Title Page
ABSTRACT
국문 초록
PREFACE
Contents
CHAPTER 1. LITERATURE REVIEW 19
1.1. Deinococcus radiodurans 19
1.1.1. Radiation resistance mechanism of Deinococcus radiodurans 21
1.1.2. Application of D. radiodurans 27
1.2. Radiation 28
1.2.1. Biological effects of ionizing radiation 29
1.2.2. Radiation protection 33
1.3. Extracellular vesicles 35
1.3.1. History and biology of extracellular vesicles 35
1.3.2. Application of EVs 37
1.3.3. Bacterial extracellular vesicles 38
1.3.4. Radioprotective effect of extracellular vesicles 43
1.4. Dissertation overview 44
CHAPTER 2. Characterization of Deinococcus radiodurans-derived extracellular vesicles 46
2.1. Abstract 46
2.2. Introduction 47
2.3. Methods and Materials 49
2.3.1. Culture of Deinococcus radiodurans 49
2.3.2. Isolation, purification, and optimization of R1-EVs 49
2.3.3. Biophysical analysis of R1-EVs 50
2.3.4. Antioxidant analysis of R1-EVs 51
2.3.5. Proteome analysis of R1-EVs 52
2.3.6. Statistical analysis 54
2.4. Results 54
2.4.1. Isolation of R1-EVs 54
2.4.2. Biophysical characterization and antioxidant capacity of R1-EVs 57
2.4.3. Proteome profiles of R1-EVs 60
2.5. Discussion 63
2.6. Conclusion 64
CHAPTER 3. Deinococcus radiodurans-derived extracellular vesicles protect HaCaT cells against H₂O₂-induced oxidative stress via modulation of MAPK and Nrf2/ARE pathways 66
3.1. Abstract 66
3.2. Introduction 67
3.3. Methods and Materials 68
3.3.1. Culture condition of HaCaT cells 68
3.3.2. Effects of R1-EVs and H₂O₂ on the cell viability of HaCaT cells 69
3.3.3. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay 70
3.3.4. Determination of intracellular ROS contents in HaCaT cells 70
3.3.5. Measurement of the intracellular antioxidant molecules and malondialdehyde (MDA) levels 71
3.3.6. Measurement of mitochondrial membrane potential (MMP) 71
3.3.7. Western blotting analysis 72
3.3.8. Statistical analysis 73
3.4. Results 73
3.4.1. R1-EVs inhibit H₂O₂-induced cytotoxicity in HaCaT cells 73
3.4.2. Effects of R1-EVs on the production of intracellular ROS 77
3.4.3. Effects of R1-EVs on MDA content, SOD and CAT activities, and GSH level 79
3.4.4. Effects of R1-EVs on mitochondrial membrane potential and apoptotic pathways in HaCaT cells 82
3.4.5. Effects of R1-EVs on MAPK signaling pathways associated with oxidative stress 86
3.4.6. R1-EVs stimulated the level of Nrf2 in H₂O₂-induced oxidative stress in HaCaT cells 88
3.5. Discussion 90
3.6. Conclusion 92
CHAPTER 4. Deinococcus radiodurans-derived extracellular vesicles as a radioprotector for acute radiation syndrome 93
4.1. Abstract 93
4.2. Introduction 94
4.3. Methods and Materials 96
4.3.1. Animals 96
4.3.2. Irradiation study 96
4.3.3. Intracellular ROS assay of bone marrow cells 97
4.3.4. Viability assay of bone marrow cells and splenocytes 98
4.3.5. MDA assay for plasma 98
4.3.6. Hematoxylin and eosin staining and immunohistochemistry 98
4.3.7. Intestinal permeability assay 99
4.3.8. Analysis of short-chain fatty acids in mouse feces 99
4.3.9. Statistical analysis 101
4.4. Results 101
4.4.1. Administration of R1-EVs protects against radiation-induced mortality in mice 101
4.4.2. R1-EVs improve hematopoietic toxicity after 8 Gy TBI 104
4.4.3. R1-EVs alleviate TBI-induced intestinal structural damage 106
4.4.4. R1-EVs induced the production of short chain fatty acids in TBI-induced ARS mice 109
4.5. Discussion 111
4.6. Conclusion 115
CHAPTER 5. Overall conclusions 116
REFERENCES 119
SUPPLEMENTARY MATERIALS 135
APPENDICES 138
A. Quantifiable proteins identified from R1-EVs 138
B. Hypothetical proteins from R1-EVs 145
Table 1. Species of ionizing-radiation-resistant bacteria 20
Figure 1.1. Ionizing radiation survival curves for various bacteria. 20
Figure 1.2. Radiation and oxidative stress resistance mechanism of Deinococcus radiodurans. 21
Figure 1.3. An overview of the two types of ionizing radiation (IR) effect on the living organisms. 30
Figure 1.4. Classification of the different extracellular vesicles (EVs) subtypes. 37
Figure 1.5. Biogenesis of bacterial extracellular vesicles. 41
Figure 2.1. Schematic isolation flow of R1-EVs. 55
Figure 2.2. Growth curve of D. radiodurans and yield of R1-EVs. 56
Figure 2.3. Biophysical characterization of R1-EVs. 58
Figure 2.4. Antioxidant activities of R1-EVs. 59
Figure 2.5. Proteomic analysis of R1-EVs. 62
Figure 3.1. Effects of R1-EVs on H₂O₂-induced HaCaT cell damage. 75
Figure 3.2. Effects of R1-EVs on intracellular ROS (fluorescence intensity) in H₂O₂-treated HaCaT cells. 78
Figure 3.3. Effect of R1-EVs on the activities of antioxidant enzymes (SOD and CAT), level of GSH, and MDA content in H₂O₂-treated HaCaT cells. 80
Figure 3.4. Effect of R1-EVs on MMP and expression of apoptotic proteins in H₂O₂-treated HaCaT cells. 84
Figure 3.5. Effects of R1-EVs on the activation of the MAPK pathway in H₂O₂-treated HaCaT cells. 87
Figure 3.6. Effect of R1-EVs on the Nrf2/ARE signaling pathway in H₂O₂-treated HaCaT cells. 89
Figure 4.1. R1-EVs protect the mice from radiation-induced death. 103
Figure 4.2. R1-EVs suppress TBI-induced hematopoietic ARS. 105
Figure 4.3. Administration of R1-EVs promotes intestinal structure regeneration in mice after 8 Gy TBI. 107
Figure 4.4. Administration of R1-EVs induces production of short-chain fatty acids. 110