Title Page
Contents
ABBREVIATIONS 10
ABSTRACT IN KOREA 12
Ⅰ. INTRODUCTION 14
Ⅱ. MATERIALS AND METHODS 21
2.1. Animals 21
2.2. Cell culture 21
2.3. Transient middle cerebral artery occlusion ischemic stroke model (tMCAO) 22
2.4. Ovariectomized mouse model (OVX) 23
2.5. Oxygen-glucose deprivation (OGD) 24
2.6. Drug administration 24
2.7. Neurological severity score (NSS) 26
2.8. Measurement of infarct volume by cresyl violet and 2,3,5-Triphenyltetrazolium chloride (TTC) staining 28
2.9. Cell viability assay 29
2.10. Immunofluoresecense 29
2.11. Proximity Ligation assay (PLA) 30
2.12. RNA extraction and Quantitative polymerase chain reaction (qPCR) 31
2.13. Western Blot Analysis 33
2.14. Statistical analysis 34
Ⅲ. RESULTS 35
3.1. Sex showed different infarction after tMCAO in mice 35
3.2. Aging or severity of ischemic damage gave different infarction after tMCAO in mice 39
3.3. Sex differences showed different autophagic response 41
3.4. Different E2 level by OVX or aging showed different autophagic response 44
3.5. TFEB mRNA expressions were changed depending on sex and aging 51
3.6. Protein levels of TFEB and its modification in male and female after tMCAO 53
3.7. CARM1 increased in the nucleus after tMCAO 64
3.8. Interaction between TFEB and E2 was found out colocalization and proximity in vivo and in vitro 66
3.9. Role of E2 in stage of autophagic response in vitro 68
Ⅳ. DISCUSSION 72
Ⅴ. REFERENCES 77
ABSTRACT 86
Figure 1. Stroke incidence rate according to age and sex. 14
Figure 2. Autophagy process illustration. 17
Figure 3. Experimental procedure to examine the autophagy responce according to estrogen in mice. 26
Figure 4. Schematic representation of the experimental of surgery (tMCAO and OVX) and administration in mice. 36
Figure 5. Infarct size following E2 treatment or ovariectomized(OVX). 38
Figure 6. Infarct size following E2 treatment, ovariectomized (OVX) and aged (3 months or 12 months). 40
Figure 7. Changes in autophagy flux according to sex and estrogen level after 30 min tMCAO in mice. 43
Figure 8. Changes in autophagy flux according to E2 level after 40 min of tMCAO in mice. 45
Figure 9. Changes in autophagy flux according to aging after 40 min of tMCAO in mice. 46
Figure 10. E2 reduced autophagosome-lysosome fusion after 40 min tMCAO in young (3 months) female mice. 48
Figure 11. E2 induced autophagosome-lysosome fusion in aged female mice after tMCAO. 50
Figure 12. Expression of TFEB according to aging and estrogen level. 52
Figure 13. Changes in TFEB, autophagy regulator, according to sex and estrogen after 30 min tMCAO in mice. 55
Figure 14. Changes in protein level of TFEB according to estrogen in stroke mice. 57
Figure 15. Changes in protein level of TFEB according to estrogen in stroke mice. 59
Figure 16. Photomicrographs of TFEB by cell type. 61
Figure 17. Differential levels of me-TFEB in different locations of the brain after tMCAO in males and females after tMCAO. 63
Figure 18. Increased nuclear translocation of CARM1 over time after tMCAO in mice. 65
Figure 19. Colocalization and interaction of TFEB and estrogen receptors (ERs). 67
Figure 20. Cell viability of N2a cells with E2 and CQ treatment using MTT assay. 69
Figure 21. Changes in autophagy flux according to sex and estrogen after tMCAO (30 min occlusion) in mice (3 months). 71