Title Page

1. Abstract

Contents

2. Introduction 12

3. Materials and Methods 20

3.1. Cell Culture and Viral Infection 20

3.2. Apoptosis Assay 21

3.3. Plaque Assay 22

3.4. Cell Survival Assay 22

3.5. Analysis of cellular lipofuscin levels using flow cytometry 24

3.6. Quantifying Autophagic Flux 24

3.7. Analysis of the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) is conducted. 28

3.8. Metabolite extraction and gas chromatography-mass spectrometry (GC-MS) analysis are performed 28

3.9. Western blot analysis is performed. 29

3.10. Drug Screening 30

3.11. Duration of Drug Treatment 30

3.12. Immunofluorescence Technique 31

3.13. The Senescence-associated (SA)-β-galactosidase assay 31

3.14. Statistical Analyses 32

4. Result 33

4.1. Metabolic reprogramming as a novel therapeutic target for Coxsackievirus B3 34

4.1.1. CVB3 induces metabolic changes through an increase in glycolysis 34

4.1.2. Confirmation of glycolysis induction by CVB3 through a metabolic analysis 36

4.1.3. Glycolysis inhibitors as novel therapeutic strategies for CVB3 treatment 40

4.2. Mitochondrial dysfunction caused by the 3C protease of coxsackievirus B3 in HeLa cell 42

4.2.1. Effects of CVB3 3Cpro on mitochondrial function and cell survival in HeLa cells 42

4.2.2. Impact of 3CproWT transfection on mitochondrial function in HeLa cells 44

4.2.3. 3Cpro of CVB3 impairs mitochondrial respiratory capacity in HeLa cells 46

4.2.4. 3Cpro of CVB3 reduces glycolytic activity and alters metabolic profiling in HeLa cells[이미지참조] 48

4.2.5. 3Cpro of CVB3 induces energy shift and mitochondrial dysfunction in HeLa cells[이미지참조] 51

4.3. Functional restoration of lysosomes and mitochondria through modulation of AKT activity ameliorates senescence 53

4.3.1. Chemical screening to find agents capable of senescence amelioration by inhibiting AKT 53

4.3.2. AKT inhibition yields lysosomal functional recovery 56

4.3.3. Elimination of dysfunctional mitochondria by GDC0068-mediated mitophagy activation 60

4.3.4. AKT inhibition yields mitochondrial functional recovery 64

4.3.5. AKT inhibition regulates mitochondrial function by increasing OXPHOS efficiency 68

4.3.6. Amelioration of senescence-associated phenotype by AKT inhibition 70

4.3.7. Optimal duration of AKT inhibition to induce senescence amelioration 72

5. Discussion 74

6. Reference 78

7. 국문초록 81

Figure 4.1.1.1. CVB3 induces metabolic alterations by promoting an upregulation of glycolysis. 35

Figure 4.1.2.1. Verification of metabolic alterations induced by CVB3 via metabolite analysis. 38

Figure 4.1.2.2. Confirmation of metabolic changes induced by CVB3 through metabolite analysis. 39

Figure 4.1.3.1. Glycolysis inhibitors as emerging therapeutic approaches for the treatment of Coxsackievirus B3 (CVB3). 41

Figure 4.2.1.1. The effect of 3C pro viral protein on the survival of HeLa cells. 43

Figure 4.2.2.1. Overexpression of viral protein 3C pro deteriorates mitochondrial function. 45

Figure 4.2.3.1. Reduction of oxidative phosphorylation by 3Cpro of CVB3. 47

Figure 4.2.4.1. Reduction of glycolysis by 3Cpro of CVB3.[이미지참조] 49

Figure 4.2.5.1. Reduced energy phenotype by 3Cpro of CVB3.[이미지참조] 52

Figure 4.3.1.1. Chemical screening was conducted to identify agents that have the potential to ameliorate senescence by inhibiting AKT. 54

Figure 4.3.1.2. The Specificity of GDC0068 as an AKT Inhibitor. The protein expression levels of mechanistic target of rapamycin (mTOR) and phosphorylated 4E-... 55

Figure 4.3.2.1. AKT inhibition leads to the restoration of lysosomal functionality. 57

Figure 4.3.2.2. The representative histogram of autofluorescence (A) and the representative histogram of lysosomal mass (B). 58

Figure 4.3.2.3. The representative histogram of autophagic flux. 59

Figure 4.3.3.1. Elimination of dysfunctional mitochondria through the activation of mitophagy by GDC0068. 61

Figure 4.3.3.2. The representative histogram of mitochondrial mass. 63

Figure 4.3.4.1. AKT inhibition leads to the restoration of mitochondrial function. 65

Figure 4.3.4.2. The representative histogram depicting the mitochondrial membrane potential (MMP). 66

Figure 4.3.4.3. The representative histogram of mitochondrial using DHR123 (A) and MitoSOX (B). 67

Figure 4.3.5.1. AKT inhibition has been shown to modulate mitochondrial function by enhancing the efficiency of oxidative phosphorylation (OXPHOS). 69

Figure 4.3.6.1. AKT inhibition ameliorates the senescence phenotype. 71

Figure 4.3.7.1. Appropriate duration of AKT inhibition for the facilitation of senescence amelioration. 73