Sickle cell anemia is caused by a single mutation in the β-hemoglobin gene, HBB. The disease originated in Africa and affects millions of people worldwide. Sickle hemoglobin tetramers polymerize upon deoxygenation and lead to hemolysis and vaso-occlusion. Patients with high fetal hemoglobin (HbF) can have milder disease. The only FDA-approved drug is hydroxyurea that increases HbF. HbF modulates the disease by preventing the polymerization of sickle hemoglobin and reduces the pain episodes, anemia, and organ damage associated with the disease. There are five common haplotypes associated with the HbS gene and that are very loosely associated with disease severity and HbF. Understanding the genetic bases of HbF regulation is a key factor to identify potential drug targets to induce HbF for therapeutic purposes. To fully understand the mechanism behind HbF regulation, developing a fast and accurate computational method for sickle cell haplotype classification is useful for examining the variability of HbF among sickle cell patients. Moreover, investigating the cis and trans-acting regulators of HbF gene expression to pinpoint the mechanism through which they regulate HbF is essential to develop a successful treatment. The availability of high-throughput genetic data provides an excellent opportunity to study HbF regulation in sickle cell patients and normal people comprehensively. The work reported in this thesis describes a fast and accurate method for sickle cell HBB haplotype classification. I also examine the differential effect of cis and trans-acting HbF hemoglobin regulators on -globin gene expression using the GTEx database and identify BCL2L1 as a new potential trans-regulator of HbF.

Abstract Introduction Sickle cell trait carriers may experience sickling events, which can cause severe health problems. Some sickle cell haplotypes contain genetic modifiers that are associated with increased levels of fetal hemoglobin, which is resistant to sickling. The aim of this study is to determine if sickle cell trait individuals who do not carry these modifiers are more likely to experience sickling episodes than those who do carry the modifiers. Methods: Participants were eligible for inclusion in this study if they were male, 18 years of age or older, a sickle cell trait carrier, and had previously played any level of organized football. Participants were recruited via Facebook, www.clinicaltrials.gov, e-mail, phone calls, and word of mouth. They were asked to complete a survey and return a buccal swab for genetic analysis to look for alleles associated with fetal hemoglobin persistence. To date, no genetic analyses have been run. Data from the surveys was analyzed using Fisher's Exact Test with the SAS 9.2 software. Results: Twenty participants were included in this phase of the study and all returned both the survey and buccal swab. Five of the 20 participants had been diagnosed with exertional sickling, 2 with heat illness, and 12 had experienced dehydration. Conclusion: Data in this study is purely observational, as no genetic analyses have been performed at this point. Early results indicate that the probability a player feels their muscle pain lasts longer than their peers' is greater among those who feel it takes their muscles longer to recover than their peers'.

Hemoglobin is an oxygen binding protein in erythrocytes. Hemoglobin is composed of four polypeptide chains. During the fetal stage the type of hemoglobin called fetal hemoglobin (HbF) dominates. After birth HbF is replaced by adult hemoglobin (HbA). HbF persists in concentrations less than 1%. Elevated concentration of HbF in adults exists in different conditions, Talassemi for example. When the uterus is damaged and the fetus doesn’t feel well its blood can pass the placenta barrier and enter the blood stream of the mother. A venous blood sample from the mother is analyzed to determine the status of the fetus. Laboratory Medicine Västernorrland already has two methods for analyzing HbF, one routine and one on call. The routine method needed to be replaced and the possibility to use flow cytometry was investigated. In this study, results from flow cytometry using Fetal Cell Count™ kit was compared to the results from the presently used methods, Kleihauer-Betke and HPLC. Cord blood was diluted with venous blood from an adult with the same blood group in various concentrations. A number of tests were performed and showed a fairly good correlation between the different methods. However more tests will be necessary to draw any clear conclusion.

