粒線體是一個高度動態平衡的胞器，經由不斷的分裂與融合可以幫助維持粒線體的型態與功能。在文獻中也可以看到參與粒線體動態平衡的蛋白會影響細胞凋亡。因此，粒線體的動態平衡與粒線體的功能和細胞的代謝反應息息相關。近期的研究發現，Metformin對許多癌症細胞皆有抑制其生長的效果，然而，AICAR也被報導過可以抑制癌細胞的生長並且影響其粒線體的功能。但是AICAR 和Metformin是否會影響粒線體的動態平衡及如何影響粒線體功能的機制尚待釐清，本篇論文將探討AICAR 和Metformin對於粒線體的動態平衡的影響。
我們的實驗結果發現，AICAR會透過融合與分裂的蛋白改變粒線體的型態。
此外，我們也觀察到AICAR和Metformin皆具有調控分裂與融合相關基因及蛋白的能力。最後，我們也發現AICAR會影響粒線體膜電位及粒線體氧化物質的生成。在我們的研究初步釐清AICAR 和Metformin在粒線體動態平衡的調控機制的影響，這些結果同時可以讓我們對於這兩種藥物的作用機制有更進一步的了解。

Mitochondria are highly dynamic organelles that play essential roles in energy supplying and cell metabolism. Dynamic fission and fusion are necessary for maintaining mitochondrial function and cell viability. Disrupted mitochondrial dynamics leads to mitochondria defect and disease. Metformin is the first-line drug for type 2 diabetes and reported to inhibit complex I in the mitochondrial respiratory chain. Previous study showed that both Metformin and 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) promote AMPK activity and mitochondrial activity. However, the mechanisms of how AICAR and Metformin affect mitochondria remain to be examined. In this project, we aim to clarify the underlying mechanisms of whether mitochondrial dynamics is affected by AICAR and Metformin. We found AICAR caused mitochondria hyperfusion but not Metformin. Also, we monitored mitochondrial dynamics related parameters such as promotor activities of fusion and fission genes, mitochondrial dynamics related proteins and activities. Finally, we found AICAR affected mitochondrial membrane potential and superoxide production. Elucidating the effects of AICAR and Metformin not only will help us to better understand the regulation of mitochondrial dynamics, but also potential mechanisms for therapeutic drugs for related diseases.

口試委員審定書………………………………………………………………………………………………………#
誌謝………………………………………………………………………………………………………….…………….. II
ABSTRACT………………………………………………………………………………………….………………….....II中文摘要…………………………………………………………………………………………………………………IV
LIST OF FIGURES……………………………………………………………………….……………………………….V
LIST OF TABLES………………………………………………………………………………….……………..………VI
CONTENTS…………………………………………………………………………………………..………….VII-IX
Chapter 1 Introduction
1.1 Overview of mitochondrial functions and structure…………………………………..1
1.2 Mitochondria are dynamic organelles………………………………………………..………1
1.3 Mitochondria fission and fusion machinery…………………………….…………………2
1.3.1 Mitochondrial dynamics in fission……………………………………….………......2
1.3.2 Mitochondrial dynamics in fusion……………………………………………………..3
1.4 Mitochondrial dynamics abnormality leads to disorders…………………………...4
1.4.1 Mitochondrial dynamics and neurodegenerative disease……….……..…4
1.4.2 Mitochondrial dynamics in cancer…………………………………..…….…………4
1.4.3 Mitochondrial dynamics in diabetes…………………………..…………………….5
1.5 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) acts as an AMP mimetic compound……………………………………………………………………….………….6
1.6 1,1-dimethylbiguanide hydrochloride (Metformin) is an AMPK activator compound…………………………………………………………………………..………….………..6
1.7 The specific aim of this study…………………………………………………………….…..…..7
Chapter 2 Materials and Methods
2.1 Cell culture and chemicals treatment ………………………………….…………….……..8
2.2 Cell viability assay ………………………………………….…….…………………………………..8
2.3 DNA extraction ………………………………………….….….………………………….…………..9
2.4 Plasmids construct ……………………………………………….……..……………………………9
2.5 Cell transfection……………………………………………….……………………..……….……….9
2.6 Luciferase assay……………………………………………….……………………………..………10
2.7 RNA extraction……………………………………………….………………………………….……10
2.8 Reverse transcription……………………………………………….…………………..…………11
2.9 Real-Time PCR…………………………………………………………………………………………11
2.10 Protein extraction……………………………………………….……………..……………………12
2.11 SDS-PAGE and western blot……………………………………….……………………………12
2.12 Immunofluorescence staining………………………………..…………….…………………13
2.13 Antibodies……………………………………………….……………………………………………..13
2.14 Measurement of mitochondrial membrane potential……………………………..13
2.15 Measurement of mitochondrial reactive oxygen species (ROS)……………….14
2.16 Measurement of mitochondrial superoxide (O2・-)…………………………………..14
2.17 Statistical analysis…………………………………….……………………………………………..15
Chapter 3 Results
3.1 High concentration of AICAR attenuated cell viability of human neuroblastoma cells but not Metformin ………………………………………….………16
3.2 AMP protein kinase (AMPK) were activated by AICAR and Metformin in SH-SY5Y cell………………………………………….………………………………………………………16
3.3 AICAR induced mitochondrial dynamics changes but not Metformin.……..17
3.4 AICAR and Metformin modulated mitochondrial fission and fusion machineries…………………………….………………………………………………………………18
3.4.1 AICAR and Metformin modulated mitochondrial fission and fusion protein content.……………………………………………….………………………………18
3.4.2 AICAR and Metformin treatment induced changes of mitochondrial fission and fusion gene expression..……………………………........……….……19
3.5 Examining the effects of AICAR and Metformin on mitochondrial activity by Flow cytometry…………………………………………………………………………………..…..19
3.5.1 Mitochondrial membrane potential in SH-SY5Y cells were affected by AICAR and Metformin……………………………….……………………………….…….20
3.5.2 AICAR increased cellular superoxide but not Metformin…………….…….20
3.5.3 Mitochondrial superoxide level was affected by AICAR but not Metformin………………………………………………………………………………….…….21
Chapter 4 Discussion and Conclusion
4.1 Cell viability on AICAR and Metformin…………………………………….……………….22
4.2 Mitochondrial morphology changes in different status. …………….…..……….23
4.3 AICAR and Metformin modulate mitochondrial fission and fusion machinery……………………………………………………………………………………………….23
4.4 Mitochondrial function in AICAR and Metformin…………………………………….24
References…………………………………………………………..………………………………………26-33

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