아주대학교

목차

CHAPTER 1 1
1. Introduction 2
2. Methods 4
2.1 Sequence alignment 4
2.2 Cell lines and reagents 4
2.3 Cell viability assay 5
2.4 Western blot analysis 5
2.5 Immunofluorescence 6
2.6 Cytokine detection assays 6
2.7 Statistical analysis 7
3. Results and discussion 8
3.1. Results 8
3.1.1. Activation of immune signaling in human macrophages 8
3.1.2. Activation of immune signalling in primary human dermal fibroblasts (HDFs) 10
3.1.3. FLA-AA–mediated signaling is specific to TLR5 11
3.2. Discussion 13
3.3. Conclusion 16
CHAPTER 2 17
4. Introduction 18
5. Methods 21
5.1. Cell lines and reagents 21
5.2. Cell viability assay 21
5.3. IL-8 and IL-6 cytokine assays 22
5.4. Cell-death recovery assay 22
5.5. Western blot analysis 22
5.6. Dissociation of TNF-α trimerization assembly 23
5.7. Surface plasmon resonance (SPR) spectroscopy 23
5.8. Murine CIA model and grouping 24
5.9. Behavioral and histological analysis of animals 24
5.10. Statistical analysis 25
6. Results and discussion (1): The effect of DMSO on the biological activity of TNF-α 26
6.1. Results 26
6.1.1. DMSO Inhibits TNF-mediated Cytokine Release and Cell Proliferation 26
6.1.2. DMSO inhibits TNF-mediated cell death 27
6.1.3. DMSO deactivates TNF-mediated pathways 28
6.1.4. DMSO prevents TNF oligomerization 29
6.2. Discussion 29
6.3. Conclusion 31
7. Results and discussion (2): A series of decoy peptides that disrupt TNF-alpha oligomeric configuration 32
7.1. Results 32
7.1.1. Identification of TNF-inhibitory peptides 32
7.1.2. Designing derivatives of shortlisted peptides 36
7.1.3. TID3c inhibits TNF-mediated downstream signaling pathways 39
7.1.4. TID3 and TID3c distort trimeric form of TNF 40
7.1.5. Biophysical interaction between TNF and peptides 41
7.2. Discussion 43
7.3. Conclusion 45
8. Results and discussion (3): Designing an orally active small molecule inhibitor of TNF-alpha to alleviate rheumatoid arthritis 45
8.1. Results 45
8.1.1. Identification of TIM1 as a potential TNF inhibitor 45
8.1.2. TIM1 attenuates TNF–mediated death of cells 46
8.1.3. TIM1 deactivates TNF-dependent pathways 48
8.1.4. Derivatives of TIM1 showed improved activity against TNF signaling 49
8.1.5. TIM1 and TIM1c disrupt TNF trimer by binding to TNF 53
8.1.6. TIM1/TIM1c reduces arthritis symptoms in murine model through oral- administration 53
8.2. Discussion 54
8.3. Conclusion 58
CHAPTER 3 59
9. Introduction 60
10. Methods 63
10.1. Design and cloning of gRNAs 63
10.2. Cell culture and reagents 66
10.3. Off-target prediction 67
10.4. T7E1 endonuclease assay 67
10.5. Reprogramming-plasmid construction 68
10.6. Optimizing knock-in of donor cassette 68
10.7. Junction PCR 69
10.8. Flow cytometry 69
10.9. Cell viability assay 69
10.10. Cytokine detection assay 70
10.11. Generation and maintenance of iPSCs 70
10.12. In vitro differentiation of iPSCs 71
10.13. Immunocytochemistry 71
10.14. RT-PCR and quantitative RT-PCR 72
10.15. Western blotting 72
10.16. Statistical analysis 73
11. Results and Discussion 74
11.1. Results 74
11.1.1. Design and validation of guide RNAs 74
11.1.2. Knock-in of reprogramming-cassette 78
11.1.3. Proliferative and immunological validation 82
11.1.4. Generation and characterization of iPSCs 83
11.2. Discussion 86
11.3. Conclusion 88
12. References 89