Thalassemia, a genetic blood disorder, occurs as a result of deformations of hemoglobin structures. Patients with thalassemia develop iron overload from chronic blood transfusions and require regular iron chelation to prevent potentially fatal iron-related complications. Deferiprone is a commercially available drug used as iron chelator for the treatment of thalassemia but the very long-term effectiveness is not clearly known yet. Therefore, some studies were carried out to find effective alternative drugs or delivery methods for treatment of thalassemia. Controlled delivery, which offers safer, more convenient, and more effective means of administering actives, seems promising with this respect. Chitosan, a natural biopolymer produced from deacetylation of chitin, has a variety of promising pharmaceutical uses and is presently considered as a novel carrier material in drug delivery systems. In this study, chitosan microspheres having different degree of deacetylation (DDA) and containing Deferiprone were prepared by oil/water emulsion method and by crosslinking with gluteraldehyde. Particle size, SEM, and in vitro drug release analysis were performed. The average sizes of the prepared microspheres increased with increasing degree of deacetylation of chitosan and with decreasing crosslinking degree. In vitro drug release studies showed that, the release rate of Deferiprone increased as the crosslinking degree increased, contrary to the expectations. This is explained by the crystalline structure of lightly crosslinked chitosan which have ordered and dense structure causing slower release rate for Deferiprone compare to highly crosslinked structures. In the second stage of the study, chitosan films hardened with gluteraldehyde were prepared by film casting method. IR, DSC and mechanical analysis were performed. For the films with various crosslinking degrees, it was found that UTS values differed from 50.6 MPa to 102.7 MPa, mean elastic modulus values differed from 3328.7 MPa to 3790.1 MPa and SAB values differed from 2.06% to 4.29%.

Οι ασθενείς με ομόζυγο β-μεσογειακή αναιμία εμφανίζουν αυξημένη καρδιακή νοσηρότητα και θνητότητα. Μία παρακλινική, μη επεμβατική μέθοδος ικανή να αποκαλύψει καρδιακή δυσλειτουργία σε ασυμπτωματικό στάδιο θα ήταν χρήσιμη για την πρώιμη ενίσχυση της αποσιδήρωσης και ενδεχόμενη χορήγηση καρδιοπροστατευτικής αγωγής στους ασθενείς αυτούς. Η ινοτροπική διέγερση με δοβουταμίνη μπορεί να αποκαλύψει λανθάνουσα δυσλειτουργία της αριστερής κοιλίας και πιθανώς αυξημένο μελλοντικό καρδιακό κίνδυνο μέσω της κατάδειξης ανεπαρκούς συστολικής εφεδρείας σε ασυμπτωματικούς, μη θαλασσαιμικούς ασθενείς. Μέθοδοι: 36 καρδιολογικά ασυμπτωματικοί ασθενείς με μείζονα β-μεσογειακή αναιμία και φυσιολογική καρδιακή λειτουργία (ηλικίας 25±6 έτη, φερριτίνη ορού 3574±2393 νανογρ / κυβ. εκατ.) και 25 υγιείς μάρτυρες συγκρίσιμοι σε ηλικία, φύλο και επιφάνεια σώματος μελετήθηκαν υπερηχοκαρδιογραφικά σε ηρεμία και μετά φόρτιση με ενδοφλέβια δοβουταμίνη σε αυξανόμενες δόσεις. Η μελέτη επαναλήφθηκε σε 23 ασθενείς μετά 24±12 μήνες. Το σύνολο των ασθενών εκτιμήθηκε κλινικά κατά τη δεκαετία που ακολούθησε την αρχική μελέτη. Αποτελέσματα: Στην ομάδα των θαλασσαιμικών υπήρχε ελαττωμένη ποσοστιαία πάχυνση του οπισθίου τοιχώματος της αριστερής κοιλίας σε σύγκριση με τους υγιείς μάρτυρες. Στην επαναληπτική μελέτη διαπιστώθηκε ότι η αριστερή κοιλία εμφάνιζε στη ηρεμία δυσμενή αναδιαμόρφωση αλλά επαρκή συστολική εφεδρεία στη φόρτιση με δοβουταμίνη. Κατά τη δεκαετή κλινική παρακολούθηση 7 ασθενείς απεβίωσαν, όλοι από καρδιακά αίτια. Η μέθοδος πολλαπλής εξάρτησης αναλογικού κινδύνου έδειξε ότι η φερριτίνη ορού και η ηλικία, με ανάστροφη συσχέτιση, όχι όμως οι υπερηχοκαρδιογραφικοί παράμετροι ήταν οι μοναδικοί προβλεπτικοί δείκτες θνητότητας. Συμπέρασμα : Η φερριτίνη ορού και η ηλικία φέρουν σημαντική προγνωστική αξία σε ασθενείς με μείζονα