Different cucurbitacin derivatives have been compared for the gamma globin induction potential. Cucurbitacin D turned out to be the most potential inducer among the derivatives had been tested. Later I had screened more herbs for the gamma globin induction activities. One of the herbs showed a higher activity than Fructus Trichosanthis, which could be the potential candidate to isolate more potent inducer. In the toxicity study, cucurbitacin D only have a mild toxic effect on the normal cell lines and transgenic mice. Finally, the efficacy of cucurbitacin D was tested on a sickle cell anemia mouse model and demonstrated a significant induction of fetal haemoglobin production. Cucurbitacin D may be a potential drug candidate for treating beta globinopathies. / Thalassemia is a global disease. It was report in 2001 that there were 270 million people who carried the severe disease. Most of the cases were found in Africa and south-east Asia. China has a high incidence rate of 0.66% in 2001. In the past, the treatments of the disease were blood transfusion and bone marrow transplantation. However, many defects in such kinds of treatments were reported. The balance of relieving the syndrome of the disease and the adverse effects of the drugs was the consideration to the physician. The drug, hydroxyurea, can activate the gamma globin gene and produce hemoglobin F to replace the beta globin as an oxygen transporter is considered as an better treatment to ameliorate the syndrome. Safety and effectiveness in the long-term treatment using hydroxyurea are questionable. Cucurbatacin D purified from a Chinese herb demonstrates 2000 folds more potent than hydroxyurea. It can activate the gamma globin gene and produce hemoglobin F shown in ELISA and confocal microscopy. The fundamental work for drug development is carrying out through this project. In this project the biological property and toxicity were studied. / Liu, Shuk Ming. / Adviser: M.C. Tung. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 245-270). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed Feb. 13, 2009). Includes bibliographical references.

Thesis (Ph.D.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 125 pages. Includes Vita. Includes bibliographical references.

Orientador: Maria de Fátima Sonati / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-19T19:29:31Z (GMT). No. of bitstreams: 1 Wobeto_VaniaPerettideAlbuquerque_D.pdf: 2287567 bytes, checksum: ff964a9410a59697f79714370d17c0c7 (MD5) Previous issue date: 2012 / Resumo: As fosfatidilinositol-fosfato quinases (PIPKs) pertencem a uma família de enzimas lipídio-quinases que geram vários mensageiros lipídicos, incluindo um importante segundo mensageiro denominado fosfatidilinositol-4,5-bifosfato, que participa da regulação de diversas atividades celulares, como a modulação do citoesqueleto de actina, o transporte de vesículas, a formação de adesão focal e diversos eventos nucleares, incluindo a expressão gênica. A subfamília da PIPK está dividida, conforme a especificidade de sinalização, em tipo I (isoformas ?, ? e ?), tipo II (isoformas ?, ? e ?) e tipo III. Em estudo recentemente realizado em nosso laboratório, o gene da PIPKII? mostrou-se diferencialmente expresso em reticulócitos de dois irmãos com Doença de Hemoglobina H, sendo maior no paciente com maior nível de hemoglobina anômala, paralelamente à expressão do gene da globina ?, sugerindo uma possível relação entre a PIPKII? e a produção de globinas. Além disso, análises realizadas durante a diferenciação eritróide de células CD34+, em cultura, demonstraram que as expressões dos genes das PIPKs aumentam à medida que essas células se tornam mais diferenciadas. O papel da PIPK no processo hematopoiético, no entanto, tem sido pouco explorado. No presente trabalho, investigamos a localização celular da PIPKII? e sua expressão gênica e protéica durante a diferenciação eritroide, megacariocítica e granulocítica de células da linhagem hematopoiética e demonstramos que o silenciamento do gene da PIPKII?, em células K562, resulta em diminuição da proliferação deste tipo celular, aumento de expressão do gene da globina ? e uma tendência de elevação da expressão do gene da globina ?. Esses resultados corroboram a sugestão prévia de existência de relação entre a PIPKII? e a expressão dos genes de globinas, relação que deve continuar a ser investigada em células eritróides normais e de pacientes com hemoglobinopatias / Abstract: Phosphatidylinositol-phosphate kinases (PIPKs) belong to a family of lipid kinase enzymes that generate various lipid messengers, including an important second messenger known as phosphatidylinositol-4,5-biphosphate, which participates of the regulation of several cell activities, such as modulation of the actin cytoskeleton, vesicle transport, formation of focal adhesions and various nuclear events, including regulation of gene expression. The PIPK subfamily is divided into types I (isoforms ?, ? and ?), II (?, ? and ?) and III according to signaling specificity. In a recent study in our laboratory, the PIPKII gene was differentially expressed in reticulocytes from two siblings with hemoglobin H disease: expression of this gene, as well as that of the 'beta'-globin gene, was greater in the patient with the higher level of the abnormal hemoglobin, suggesting a possible relationship between PIPKII and the production of globins. In addition, analyses carried out using CD34+ cells from cultures undergoing erythroid differentiation showed that PIPK gene expression increased as the cells became more differentiated. However, there has been little research into the role of PIPK in the hematopoietic process. In this study we investigate the cellular location of PIPKII and the gene and protein expression of this enzyme during erythroid, megakaryocytic and granulocytic differentiation of hematopoietic lineage cells. We show that PIPKII gene silencing in K562 cells results in reduced proliferation of this cell type, increased 'gama'-globin gene expression and a tendency towards increased 'alfa'-globin gene expression. These results corroborate the previous suggestion of a relationship between PIPKII and globin gene expression, a relationship that should be the subject of further investigation in normal erythroid cells and erythroid cells from patients with hemoglobinopathies / Doutorado / Ciencias Biomedicas / Doutor em Ciências Médicas

