rôle des IgA1 dans l’érythropoïèse. Nous montrons que le RTf1 lié aux polymères d'IgA1 (pIgA1) induit la croissance et une augmentation de la prolifération des érythroblastes en concentration sous-optimale d'érythropoïétine (Epo). De même, l'expression transgénique d’IgA1 humaine (souris alpha1-KI), ou le traitement de souris de type sauvage avec les pIgA1 permettent une récupération accélérée de l’anémie aiguë. L’engagement du RTf1 module la sensibilité à l'Epo, en diminuant le seuil d'activation cellulaire, et en induisant les voies de signalisation MAPK/ERK et phosphatidylinositol-3-kinase (PI3K/AKT). Ces données mettent en évidence un nouveau rôle du RTf1 en tant que régulateur positif de l'érythropoïèse. Parallèlement au RTf1, nous avons identifié un autre récepteur pouvant constituer une cible thérapeutique pour corriger une érythropoïèse inefficace : le récepteur de l’activine de type IIA (ActRIIA). Dans un modèle murin de Beta-thalassémie intermédiaire (Hbbth1/th1), résultant d'une déficience génétique de la chaîne Beta de la globine, nous montrons que l'administration d'une protéine de fusion constituée du domaine extracellulaire de l’ActRIIA lié à un fragment Fc d’IgG de souris (RAP-011), corrige l'anémie, augmente le taux d'hémoglobine et diminue la splénomégalie. Ce traitement favorise l’érythropoïèse splénique et diminue la saturation de la transferrine et l’hémolyse. Fait intéressant, des niveaux élevés de GDF11 (Growth Differentiation Factor 11) sont observés sur des coupes spléniques de souris thalassémiques ainsi que dans le sérum de patients thalassémiques. In vivo, l’inhibition de l’interaction GDF11/ActRIIa par le RAP-011 favorise l’apoptose des érythroblastes précoces par la voie Fas/FasLigand. Ces résultats suggèrent que l’activation constitutive des signaux GDF11/ActRIIA contribue à l’établissement d’une érythropoïèse inefficace caractéristique de la Beta-thalassémie. La neutralisation de cette signalisation inverse ce processus. En conclusion, nos travaux ouvrent de nouvelles perspectives dans la compréhension de l'hématopoïèse normale et pathologique, et pourraient conduire à envisager des traitements innovants pour l'anémie. / Anemia produced by a variety of underlying causes is the most common disorder of the blood, and remains a major global public health problem associated with a poor quality of life for many patients. Thus, better understanding the erythroid process in physiological and pathological conditions, and developing new strategies to boost erythropoiesis appear of great interest. Transferrin receptor 1 (CD71/TfR1) plays an essential role in erythropoiesis, and investigations of TfR1 functions have been focused on their undeniable role in iron metabolism. However, recent data demonstrate that TfR1 is a multi-ligand receptor that participates in a wide array of cellular functions. We have identified TfR1 as a receptor for A1 isotype immunoglobulins (IgA1). In this work, we show that pIgA1s are able through their interaction with the TfR1, to stimulate erythropoiesis by sensitizing erythroblasts to Epo. Likewise, transgenic expression of human IgA1 (Alpha1-KI mice) or treatment of wild-type mice with pIgA1 accelerated recovery from acute anemia. TfR1 engagement by pIgA1 increased cell sensitivity to Epo by inducing activation of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways. These findings unveiled a new role of TfR1 as a signaling competent molecule positively regulating erythropoiesis. In addition to TfR1, our work identifies another receptor as a putative target for correcting ineffective erythropoiesis: the activin receptor IIA (ActRIIA). Indeed, using a mouse model of Beta-thalassemia intermedia (Hbbth1/th1) resulting from a genetic deficiency of Beta-globin chain, we show that administration of a ligand trap (named RAP-011), consisting in a fusion protein between the extracellular domain of ActRIIA and the Fc fragment of a mouse IgG, improves anemia, increases total hemoglobin levels and decreases splenomegaly. In addition, targeting ActRIIa signaling corrects ineffective erythropoiesis in the spleen, reduces hemolysis and transferrin saturation. Interestingly, high levels of Growth Differentiation Factor 11 (GDF11) are detected in spleen sections from Beta-thalassemic mice, as well as in sera from thalassemic patients. In addition, the inactivation of GDF11 promotes terminal erythroblast differentiation. Finally, blockade of the GDF11/ActRIIa signaling, promotes premature apoptosis of early erythroblasts through induction of Fas/FasLigand pathway. Therefore, these results first suggest that constitutive GDF11/ActRIIa signaling pathway may promote ineffective erythropoiesis in Beta-thalassemia intermedia, and secondly, support the use of ActRIIa traps for the treatment of chronic anemia and ineffective erythropoiesis. Altogether, these results open new perspectives in the understanding of normal and pathological hematopoiesis and lead to propose innovative treatments for anemia.