β-μεσογειακή αναιμία. Το υπερηχοκαρδιογράφημα δοβουταμίνης δεν συνεισφέρει στην διαβάθμιση κινδύνου στους ασθενείς αυτούς. / Patients with beta-thalassemia major demonstrate increased cardiac morbidity and mortality. A non-invasive method, capable of revealing cardiac dysfunction at an asymptomatic stage would be useful for the early intensification of chelation therapy and possible application of cardioprotective treatment in these patients. The inadequate, left ventricular inotropic response upon dobutamine challenge has prognostic implications in studies on asymptomatic, non-thalassemic patients. Methods: 36 asymptomatic patients with beta-thalassemia major and normal biventricular systolic function (mean age 25±6 years, mean serum ferritin 3574±2393ng/ml) and 25 healthy controls matched for age, sex, and body surface area were examined echocardiographically at rest and after intravenous dobutamine infusion in incremental doses. Echocardiographic dobutamine examination was repeated in 23 thalassemic patients after 24±12 months. All patients were clinically evaluated during the decade following the first study. Results: Compared to the healthy controls, thalassemic patients exhibited a reduced thickening of the left ventricular posterior wall. At follow up, both an unfavorable left ventricular remodeling at rest and sufficient systolic reverse were evident. During the ten-year clinical evaluation seven patients died, all of cardiac causes. Cox regression analysis showed that higher serum ferritin and lower age, but not the echocardiographic parameters, were predictive of mortality. Conclusions: Serum ferritin and age, but not the dobutamine stress echocardiogram, are independently associated with cardiac mortality among patients with beta-thalassemia major.

The primary aim of this thesis was to outline an approach for the prenatal diagnosis of β-thalassaemia in the Asian Indian population by DNA analysis. A polymerase chain reaction (PCR) based, nonradioactive and rapid technique, allele specific PCR, was successfully developed for the detection of β-thalassaemia mutations. A large sample of 656 unrelated carriers from seven different regions of the Indian subcontinent was studied by allele specific PCR and DNA sequence analysis. Sixteen different β-thalassaemia mutations were identified, two of which were new mutations. Of these five common mutations accounted for 91.7% of β-thalassaemia alleles. The β-globin gene haplotypes of 419 β-Th and 196 β-A chromosomes were constructed. On analysis of which it was inferred that β-thalassaemia mutations occurred relatively recently on existing chromosomal backgrounds and then they experienced positive selection. A strong but not invariant haplotype-mutation linkage was observed. A regional variation in the distribution of β-thalassaemia mutations was found. a-Globin gene mapping studies identifed the single a-globin gene deletion in 24 out of 51 unrelated Asian Indians who were suspected to have a-thalassaemia. It is likely that the remaining carriers have nondeletional a-thalassaemia determinants. To perform preimplantation diagnosis of β-thalassaemia, by analysis of a 10-30 cell embryonic biopsy, a PCR protocol was developed. Using two rounds of PCR with nested primers, successful amplification of a 597 bp fragment of the β-globin gene was achieved from as few as two embryonic cells. The problem of false positive amplification was encountered which appeared to be resolved by UV transillumination of the pre-amplification PCR mix. By allele specific PCR with nested primers it was possible to identify the presence or absence of five β-thalassaemia mutations from 10 pg of template DNA (equivalent to approximately two diploid cells). Thalassaemia control in India is a complex issue; the financial, social and demographic factors involved were considered and recommendations made.

