Chiu, Wui Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 107-128). / Abstracts in English and Chinese. / Abstract --- p.i / 中文摘要 --- p.iv / Acknowledgements --- p.vi / Publications --- p.viii / Table of Contents --- p.ix / List of Tables --- p.xiv / List of Figures --- p.xv / List of Abbreviations --- p.xviii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Hypoxic-ischemic encephalopathy in human infants --- p.1 / Chapter 1.1.1 --- Incidence --- p.1 / Chapter 1.1.2 --- Biphasic development of HI brain damage --- p.2 / Chapter 1.1.2.1 --- Initiating mechanism: energy failure in immature brain --- p.3 / Chapter 1.1.2.2 --- Biochemical cascades --- p.4 / Chapter 1.1.2.2.1 --- Excitatory amino acid receptor activation by glutamate --- p.4 / Chapter 1.1.2.2.2 --- Intracellular calcium accumulation --- p.4 / Chapter 1.1.2.2.3 --- Formation of free radicals --- p.5 / Chapter 1.1.2.2.3.1 --- Reactive oxygen species (ROS) --- p.5 / Chapter 1.1.2.2.3.2 --- Nitric oxide (NO) --- p.6 / Chapter 1.1.2.3 --- Release of inflammatory mediators --- p.6 / Chapter 1.1.2.4 --- Mitochondrial dysfunction --- p.7 / Chapter 1.1.2.5 --- Final path to death: necrosis or apoptosis --- p.8 / Chapter 1.1.2.6 --- Ways to change: neuronal survival and proliferation signaling --- p.8 / Chapter 1.1.3 --- Interventions for neonatal hypoxia-ischemia --- p.9 / Chapter 1.2 --- Animal models mimicking hypoxia-ischemia brain injury --- p.12 / Chapter 1.2.1 --- Comparisons of animal models of hypoxia-ischemia --- p.12 / Chapter 1.2.2 --- Development of neonatal rat model with hypoxic-ischemic damage --- p.14 / Chapter 1.3 --- Neural stem/progenitor cells --- p.15 / Chapter 1.3.1 --- Effect of hypoxic-ischemia on neural stem/progenitor cells --- p.17 / Chapter 1.4 --- Thrombopoietin --- p.18 / Chapter Chapter 2 --- Objectives --- p.23 / Chapter Chapter 3 --- Materials and Methodology --- p.24 / Chapter 3.1 --- Establishment of neonatal rat model of HI brain damage and effects of TPO on neural protection --- p.24 / Chapter 3.1.1 --- Animal protocols --- p.24 / Chapter 3.1.2 --- Induction of HI brain damage in neonatal rats --- p.24 / Chapter 3.1.3 --- Treatment with TPO --- p.25 / Chapter 3.1.4 --- Sacrifice of rats --- p.25 / Chapter 3.1.5 --- Read-out measurements --- p.26 / Chapter 3.1.5.1 --- Brain weight --- p.26 / Chapter 3.1.5.2 --- Gross injury assessment of the right hemisphere --- p.26 / Chapter 3.1.5.3 --- Histology --- p.27 / Chapter 3.1.5.4 --- Blood cell count --- p.27 / Chapter 3.1.5.6 --- Functional assessments --- p.28 / Chapter 3.1.5.6.1 --- Grip traction test --- p.28 / Chapter 3.1.5.6.2 --- Elevated body swing test --- p.28 / Chapter 3.1.5.7 --- Statistical analysis --- p.28 / Chapter 3.2 --- Establishment of in vitro model of primary mouse NSPs and the effect of TPO on their proliferation --- p.29 / Chapter 3.2.1 --- Mouse embryo dissection for the extraction of NSP --- p.29 / Chapter 3.2.2 --- Culturing of NSP --- p.30 / Chapter 3.2.3 --- Immunofluorescence staining for stem cell markers --- p.31 / Chapter 3.2.4 --- Neurosphere assay with different combinations of mitogens --- p.31 / Chapter 3.2.5 --- Neurosphere assay with different concentrations of TPO --- p.32 / Chapter 3.2.6 --- Neurosphere