Resumo: A beta talassemia é um dos mais freqüentes distúrbios genéticos no mundo. Estima-se que 1,5% a 3% da população mundial seja portadora do traço talassêmico. Esses portadores geralmente são oligo ou assintomáticos e têm uma expectativa de vida semelhante à dos não portadores. Entretanto, níveis elevados de ferritina sérica têm sido observados em alguns estudos comparativos entre beta talassemia heterozigota e não portadores e, alguns indivíduos, que nunca foram transfundidos, apresentam sinais clínicos e laboratoriais de sobrecarga de ferro. A fisiopatologia dessa complicação continua em discussão. Vários pesquisadores têm sugerido um efeito modulador da mutação do gene da beta globina e mutações em genes codificadores de proteínas relacionadas ao metabolismo do ferro. Mutações no gene HFE são as mais freqüentemente associadas à hemocromatose hereditária. O objetivo do presente trabalho foi avaliar a freqüência das mutações C282Y e H63D no gene HFE em portadores de beta talassemia heterozigota e analisar sua influência no metabolismo do ferro. Foram estudados 162 portadores de beta talassemia heterozigota, residentes na cidade de São Carlos ou região, caucasóides e, acompanhados no serviço de Hematologia. O diagnóstico de traço talassêmico foi confirmado em todos por meio do eritrograma e da quantificação da Hb A2 e Hb fetal por HPLC. O metabolismo do ferro foi avaliado pelas dosagens de ferro sérico, capacidade total de ligação do ferro, ferritina e saturação da transferrina e, a análise molecular das mutações no gene HFE, pela técnica de PCR-RLFP. Foram realizadas análises de correlação linear de Pearson por idade e gênero entre hemoglobina... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Beta thalassemia is one of the most frequent genetic disorder in the world. It is estimated that 1.5% to 3% of the world population is a thalassemia carrier. These individuals are generally slightly symptomatic or asymptomatic and they have a life expectancy similar to those who are non-carriers. However, high levels of serum ferritin have been observed in some comparative studies between heterozygous for beta thalassemia and non-carriers, and some individuals that were never transfused, present clinic and laboratories signs of iron overload. The physiopathology of this disease continues in discussion. Several researchers have suggested a modulator effect from the mutation of the beta globin gene and mutations in genes related with the iron metabolism. Mutations of the gene HFE are the most frequently associated to the hereditary hemochromatosis. The aim of this study was evaluate the frequency of C282Y and H63D mutations in the HFE gene in beta thalassemia carriers, and analyze its influence in the iron metabolism. 162 beta thalassemia carriers, Caucasoid, residing in the city of Sao Carlos or region and accompanied in the Hematology service were studied. The diagnostic of thalassemia trait was confirmed in every one through a complete erythrogram and quantification of Hb A2 and Hb fetal by HPLC. The iron metabolism was evaluated by serum iron, total iron-binding capacity, serum ferritin and percent saturation of transferring. The molecular analysis of the mutations in the HFE gene was made by PCR-RLFP. There were made analysis of linear Pearson' correlation, by age and gender, among hemoglobin, Hb A2, VCM and among reticulocytes count and the values of saturation of transferrin and serum ferritin. / Orientador: Claudia Regina Bonini Domingos / Coorientador: Antonio José Manzato / Banca: Celso Carlos de Campos Guerra / Banca: Paula Rahal Liberatore / Mestre