Thesis ( Ph.D.) -- McMaster University, 1998. / Includes bibliographical references (leaves 191-211). Also available via World Wide Web.

Thesis (Ph. D.)--Victoria University (Melbourne, Vic.), 2006. / Includes bibliographical references.

Thesis (Ph. D.)--Cornell University, March, 2008. / Vita. Includes bibliographical references (leaves 376-419).

Made available in DSpace on 2014-11-10T11:09:51Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-02-17Bitstream added on 2014-11-10T11:57:53Z : No. of bitstreams: 1 000790770_20150217.pdf: 705337 bytes, checksum: 76b2351a6e2dbcbb447e928581f178da (MD5) Bitstreams deleted on 2015-02-18T15:02:19Z: 000790770_20150217.pdf,Bitstream added on 2015-02-18T15:02:50Z : No. of bitstreams: 1 000790770.pdf: 1719832 bytes, checksum: 2e325f95cdb92f6b81892ae8e36726eb (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / As talassemias do tipo beta são afecções genéticas frequentes na população mundial e seus portadores podem apresentar elevação nos níveis de hemoglobina A2 (Hb A2) e hemoglobina fetal (Hb F). As mutações presentes nos indivíduos podem estar associadas a diferentes haplótipos do grupamento da β-globina. Os objetivos do trabalho consistiram em investigar as frequências do polimorfismo XmnI (-158 CT) e do padrão de haplótipos da β-globina em indivíduos heterozigotos e homozigotos para a beta talassemia, relacioná-las com os níveis de Hb F, e compará-las com indivíduos sem hemoglobinopatias. Foram analisadas 150 amostras de indivíduos beta talassêmicos heterozigotos; 22 de indivíduos homozigotos e 150 de indivíduos sem hemoglobinopatias (grupo controle). Todas as amostras foram submetidas aos testes clássicos de diagnóstico de hemoglobinopatias e à análises moleculares por PCR Alelo Específico (PCR-AE) para confirmação da mutação de beta talassemia, e PCR para polimorfismos de comprimento de fragmentos de restrição (PCR-RFLP) para a identificação do polimorfismo XmnI e dos sítios de haplótipos da β-globina. Os testes estatísticos foram realizados com o software STATISTICA 8.0 e Haploview 4.2. Nos indivíduos beta talassêmicos heterozigotos, a mutação CD39 foi a mais frequente (62%) e nos homozigotos, a IVS-I-6 (22%). Foi observada diferença significativa nos níveis de Hb F entre os indivíduos que possuem mutações ainda não identificadas, em relação aos que possuem a mutação IVS-I-110 (p<0,05), no grupo de heterozigotos. A presença do polimorfismo XmnI foi observada em todos os grupos estudados, porém, somente para os indivíduos com talassemia beta heterozigota houve diferença estatística em relação aos níveis aumentados de Hb F (p=0,007). Nos três grupos foram observados os padrões típicos de haplótipos I, II, IV, VI, VII e IX. Para os indivíduos com beta talassemia ... / The beta thalassemia are frequent genetic disorders and sufferers may have increased levels of hemoglobin A2 (Hb A2) and fetal hemoglobin (Hb F). The mutations present in individuals may be associated with different haplotypes of β–globin grouping. The objectives of this study consisted in investigating the frequencies of the XmnI polymorphism (-158 CT) and the pattern of the β-globin haplotypes in heterozygous and homozygous individuals for beta thalassemia, relate them to the levels of Hb F, and compare them with individuals without hemoglobinopathies. We analyzed 150 samples from heterozygous