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Orientador: Maria de Fatima Sonati / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-09T04:43:04Z (GMT). No. of bitstreams: 1 Ribeiro_DanielaMaria_D.pdf: 2122347 bytes, checksum: 625fa24f0c7882709f12ba0054267aff (MD5) Previous issue date: 2007 / Resumo: A expressão dos genes do cluster da globina a em humanos é regulada pelo a-MRE ( a-Major Regulatory Element), um elemento localizado 40 kb à montante do respectivo cluster na região telomérica do braço curto do cromossomo 16. O a-MRE é geneticamente polimórfico e seis diferentes haplótipos, nomeados de A a F, foram determinados em alguns grupos populacionais da África, Europa e Ásia e em indivíduos pertencentes a duas populações indígenas brasileiras. As substituições de base que resultaram nestes haplótipos estão localizadas entre sítios de ligação para fatores nucleares ou em um sítio não ocupado in vivo, exceto no caso do haplótipo D, em que o polimorfismo altera a seqüência de um dos sítios ocupado in vivo pelo fator NF-E2. Não há, de nosso conhecimento, nenhum estudo experimental feito para avaliar se essa variabilidade pode influenciar a expressão gênica. Assim, no presente trabalho foi analisada e comparada a expressão do gene repórter da luciferase em células K562 transientemente transfectadas com construções que tiveram como enhancers os diferentes haplótipos do a-MRE, além de 3 dos polimorfismos isoladamente (+130, +199 e +209). Os resultados revelaram redução da expressão do gene da luciferase com todas as construções em relação à do haplótipo selvagem A: os haplótipos B e C corresponderam a 19% do grau de expressão do haplótipo A, o D a 21%, o E a 15%, o F a 3%, o polimorfismo +130 a 24%, o +199 a 32% e o +209 a 3%. Em seu conjunto, eles demonstram que os polimorfismos responsáveis pelos diferentes haplótipos do a-MRE, em sua maioria situados nas seqüências flanqueadoras dos sítios de ligação para proteínas regulatórias, reduzem a expressão gênica in vitro / Abstract: The expression of human a-like globin genes is regulated by the a-MRE (a-Major Regulatory Element), an element located 40 kb upstream of the a cluster in the short arm of chromosome 16. The a-MRE is genetically polymorphic and six different haplotypes, named A to F, have been identified in some population groups from Europe, Africa and Asia and in native Indians from two Brazilian Indian tribes. The base substitutions that resulted in these haplotypes are located between binding sites for nuclear factors or in a site that is considered not to be active in vivo, with the exception of haplotype D, in which the polymorphism changes the first binding site for the NF-E2 factor occupied in vivo. To our knowledge, there are no experimental studies evaluating whether this variability may influence gene expression. Thus, the present work analyzed and compared the expression of the luciferase reporter gene in K562 cells transiently transfected with constructs that have, as enhancers, the different a-MRE haplotypes, besides three isolated polymorphisms (+130, +199 and +209). The results demonstrated a reduction in luciferase gene expression with all the constructs compared with the wild type a-MRE (A haplotype): the B and C haplotypes corresponded to 19% of the A haplotype expression, the D to 21%, the E to 15%, the F to 3%, the polymorphism +130 to 24%, the +199 to 32% and the +209 to 3%. They demonstrate that the polymorphisms responsible for the a-MRE haplotypes, most located in the flanking sequences of the regulatory protein binding sites, decrease in vitro gene expression / Doutorado / Ciencias Biomedicas / Doutor em Ciências Médicas

Thesis (Ph. D.) -- Virginia Commonwealth University, 2004. / Prepared for: Dept. of Microbiology and Immunology. Bibliography: leaves 130-148. Also available online.

