Title Page
Contents
국문요약 13
ABSTRACT 15
1. INTRODUCTION 17
1.1. Aging 17
1.2. Cellular senescence 18
1.3. Saccharomyces cerevisiae 20
1.4. Chronological lifespan 21
1.5. Diets and aging 22
1.6. Caloric restriction 23
1.7. Reactive oxygen species (ROS) 26
1.8. Branched-chain amino acids (leucine) 28
1.9. Leucine biosynthesis pathway 33
1.10. Aim and Objective 35
1.11. Summary 35
2. MATERIALS AND METHODS 37
2.1. Yeast strains and plasmid 37
2.2. Growth media 37
2.3. Cell growth 41
2.4. Colony formation rate 41
2.5. Chronological lifespan (CLS) assay 42
2.6. Vital dye exclusion assay 43
2.7. Western blot analysis 43
2.8. Spontaneous mutagenesis 45
2.9. Hydrogen peroxide sensitivity 47
2.10. Hydrogen peroxide mutagenesis 48
3. RESULTS 49
3.1. Leucine supplementation increased the cell density of leu2 yeast strain 49
3.2. Leucine supplementation increase CLS of leu2 yeast strain 56
3.3. Leucine auxotrophic leu2 strain has increased lifespan in CR medium supplemented with exogenous leucine 57
3.4. CR extends CLS of both prototrophic LEU2 and auxotrophic leu2 yeast strains 58
3.5. Leucine and CR increased Cox2p levels in both LEU2 and leu2 yeast strains 63
3.6. Leucine supplementation and CR reduce cell death rate in LEU2 and leu2 yeast strains 67
3.7. Leucine prototrophy, exogenous leucine and CR reduce rate of spontaneous mutation in LEU2 and leu2 yeast strains 73
3.8. Leucine prototrophy, exogenous leucine and CR reduced spontaneous mutation rate in ogg1△ yeast strain 77
3.9. Hydrogen peroxide sensitivity of LEU2 and leu2 yeast strains 79
3.10. Leucine supplementation reduced rate of hydrogen peroxide mutagenesis 82
4. DISCUSSION 86
4.1. Sufficient leucine is required for optimal cell growth 86
4.2. Leucine and CR efficiently extended CLS 90
4.3. Increase mitochondrial metabolism by leucine and CR promoted CLS 96
4.4. Leucine and CR decreased rate of cell death 98
4.5. Leucine and CR contributed to decreased spontaneous mutagenesis 100
4.6. LEU2 and leu2 yeast cells exhibited similar sensitivity pattern to H₂O₂ at early growth phase in leucine supplemented and CR medium 102
4.7. Leucine promotes human muscle maintenance needed in preventing decrepit thereby extending lifespan 104
5. CONCLUSION 108
6. REFERENCES 110
Table 2.1. Yeast strains used in this study 39
Table 2.2. Composition of Synthetic Complete media 40
Figure 1. Leucine concentration for optimal growth of LEU2 and leu2 strains in SC medium. 52
Figure 2. Growth of LEU2 and leu2 yeast strains. 53
Figure 3. Growth of LEU2 and leu2 strains on solid medium. 55
Figure 4. CLS of LEU2 strains in SC, CR and exogenous leucine supplemented medium. 60
Figure 5. CLS of leu2 strains in SC, CR and exogenous leucine supplemented medium. 62
Figure 6. Cox2p expression. 65
Figure 7. Cell death rate of LEU2 yeast strain. 69
Figure 8. Cell death rate of leu2 yeast strain. 71
Figure 9. Spontaneous mutation rate in LEU2 strain. 75
Figure 10. Spontaneous mutation rate in leu2 strain. 76
Figure 11. Spontaneous mutation rate in ogg1△ strains. 78
Figure 12. Hydrogen peroxide sensitivity of LEU2 yeast strain. 80
Figure 13. Hydrogen peroxide sensitivity of leu2 yeast strain. 81
Figure 14. Hydrogen peroxide mutagenesis of LEU2 yeast strain. 83
Figure 15. Hydrogen peroxide mutagenesis of leu2 yeast strain. 84
Figure 16. Hydrogen peroxide mutagenesis of ogg1△ yeast strain. 85