粒線體向來被視為是細胞中重要的能量生產者，同時它也是一個具高度動態的胞器。粒線體分裂及融合被稱為粒線體的動態平衡，粒線體在細胞中參與許多重要的生化反應，包括訊息傳遞、氧化壓力下的調節及鈣離子的儲存等等。在許多文獻中提到粒線體的功能異常被發現和老化的過程及其衍生出的疾病有極高的相關性．粒線體利用動態平衡的調控來維持正常的功能運作，我們實驗室過去的研究中也發現，衰老的酵母菌其粒線體大多呈現較為破碎、不完整的型態、且這個情形將隨培養時間增加而加劇。紅茶萃取物茶黃素(Theaflavin)屬於多酚類的一種，同時也是紅茶茶湯中比例最高的化合物。在我們實驗室先前的研究中，同樣屬於多酚類的白藜蘆醇(Resveratrol)被證明能有效影響粒線體動態平衡並提升粒線體功能。我的研究以同為多酚類的茶黃素為主題，目的為觀察將其使用在衰老酵母菌模型下對於粒線體動態平衡調控的影響。我們發現在生長前期的酵母菌中粒線體的型態比例不會受加入茶黃素與否影響，但在衰老酵母菌中加入茶黃素會明顯降低破碎粒線體的比例。我們更進一步針對粒線體膜電位和其氧化壓力作分析。結果顯示茶黃素會對衰老酵母菌細胞中的粒線體動態調控產生影響，而在過去曾使用過茶黃素的研究中、也曾證實茶黃素能幫助衰老哺乳動物細胞抵抗老化過程造成的傷害。另外經過即時定量PCR的分析，發現茶黃素會影響粒線體動態平衡蛋白Fis1、Dnm1和Fzo1的mRNA表現。這個結果不僅支持前面我們所觀察到茶黃素對於減少衰老酵母菌粒線體破碎化的統計結果，同時也為探究茶黃素在對抗衰老過程中的作用機制提供了方向。

Mitochondria are highly-dynamic organelles. They involved in several cellular functions like respiration and programmed cell death. Mitochondria dynamics determined mitochondria morphology with fission/fusion process. I classified mitochondrial morphology into three types: Hyperfused, Tubular and Fragmented. Theaflavins are the major compounds which are extracted from black tea. In our previous finding, the polyphenol compound Resveratrol from wine was shown to affect mitochondrial dynamics and improve mitochondria functions. In my works, I aim to clarify the effects of theaflavin, also one of the polyphenols from black tea, on mitochondria dynamics in senescent yeast. After theaflavin treatment, the results showed it can reduce the ratio of senescent yeast cells with fragmented mitochondria. I also went further to examine the effects of theaflavin on mitochondria membrane potential and ROS level regulation. Besides, theaflavin can regulate mitochondrial dynamic-related proteins effectively. With qPCR analysis, the morphology result after theaflavin treating can be proved that may related with dynamic-related protein gene expression level. My results provide more details about how theaflavin affects mitochondria in senescent cells. In addition, these results also provide insights of the effects of polyphenol in senescent cells.

指導教授審定書
口試委員審定書
ACKNOWLEDGEMENT
中文摘要 ……………………………………………………………………………..1
ABSTRACT …………………………………………………………………………..2
CONTENTS …………………………………………………………………………..3
LIST OF FIGURES …………………………………………………………………...6
Chapter 1 Introduction ……………………………………………………………...7
1.1 The role of mitochondria in cell ………………………………………………………7
1.1.1 Mitochondrial dynamics ………………………………………………………….....7
1.2 Aging ………………………………………………………………………………….7
1.2.1 Aging affects mitochondrial functions ……………………………………………...8
1.2.2 Replicative life span and chronological life span in yeast model ………………..…8
1.2.3 Definition of senescence …………………………………………………………....9
1.3 The benefits of theaflavin ……………………………………………………………9
1.3.1 The role of tea polyphenols play in aging …………………………………...……...9
1.3.2 The relationship between theaflavin and mitochondria ……………………...……..9
1.4 Specific aim ………………………………………………………………….10
Chapter 2 Materials and methods …………..…………………………………….12
2.1 Yeast strain and culture medium ….…………………………………………….12
2.2 Experiment protocol ……………………………………………………………........12
2.2.1 Theaflavin preparation ……………………………………………..……………...12
2.2.2 Measurement of yeast cell growth curve ……………………………...…………...12
2.2.3 Log phase yeast cell labeling with biotin ………………………………..………...13
2.2.4 Senescent yeast cell sorting ………………………………………………..……....13
2.2.5 Observation of mitochondrial morphology …………………………………….…13
2.2.6 Mitochondrial membrane potential measurement …………………...…………...14
2.2.7 Mitochondrial ROS level measurement …………………………………………..14
2.2.8 Cytosolic ROS level measurement ………………………………………………..14
2.2.9 Flow cytometry ………………………………………………………………........15
2.2.10 Real time PCR ……………………………………………………………………..15
2.3 Data analysis …………………………………………………………..……….........16
2.3.1 T-test ………...……………………………………………………………………..16
Chapter 3 Results ………………..…………………………………………………17
　3.1 Different concentrations of theaflavin would not effect on yeast growth. ……......…17
　3.2 Theaflavin treatment reduced mitochondrial fragmentation in senescent yeast cells .17
　　3.2.1 Senescence leads mitochondrial fragmentation ……………………….…………….18
　　3.2.2 Mitochondrial fragmentation reduced by theaflavin in senescent yeast cell ….18
3.3 Theaflavin treatment affects mitochondrial functions in senescent yeast cells ……..18
　　3.3.1 Theaflavin treatment effects on mitochondrial membrane potential and superoxide level ………………………………………………………………….……….…………………...18
　　3.3.2 Theaflavin treatment has no effects on mitochondrial membrane potential in senescent yeast cells ….………………………………………………………..………………….…………19
3.3.3 Theaflavin treatment affects cytosolic superoxide level in senescent yeast cells …...19
3.4 The effects of theaflavin treatment in fusion/fission gene mRNA expression level ..19
3.4.1 The effect of theaflavin on fusion gene Fzo1 mRNA level ……………………….….19
3.4.2 The effect of theaflavin on fission gene Dnm1 and Fis1 mRNA level ……...20
Chapter 4 Conclusion and discussion ……………………………………………..21
4.1 Conclusion ………………………………………………………………………..…21
4.2 Discussion …………………………………………………………………………...21
4.2.1 Theaflavin reduced mitochondrial fragmentation in senescent yeast cells .……..21
4.2.2 Theaflavin would not disturb on mitochondria but increasing cytosolic superoxide level slightly …………………………………………………...………………………….22
4.2.3 Theaflavin induce fusion gene Fzo1 mRNA level increasing …………..22
Chapter 5 Perspective ……………………………………………………………...23
References …………………………………………………………………………..24
Figures ………………………………………………………………………………26
 

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