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

Thesis (Ph. D.)--Virginia Commonwealth University, 2006. / Prepared for: Dept. of Microbiology and Immunology. Bibliography: leaves 159-173. Also available online.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / IntroduÃÃo: A anemia falciforme à o resultado de uma mutaÃÃo pontual (GAG&#61614;GTG) no cÃdon do gene da globina &#946;, conduzindo a uma substituiÃÃo de Ãcido glutÃmico por valina na sexta posiÃÃo da cadeia polipeptÃdica. A anemia falciforme apresenta manifestaÃÃes clÃnicas heterogÃneas, que podem ser relacionadas ao tipo de haplÃtipo associado ao gene da HbS e aos nÃveis de HbF. Objetivo: CaracterizaÃÃo clÃnica, hematolÃgica e molecular dos pacientes com anemia falciforme em Fortaleza, CearÃ. Metodologia: Foram analisados 47 pacientes com anemia falciforme, adultos e de ambos os sexos. A determinaÃÃo dos valores hematolÃgicos foi realizada em contador automÃtico de cÃlulas sangÃÃneas; a determinaÃÃo da presenÃa de HbSS foi realizada por eletroforese em pH alcalino em fitas de acetato de celulose e por eletroforese de diferenciaÃÃo em Ãgar-fostato pH 6.2; os nÃveis de HbF foram determinados pela tÃcnica da desnaturaÃÃo alcalina; e a anÃlise dos haplÃtipos da mutaÃÃo ÃS foi realizada por meio da tÃcnica da reaÃÃo em cadeia mediada pela polimerase para polimorfismo dos comprimentos dos fragmentos de restriÃÃo (PCR-RFLP). As anÃlises estatÃsticas foram desenvolvidas no programa GraphPad Prism (versÃo 5.0) e o nÃvel de significÃncia estabelecido foi p < 0,05. Resultados: A distribuiÃÃo dos haplÃtipos do gene da &#946;S-globina, 63% do tipo Bantu, 25% do tipo Benin e 12% do tipo AtÃpico, està em conformidade com a observada para a populaÃÃo brasileira, em que o haplÃtipo Bantu à o mais prevalente, seguido pelo Benin e Senegal. NÃo houve diferenÃa significativa entre o presente estudo e os resultados encontrados no Rio de Janeiro, Porto Alegre, Campinas e RibeirÃo Preto; porÃm uma diferenÃa significativa foi observada quando o estudo foi comparado aos resultados obtidos em Salvador, BelÃm, Amazonas e por outros pesquisadores no CearÃ. A distribuiÃÃo das freqÃÃncias dos haplÃtipos do gene da &#946;S-globina nos diferentes estudos està condizente com a histÃria da formaÃÃo da populaÃÃo brasileira, exceto nos resultados do estudo anterior realizado no CearÃ, que obteve o haplÃtipo Benin com maior prevalÃncia. Conforme os dados histÃricos sobre as origens da populaÃÃo negra trazida ao estado do CearÃ, o haplÃtipo Bantu seria o mais prevalente. Na comparaÃÃo entre os haplÃtipos e as caracterÃsticas hematolÃgicas estudadas, apenas os valores de HbF e Ht apresentaram diferenÃa estatisticamente significativa. Os nÃveis de HbF foram maiores no haplÃtipo Benin, seguido do haplÃtipo Bantu, o que està em conformidade com os dados da literatura. Foi demonstrada maior presenÃa de crises vaso-oclusivas e episÃdios de pneumonia no haplÃtipo Benin/AtÃpico do que no haplÃtipo Bantu/AtÃpico; e maior presenÃa de crises de infecÃÃo urinÃria no haplÃtipo Benin/AtÃpico do que no haplÃtipo Benin/Benin. NÃo houve diferenÃa estatisticamente significativa entre os haplÃtipos Bantu/Bantu e Benin/Benin em relaÃÃo Ãs complicaÃÃes clÃnicas, entretanto foi observado que o haplÃtipo Bantu/Bantu tem uma maior freqÃÃncia em todos os eventos clÃnicos estudados quando comparado ao Benin/Benin. Dentre os resultados foi demonstrada uma tendÃncia de menor nÃmero de pacientes com crises vasos-oclusivas e Ãlceras de perna com o aumento dos nÃveis de HbF. NÃo houve diferenÃa estatisticamente significativa na comparaÃÃo entre os nÃveis de HbF e os valores de HemÃcias, Hemoglobina, HematÃcrito, LeucÃcitos e Plaquetas. ConclusÃes: A determinaÃÃo dos haplÃtipos da anemia falciforme à de grande importÃncia nÃo sà para o acompanhamento e prognÃstico dos pacientes, como tambÃm como ferramenta para estudos antropolÃgicos que contribuam no esclarecimento da origem dos africanos que tanto contribuÃram na formaÃÃo