L’hémoglobine est une protéine contenue dans les globules rouges dont la principale fonction est le transport de l’oxygène. Chaque molécule d’hémoglobine est un tétramère constitué de deux paires de globines identiques de type α et β. La β-thalassémie est une maladie génétique hématopoïétique provenant de mutations du gène encodant l'hémoglobine. Ce désordre se caractérise par une diminution ou une absence totale de la synthèse de la chaîne β-globine résultant principalement en une anémie hémolytique sévère ainsi que des complications multisystémiques, telles que la splénomégalie, des déformations osseuses et une dysfonction hépatique et rénale. Actuellement, les transfusions sanguines chroniques représentent le traitement standard des patients β-thalassémiques. Cette thérapie nécessite l’administration conjointe d’un traitement chélateur de fer puisqu’elle entraîne une accumulation pathologique du fer, considéré à ce jour comme la source principale des complications cardiovasculaires de la β-thalassémie. Néanmoins, malgré le traitement efficace de la surcharge de fer transfusionnelle, l’insuffisance cardiaque demeure encore la principale cause de mortalité chez les patients atteints de β-thalassémie. Cette observation indique possiblement la présence d’un mécanisme complémentaire dans le développement de la physiopathologie cardiaque β-thalassémique. L’objectif du présent projet consistait donc à étudier les altérations cardiovasculaires de la β-thalassémie indépendamment de la surcharge de fer transfusionnelle. En utilisant un modèle murin non-transfusé de la β-thalassémie majeure, nous avons d’abord évalué in vivo, par méthode d’imagerie novatrice échographique à haute fréquence, les propriétés hémodynamiques vasculaires. Nos résultats d’index de Pourcelot ainsi que de résistance vasculaire périphérique totale ont démontré une perturbation de l’écoulement microcirculatoire chez les souris β-thalassémiques non-transfusées. Subséquemment, nous avons étudié la fonction endothéliale de régulation du tonus vasculaire de vaisseaux mésentériques isolés. Nos résultats ont révélé un dysfonctionnement de la réponse vasodilatatrice dépendante de l’endothélium chez les souris β-thalassémiques malgré une augmentation de l’expression de l’enzyme de synthèse du monoxyde d’azote ainsi qu’un remodelage de la carotide commune caractérisé par un épaississement de la paroi vasculaire. Finalement, notre étude échocardiographique de la fonction et la morphologie cardiaque a montré, chez les souris β-thalassémiques, le développement d’une hypertrophie et une dysfonction ventriculaire gauche en l’absence de transfusions sanguines chroniques ou de dépôts directs de fer dans le myocarde. L’ensemble des résultats présentés dans le cadre de cette thèse indique la présence d’une pathologie cardiovasculaire chez les souris β-thalassémiques non-transfusés. Nos travaux permettent de proposer un mécanisme de la pathophysiologie cardiovasculaire β-thalassémique, indépendant de la charge de fer transfusionnelle, impliquant les effets compensatoires d’une anémie chronique combinés à une vasculopathie complexe initiée par les érythrocytes endommagés et l’hémolyse intravasculaire. / Hemoglobin is the major protein in red blood cells and is responsible of the oxygen transport. Each hemoglobin molecule is a tetramer consisting of two identical α- and β-globin subunits. β-thalassemia is a genetic hematopoietic disease caused by mutations in hemoglobin genes. This disorder is characterized by a decrease or absence of production of β-globin chain leading mainly to a severe hemolytic anemia and several systemic manifestations, including splenomegaly, skeletal deformities as well as hepatic and renal dysfunctions. Chronic blood transfusions remain the standard treatment for β-thalassemic patients. This therapy requires iron chelating management since it leads to pathological iron accumulation which is currently considered the main cause of cardiovascular complications of β-thalassemia. However, despite adequate control of transfusional iron loading, heart failure remains the leading cause of mortality in β-thalassemia. This issue is possibly indicative of additional pathogenic mechanisms underlying the development of the β-thalassemic cardiac pathology. The objective of the present research project was to study cardiovascular alterations of β-thalassemia independently of transfusional iron overloading. Using an untransfused murine model of β-thalassemia major, we have evaluated in vivo, by non-invasive high-frequency ultrasound imaging, vascular hemodynamic properties. Our results of Pourcelot indices and total