粒線體是一個動態胞器，不停地進行融合與分裂，這個過程又稱為粒線體動態平衡。在細胞中，粒線體最主要的功能是藉由呼吸作用來提供能量；除此之外，還參與了許多的代謝與訊息傳遞的調控。先前研究指出，老化、疾病與粒線體功能異常有關，且粒線體會藉由融合與分裂來維持其正常的功能。在我們先前的研究中發現，老化的酵母菌其粒線體呈現較破碎的型態，並且會藉由提升粒線體功能來維持正常的細胞功能。而在酵母菌及線蟲的生物研究中發現，白藜蘆醇可提高粒線體的活性來延長壽命並且延緩老化的發生。我的實驗針對白藜蘆醇在老化的酵母菌中的作用進行研究。我們發現在老化的酵母菌細胞中加入白藜蘆醇可以減少衰老細胞族群中帶有破碎狀粒線體的比例，更進一步發現白藜蘆醇會改變衰老細胞中粒線體融合與分裂的基因表現、粒線體DNA的總量，以及提高氧氣的消耗量。我們的解果顯示白藜蘆醇在老化的細胞中會改變粒線體融合與分裂的平衡，並且進一步提升粒線體的功能。

Mitochondria are important organelles that play key roles in cellular functions, such as energy production, oxidative stress responses and programmed cell death. Proper mitochondria dynamics fission/fusion processes are critical for regulating their shape, size and integrity. The cellular activity declines gradually during the senescence process. Previous studies in our lab demonstrated that mitochondria not only become fragmented by shifting dynamic balance toward fission, but also alter their activity in replicative senescent yeast cells. Resveratrol is known to be an effective antioxidant and be able to extend lifespan by boosting mitochondria biogenesis and activity. However, the effects of resveratrol on mitochondria dynamics in replicative senescent yeast cells remain to be explored. We applied biotin-streptavidin labeling method to isolate resveratrol-treated senescent cells to clarify related issues. Our results demonstrated that treatment of resveratrol can reduce mitochondria fragmentation in replicative senescent yeast cells and increase fusion and fission gene expression levels. Furthermore, resveratrol increased mtDNA and enhanced the oxygen consumption. Our finding indicated that resveratrol possesses the ability to adjust the fusion and fission balance of mitochondria and further facilitates mitochondrial functions in senescent yeast cells.

口試委員會審定書 ＃
致謝 i
中文摘要 iii
ABSTRACT iv
CONTENTS v
LIST OF FIGURES viii
LIST OF TABLES ix
Chapter 1 Introduction 1
1.2 Mitochondria 1
1.1.1 Mitochondria are dynamic organelles 1
1.1.2 The roles of mitochondrial fission and fusion 2
1.1.3 The machinery of mitochondrial dynamics in yeast 3
1.2 Aging 4
1.2.1 Aging in yeast 4
1.2.2 Mitochondria and aging 5
1.3 Resveratrol 6
1.3.1 Aging and resveratrol 6
1.3.2 Resveratrol and mitochondria 7
1.4 Specific aim 8
Chapter 2 Materials and methods 9
2.1 Yeast strains and medium 9
2.2 Experiment protocol 9
2.2.1 Yeast transformation 9
2.2.2 Measurement of cytotoxicity 10
2.2.3 Senescent yeast cells sorting 10
2.2.4 Fixation of yeasts to observe the mitochondrial morphology 11
2.2.5 Yeast genomic DNA extraction 11
2.2.6 Yeast RNA extraction 12
2.2.7 Reverse transcription polymerase chain reaction 12
2.2.8 Real time PCR 13
2.2.9 Mitochondrial membrane potential detection 14
2.2.10 Mitochondrial ROS level measurement 14
2.2.11 Cytosolic ROS level measurement 14
2.2.12 Flow cytometry 15
2.2.13 Measurement of mitochondrial bioenergetics 15
Chapter 3 Results 16
3.1 The mitochondrial network turns into fragmented in senescent yeast cells 16
3.1.1 Mitochondrial morphology becomes fragmented in senescent cells. 17
3.1.2 Mitochondria morphology becomes fragmented with the culture time 17
3.2 Resveratrol changes the balance of mitochondrial dynamics. 18
3.2.1 Resveratol affects mitochondrial network morphology in senescent cells 18
3.2.2 Resveratrol affects fusion/fission gene expression profiles 19
3.2.3 Resveratrol-induced mitochondrial morphology change is SIR2-independent. 19
3.3 Resveratrol promotes mitochondrial activity 21
3.3.1 Resveratrol treatment causes mtDNA copy numbers increase. 21
3.3.2 Mitochondrial membrane potential is not affected by resveratrol. 21
3.3.3 The oxygen consumption rate level increases after resveratrol treatment. 22
3.3.4 The cytosolic ROS level significantly decreases after resveratrol treatment.. 22
Chapter 4 Conclusions and discussion 24
4.1 Conclusion. 24
4.2 Discussion. 25
4.2.1 Resveratrol provides a new balance of fission and fusion in mitochondria. 25
4.2.2 Resveratrol regulates mitochondrial dynamic by another pathway. 25
4.2.3 Resveratrol slows aging process by promoting mitochondrial function and removing intracellular ROS 25
Chapter 5 Perspective 27
REFERENCE 28

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