individuals with beta thalassemia, 22 homozygous and 150 individuals without hemoglobinopathies (control group). All samples were tested for classical hemoglobinopathies diagnosis and molecular analyzes Allele Specific PCR (AE-PCR) to confirm the mutation of beta thalassemia and PCR length polymorphism restriction fragment (PCR-RFLP) for identification of the polymorphism XmnI and sites of the β-globin haplotypes. Statistical tests were performed using STATISTICA 8.0 and Haploview 4.2 software. In beta thalassemia heterozygous individuals, mutation CD39 was the most common (62%) and in homozygous was the IVS-I-6 (22 %). We observed a significant difference in the levels of Hb F among the unidentified mutations and IVS-I-110 (p<0.05) in heterozygous individuals. The presence of the XmnI polymorphism was observed in all groups, but only heterozygous individuals for beta thalassemia was statistical difference in relation to increased levels of Hb F (p = 0.007). In the three groups were observed haplotype patterns I, II, IV, VI, VII and IX. For individuals heterozygous to beta thalassemia, the patterns II (24.3 %) and VII (32 %) were the most frequent, for individuals with beta thalassemia homozygous, the patterns I (29.5 %) and VII (38.6 %) and individuals without hemoglobinopathies, the patterns I (31%) and VII (28.3%). There was ...

IntroduÃÃo: A beta talassemia à um grupo de distÃrbios, cada um resultando de um defeito genÃtico, na velocidade de sÃntese de uma ou mais cadeias globÃnicas da hemoglobina (Hb). A desproporÃÃo na produÃÃo das cadeias globÃnicas pode resultar em eritropoese ineficaz, produÃÃo insuficiente de Hb, hemÃlise e anemia de grau variado. A beta talassemia à mais frequente nos paÃses banhados pelo mar MediterrÃneo, refletindo a participaÃÃo desses povos na formaÃÃo da populaÃÃo brasileira. As mutaÃÃes predominantes no Brasil sÃo a IVS-I-1, IVS-I-6, IVS-I-110 e o CD 39, as quais estÃo associadas a diversos quadros clÃnicos e sÃo, em sua maioria, regionalmente especÃficas. Objetivo: Caracterizar o perfil clÃnico, hematolÃgico e molecular dos indivÃduos adultos com beta talassemia do Hospital UniversitÃrio Walter CantÃdeo, em acompanhamento no centro de referÃncia de Hematologia e Hemoterapia do estado do Cearà (HEMOCE). Metodologia: Foram analisados 22 indivÃduos portadores de beta talassemia, sendo 7 intermediÃria e 15 menor, de ambos os sexos, no perÃodo de fevereiro de 2008 a setembro de 2009. Os dados clÃnicos e laboratoriais: hemograma, nÃveis de Hb A2 e de Hb F; ferro sÃrico; capacidade total e latente de ligaÃÃo do ferro (CTLFe, CLLFe), ferritina e Ãndice de saturaÃÃo da transferrina (IST), foram obtidos dos prontuÃrios, ao diagnÃstico. Cerca de 5 mL de sangue venoso foi coletado em tubo contendo o anticoagulante EDTA para o estudo molecular. A anÃlise das mutaÃÃes foi realizada por meio da tÃcnica da reaÃÃo em cadeia mediada pela polimerase alelo especÃfico (PCR-AE), onde foram analisadas as seguintes mutaÃÃes: IVS-I-1, IVS-I-6, IVS-I-110 e o CD 39. As anÃlises estatÃsticas foram desenvolvidas no software livre R (versÃo 2.7.0) e o nÃvel de significÃncia estabelecido foi 5%. Resultados: Dos 22 pacientes estudados, 15 eram portadores de &#946; talassemia menor e sete intermediÃria. A idade variou de 18 a 68 anos, com mÃdia de 44,7 anos. 18,2 % do sexo masculino e 81,8% do