assay under hypoxia --- p.32 / Chapter 3.2.7 --- Statistical analysis --- p.33 / Chapter Chapter 4 --- Effects of thrombopoietin on neonatal rat models of hypoxia-ischemia brain damage --- p.39 / Chapter 4.1 --- Summary of experimental settings --- p.39 / Chapter 4.2 --- Results --- p.39 / Chapter 4.2.1 --- Mortality --- p.39 / Chapter 4.2.2 --- Effects of TPO on p7 mild damage model 1 week post-surgery --- p.40 / Chapter 4.2.2.1 --- Body and brain weights --- p.40 / Chapter 4.2.2.2 --- Gross injury score --- p.41 / Chapter 4.2.2.3 --- Cortex and hippocampus area --- p.41 / Chapter 4.2.2.4 --- Blood cell counts --- p.42 / Chapter 4.2.3 --- Effects of TPO on p7 severe damage model 1 week post-surgery --- p.43 / Chapter 4.2.3.1 --- Body and brain weights --- p.43 / Chapter 4.2.3.2 --- Gross injury score --- p.43 / Chapter 4.2.3.3 --- Cortex area --- p.44 / Chapter 4.2.3.4 --- Blood cell counts --- p.44 / Chapter 4.2.4 --- Effects of TPO on p7 severe damage model 3 week post-surgery --- p.45 / Chapter 4.2.4.1 --- Body and brain weights --- p.45 / Chapter 4.2.4.2 --- Gross injury score --- p.46 / Chapter 4.2.4.3 --- Blood cell counts --- p.46 / Chapter 4.2.4.4 --- Functional outcomes --- p.46 / Chapter 4.2.5 --- Effects of TPO on pl4 severe damage model 1 week post-surgery --- p.47 / Chapter 4.2.5.1 --- Body and brain weights --- p.47 / Chapter 4.2.5.2 --- Gross injury score --- p.48 / Chapter 4.2.5.3 --- Cortex area --- p.48 / Chapter 4.2.5.4 --- Blood cell counts --- p.49 / Chapter 4.3 --- Discussion --- p.49 / Chapter Chapter 5 --- Effects of thrombopoietin on the proliferation of primary mouse neural stem/ progenitor cells in culture --- p.83 / Chapter 5.1 --- Summary of experimental settings --- p.83 / Chapter 5.2 --- Results --- p.83 / Chapter 5.2.1 --- Effect of EGF or bFGF withdrawal on NSP proliferation --- p.84 / Chapter 5.2.2 --- Dose effect of TPO treatment on NSP proliferation --- p.85 / Chapter 5.2.3 --- Effect of hypoxia --- p.85 / Chapter 5.2.4 --- Effect of TPO treatment in combination with hypoxia --- p.86 / Chapter 5.2.5 --- Detection of neural progenitor cell marker --- p.87 / Chapter 5.3 --- Discussion --- p.88 / Chapter Chapter 6 --- General discussion --- p.101 / Bibliography --- p.106

L’objectif de notre travail était de déterminer le rôle joué par l’érythropoïétine et la thrombopoïétine, respectivement, dans l’anémie et la thrombocytopénie observées chez des enfants souffrant d’une hémopathie maligne. Par le dosage simultané de la forme soluble du récepteur de la transferrine et de l’érythropoïétine dans le sérum nous avons montré que l’anémie observée chez ces patients est bien la conséquence d’une réduction du nombre de progéniteurs érythropoïétiques (atteinte médullaire centrale) mais que celle-ci n’est pas la conséquence d’une production insuffisante d’érythropoïétine. Nous avons fait la même observation chez des enfants souffrant d’une tumeur solide non hématologique et chez des patients en cours de traitement par chimiothérapie. Chez ces derniers patients, en appliquant un modèle de culture de moelle à long terme, nous avons pu démontrer l’existence d’une altération du microenvironnement médullaire, probablement induite par la chimiothérapie, se