Sickle cell disease (SCD) is a severe hemoglobin disorder caused by co-inheritance of a single mutation in the β-globin gene of adult hemoglobin (HbA; α2β2). This alteration leads to the formation of sickle hemoglobin (HbS; α2βS2) and deformed, sickle-shaped red blood cells (RBCs). Sickle RBCs obstruct small blood vessels resulting in anemia, excruciating pain crises, organ damage, and stroke. For the millions of people affected by this disease, life expectancy is only 40-60 years of age. The only cure for SCD is hematopoietic stem cell (HSC, CD34+) transplantation, which requires a human leukocyte antigen (HLA)-matched donor. However, this option runs the risk of complications associated with graft versus host disease and infection. Before birth, individuals with SCD do well because their RBCs are filled with γ-globin containing fetal hemoglobin (HbF; α2γ2), which inhibits the formation of HbS. In fact, some SCD patients who co-inherit mutations that allow for high-level expression of HbF into adulthood are asymptomatic. This suggests that genetic modification of the patient’s own HSCs to permit HbF production would be a viable therapeutic alternative to HSC transplantation. Our work has focused on the use of lentiviral vectors to introduce an exogenous γ-globin gene or shRNA sequences designed to knockdown repressors of γ-globin, such as the zinc-finger transcription factor, BCL11A, to prevent silencing of the endogenous γ-globin genes allowing for persistent expression of HbF. Despite significant progress using both approaches, we have been unable to increase the level of HbF > 30%; a curative threshold for SCD patients who continue to produce HbF into adulthood. The goal of my project was to combine these approaches into a single lentiviral vector to achieve co-regulated, erythroid-specific expression and augmented levels of HbF. I successfully modified the insulated, erythroid-specific γ-globin vector (termed V5m3-400) to include microRNA (miR)-adapted shRNAs (or shmiRs) targeting BCL11A (based on miR-30 and miR-E architectures) in the first and second noncoding introns of the γ-globin genomic sequences. Inclusion of shmiRs had no appreciable effect on integrity of the integrated provirus or vector titer. Vector performance was initially tested using human K562 erythroleukemia cells expressing a flag-tagged version of BCL11A. In this cell line, BCL11A knockdown was significantly improved using miR-E-shRNAs due to a dramatic increase (up to 350-fold) in processing of mature shRNA sequences. The miR-E vectors also provided high-level expression of γ-globin. Erythroid-specific expression of the γ-globin transgene and BCL11A knockdown was confirmed in maturing erythroid cells derived from transduced CD34+ cells of a healthy donor resulting in a 50% increase in HbF levels compared with cells transduced with V5m3-400 as a control. While encouraging, I was unable to discriminate HbF derived from the vector-encoded versus endogenous γ-globin genes. To address this, I introduced a single base change in exon 2 of the γ-globin gene encoded by V5m3-400 such that threonine replaces isoleucine at amino acid 75 (I75T). This variant was successfully distinguished from endogenous γ-globin gene products by reverse phase high performance liquid chromatography (HPLC) in culture-differentiated erythroid cells. Based on these findings, I created compound γ-globin/shmiR-E vectors that include the I75T substitution (I75Tγ-globin/shmiR-E). Future studies will focus on testing this novel vector design in erythroid cells derived from transduced CD34+ cells of healthy donors and patients with SCD. I anticipate that this compound vector has the potential to maximize γ-globin expression and promote levels of HbF that are unlikely to be safely and effectively achieved by conventional globin gene addition or shRNA knockdown approaches alone.

