粒線體為細胞內負責能量產生與調控細胞生長及活性的重要胞器。粒線體的形態透過不斷的融合與分裂達到動態平衡，許多研究指出粒線體的形態改變與其活性具有高度的關聯性。茶黃素為多酚類化合物，是紅茶中色素與抗氧化的主要成份，茶黃素的研究指出其對於生物具有抗老化、抑制腫瘤生長與降低血膽固醇等功能。然而，目前茶黃素對於老化細胞粒線體動態的影響所知甚少。因此，本篇論文透過酵母菌來研究茶黃素與粒線體之間的關係。本論文的研究中發現，在培養過程中加入茶黃素的衰老酵母菌擁有更多碎裂形態的粒線體。我發現最常存在於粒線體中的活性氧化物質並非造成衰老細胞粒線體碎裂的主因。將負責粒線體分裂的基因DNM1剔除掉後可以抑制衰老細胞與茶黃素所造成的碎裂形態。此外，我的研究結果也指出茶黃素能夠減緩細胞分裂的速度。透過檢測粒線體的呼吸能力也發現茶黃素能減緩老化細胞中粒線體功能的衰退。在本研究中，我的結果提供了更多資訊去了解茶黃素對衰老酵母菌中的粒線體的影響，將有助於進一步的了解粒線體動態調控機制。

Mitochondria are highly dynamic organelles. Their morphology were mainly determined by continuous fusion and fission. Many research studies indicated that aging process is associated with mitochondrial disintegration. Theaflavins are the major compounds extracted from black tea. Theaflavins dominated the pigment and fragrance. The benefits of theaflavins, such as antiaging, inhibition of tumor proliferation and blood cholesterol reduction have been suggested in previous studies. However, little is known about the effect of theaflavins on mitochondria in senescent yeasts, so I aim to clarify the effects of theaflavins on mitochondrial dynamics and activity. Previous work found that mitochondria networks turn into fragmented in senescent yeast cells. Surprisingly, my results demonstrated that treating theaflavins caused mitochondrial morphology more fragmented than untreated senescent cells. In addition, my results indicated that the fragmented mitochondria in senescent yeasts are not caused by reactive oxygen species. The fragmented mitochondria in senescent cells under theaflavins treatment derived from fission protein Dnm1. Furthermore, my experiment results showed that theaflavins increase mitochondrial respiratory capacity and reduced budding scars. Taken together, the results suggested that theaflavins may slow down cell cycle progression and aid to maintain proper mitochondrial activity. These information shed light on the underlying mechanisms of theaflavins effects on mitochondria in senescent yeasts.

口試委員審定書...................................................#
致謝............................................................i
ABSTRACT......................................................iii
中文摘要........................................................iv
CONTENTS........................................................v
LIST OF FIGURES...............................................vii
LIST OF TABLES................................................vii
Chapter 1 Introduction.........................................1
1.1 Mitochondria are indispensable double membrane organelles in cells...........................................................1
1.2 Mitochondrial dynamics is associated with cellular function adjustments.....................................................2
1.3 Mitochondria dynamics in Saccharomyces cerevisiae...........3
1.4 Aging process is irreversible and unavoidable...............4
1.5 Two aging models: replicative lifespan and chronological lifespan in Saccharomyces cerevisiae............................5
1.6 Mitochondria plays a pivotal role in aging process..........5
1.7 Theaflavins, extracts from black tea, are beneficial for health..........................................................6
1.8 Three major compounds of theaflavins have similar effects in cells...........................................................7
1.9 Specific Aim................................................8
Chapter 2 Materials and methods................................9
2.1 Yeast strains, plasmid construct, medium and reagents.......9
2.2 Experimental protocol.......................................9
2.2.1 Senescent yeasts sorting procedure........................9
2.2.2 Budding scar counting....................................10
2.2.3 Exponential growth curves analysis.......................11
2.2.4 Yeast genomic DNA extraction.............................11
2.2.5 Yeast RNA extraction, DNase treatment and reverse transcription..................................................12
2.2.6 Real-time quantitative PCR...............................13
2.2.7 Detection of mitochondrial membrane potential and ROS level..........................................................14
2.2.8 Oxygen consumption rate measurements.....................15
2.2.9 Statistical analysis.....................................15
Chapter 3 Results.............................................16
3.1 Theaflavins have effects on mitochondria dynamics in senescent yeast cells....................................................16
3.2 Theaflavins may affect cell cycle progression and senescence progress in yeast cells........................................17
3.3 Theaflavins preserve respiration capacity in senescent cells..........................................................18
3.4 Theaflavin reduces mitochondrial genome transcription......19
3.5 Theaflavins cause lower mitochondrial membrane potential in senescent yeast................................................20
3.6 Theaflavins are capable to scavenge reactive oxygen species........................................................21
3.7 Mitochondrial fragmentation under theaflavin treatments can be blocked by deletion of DNM1....................................22
Chapter 4 Conclusion and discussion...........................23
4.1 Theaflavin monogallate, other than theaflavin, theaflavin-3, 3’-digallate, has more impacts on senescent yeasts.............23
4.2 Theaflavins reduced the budding rate in yeasts.............24
4.3 The fragmented mitochondria in senescent yeasts are not caused by reactive oxygen species.....................................25
4.4 The effects of theaflavins on mitochondrial activity in senescent yeasts...............................................26
Chapter 5 Perspective.........................................27
REFERENCE......................................................28

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