traduisant par une réduction de son aptitude à supporter le développement de la lignée érythroïde. Ceci expliquant au moins partiellement l’inadéquation de la réponse érythropoïétique observée chez ces patients en réponse à l’anémie. Dans la dernière partie du travail, nous avons montré que la thrombocytopénie très fréquemment observée chez les patients leucémiques s’accompagne dans la majorité des cas d’une élévation exponentielle de la concentration de thrombopoïétine, excepté dans les cas de leucémies de la lignée myéloïde. Chez ces derniers la concentration de thrombopoïétine est proche des valeurs observées chez des sujets normaux alors qu’elle devrait être 10 à 100 fois plus élevée compte tenu du nombre de plaquettes extrêmement bas. Nous avons pu montrer que ces taux très bas sont la conséquence de la liaison de la thrombopoïétine à un récepteur spécifique et fonctionnel présent à la surface des cellules leucémiques myéloïdes qui, en l’utilisant comme facteur de croissance, (stimulant leur prolifération et retardant leur mort cellulaire) « consomment » la thrombopoïétine présente dans le sérum.

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Made available in DSpace on 2014-06-11T19:32:51Z (GMT). No. of bitstreams: 0 Previous issue date: 2005-10Bitstream added on 2014-06-13T21:05:04Z : No. of bitstreams: 1 bulla_c_dr_botfmvz.pdf: 626020 bytes, checksum: d96c8e4feb7120d23a1c75d9cd2666f7 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Clicar acesso eletrônico abaixo. / Click electronic access below.

Resumo: Clicar acesso eletrônico abaixo. / Abstract: Click electronic access below. / Orientador: Regina Kiomi Takahira / Coorientador: João Pessoa Araújo Júnior / Banca: Sheila Canavese Rahal / Banca: Luis Artur Loyola Chardulo / Banca: Luis Fernando Pita Gondim / Banca: Paulo Ricardo Oliveira Paes / Doutor

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Hematopoietic stem cells (HSCs) lie at the top of the hematopoietic hierarchy and give rise to all mature blood cells. They are tightly regulated not only by cell-intrinsic but also cell-extrinsic mechanisms that allow HSCs to respond to dynamic physiological demands of the body. HSCs build the hematopoietic system during development and maintain homeostasis in adults by changing their properties according to different needs. A niche is the microenvironment where HSCs reside and receive extrinsic regulation. Understanding the niche is crucial for elucidating how HSCs are regulated by extrinsic cues. During mammalian development, HSCs pass through several different niches, among which the liver is the major site for their rapid expansion and maturation. The fundamental question of what cells constitute the fetal liver niche in vivo remains largely elusive. It is also unclear whether and how cell-extrinsic maintenance mechanisms accompany changes in HSC properties during ontogeny. Here, I genetically dissected the cellular components of the HSC niche in the fetal liver by identifying the cellular source of a key cytokine, stem cell factor (SCF). In addition, I found that HSCs switch to depend on thrombopoietin (TPO), another key factor, during ontogeny and uncovered the mechanism by which HSCs gain this dependence.