Sickle cell disease (SCD) is one of the most common hemoglobinopathies worldwide. It is caused by a homozygous mutation in codon 6 of the beta globin gene (HBB), which leads to polymerization of the variant hemoglobin and sickled red blood cells that obstruct blood vessels and reduce oxygen delivery to tissues. Patients with SCD have multiple clinical problems, including pain crises, anemia and organ damage. However, not all patients with SCD display all these clinical manifestations. One major factor for reduced occurrences of symptoms is fetal hemoglobin (HbF). HbF is the main hemoglobin in the fetus, and declines one year after birth to less than one percent of total hemoglobin. Nevertheless, there are individuals who continue to have high levels of HbF into adulthood, which is beneficial for an individual with SCD because HbF reduces the amount of sickle polymer in red blood cells. There are three major quantitative trait loci (QTL) associated with high HbF. However, these QTL account for 20-45% of HbF variance. Therefore, further investigation is required to fully understand how HbF is regulated. The HBS1L-MYB intergenic polymorphism (HMIP) on chromosome 6q23 is one of the major QTL associated with high HbF. This region is also known to regulate other erythroid-specific traits due to an enhancer element that promotes the expression of the downstream gene, MYB, which controls hemoglobin expression and erythroid proliferation and maturation. The presence of RNA polymerase II binding and a 50-bp transcript suggested that a long noncoding RNA (lncRNA) is transcribed from this region. LncRNAs are non-protein-coding transcripts greater than 200 nucleotides and are involved in gene regulation. Therefore, it was hypothesized that a lncRNA is transcribed from the enhancer of MYB and regulates hemoglobin expression. I characterized a novel lncRNA, 1283 bp in length that was differentially expressed among various tissue types, among erythroid progenitor cells with different hemoglobin makeup, and also during erythroid differentiation. Furthermore, knockdown of this lncRNA, named the HBS1L-MYB intergenic long noncoding RNA (HMI-LNCRNA), significantly increased HbF. Taken together, these observations suggest that HMI-LNCRNA can be a possible therapeutic target to increase HbF expression in patients with SCD and β-thalassemia. / 2018-05-01T00:00:00Z

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

Choi Wai-wah. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 120-138). / Abstracts in English and Chinese. / Statement --- p.i / Acknowledgements --- p.ii / Abstract --- p.iii / Abstract (Chinese Version) --- p.v / Table of Contents --- p.vii / List of Tables --- p.xii / List of Figures --- p.xiii / List of Abbreviations --- p.xv / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- "Hemoglobin ´ؤ Structures, Types and Functions" --- p.1 / Chapter 1.1.1 --- Structures of Hemoglobin --- p.1 / Chapter 1.1.2 --- Types of Hemoglobin --- p.2 / Chapter 1.1.3 --- Functions of Hemoglobin --- p.3 / Chapter 1.2 --- Human Globin Genes and Their Regulation --- p.5 / Chapter 1.2.1 --- Organization of the Human Globin Genes --- p.5 / Chapter 1.2.2 --- Regulation of Globin Gene Expression --- p.6 / Chapter 1.2.2.1 --- The Locus Control Region (LCR) --- p.6 / Chapter 1.2.2.2 --- Cis-Regulatory Elements --- p.7 / Chapter 1.2.2.2.1 --- Promoters --- p.7 / Chapter 1.2.2.2.2 --- Enhancers --- p.7 / Chapter 1.2.2.2.3 --- Silencers --- p.8 / Chapter 1.2.2.3 --- Trans-Acting Factors --- p.8 / Chapter 1.2.2.3.1 --- GATA Family --- p.9 / Chapter 1.2.2.3.2 --- Kruppel-like Factors --- p.9 / Chapter 1.2.2.3.3 --- Nuclear Factor-Erythroid (NF-E) --- p.9 / Chapter 1.2.2.4 --- Chromatin Remodelling --- p.10 / Chapter 1.2.2.5 --- Intergenic Sequences --- p.11 / Chapter 1.3 --- Mechanisms of Hemoglobin Switching --- p.12 / Chapter 1.3.1 --- Autonomous Silencing --- p.12 / Chapter 1.3.2 --- LCR and Globin Gene Interaction --- p.12 / Chapter 1.4 --- Hemoglobinopathies --- p.14 / Chapter 1.4.1 --- α -thalassemia --- p.14 / Chapter 1.4.2 --- β -thalassemia --- p.14 / Chapter 1.4.3 --- Sickle Cell Anemia --- p.16 / Chapter 1.5 --- Therapies for β-thalassemia --- p.16 / Chapter 1.5.1 --- Blood Transfusion --- p.16 / Chapter 1.5.2 --- Bone Marrow Transplantation --- p.17 / Chapter 1.5.3. --- Gene Therapy --- p.17 / Chapter 1.6 --- Gene Switch Therapy --- p.18 / Chapter "1.6,1" --- Pharmacological Induction of HbF --- p.18 / Chapter 1.6.1.1 --- Hydroxyurea --- p.19 / Chapter 1.6.1.2 --- Butyrate --- p.20 / Chapter 1.6.1.3 --- Summary --- p.21 / Chapter 1.7 --- Objectives --- p.22 / Chapter Chapter 2 --- Induction of HbF by LC978 in K562 / Chapter 2.1 --- Introduction --- p.23 / Chapter 2.2 --- Materials --- p.26 / Chapter 2.2.1 --- Chemicals and Reagents --- p.26 / Chapter 2.2.2 --- Kits --- p.27 / Chapter 2.2.3 --- Buffers and Solutions --- p.27 / Chapter 2.2.4 --- Primers --- p.30 / Chapter 2.2.5 --- Equipment and Other Consumables --- p.30 / Chapter 2.2.6 --- Maintenance of K562 --- p.31 / Chapter 2.2.7 --- Handling and Treatment of utilities for RNA isolation --- p.31 / Chapter 2.3 --- Methods --- p.32 / Chapter 2.3.1 --- Dose-response and time-response study of LC978 in K562 by TMB assay --- p.32 / Chapter 2.3.2 --- Detection of γ -Globin Gene Expression in LC978-induced K562 by RT-PCR --- p.33 / Chapter 2.3.3 --- Fetal Hemoglobin Analysis by Human Fetal Hemoglobin (HbF) ELISA Quantitation Kit --- p.36 / Chapter 2.3.4 --- Statistical Analysis --- p.38 / Chapter 2.4 --- Results --- p.39 / Chapter 2.4.1 --- Dose-response and time-response study of LC978 in K562 by TMB assay --- p.39 / Chapter 2.4.2 --- Detection of γ -Globin Gene Expression in LC978-induced K562 by RT-PCR --- p.45 / Chapter 2.4.3 --- Fetal Hemoglobin Analysis by Human Fetal Hemoglobin (HbF) ELISA Quantitation Kit --- p.48 / Chapter 2.5 --- Discussions --- p.51 / Chapter Chapter 3 --- Signal Transduction Pathways Modulated by LC978 / Chapter 3.1 --- Introduction --- p.54 / Chapter 3.2 --- Materials --- p.57 / Chapter 3.2.1 --- Chemicals and Reagents --- p.57 / Chapter 3.2.2 --- Kits --- p.57 / Chapter 3.2.3 --- Buffers and Solutions --- p.58 / Chapter 3.2.4 --- Primers --- p.59 / Chapter 3.2.5 --- Equipment and Other Consumables --- p.60 / Chapter 3.2.6 --- Maintenance of K562 --- p.60 / Chapter 3.2.7 --- Handling and Treatment of utilities for RNA isolation --- p.60 / Chapter 3.3 --- Methods --- p.61 / Chapter 3.3.1 --- Identification of Signaling Pathways by Microarray --- p.61 / Chapter 3.3.2 --- Real-time RT-PCR --- p.65 / Chapter 3.4 --- Results --- p.67 / Chapter 3.4.1 --- Identification of Signaling Pathways by Microarray --- p.67 / Chapter 3.4.2 --- Real-time RT-PCR --- p.74 / Chapter 3.5 --- Discussions --- p.80 / Chapter Chapter 4 --- MAPK pathways and HbF induction by LC978 / Chapter 4.1 --- Introduction --- p.84 / Chapter 4.2 --- Materials --- p.87 / Chapter 4.2.1 --- Chemicals and Reagents --- p.87 / Chapter 4.2.2 --- Kits --- p.88 / Chapter 4.2.3 --- Buffers and Solutions --- p.88 / Chapter 4.2.4 --- Equipment and Other Consumables --- p.90 / Chapter 4.2.5 --- Maintenance of K562 --- p.90 / Chapter 4.3 --- Methods --- p.91 / Chapter 4.3.1 --- "Roles of three MAPKs ´ؤ ERK, JNK and p38 in LC978-mediated γ -globin gene induction in K562 using CASE´ёØ Kits" --- p.91 / Chapter 4.3.2 --- Effect of p38 inhibitor SB203580 on HbF induction --- p.94 / Chapter 4.3.3 --- Statistical Analysis --- p.97 / Chapter 4.4 --- Results --- p.98 / Chapter 4.4.1 --- "Roles of three MAPKs - ERK, JNK and p38 in LC978-mediated γ -globin gene induction in K562 using CASETM Kits" --- p.98 / Chapter 4.4.2 --- Effect of p38 inhibitor SB203580 on HbF induction --- p.106 / Chapter 4.5 --- Discussions --- p.110 / Chapter Chapter 5 --- Summary and Prospects / Appendix / References

