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The role of reactive oxygen species during erythropoiesis: an in vitro model using TF-1 cells.

Ge, Tianfang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 87-93). / Abstract also in Chinese. / EXAMINATION COMMITTEE LIST --- p.ii / DECLARATION --- p.iii / ACKNOWLEDGEMENTS --- p.iv / ABSTRACT --- p.v / ABSTRACT IN CHINESE --- p.vii / ABBREVIATIONS --- p.ix / TABLE OF CONTENTS --- p.xiii / Chapter 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Erythropoiesis --- p.2 / Chapter 1.2 --- The TF-1 model --- p.3 / Chapter 1.3 --- The erythroid marker glycophorin A (GPA) --- p.4 / Chapter 1.4 --- Reactive oxygen species (ROS) --- p.4 / Chapter 1.5 --- Oxidative stress in human erythrocytes --- p.6 / Chapter 1.6 --- Antioxidant defense systems --- p.6 / Chapter 1.7 --- Glucose provides the majority of reducing equivalents in human erythrocytes --- p.9 / Chapter 1.8 --- Glucose transporter type 1 (Glut l) transports glucose and vitamin C into human erythrocytes --- p.10 / Chapter 1.9 --- Hypothesis and objectives --- p.11 / Chapter 1.10 --- Long-term significance --- p.12 / Figure 1.1 Stages of mammalian erythropoiesis. Adapted from (Koury et al.,2002) --- p.13 / "Figure 1.2 Conversion of major ROS. Adapted from (Ghaffari," --- p.14 / Figure 1.3 Major oxidative defense in human erythrocytes --- p.15 / "Figure 1.4 Peroxide scavenging systems. Adapted from (Day," --- p.16 / Chapter 2 --- MATERIALS AND METHODS --- p.17 / Chapter 2.1 --- Cell culture --- p.18 / Chapter 2.1.1 --- Culture media --- p.18 / Chapter 2.1.2 --- Cell maintenance --- p.19 / Chapter 2.1.3 --- Cell cryopreservation --- p.19 / Chapter 2.1.4 --- Cell differentiation --- p.20 / Chapter 2.1.5 --- Cell treatments --- p.20 / Chapter 2.1.5.1 --- Antioxidant treatments --- p.21 / Chapter 2.1.5.2 --- H2O2 challenging --- p.22 / Chapter 2.1.5.3 --- Antibiotic treatment --- p.22 / Chapter 2.2 --- Flow cytometry --- p.23 / Chapter 2.2.1 --- Flow cytometers --- p.23 / Chapter 2.2.2 --- Analysis of erythroid differentiation --- p.23 / Chapter 2.2.3 --- Analysis of cell lineage --- p.24 / Chapter 2.2.4 --- Analysis of intracellular ROS --- p.24 / Chapter 2.2.5 --- Analysis of mitochondrial transmembrane potential (Δψm) --- p.25 / Chapter 2.2.6 --- Analysis of mitochondrial mass --- p.25 / Chapter 2.2.7 --- Analysis of cell death --- p.26 / Chapter 2.2.8 --- Analysis of caspase-3 activity --- p.27 / Chapter 2.2.9 --- FACS cell sorting --- p.27 / Chapter 2.2.10 --- Two-variant flow cytometric experiments --- p.28 / Chapter 2.2.11 --- Analysis of flow cytometry data --- p.28 / Chapter 2.2.12 --- Compensation --- p.29 / Chapter 2.2.12.1 --- Compensation matrix for Annexin V-PI double-staining --- p.29 / Chapter 2.2.12.2 --- Compensation matrix for Annexin V-TMRM double-staining --- p.30 / Chapter 2.2.12.3 --- Compensation matrix for CFSE- GPA double-staining --- p.31 / Chapter 2.2.12.4 --- Compensation matrix for CFSE- TMRM double-staining --- p.31 / Chapter 2.2.12.5 --- Compensation matrix for CM- H2DCFDA-GPA double-staining --- p.32 / Chapter 2.2.12.6 --- Compensation matrix for GPA- TMRM double-staining --- p.33 / Chapter 2.3 --- Western blot --- p.35 / Chapter 2.4 --- Statistical analysis --- p.37 / Chapter 3 --- RESULTS AND DISCUSSION --- p.38 / Chapter 3.1 --- The cells with high GPA staining were younger in cell lineage --- p.39 / Chapter 3.2 --- ROS was produced during TF-1 erythropoiesis --- p.40 / Chapter 3.3 --- ROS production was not essential for TF-1 erythropoiesis --- p.41 / Chapter 3.4 --- ROS production was not the cause of cell proliferation during TF-1 erythropoiesis --- p.41 / Chapter 3.5 --- ROS production was not the cause