sexo feminino. As mutaÃÃes foram caracterizadas em 68,2% dos casos, que teve como mais frequente a IVS-I-6, seguida do cÃdon 39. A mutaÃÃo IVS-I-I foi caracterizada em um paciente e a IVS-I-110 nÃo foi encontrada. NÃo houve diferenÃa clÃnica significante entre os parÃmetros hematolÃgicos e bioquÃmicos. Houve discrepÃncia entre o fenÃtipo e o genÃtipo em alguns pacientes, porÃm nÃo houve diferenÃa significativa entre as mutaÃÃes e as manifestaÃÃes clÃnicas. ConclusÃes: Os resultados do presente estudo reforÃam o predomÃnio da mutaÃÃo IVS-I-6 no nordeste do Brasil. Recomendam-se estudos posteriores para investigaÃÃo da co-heranÃa com a &#945; talassemia nesses pacientes para justificar a discrepÃncia entre genÃtipos e fenÃtipos. / Background: Beta thalassemia is a group of disorders, each resulting from a genetic defect in the rate of synthesis of one or more globin chains of hemoglobin (Hb). The imbalance in the production of globin chains can result in ineffective erythropoiesis, insufficient production of hemoglobin, hemolysis and anemia of varying degree. Beta thalassemia is more common in countries bordering the Mediterranean Sea, reflecting the participation of these peoples in the formation of the Brazilian population. The predominant mutations in Brazil are the IVS-I-1, IVS-I-6, IVS-I-110 and CD 39, which are associated with different clinical conditions and are mostly regionally specific. Objective: To characterize the clinical, hematological and molecular adults with beta thalassemia of the University Hospital CantÃdeo Walter and followed at a referral center for Hematology of the state of Cearà (Hemoce). Methods: We analyzed 22 individuals with beta thalassemia, 7 intermediate and 15 minor, in both sexes, from February 2008 to September 2009. Clinical data and laboratory tests: blood count, levels of Hb A2 and Hb F, serum iron, total capacity and latent iron binding (CTLFe, CLLFe), ferritin and transferrin saturation index (IST) were obtained from medical records, diagnosis. About 5 mL of venous blood was collected in tubes containing EDTA anticoagulant for molecular study. The analysis of mutations was performed using the technique of chain reaction mediated by allele specific polymerase (PCR-AE), where we analyzed the following mutations: IVS-I-1, IVS-I-6, IVS-I-110 and CD 39. Statistical analysis was carried out in software R (version 2.7.0) and the level of significance was 5%. Results: Of 22 patients studied, 15 were patients with &#946; thalassemia minor and seven intermediate. The age ranged 18-68 years with a mean of 44.7 years. 18.2% male and 81.8% female. The mutations were characterized in 68.2% of cases, which had the most frequent IVS-I-6, followed by the codon 39. The mutation IVS-I-1 was found in one patient and IVS-I-110 was not found. There was no significant clinical differences between the hematological and biochemical parameters. There was a discrepancy between phenotype and genotype in some patients, but no significant difference between mutations and clinical manifestations. Conclusions: The results of this study reinforce the dominance of the mutation IVS-I-6 in northeastern Brazil. Are recommended further studies to investigate the co-inheritance with &#945;-thalassemia in these patients to justify the discrepancy between genotypes and phenotypes.

No description available.