etnolÃgica, econÃmica, cultural e social do Brasil. / Introduction: The sickle cell anemia is the result of a point mutation in the &#946;-globin gene, leading to a substitution of glutamic acid by valine at the sixth position of the polypeptide chain. The sickle cell anemia presents heterogeneous clinical manifestations, which may be related to the type of haplotype associated with the gene for HbS and HbF levels. Objective: Clinical characterization, molecular and haematological patients with sickle cell anemia in Fortaleza, CearÃ. Methods: We analyzed 47 patients with sickle cell anemia, adults of both sexes. The determination of hematological values was performed on blood cells automated meter; the determination of the presence of HbSS was performed by alkaline hemoglobin electrophoresis on cellulose acetate tapes and by differentiation electrophoresis on agar-phosphate pH 6.2; the levels of HbF were determined by alkali denaturation technique; and the analysis of the haplotypes of the ÃS mutation was done by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Statistical analysis was developed in the program GraphPad Prism (version 5.0) and the level of significance was set p <0.05. Results: The distribution of the haplotypes of &#946;S-globin gene - 63% of the Bantu type, 25% of the Benin type and 12% atypical â was in conformity with that observed for the entire Brazilian population, in which the Bantu haplotype is most prevalent, followed by the Benin and Senegal. There was no significant difference between the results found in this study and those found for the cities of Rio de Janeiro, Porto Alegre, Campinas and RibeirÃo Preto; but there was a significant difference with the results obtained for the cities of Salvador and BelÃm and the state of Amazonas, and by other researchers in CearÃ. The distribution of haplotype frequencies of the &#946;S-globin gene in the different studies is in line with the history of the formation of the Brazilian population, except for the results of a previous study carried out in CearÃ, in which the Benin haplotype was found to be most prevalent. According to the historical information on the origins of the slave population brought to CearÃ, the Bantu haplotype should be the most prevalent. In the comparison between the haplotypes and the haematological characteristics studied, only the values of HbF and Ht showed statistically significant difference. The levels of HbF were higher in the Benin haplotype, followed by the Bantu haplotype, which is in accordance with the literature. Was demonstrated greater presence of painful episodes and episodes of pneumonia in Benin haplotype/Atypical haplotype than in Bantu/Atypical and increased presence of urinary infection crises in Benin haplotype/Atypical haplotype than in Benin/Benin. There was no statistically significant difference between the haplotypes Bantu/Bantu and Benin/Benin for clinical complications, however it was observed that the haplotype Bantu/Bantu has a higher frequency in all studied clinical events when compared to Benin/Benin. Among the results was shown a trend of fewer patients with painful episodes and of leg ulcers with increased levels of HbF. There was no statistically significant difference in the comparison between the levels of HbF and the values of red blood cells, hemoglobin, hematocrit, leukocytes and platelets. Conclusions: The determination of haplotypes of sickle cell anemia is of great importance not only for monitoring and prognosis of patients, but also as a tool for anthropological studies which help in clarifying the origin of Africans who have contributed so much in training ethnological, economic, cultural and social Brazil.

Thesis (Ph.D.)--University of Florida, 2005. / Typescript. Title from title page of source document. Document formatted into pages; contains 103 pages. Includes Vita. Includes bibliographical references.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on July 13, 2010). Includes bibliographical references.