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A anemia falciforme (AF) à uma hemoglobinopatia hereditÃria autossÃmica causada por uma mutaÃÃo pontual no gene da beta globina gerando uma hemoglobina anormal denominada de hemoglobina S (HbS), em homozigose. A doenÃa se caracteriza por apresentar uma variabilidade do quadro clÃnico, que se deve à mÃltiplos fatores, dentre eles a concentraÃÃo de hemoglobina fetal (HbF), os haplÃtipos do gene da beta globina e os polimorfismos do gene BCL11A, entre outros. A avaliaÃÃo dos moduladores genÃticos na AF tem sido desenvolvida com a finalidade de melhorar o entendimento da sua fisiopatologia e direcionar a abordagem terapÃutica objetivando sua individualizaÃÃo. A pesquisa se propÃs a determinar a modulaÃÃo genÃtica dos polimorfismos do gene BCL11A (rs4671393, rs7557939 e rs1186868) sobre o perfil inflamatÃrio, hemolÃtico, no estresse oxidativo e nas concentraÃÃes das HbF, HbS nos pacientes portadores de AF, em estado estacionÃrio. O estudo foi do tipo transversal e analÃtico com 42 pacientes adultos, em acompanhamento ambulatorial no Hospital UniversitÃrio Walter CantÃdio (HUWC), com diagnÃstico molecular e haplÃtipos do gene da beta globina S previamente realizados. Os pacientes estavam em uso de HidroxiurÃia (HU), em mÃdia, 20mg/kg de peso corporal. Amostras biolÃgicas de sangue perifÃrico foram obtidas para a realizaÃÃo dos exames laboratoriais: as dosagens das citocinas prà inflamatÃrias IL-6, IL-17, TNF-alpha e das antiinflamatÃrias IL-10 e TGF-beta, por Elisa; contagem de reticulÃcitos por metodologia manual, dosagem de metemoglobina (MetHb) e lactato desidrogenase (LDH), por espectrofotometria; do nitrito (NOx), malonaldeÃdo (MDA) sÃricos, as enzimas antioxidantes eritrocitÃrias, catalase (CAT) e da glutationa peroxidase (GPx) por kits e espectrofotometria. Os polimorfismos genÃticos do gene BCL11A nas regiÃes, rs4671393, rs7557939 e rs1186868 foram determinados por Real Time PCR. As dosagens da HbF e HbS foram realizadas por HPLC (High Performance Liquid Chromatography). Os dados idade, sexo e eventos clÃnicos foram obtidos dos prontuÃrios. Toda a anÃlise estatÃstica foi realizada usando o software livre R, na versÃo 3.1.2. Para anÃlise da frequÃncia do sexo e dos genÃtipos, por regiÃo e das associaÃÃes entre o tipo de haplÃtipo e dos eventos clÃnicos com as regiÃes do BCL11A, foram usados os testes de Qui-quadrado e o exato de Fisher. Realizou-se o teste paramÃtrico de ANOVA (obtido sob suposiÃÃes distribucionais), bem como o teste nÃo-paramÃtrico de Kruskal-Wallis para a anÃlise da associaÃÃo dos genÃtipos do gene BCL11A com a idade, os nÃveis de HbS, HbF, perfil inflamatÃrio, hemolÃtico e do estresse oxidativo. Foi considerado significante ao nÃvel de 5%. A maioria dos pacientes (57,14%) era do sexo feminino. A idade dos pacientes incluÃdos foi de 18 a 65 anos, com valor mÃdio e mediano de 35,1 e 33 anos, respectivamente. Somente a rs7557939 do BCL11A, o genÃtipo A/G foi o mais prevalente e a prevalÃncia do genÃtipo A/G foi maior nas mulheres , enquanto nos homens a prevalÃncia maior foi do genÃtipo A/A. No entanto, a rs1186868 do BCL11A, a maioria (56,52%) das mulheres apresentaram o genÃtipo C/T e a metade dos homens apresentaram o genÃtipo T/T. Nenhuma regiÃo do gene BCL11A apresentou associaÃÃo significativa com os haplÃtipos do gene da beta globina S. Em relaÃÃo a moduÃÃo do gene BCL11A com os nÃveis de HbS e HbF, verificou-se que na rs1186868 houve resultado significativo do genÃtipo mutante T/T, que apresentou maiores nÃveis de HbS e menores nÃveis de HbF. Na rs7557939 houve uma diminuiÃÃo significante de HbF no alelo mutante A/A, porÃm, nÃo houve relaÃÃo com a HbS. NÃo houve associaÃÃo entre os SNPs, nas trÃs regiÃes estudadas, com relaÃÃo ao nÃmero mÃdio/mediano dos moduladores inflamatÃrios, marcadores de hemÃlise, do estresse oxidativo e dos eventos clÃnicos, ao nÃvel de 5%.Os achados reforÃam a hipÃtese da moduÃÃo genÃtica dos polimorfismos do gene BCL11A em relaÃÃo aos nÃveis de HbF, onde os alelos selvagens, nas regiÃes rs7557939 e rs1186868 apresentaram um carÃter protetor no prognÃstico em decorÃncia de terem apresentado aumento dos nÃveis de HbF, nos pacientes com AF do estudo.