Talassemias são as mais comuns desordens monogenéticas em humanos; são caracterizadas pela presença de anemia microcíticas e hipocrômicas que resultam da redução ou ausência na síntese de uma ou mais cadeias globínicas. No Brasil a beta-talassemia tem importante significado clínico e sua ocorrência pode ser explicada como conseqüência da grande mistura étnica. A beta-talassemia é extremamente heterogênea e mais de 200 mutações têm sido descritas causando diferentes graus de anemia. Essas mutações são regionalmente específicas e cada país possui seu grupo de alterações característico. No estado homozigoto são classificadas clinicamente como major e no estado heterozigoto como intermédia e minor. Os pacientes com talassemia intermédia podem não apresentar sintomas clínicos, mas constatam-se características laboratoriais específicas. A avaliação dos índices hematológicos, dosagens dos parâmetros bioquímicos e dos valores das hemoglobinas fetal e HbA2 em combinação com o entendimento das possíveis interações gênicas são os procedimentos recomendados para o diagnóstico laboratorial. O objetivo deste estudo foi determinar a presença de mutações em pacientes que utilizam a Divisão de Laboratório Central do Hospital das Clínicas da Faculdade de Medicina da Universidade São Paulo (DLC-HC-FMUSP) e que apresentavam perfil laboratorial sugestivo para beta-talassemia. Estudou-se a região do gene da globina em onde estão descritas as mutações mais freqüentes para nosso meio através da técnica de PCR, e posterior seqüenciamento para identificação das mutações. As mutações observadas na população avaliada (N=40), em ordem crescente de freqüência foram: Codon 39 (CT), IVS-I-110 (GA), IVS-I-1(GA), IVS-I-5 (GC), IVS-I-6 (TC), IVS-I-Exon-II (GA), Exon-II-IVS-II (GA), Stop Cd 6 (GT), Stop Cd15 (GA), Del C Cd 44 e 5UTR+20. Os primeiros quatro genótipos representaram 82,5% das mutações observadas neste trabalho. O uso combinado dos índices hematimétricos, parâmetros bioquímicos e avaliação das características morfológicas das hemácias demonstraram eficiência no rastreamento de pacientes portadores de beta-talassemia, assintomáticos A técnica do PCR e posterior processo de seqüenciamento demonstraram elevada eficiência na determinação das alterações moleculares no grupo de pacientes avaliados. / Thalassemias are the most common monogenic disorders in humans; are characterized by hypochromic and microcytic anemia arising from the reduction or absence in the synthesis of one or more chains globínicas. In Brazil, beta-thalassemia has important clinical significance and its occurrence can be explained as a result of the large ethnic mix. The beta-thalassemia is extremely heterogeneous and more than 200 mutations have been described causing varying degrees of anemia, these mutations are regionally specific and each country has its characteristic group of amendments. In the homozygous state are clinically classified as major and the heterozygous state as intermediate and minor. Patients with thalassemia can not present interim clinical symptoms, but have characteristics specific laboratory. The evaluation of hematologic indices, strengths and biochemical parameters of fetal hemoglobin and HbA2 values in combination with an understanding of possible interactions genes, are the recommended procedures for laboratory diagnosis. The purpose of this study was to determine the presence of mutations in patients who use the Division of Central Laboratory of the Hospital of the Faculty of Medicine of the University Sao Paulo (DLC-HCFMUSP) who presented suggestive profile laboratory for beta-thalassemia. It was studied the region of the gene of beta globin where they are frequently described the changes to our region through the PCR technique, and subsequent sequencing to identify the mutations. Of the 40 patients evaluated were found the following changes: Codon 39 (CT), IVS-I-110 (GA), IVS-I-1 (GA), IVS-I-5 (GC), IVS-I-6 (TC), IVS-I-Exon-II (GA), Exon-II-IVS-II (GA), Stop Cd 6 (GT), Stop Cd15 (G A), Del Cd 44 and 5UTR+20. The first four genotypes accounted for 82.5% of mutations observed in this study. The combined use of hematologic indices, biochemical parameters and evaluation of morphological characteristics of red blood cells demonstrated efficiency in the tracking of patients with beta- thalassemia, asymptomatic. The technique of PCR and subsequent process of sequencing demonstrated high efficiency in determining the molecular changes in the group of patients evaluated.