Chan Kai Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 109-119). / Abstracts in English and Chinese. / Title --- p.i / Thesis committee --- p.ii / Statement --- p.iii / Acknowledgement --- p.iv / Abbreviations --- p.v / Abstract (English) --- p.vii / Abstract (Chinese) --- p.ix / Table of contents --- p.xi / List of Figures --- p.xvi / Chapter Chapter 1: --- General Introduction --- p.1 / Chapter 1.1 --- Human Hemoglobin --- p.1 / Chapter 1.2 --- Hemoglobinopathies --- p.4 / Chapter 1.3 --- Hereditary Persistence of Fetal Hemoglobin (HPFH) and β - Thalassemias --- p.6 / Chapter 1.4 --- Globin Genes Switching --- p.7 / Chapter 1.5 --- Pharmaceutical Agents for γ-Globin Gene Reactivation --- p.9 / Chapter 1.6 --- Discovery of LC978: A Novel Fetal Hemoglobin Inducing Agent --- p.10 / Chapter 1.7 --- Aim of Study --- p.11 / Chapter Chapter 2: --- Specific Induction of Gamma Globin Gene Transcription in K562 Leukemia Cell Line by Lead Compound LC978 --- p.12 / Chapter 2.1 --- K562 Cell Line as a Model for Gamma Globin Gene Induction Studies --- p.12 / Chapter 2.2 --- LC978-Induced Fetal Hemoglobin Expression in K562 Cell Line --- p.12 / Chapter 2.3 --- Materials --- p.14 / Chapter 2.3.1 --- "Chemicals, Kits and Reagents" --- p.14 / Chapter 2.3.2 --- Buffers and Solutions --- p.15 / Chapter 2.3.3 --- Cell Line --- p.16 / Chapter 2.3.4 --- Instruments and Equipments --- p.16 / Chapter 2.3.5 --- Enzymes --- p.16 / Chapter 2.3.6 --- Nucleic Acids --- p.17 / Chapter 2.3.7 --- Oligo Primers --- p.17 / Chapter 2.4 --- Methods --- p.17 / Chapter 2.4.1 --- In vitro Bioassay for Total Hemoglobin Production --- p.17 / Chapter (a) --- Preparation of Treatment Cell Culture Plates --- p.17 / Chapter (b) --- Treatment of K562 Cells by LC978 --- p.18 / Chapter (c) --- Signal Development --- p.18 / Chapter 2.4.2 --- Detection of Fetal Hemoglobin Production by HbF Sandwich ELISA --- p.18 / Chapter (a) --- Treatment of K562 Cells by LC978 --- p.18 / Chapter (b) --- Preparation of Capture Antibody-Coated and BSA-Blocked ELISA Plate --- p.19 / Chapter (c) --- Preparation of K562 Cell Lysates --- p.19 / Chapter (d) --- Antigen Capture and Signal Development --- p.19 / Chapter 2.4.3 --- Detection of Gamma Globin mRNA Level by Real-time RT-PCR --- p.20 / Chapter (a) --- Treatment of K562 Cells by LC978 --- p.20 / Chapter (b) --- Preparation of K562 Cell Lysate in Guanidium Thiocyanate (GT) Solution --- p.20 / Chapter (c) --- Isolation of Total RNA from LC978-treated K562 Cells --- p.21 / Chapter (d) --- cDNA Synthesis from mRNA by Reverse Transcriptase (RT) --- p.22 / Chapter (e) --- Real-Time Quantitative Polymerase Chain Reaction (PCR) --- p.23 / Chapter 2.5 --- Results --- p.24 / Chapter (a) --- In vitro Bioassay for Total Hemoglobin Production --- p.24 / Chapter (b) --- Fetal Hemoglobin Sandwich ELISA --- p.24 / Chapter (c) --- LC978-Induced Gamma Globin mRNA Accumulation --- p.25 / Chapter 2.6 --- Discussion --- p.31 / Chapter Chapter 3: --- Construction of Promoter-Reporter Plasmid Constructs --- p.33 / Chapter 3.1 --- The Human Gamma Globin Gene Promoter --- p.33 / Chapter 3.2 --- SEAP as a Reporter Gene for Promoter Deletion Study --- p.34 / Chapter 3.3 --- Materials --- p.35 / Chapter 3.3.1 --- "Chemicals, Kits and Reagents" --- p.35 / Chapter 3.3.2 --- Buffers and Solutions --- p.35 / Chapter 3.3.3 --- Bacterial Strain --- p.36 / Chapter 3.3.4 --- Cell Line --- p.36 / Chapter 3.3.5 --- Enzymes --- p.37 / Chapter 3.3.6 --- Nucleic Acids --- p.37 / Chapter 3.3.7 --- Oligo Primers --- p.37 / Chapter 3.4 --- Methods --- p.38 / Chapter 3.4.1 --- Molecular Cloning of A-Gamma Globin Gene Promoter and 3' Enhancer into pBlueScript II SK (-) --- p.38 / Chapter (a) --- Design and Synthesis of Oligo Primers --- p.38 / Chapter (b) --- Isolation of Genomic DNA from K562 Cells --- p.39 / Chapter (c) --- PCR Amplification of Gamma Globin Gene Promoter and 3' Enhancer --- p.40 / Chapter (d) --- Ligation of PCR Fragments into EcoR V-cut pBlueScript II SK (-) --- p.41 / Chapter (e) --- Preparation of E coli DH5a Competent Cells --- p.43 / Chapter (f) --- Heat-Shock Transformation of E. coli DH5a Competent Cells --- p.44 / Chapter (g) --- PCR Screening and Plasmid Purification of Putative pBlu2SKM-γAP and pBlu2SKM-γAE --- p.44 / Chapter (h) --- Isolation of Putative pBlu2SKM-γAP and pBlu2SKM-γAE Plasmid DNA --- p.45 / Chapter (j) --- Nucleotide Sequencing of Putative pBlu2SKM-yAP and pBlu2SKM-γAE --- p.47 / Chapter (j) --- Graphical Summary of Section 3.6.1 Sub-cloning Procedures --- p.49 / Chapter 3.4.2 --- Molecular Cloning of A-Gamma Globin Gene Promoter and 3' Enhancer into pSEAP2-Enhancer --- p.51 / Chapter (a) --- Sub-cloning of Promoter Fragment into pSEAP2-Enhancer --- p.51 / Chapter (b) --- Sub-cloning of 3' Enhancer Fragment into p 1224γAP-SEAP2 --- p.52 / Chapter (c) --- Graphical Summary of Section 3.6.2 Sub-cloning Procedures --- p.54 / Chapter 3.4.3 --- Construction of p 1224γAP-SEAP2-γAE Promoter Deletions Constructs --- p.56 / Chapter (a) --- Restriction Digestion at 5' End of A-Gamma Promoter Deletions --- p.56 / Chapter (b) --- Restriction Digestion at 3' Ends of A-Gamma Promoter Deletions --- p.56 / Chapter (c) --- Blunting 5'-overhangs and Self-Ligation of Linearized Plasmid Constructs --- p.57 / Chapter (d) --- Graphical Summary of Section 3.6.3 5，Deletions Procedures --- p.58 / Chapter 3.5 --- Results --- p.59 / Chapter (a) --- Nucleotide Sequence Confirmed by Cycle Sequencing --- p.60 / Chapter (b) --- "Resulting Plasmid Constructs p 1224γAP-SEAP2-yAE, p754yAP-SEAP2-yAE and p205yAP-SEAP2-γAE" --- p.64 / Chapter 3.6 --- Discussion --- p.67 / Chapter Chapter 4: --- Mapping of LC978-Responsive Elements on Human A-Gamma Globin Gene Promoter --- p.69 / Chapter 4.1 --- Introduction --- p.69 / Chapter 4.2 --- pSV-β-Galactosidase as a Transfection Normalization Standard --- p.69 / Chapter 4.3 --- pSV-β-Galactosidase as a Transfection Normalization Standard --- p.70 / Chapter 4.4 --- Materials --- p.72 / Chapter 4.4.1 --- "Chemicals, Kits and Reagents" --- p.72 / Chapter 4.4.2 --- Buffers and Solutions --- p.73 / Chapter 4.4.3 --- Cell Line --- p.74 / Chapter 4.4.4 --- Nucleic Acids --- p.74 / Chapter 4.4.5 --- Instruments and Equipments --- p.74 / Chapter 4.5 --- Methods --- p.74 / Chapter 4.5.1 --- Determination of Optimal Dose of Transfection Reagent for --- p.74 / Chapter (a) --- Sterilization of Plasmid DNA for Transfection --- p.74 / Chapter (b) --- Transient Transfection of K562 Cells by pEGFP-N 1 --- p.75 / Chapter (c) --- Examination of EGFP Expression Level --- p.76 / Chapter 4.5.2 --- β-Galactosidase as Normalization Standard for K562 Transfections --- p.76 / Chapter (a) --- Transient Transfection of K562 Cells by pSV-β-Gal --- p.76 / Chapter (b) --- Determination of β-Galactosidase Expression Level --- p.76 / Chapter 4.5.3 --- Mapping of LC978-Responsive Elements on Human Gamma Globin Gene Promoter --- p.77 / Chapter (a) --- Co-Transfection of K562 Cells by p1224/754/205γAP-SEAP2 -γAE and pSV-β-Gal --- p.77 / Chapter (b) --- Treatment of Co-Transfected K562 Cells by LC978 --- p.77 / Chapter (c) --- Determination of β-Galactosidase Expression Level --- p.78 / Chapter (d) --- Determination of Secreted Alkaline Phosphatase (SEAP) Expression Level --- p.78 / Chapter (e) --- Determination of Fetal Hemoglobin Expression Level --- p.79 / Chapter 4.5.4 --- Mapping of Hydroxyurea-Responsive Elements on Human Gammm Globin Gene Promoter --- p.80 / Chapter (a) --- Determination of Optimal Biological Dose (OBD) of Hydroxyurea --- p.80 / Chapter (b) --- Co-Transfection of K562 Cells and Subsequent Treatment by Hydroxyurea --- p.80 / Chapter (c) --- "Assay for β-Galactosidase (β-Gal), Secreted Alkaline Phosphatase (SEAP) and Fetal Hemoglobin (HbF) Expression Level" --- p.81 / Chapter 4.5.5 --- Sodium Butyrate-Induced SEAP Expression --- p.81 / Chapter (a) --- Determination of Optimal Biological Dose (OB(d) of Sodium Butyrate --- p.81 / Chapter (b) --- Co-Transfection of K562 Cells and Treatment by Sodium Butyrate --- p.82 / Chapter (c) --- "Assay for p-Galactosidase (β-Gal), Secreted Alkaline Phosphatase (SEAP) and Fetal Hemoglobin (HbF) Expression Level" --- p.83 / Chapter 4.5.6 --- Data Analysis --- p.83 / Chapter (a) --- "Data Processing, Normalization and Graphing" --- p.83 / Chapter (b) --- Statistical Analysis --- p.83 / Chapter 4.6 --- Results --- p.84 / Chapter 4.6.1 --- Optimal Dose of Transfection Reagent for K562 --- p.84 / Chapter 4.6.2 --- β-Galactosidase as Normalization Standard for K562 Transfections --- p.84 / Chapter 4.6.3 --- LC978 Induction on Co-Transfected K562 Cells --- p.84 / Chapter 4.6.4 --- Hydroxyurea Induction on Co-Transfected K562 Cells --- p.85 / Chapter 4.6.5 --- Sodium Butyrate Induction on Co-Transfected K562 Cells --- p.86 / Chapter 4.7 --- Discussion --- p.98 / Chapter 4.7.1 --- Theme Question to be Answered --- p.98 / Chapter 4.7.2 --- Optimal Dose of DMRIE-C Transfection Reagent on K562 Cell Line --- p.98 / Chapter 4.7.3 --- pSV-β-gal as an Internal Normalization Control --- p.99 / Chapter 4.7.4 --- Responsive Element Mapping --- p.99 / Chapter (a) --- LC978-Induced Response --- p.100 / Chapter (b) --- Hydroxyurea-Induced Response --- p.100 / Chapter (c) --- Sodium Butyrate-Induced Response --- p.101 / Chapter 4.7.5 --- LCR-Dependent Gamma Globin Gene Reactivation --- p.101 / Chapter 4.7.6 --- Induction of Gamma Globin by Histone Deacetylase Inhibitor --- p.104 / Chapter 4.7.7 --- Basal SEAP Expression Levels of the Promoter-Reporter Constructs --- p.105 / Chapter 4.7.8 --- Summary --- p.105 / Chapter Chapter 5: --- General Discussion --- p.106 / References Cited --- p.109