Liu, Kan. / "November, 2010"--Abstract. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 116-129). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Le but de cette thèse est de mettre en relation les propriétés structurale, dynamique et fonctionnelle de la forme humaine d’une nouvelle protéine découverte dans le cerveau des vertébrés en 2000 : la Neuroglobine. Dans un premier temps, j’ai réalisé une étude théorique dans laquelle un mécanisme à deux voies menant à la forme pentacoordinée avec cystéines oxydées a été mis en avant. A travers ce mécanisme, un conformère de la Neuroglobine au sein duquel le groupe prosthétique hème a basculé au cœur de la structure protéique a été déterminé. A partir des structures de ce mécanisme, une étude sur la diffusion de petits ligands au sein des cavités internes de la protéine à l’aide de la méthode de métadynamique a mis en évidence que la formation du pont disufure intramoléculaire favorisait la poche de ligation. De plus un certain nombre de voies de sortie pour les ligands a pu être obtenu. Pour compléter ce premier aspect de la thèse, une étude des propriétés mécaniques, communes avec les autres globines, a montré l’importance de quatre résidus centraux, dit mécaniquement sensibles, qui régulent les canaux d’accès aux différentes poches internes de la protéine, appelé phénomène de respiration. Dans un second temps, je me suis intéressé à l’interaction de la Neuroglobine avec un petit ligand via une étude expérimentale par ITC. La première conclusion importante est que la cinétique de ligation est plus importante lorsque le pont disulfure est formé. De plus j’ai observé une diminution de la cinétique lors du passage Wild Type vers C120S puis réaugmentation de la cinétique lors du passage C120S vers C46G/C55S/C120. Afin de comprendre ce phénomène, une simulation de la Neuroglobine triplement mutée a été réalisée au cours de laquelle un réseau de deux liaisons hydrogènes a été mis en avant. Ce réseau change considérablement les voies d’entrée/sortie pour les ligands. Ainsi la mutation 120 ferme une/ou plusieurs voies de sortie alors que la mutation 46 ouvre la voie naturelle des globines. Le changement observé étant important, une étude par RMN de Ngb TM et WT cystéines réduites a montré qu’il y avait une différence de structure entre ces formes pas seulement au niveau des points de mutation mais sur l’ensemble de la structure. Ces nouveaux résultats mettent ainsi en évidence le rôle important des trois cystéines chez la Neuroglobine humaine. / In this PhD work, I tried to link together the different structural, dynamic and fonctional properties of a new human protein discovered in the mamals brain in 2000: the Neuroglobin. First of all, I established a new two ways mecanism in order to get the pentacoodinated oxydized cysteins state using theoritical method. One of this mecanism’s conformer shows an important heme sliding inside of the proteic structure. Furthermore with help of metadynamic method, I studied the small ligand diffusion and migration in the internal cavity network. I showed the higher ligand affinity when the disulfide bridge is bond and we proposed an important number of exit pathways. Then we developed a method to understand the mechanical properties of the globins and we found four residues mechanically sensitive which form together a control access pathway between internal cavities, called breath phenomenon. Secondly I used ITC method in order to characterize the interaction between the Neuroglobin and a small ligand. From this experiment we highlighted that the kinetic ligation is faster when the disulfide bridge is formed. Then I noticed a relative decrease of the velocity when the mutation C120S is operated followed by a relative increase of the velocity for the triple mutation C46G/C55S/C120 compared to the Wild Type data. To understand these results, I performed a molecular simulation of the triple mutation Neuroglobin form. During this trajectory, I discovered a structure with a two hydrogen bonds network, which significantly changes the ligand entry/exit pathways. The 120 mutation closes one/several exit pathways while the 46 mutation opens the natural globin exit pathway. Because of the considerable structural change observed in the triple mutation Neuroglobin form, I decided to produce NMR results. These last points reveal a relative structure difference between the Wild Type oxidized cysteins form and the triple mutation form not only on the mutation points but also on the global structure. All these new results highlight the essential role of the three cysteins in the human Neuroglobin.

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