粒線體在細胞中扮演很重要的角色，特別是在能量調控上。藉由粒線體分裂和融合的過程可以幫助粒線體維持正常的功能。失去功能的粒腺體可能跟老化的過程有關，並且在衰老的細胞中可以發現比較多碎裂型態的粒線體。白藜蘆醇是一種天然的多酚抗氧化物，根據我們實驗室的研究結果，白藜蘆醇可以減少碎裂型態的粒線體並且降低衰老細胞中活性氧化物的含量。然而白藜蘆醇的作用機制仍然在研究領域中有不同的爭辯。我們實驗室過去發現缺乏磷酸二酯酶後會抑制住白藜蘆醇對衰老的酵母菌細胞的效果，因此我們想進一步去探討磷酸二酯酶在白藜蘆醇對粒線體動態平衡的調控所扮演的角色。在我們的研究結果中發現將磷酸二酯酶過度表現會影響白藜蘆醇調控粒線體的型態，但卻不會影響白藜蘆醇降低活性氧化物的機制。與缺乏磷酸二酯酶的結果比較後，我們可以推論出白藜蘆醇在調控衰老酵母菌細胞中的粒腺體型態及降低活性氧化物的過程中是需要磷酸二酯酶來作用的。這些結果提供更多資訊讓我們了解白藜蘆醇在細胞中的作用機轉。

Mitochondria play critical roles in cells, especially in energy homeostasis. One way to regulate mitochondrial homeostasis and quality is via dynamic fission and fusion processes. Mitochondrial dysfunction may associate with aging process. Fragmentation of mitochondria was found in senescent cells. In addition, Resveratrol, one of the ROS scavengers, can reduce the ratio of the senescent yeast cells with fragmented mitochondria. Studies indicated that resveratrol inhibits the activity of phosphodiesterase to ameliorate the aging phenotype in mammalian cells. Whether resveratrol affects cellular activity through phosphodiesterase is still under extensive debate. Our previous results suggested that inhibiting phosphodiesterase compromise the effects of resveratrol in senescent yeast cells. We aim to elucidate the role of phosphodiesterase in the mechanism of resveratrol mediated-mitochondrial dynamics in senescent cells. The results showed overexpression of PDE2 affects resveratrol to reduce the ratio of senescent cells with fragmented mitochondria, but not interfere resveratrol to mediate superoxide removal. Comparing to the wild type and Δpde2 cells, PDE2 is required for the effect of resveratrol on mitochondrial dynamics in senescent yeast cells. The results provide more understanding of underlying mechanisms of how resveratrol acts in senescent yeast cells.

CONTENTS
口試委員審定書………………………………………………………………………#
致謝…………………………………………………………………………………….i
ABSTRACT…………………………………………………………………………ii
中文摘要…………………………………………………………………………….iii
CONTENTS………………………………………………………………………..iv
LIST OF FIGURES………………………………………………………………..viii
LIST OF TABLE…………………………………………………………………..xi
Chapter 1 Introduction……………………………………………………………...1
1.1 Mitochondria play critical roles in cells………………………………………....1
1.1.1 Mitochondrial dynamics regulates mitochondrial homeostasis and quality. .1
1.1.2 Mitochondrial dynamic in yeast……………………………………………..2
1.2 Aging process………………………………………………………………….....3
1.2.1 Senescence is the phenotype of aging. ………………………………….......3
1.2.2 Replicative & chronological senescence yeast model………………..….......3
1.2.3 Aging and mitochondria…………………………………………………......4
1.3 Resveratrol offers a broad range of health benefits………………………………..5
1.3.1 Resveratrol and senescence………………………………………….............5
1.3.2 The effect of resveratrol on mitochondrial function………………………...6
1.4 The degradation of cyclic nucleotide is mediated by phosphodiesterase. ………7
1.4.1 Phosphodiesterase regulates cAMP/PKA pathway. ………………………..7
1.4.2 Resveratrol inhibits the activity of cAMP-phosphodiesterase. …………….8
1.5 Specific aim……………………………………………………………………….9
Chapter 2 Materials and methods…………………………………………………10
2.1 Yeast strains, medium and reagent………………………………………………10
2.2 Experiment protocol ……………………………………………………………10
2.2.1 Construct PDE2 overexpression plasmid…………………………………..10
2.2.2 Yeast transformation………………………………………………………..10
2.2.3 Collection of senescent yeast cells………………………………………….11
2.2.4 DiOC6(3) staining to observe the mitochondrial morphology……………...12
2.2.5 Yeast genomic DNA extraction .……………………………………............13
2.2.6 Yeast RNA extraction .………………...……………………………............13
2.2.7 DNase I treatment…………………………………………………………...14
2.2.8 Reverse transcription polymerase chain reaction....…………………………15
2.2.9 Real time PCR………………………………………………………………15
2.2.10 Mitochondrial membrane potential detection …………………………......16
2.2.11 Mitochondrial superoxide level measurement ……………………………17
2.2.12 Cytosolic superoxide level measurement…………………………............17
2.2.13 Flow cytometry………………………………………………………….17
2.2.14 Statistical analysis…………………………………………………………18
Chapter 3 Result…………………………………………………………………….19
3.1 The effect of resveratrol on mitochondrial dynamics in senescent cells was affected by PDE2. ……………..…………………………………………………………19
3.1.1 Overexpression of PDE2 affects resveratrol to reverse mitochondrial
morphology in senescence cells. …………………………………….20
3.1.2 Resveratrol affects the mRNA level of fission/ fusion proteins in WT and PDE2
overexpressed senescent cells. ………………………………………………21
3.2 Resveratrol affects mitochondrial activities in PDE2 overexpressed senescent
yeast cells. ………………………………………………………………………22
3.2.1 PDE2 overexpressed senescent yeast cells had similar mitochondrial
membrane potential as WT cells. ………………………………………….22
3.2.2 Resveratrol decreases the mitochondrial superoxide level in both of WT and
PDE2 overexpressed senescent yeast cells. ………………………………..23
3.2.3 Resveratrol reduces the intracellular superoxide level in both of WT and
PDE2 overexpressed senescent yeast cells. ………………………………..24
Chapter 4 Discussion and conclusion……………………………………………..25
4.1 PDE2 is required in resveratrol mediated mitochondrial dynamics. ………….25
4.2 Resveratrol improves mitochondrial membrane potential not through PDE2. …26
4.3 PDE2 is required for resveratrol to mediate superoxide removal. ………………26
4.4 Conclusion………………………………………………………………………..27
Chapter 5 Perspective………………………………………………………………28
REFERENCE……………………………………………………………………….29

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