of sub-lethal mitochondrial depolarization in TF-1 erythropoiesis --- p.42 / Chapter 3.6 --- The cells showing mitochondrial depolarization were mother cells that gave rise to differentiating cells --- p.44 / Chapter 3.7 --- ROS production was not the cause of cell death in TF-1 erythropoiesis --- p.45 / Chapter 3.8 --- ROS production confers oxidative defense during TF-1 erythropoiesis --- p.47 / Chapter 3.8.1 --- Glut l inhibition partially blocked TF-1 erythropoiesis without affecting cell viability --- p.47 / Chapter 3.8.2 --- Antioxidant defense systems were established during TF-1 erythropoiesis --- p.48 / Chapter 3.8.3 --- Antioxidant treatments blocked the establishment of antioxidant defense systems during TF-1 erythropoiesis --- p.51 / Chapter 3.9 --- Conclusion --- p.55 / Chapter 3.10 --- Future work --- p.56 / Figure 3.1 Cell lineage versus erythroid marker during erythropoiesis under vitamin E treatment --- p.59 / Figure 3.2 ROS production during erythropoiesis --- p.60 / Figure 3.3 ROS production versus erythroid marker during erythropoiesis under vitamin E treatment --- p.61 / Figure 3.4 Percentage of ROS+ cells in vitamin E-treated TF-1 erythropoiesis as compared to control --- p.63 / Figure 3.5 Percentage of GPA+ cells in vitamin E-treated TF-1 erythropoiesis as compared to control --- p.64 / Figure 3.6 Cell death versus mitochondrial transmembrane potential (Δψm) during erythropoiesis under vitamin E treatment --- p.65 / Figure 3.7 Erythroid marker versus mitochondrial transmembrane potential (Δψm) during erythropoiesis under vitamin E treatment --- p.67 / Figure 3.8 Cell lineage versus mitochondrial transmembrane potential (Δψm) during erythropoiesis under vitamin E treatment --- p.69 / Figure 3.9 Change of mitochondrial mass during erythropoiesis --- p.71 / Figure 3.10 ROS production versus erythroid marker during erythropoiesis under levofloxacin treatment --- p.72 / Figure 3.11 Percentage of GPA+ cells in levofloxacin-treated TF-1 erythropoiesis as compared to control --- p.73 / Figure 3.12 Cell death versus mitochondrial transmembrane potential (Δψm) during erythropoiesis under levofloxac in treatment --- p.74 / Figure 3.13 Expression level of antioxidant enzymes during erythropoiesis --- p.75 / Figure 3.14 Expression level of Glut l during erythropoiesis --- p.76 / Figure 3.15 Expression level of Glut l in GPA positive and GPA negative populations --- p.77 / Figure 3.16 Cell death under oxidative stress challenging during erythropoiesis --- p.78 / Figure 3.17 Expression level of antioxidant enzymes and Glutl during erythropoiesis under EUK-134 treatment --- p.79 / Figure 3.18 Expression level of antioxidant enzymes and Glutl during erythropoiesis under vitamin E treatment --- p.80 / Figure 3.19 Cell death under oxidative stress challenging during erythropoiesis under vitamin E treatment --- p.82 / Figure 3.20 Expression level of antioxidant enzymes during erythropoiesis under vitamin C treatment --- p.83 / Figure 3.21 Cell death under oxidative stress challenging during erythropoiesis under vitamin C treatment --- p.84 / Figure 3.22 Cell death under oxidative stress challenging during erythropoiesis under NAC treatment --- p.85 / Figure 3.23 Summary of oxidative stress challenging during erythropoiesis --- p.86 / REFERENCES --- p.87

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_326891
Date January 2009
ContributorsGe, Tianfang., Chinese University of Hong Kong Graduate School. Division of Life Sciences.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xvii, 93 leaves : ill. (some col.) ; 30 cm.
RightsUse of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

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