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To Man Yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 85-105). / Abstracts in English and Chinese. / Abstract (in English) --- p.i / (in Chinese) --- p.iv / Acknowledgements --- p.vi / Publications --- p.viii / Table of Contents --- p.ix / List of Tables --- p.xii / List of Figures --- p.xiii / List of Abbreviations --- p.xiv / Chapter CHAPTER 1: --- General Introduction --- p.1 / Chapter Section 1.1 --- Background and Clinical Application of Anthracylines --- p.1 / Chapter Section 1.2 --- DOX-induced Cardiotoxicity --- p.3 / Chapter 1.2.1 --- Types of Cardiotoxicity --- p.4 / Chapter 1.2.1.1 --- Acute Cardiotoxicity --- p.4 / Chapter 1.2.1.2 --- Chronic Cardiotoxicity --- p.5 / Chapter 1.2.2 --- Subcellular Effects of DOX --- p.6 / Chapter 1.2.2.1 --- Ultrastructural Lesions --- p.6 / Chapter 1.2.2.2 --- Effects on Mitochondrial Functions --- p.7 / Chapter 1.2.2.3 --- Effects on Sarcoplasmic reticulum (SR) Functions --- p.8 / Chapter Section 1.3 --- Mechanisms of DOX-induced Cardiotoxicity --- p.8 / Chapter 1.3.1 --- Formation of Free Radicals --- p.9 / Chapter 1.3.1.1 --- Generation of Free Radicals by DOX --- p.10 / Chapter 1.3.1.2 --- Cardiac damage by Free radicals --- p.12 / Chapter 1.3.2 --- Induction of Apoptosis --- p.14 / Chapter 1.3.2.1 --- Characteristics and Pathway of Apoptosis --- p.14 / Chapter 1.3.2.2 --- Mitochondria and Apoptosis --- p.15 / Chapter 1.3.2.3 --- Caspases and Apoptosis --- p.17 / Chapter 1.3.2.4 --- Apoptosis and DOX-induced Cardiotoxicity --- p.18 / Chapter Section 1.4 --- Strategies to Reduce DOX-induced Cardiotoxicity --- p.19 / Chapter 1.4.1 --- Dosage optimization and Schedule modification --- p.19 / Chapter 1.4.2 --- Anthracycline Analogues --- p.21 / Chapter 1.4.3 --- Cardioprotective Agents --- p.21 / Chapter Section 1.5 --- Thrombopoietin --- p.23 / Chapter CHAPTER 2: --- Hypotheses and Objectives --- p.30 / Chapter CHAPTER 3: --- Methodology --- p.33 / Chapter Section 3.1 --- Methods --- p.33 / Chapter 3.1.1 --- Culture of Rat H9C2 Myoblast Cell Line and Primary Neonatal Rat Cardiomyocytes --- p.33 / Chapter 3.1.1.1 --- Maintenance of Cell Line --- p.33 / Chapter 3.1.1.2 --- Culture of Primary Neonatal Rat Cardiomyocytes --- p.34 / Chapter 3.1.2 --- Effects of Thrombopoietin on Cell Viability of Rat H9C2 Myoblast Cell Line and Beating Rates of Primary Rat Cardiomyocytes --- p.35 / Chapter 3.1.2.1 --- Cell Viability assay --- p.35 / Chapter 3.1.2.2 --- Beating Rate of Primary Beating Cardiomyocytes --- p.36 / Chapter 3.1.3 --- Effects of Thrombopoietin on the Protection against DOX-induced Heart Injury In Vivo --- p.36 / Chapter 3.1.3.1 --- Animals --- p.36 / Chapter 3.1.3.2 --- Experimental Protocol --- p.37 / Chapter 3.1.3.3 --- Echocardiography --- p.38 / Chapter 3.1.3.4 --- Blood Cell Counts --- p.39 / Chapter 3.1.3.5 --- Histopathology --- p.39 / Chapter 3.1.4 --- Effects of Thrombopoietin on Apoptosis and Mitochondrial Integrity of Rat H9C2 Myoblast Cell Line and Apoptosis In Vivo --- p.40 / Chapter 3.1.4.1 --- Determination of Externalized Phosphatidylserine --- p.40 / Chapter 3.1.4.2 --- Determination of Active Caspase-3 Expression --- p.41 / Chapter 3.1.4.3 --- Assessment of Mitochondrial Integrity --- p.42 / Chapter 3.1.4.4 --- TUNEL assay --- p.43 / Chapter 3.1.5 --- Statistical Analysis --- p.44 / Chapter CHAPTER 4: --- Effects of Thrombopoietin on Cell Viability of Rat H9C2 Myoblast Cell Line and Beating Rates of Primary Neonatal Rat Cardiomyocytes --- p.46 / Chapter Section 4.1 --- Results --- p.46 / Chapter 4.1.1 --- Effects of TPO on DOX-induced Cell Death --- p.46 / Chapter 4.1.2 --- Effects of TPO on the Beating Rates