peripheral vascular resistance have shown microcirculatory flow disturbances in untransfused β-thalassemic mice. Consequently, we have studied ex vivo the endothelial vasomotor function in isolated mesenteric arterioles. Our findings have pointed out endothelium-dependent vasodilator dysfunction in β-thalassemic mice despite increased expression of nitric oxide synthase, as well as remodeling of the common carotid artery wall. Lastly, our echocardiography studies of heart morphology and function in β-thalassemic mice have demonstrated the development of left ventricle hypertrophy and dysfunction in the absence of chronic blood transfusions or direct myocardial iron deposits. In conclusion, findings presented in this thesis have demonstrated for the first time development of severe cardiovascular complications in untransfused β-thalassemic mice. Based on our results, we have proposed a novel mechanism, independent of direct myocardial iron deposition, responsible for the cardiovascular complications in β-thalassemia. This model combines compensatory effects of chronic anemia with a complex vasculopathy initiated by abnormal erythrocytes and intravascular hemolysis.

Bone marrow mesenchymal stem cells (BM-MSCs) are pluripotent cells that can differentiate into a variety of non-hematopoietic tissues. They also maintain healthy heamatopoiesis by providing supportive cellular microenvironment into BM. In this thesis, MSCs were characterized in terms of their morphological, immunophenotypical and differentiation properties. Then, they were examined by attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy together with hierarchical clustering, and FTIR microspectroscopy. In the first part of this study, global structural and compositional changes in BM-MSCs during beta thallasemia major (

Bone marrow mesenchymal stem cells (BM-MSCs) are pluripotent cells that can differentiate into a variety of non-hematopoietic tissues. They also maintain healthy heamatopoiesis by providing supportive cellular microenvironment into BM. In this thesis, MSCs were characterized in terms of their morphological, immunophenotypical and differentiation properties. Then, they were examined by attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy together with hierarchical clustering, and FTIR microspectroscopy. In the first part of this study, global structural and compositional changes in BM-MSCs during beta thallasemia major (

L’hémoglobine est une protéine contenue dans les globules rouges dont la principale fonction est le transport de l’oxygène. Chaque molécule d’hémoglobine est un tétramère constitué de deux paires de globines identiques de type α et β. La β-thalassémie est une maladie génétique hématopoïétique provenant de mutations du gène encodant l'hémoglobine. Ce désordre se caractérise par une diminution ou une absence totale de la synthèse de la chaîne β-globine résultant principalement en une anémie hémolytique sévère ainsi que des complications multisystémiques, telles que la splénomégalie, des déformations osseuses et une dysfonction hépatique et rénale. Actuellement, les transfusions sanguines chroniques représentent le traitement standard des patients β-thalassémiques. Cette thérapie nécessite l’administration conjointe d’un traitement chélateur de fer puisqu’elle entraîne une accumulation pathologique du fer, considéré à ce jour comme la source principale des complications cardiovasculaires de la β-thalassémie. Néanmoins, malgré le traitement efficace de la surcharge de fer transfusionnelle, l’insuffisance cardiaque demeure encore la principale cause de mortalité chez les patients atteints de β-thalassémie. Cette observation indique possiblement la présence d’un mécanisme complémentaire dans le développement de la physiopathologie cardiaque β-thalassémique. L’objectif du présent projet consistait donc à étudier les altérations cardiovasculaires de la β-thalassémie indépendamment de la surcharge de fer transfusionnelle. En utilisant un modèle murin non-transfusé de la β-thalassémie majeure, nous avons d’abord évalué in vivo, par méthode d’imagerie novatrice échographique à