Zhang, Siwei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 87-98). / Abstracts in English and Chinese. / Chapter 1. --- General introduction --- p.1 / Chapter 1.1. --- "Types, structure and function of human hemoglobin" --- p.1 / Chapter 1.1.1. --- Structure and functions of human hemoglobin --- p.1 / Chapter 1.1.2. --- Types of human hemoglobin --- p.2 / Chapter 1.2. --- Regulatory mechanism of human hemoglobin expression --- p.3 / Chapter 1.2.1. --- The human a and β locus --- p.3 / Chapter 1.2.2. --- Development of globin genes switching concept --- p.4 / Chapter 1.2.3. --- Factors that regulate globin gene expression --- p.5 / Chapter 1.2.3.1. --- The locus control region (LCR) --- p.5 / Chapter 1.2.3.2. --- The cis-regulatory elements --- p.5 / Chapter 1.2.3.3. --- The trans-acting factors --- p.6 / Chapter 1.3. --- The human hemoglobinopathies --- p.8 / Chapter 1.3.1. --- α-thalassemia --- p.8 / Chapter 1.3.2. --- β-thalassemia --- p.9 / Chapter 1.3.3. --- Sickle cell anemia --- p.10 / Chapter 1.4. --- Current approaches towards β-thalassemia treatment --- p.11 / Chapter 1.4.1. --- Blood transfusion --- p.11 / Chapter 1.4.2. --- Bone marrow transplantation --- p.12 / Chapter 1.4.3. --- Drug-induced activation of fetal hemoglobin production --- p.12 / Chapter 1.4.3.1. --- Hydroxyurea --- p.12 / Chapter 1.4.3.2. --- Butyrate and short-chain fatty acids --- p.13 / Chapter 1.4.3.3. --- "Mutagens, DNA methyltransferase inhibitors and other HbF inducible agents" --- p.13 / Chapter 1.4.3.4. --- Cucurbitacin D --- p.14 / Chapter 1.4.4. --- Gene therapy --- p.14 / Chapter 1.5. --- Research Objectives --- p.15 / Chapter 2. --- "Analysis of CuD, Hydroxyurea and other inducers on the induction of α, β, γ, δ， ε，ζ BP-1 genes and fetal hemoglobin induction" --- p.16 / Chapter 2.1. --- Introduction --- p.16 / Chapter 2.1.1. --- Properties of human K562 cell line --- p.16 / Chapter 2.1.2. --- Induction and measurement of fetal hemoglobin --- p.16 / Chapter 2.1.3. --- "Induction of α, β, γ, δ, ε , ζ and BP-1 gene and Real-time RT-PCR analysis" --- p.17 / Chapter 2.2. --- Materials --- p.18 / Chapter 2.2.1. --- Chemicals and reagents --- p.18 / Chapter 2.2.2. --- Kits --- p.19 / Chapter 2.2.3. --- Buffers and solutions --- p.19 / Chapter 2.2.4. --- Cell lines --- p.20 / Chapter 2.3. --- Experimental procedures --- p.20 / Chapter 2.3.1. --- Hemoglobin quantity measurement by HbF ELISA --- p.20 / Chapter 2.3.1.1. --- MTT assay --- p.21 / Chapter 2.3.1.2. --- Preparation of capture-antibody coated ELISA plates --- p.21 / Chapter 2.3.1.3. --- Plate blocking --- p.22 / Chapter 2.3.1.4. --- Sample and standard preparation --- p.22 / Chapter 2.3.1.5. --- HRP antibody and colorimetric detection --- p.23 / Chapter 2.3.1.6. --- Statistical analysis --- p.23 / Chapter 2.3.2. --- Preparation of mRNA extract from K562 cells --- p.23 / Chapter 2.3.3. --- Reverse transcription and Real-time PCR analysis --- p.24 / Chapter 2.4. --- Results --- p.25 / Chapter 2.4.1. --- CuD significantly upregulates HbF expression in K562 cells --- p.25 / Chapter 2.4.2. --- "CuD augments α, β, γ, δ, ε , ζ and BP-1 genes at different level in K562 cells" --- p.28 / Chapter 2.4.3. --- Cucurbitacin D-induced γ-globin gene activation requires12-24 hours in K562 cells --- p.31 / Chapter 2.5. --- Discussion --- p.33 / Chapter 2.5.1. --- Enhancement of fetal hemoglobin production using different chemical compounds --- p.33 / Chapter 2.5.2. --- CuD increased HbF synthesis by increasing γ-globin mRNA amount --- p.35 / Chapter 2.5.3. --- CuD and HU down-regulated the BP-1 gene expression --- p.36 / Chapter 3. --- Determination of potential signal transduction pathways during CuD and HU-mediated fetal hemoglobin production --- p.36 / Chapter 3.1. --- Introductions --- p.36 / Chapter 3.1.1. --- The p38 MAPK family --- p.37 / Chapter 3.1.2. --- The JAK2-STAT3 pathway --- p.38 / Chapter 3.1.3. --- Fundamentals on inhibition assay of p38 MAPK and JAK2-STAT3 pathway --- p.39 / Chapter 3.1.4. --- Fundamentals on nuclear translocation of STAT3 --- p.41 / Chapter 3.2. --- Materials --- p.41 / Chapter 3.2.1. --- Chemicals and reagents --- p.41 / Chapter 3.2.2. --- Kits --- p.44 / Chapter 3.2.3. --- Buffers and solutions --- p.44 / Chapter 3.3. --- Experimental procedures --- p.45 / Chapter 3.3.1. --- Detection of p3 8 MAPK phosphorylation status --- p.46 / Chapter 3.3.1.1. --- Preparation of cytosolic protein extracts --- p.46 / Chapter 3.3.1.2. --- Quantitative measurement of phospho-p38 and pan-p38 by ELIS A method --- p.46 / Chapter 3.3.1.2.1. --- Antigen adsorption and establishment of standard curves --- p.46 / Chapter 3.3.1.2.2. --- Plate washing and application of detection antibody --- p.47 / Chapter 3.3.1.2.3. --- Plate washing and application of secondary antibody --- p.47 / Chapter 3.3.1.2.4. --- Plate washing and chromogen detection --- p.48 / Chapter 3.3.2. --- Detection of signal cascade on JAK2-STAT3 pathway --- p.48 / Chapter 3.3.2.1. --- Preparation of cytosolic protein extracts for Western Blot detection --- p.48 / Chapter 3.3.2.2. --- Gel running and Western Blot detection --- p.48 / Chapter 3.3.3. --- Quantitative measurement of phospho-STAT3-Tyr705 using ELISA method --- p.50 / Chapter 3.3.3.1. --- Preparation of cytosolic protein extracts --- p.50 / Chapter 3.3.3.2. --- Reconstitution and Dilution of STAT3 [pY705] Standard --- p.50 / Chapter 3.3.3.3. --- Measurement of STAT3 [pY705] concentration in cell lysates --- p.51 / Chapter 3.3.4. --- Inhibitor assay of JAK2-STAT3 and p38 MAPK pathway --- p.52 / Chapter 3.3.4.1. --- Establishment of inhibitor assay --- p.52 / Chapter 3.3.4.2. --- HbF ELISA detection --- p.53 / Chapter 3.3.5. --- Detection of STAT3 nuclear translocation and DNA binding affinity --- p.53 / Chapter 3.3.5.1. --- Preparation of nuclear extract from K562 cells --- p.53 / Chapter 3.3.5.2. --- EMS A detection of transcriptional factors binding to γ-promoter region --- p.54 / Chapter 3.3.5.2.1. --- 3´ة end-labeling of EMS A probes --- p.54 / Chapter 3.3.5.2.2. --- Dot blotting for labeling efficiency estimation --- p.56 / Chapter 3.3.5.2.3. --- EMSA binding reaction and non-denaturing gel electrophoresis --- p.57 / Chapter 3.3.5.2.4. --- Membrane development and chemiluminescence detection --- p.58 / Chapter 3.3.5.3. --- Preparation of K562 samples for immunofluorescence detection --- p.60 / Chapter 3.3.5.3.1. --- Slide coating for cell capture --- p.60 / Chapter 3.3.5.3.2. --- Preparation of cell slide --- p.60 / Chapter 3.3.5.3.3. --- Sample fixation and antibody probing treatment --- p.60 / Chapter 3.3.5.3.4. --- Sample imaging and immunofluorescence detection --- p.61 / Chapter 3.4 --- Results --- p.62 / Chapter 3.4.1. --- Activation of p38 MAPK pathway and STAT3 phosphorylation by hydroxyurea --- p.62 / Chapter 3.4.1.1. --- "The p38 MAPK pathway is activated by hydroxyurea, but not activated by Cucurbitacin D" --- p.62 / Chapter 3.4.1.2. --- Increased p38 phosphorylation level elicits STAT3 phosphorylation at Ser727 site --- p.64 / Chapter 3.4.2. --- Activation of JAK2 and STAT3 phosphorylation by Cucurbitacin D --- p.66 / Chapter 3.4.2.1. --- Cucurbitacin D promotes JAK2 activation --- p.66 / Chapter 3.4.2.2. --- Cucurbitacin D and hydroxyurea promote STAT3 phosphorylation at Tyr705 site --- p.66 / Chapter 3.4.3. --- Basal activity of signal transduction pathways is essential for HbF induction --- p.69 / Chapter 3.4.3.1. --- Activation of γ-globin gene requires presence of basal phosphorylation level of p38 MAPK --- p.69 / Chapter 3.4.3.2. --- Inhibition on JAK2-STAT3 pathway results in reduced fetal hemoglobin production --- p.71 / Chapter 3.4.4. --- Translocation and DNA binding of STAT under Cucurbitacin D induction --- p.72 / Chapter 3.4.4.1. --- Cucurbitacin D and hydroxyurea both enhance binding affinity of transcriptional factors to the Gγ/Aγ promoter --- p.72 / Chapter 3.4.4.2. --- Cucurbitacin D and hydroxyurea induces nuclear translocation of STAT3 --- p.75 / Chapter 3.5. --- Discussion --- p.77 / Chapter 3.5.1. --- The role of p38 MAPK activation during γ-globin gene activation --- p.77 / Chapter 3.5.2. --- STAT3 phosphorylation at Ser727 site promotes transcription factor activity and γ-globin gene expression --- p.77 / Chapter 3.5.3. --- The role of JAK2-STAT3 activation during γ-globin gene activation --- p.78 / Chapter 3.5.4. --- Inhibitor assay --- p.79 / Chapter 3.5.5. --- Relations between STAT3 nuclear translocation and enhanced fetal hemoglobin production --- p.82 / Chapter 4. --- Summery and Prospect --- p.83 / Chapter 5. --- References --- p.87