of Primary Cardiomyocytes --- p.47 / Chapter Section 4.2 --- Discussion --- p.47 / Chapter CHAPTER 5： --- Effects of Thrombopoietin on the Protection Against DOX-induced Heart Injury In Vivo --- p.54 / Chapter Section 5.1 --- Results --- p.54 / Chapter 5.1.1 --- General Observations and Survival --- p.54 / Chapter 5.1.2 --- Blood Cell Counts --- p.55 / Chapter 5.1.3 --- Cardiac Functions by Echocardiography --- p.56 / Chapter 5.1.4 --- Gross Anatomic Changes and Pathology of the Myocardium --- p.57 / Chapter Section 5.2 --- Discussion --- p.58 / Chapter CHAPTER 6: --- Effects of Thrombopoietin on Apoptosis and Mitochondrial Integrity of H9C2 Cell Line and Apoptosis In Vico --- p.69 / Chapter Section 6.1 --- Results --- p.69 / Chapter 6.1.1 --- Determination of Externalized Phosphatidylserine --- p.69 / Chapter 6.1.2 --- Determination of Active Caspase-3 Activity --- p.70 / Chapter 6.1.3 --- Assessment of Mitochondrial Membrane Potential --- p.70 / Chapter 6.1.4 --- Determination of Apoptosis by TUNEL assay --- p.72 / Chapter Section 6.2 --- Discussion --- p.72 / Chapter CHAPTER 7： --- General Discussion and Conclusion --- p.83 / References --- p.85

Megakaryocytes (Mks) are unique bone marrow cells, which produce platelets. Dysregulated Mk development can lead to abnormal platelet number and the production of functionally defective platelets, causing bleeding, thrombotic events, and leukaemia. Understanding the molecular mechanisms driving megakaryopoiesis may yield insights into the molecular genetics and cellular pathophysiology of a diversity of disorders. The primary aim of this thesis was to gain insight into the molecular events required for normal Mk development. As transcription factors and cytokines play a central role in driving Mk development, both of these processes were investigated. Fli-1 and GATA-1 are key transcription factors regulating Mk-gene expression, alone and co-operatively. To understand the mechanism of transcriptional synergy exerted by Fli-1 and GATA-1, in vitro assays were carried out investigating the interactions between Fli-1, GATA-1 and DNA that mediate synergy. A novel mechanism of synergy was identified, where Fli-1 DNA binding is not required, although an interaction between Fli-1 and GATA-1, and GATA-1 DNA binding is required. Importantly, the results demonstrate that Fli-1 DNA binding is not essential for promoting Mk-gene expression in primary murine bone marrow cells. Thrombopoietin (TPO) is the primary cytokine responsible for Mk and platelet development. Identifying novel TPO gene-targets may provide invaluable information to aid the understanding of the complex and unique processes required for Mk development. Using microarray technology, IFI16 was identified as a TPO-responsive gene that has not previously been studied in the Mk lineage. This work demonstrated that IFI16 is expressed in CD34+ HSC-derived Mks, and that the Jak/STAT pathway is essential for the activation of IFI16 by both TPO and IFN-??. Of biological significance, IFI16 was found to regulate both the proliferation and differentiation of primary Mks, suggesting that IFI16 may control the balance between these two essential processes. In conclusion, the data in this thesis presents a novel mechanism through which Fli-1 and GATA-1 regulate the synergistic activation of Mk genes. The identification and functional characterisation of a novel TPO-inducible gene, IFI16, involved in regulating the proliferation and differentiation of Mks is also described. These findings have implications for several congenital and malignant conditions affecting Mk and platelet development, and possibly a mechanism for IFN-induced thrombocytopaenia.