haute fréquence, les propriétés hémodynamiques vasculaires. Nos résultats d’index de Pourcelot ainsi que de résistance vasculaire périphérique totale ont démontré une perturbation de l’écoulement microcirculatoire chez les souris β-thalassémiques non-transfusées. Subséquemment, nous avons étudié la fonction endothéliale de régulation du tonus vasculaire de vaisseaux mésentériques isolés. Nos résultats ont révélé un dysfonctionnement de la réponse vasodilatatrice dépendante de l’endothélium chez les souris β-thalassémiques malgré une augmentation de l’expression de l’enzyme de synthèse du monoxyde d’azote ainsi qu’un remodelage de la carotide commune caractérisé par un épaississement de la paroi vasculaire. Finalement, notre étude échocardiographique de la fonction et la morphologie cardiaque a montré, chez les souris β-thalassémiques, le développement d’une hypertrophie et une dysfonction ventriculaire gauche en l’absence de transfusions sanguines chroniques ou de dépôts directs de fer dans le myocarde. L’ensemble des résultats présentés dans le cadre de cette thèse indique la présence d’une pathologie cardiovasculaire chez les souris β-thalassémiques non-transfusés. Nos travaux permettent de proposer un mécanisme de la pathophysiologie cardiovasculaire β-thalassémique, indépendant de la charge de fer transfusionnelle, impliquant les effets compensatoires d’une anémie chronique combinés à une vasculopathie complexe initiée par les érythrocytes endommagés et l’hémolyse intravasculaire. / Hemoglobin is the major protein in red blood cells and is responsible of the oxygen transport. Each hemoglobin molecule is a tetramer consisting of two identical α- and β-globin subunits. β-thalassemia is a genetic hematopoietic disease caused by mutations in hemoglobin genes. This disorder is characterized by a decrease or absence of production of β-globin chain leading mainly to a severe hemolytic anemia and several systemic manifestations, including splenomegaly, skeletal deformities as well as hepatic and renal dysfunctions. Chronic blood transfusions remain the standard treatment for β-thalassemic patients. This therapy requires iron chelating management since it leads to pathological iron accumulation which is currently considered the main cause of cardiovascular complications of β-thalassemia. However, despite adequate control of transfusional iron loading, heart failure remains the leading cause of mortality in β-thalassemia. This issue is possibly indicative of additional pathogenic mechanisms underlying the development of the β-thalassemic cardiac pathology. The objective of the present research project was to study cardiovascular alterations of β-thalassemia independently of transfusional iron overloading. Using an untransfused murine model of β-thalassemia major, we have evaluated in vivo, by non-invasive high-frequency ultrasound imaging, vascular hemodynamic properties. Our results of Pourcelot indices and total peripheral vascular resistance have shown microcirculatory flow disturbances in untransfused β-thalassemic mice. Consequently, we have studied ex vivo the endothelial vasomotor function in isolated mesenteric arterioles. Our findings have pointed out endothelium-dependent vasodilator dysfunction in β-thalassemic mice despite increased expression of nitric oxide synthase, as well as remodeling of the common carotid artery wall. Lastly, our echocardiography studies of heart morphology and function in β-thalassemic mice have demonstrated the development of left ventricle hypertrophy and dysfunction in the absence of chronic blood transfusions or direct myocardial iron deposits. In conclusion, findings presented in this thesis have demonstrated for the first time development of severe cardiovascular complications in untransfused β-thalassemic mice. Based on our results, we have proposed a novel mechanism, independent of direct myocardial iron deposition, responsible for the cardiovascular complications in β-thalassemia. This model combines compensatory effects of chronic anemia with a complex vasculopathy initiated by abnormal erythrocytes and intravascular hemolysis.