142f. / Submitted by Suelen Reis (suziy.ellen@gmail.com) on 2013-05-24T13:11:05Z No. of bitstreams: 1 Elisangela Adorno.pdf: 3379043 bytes, checksum: 972521d9aae8561da326946dd4da678a (MD5) / Approved for entry into archive by Flávia Ferreira(flaviaccf@yahoo.com.br) on 2013-05-30T00:18:31Z (GMT) No. of bitstreams: 1 Elisangela Adorno.pdf: 3379043 bytes, checksum: 972521d9aae8561da326946dd4da678a (MD5) / Made available in DSpace on 2013-05-30T00:18:31Z (GMT). No. of bitstreams: 1 Elisangela Adorno.pdf: 3379043 bytes, checksum: 972521d9aae8561da326946dd4da678a (MD5) Previous issue date: 2005 / Introdução. A hemoglobina S (HbS) resulta da troca de nucleotídeo (GAG GTG) no sexto codon do gene da globina beta, levando à substituição do ácido glutâmico por valina na cadeia da globina beta. A anemia falciforme ou a homozigose para a HbS, freqüentemente apresenta manifestações clínicas heterogêneas, fortemente relacionadas aos níveis de hemoglobina fetal (HbF). Objetivo. O presente estudo investigou as características fenopíticas e os marcadores moleculares presentes em portadores da anemia falciforme de Salvador-BA, identificando seqüências gênicas potencialmente importantes para a expressão dos genes gama. Métodos. O perfil de hemoglobinas e o nível de HbF foram determinados por cromatografia líquida de alta performance (HPLC). Informações sobre o perfil clínico dos pacientes foram obtidas através da análise de prontuários. A talassemia α2 3.7Kb foi investigada pela reação em cadeia da polimerase (PCR) e os haplótipos ligados ao grupo de genes da globina berta S foram investigados por PCR e análise de sítios polimórficos utilizando endonucleases de restrição (RFLP). As regiões promotoras dos genes yG e γA e o HS2-LCR foram amplificados assimetricamente e seqüenciadas no ABI Prism 3100 prism DNA Sequencer. As análises estatísticas foram desenvolvidas no software EPI-INFO versão 6.04 e a significância foi estabelecida para p < 0.05. Resultados. Foram analisados 131 pacientes, dos quais 125 tiveram identificado o genótipo beta S, tendo sido encontrado 64 (51,2%) CAR/Ben; 36 (28,8%) Ben/Ben; 18 (14,4%) CAR/CAR; dois (1,6%) CAR/Aty;dois (1,6%) Ben/Cam; um (0,8%) CAR/Cam; um (0,8%) Car/Arabia-India e um (0,8%) Sen/Aty. A talassemia 2 3.7Kb foi estudada em 110 pacientes, onde 30 (27,3%) foram heterozigotos e dois (1,8%) homozigotos. O uso de transfusão sangüínea foi maior em pacientes com HbF menor que ou igual a 10,0% (p=0,009). Pacientes com genótipos α diferentes apresentaram diferenças para os valores de Hb (p=0,018); Ht (p=0,019); VCM (p=0,0004) e HCM (p=0,039). Os níveis de HbF foram maiores entre os pacientes Ben/Ben que entre os CAR/CAR (p=0,007) e CAR/Ben (p=0,013). A análise das seqüências do HS2-LCR de dez indivíduos demonstrou a substituição G A na posição ?10.677, presente apenas entre os portadores do haplótipo Ben com nível elevado de HbF, sugerindo uma possível associação entre este polimorfismo, a expressão dos genes γ e a síntese da HbF. A análise da região promotora do gene γG demonstrou a substituição T C na posição -157, que parece ser uma seqüência característica entre os pacientes estudados. Também foi encontrada a deleção de 4 pb na posição ?222 a ?225 no gene γG e relacionado ao haplótipo Cam. Conclusões. Os dados demonstraram um novo polimorfismo localizado no HS2-LCR e na região promotora do gene γG da globina, justificando a realização de estudos adicionais, associando os níveis de HbF, marcadores biológicos e mecanismos relacionados, visando esclarecer um possível papel no desenvolvimento do fenótipo da doença. / Salvador