為探索使用ADH3p過度表現後酵母菌粒線體的型態與[NADH]/[NAD+]相關性，將已經構築好的目標酵母菌ADH3-pYES2 BJ2168(蛋白酶缺失)、ADH3-pYES2 BY4742、ADH3G211A-pYES2 BY4742(∆YLL001W)三種表現對象，進行誘導表現後蒐集量測誘導後的粒線體[NADH]/[NAD+]比值和計數每株菌的粒線體三種型態，透過統計分析尋找誘導後的粒線體[NADH]/[NAD+]比值和型態計數的相關性，結果顯示每株菌的粒線體型態的計數與[NADH]/[NAD+]比值的關聯性甚低。
本篇也提出了幾個更進一步的假設來針對實驗的事實進行探討。

To discover the correlation of mitochondrial morphology and the level of Nicotinamide adenine dinucleotide, which has been modified to over express ADH3 protein, in Saccharomyces cerevisiae.
We over express target yeast, ADH3-pYES2 BJ2168, ADH3-pYES2 BY4742, ADH3G211A-pYES2 BY4742(∆YLL001W) , and collect the level of Nicotinamide adenine dinucleotide to compare with mitochondrial morphology accounting. Through the Analysis of variance we can reveal the correlation between the level of Nicotinamide adenine dinucleotide and mitochondrial morphology accounting might not be exist. 
Overall, we still have some hypothesis to discuss the conclusion.

目錄
第一章	緒論	1
1.1	研究動機：……………………………………………………..2
第二章	材料與方法	3
2.1	實驗材料…………………………………….………………….3
2.2	實驗儀器………………………………………………………..	3
2.3	方法……………………………………………………………..	4
2.4	質體轉形：……………………………………………………..	5
2.5	轉形質體蛋白質表現：……………………………………….	6
2.6	粒線體型態觀察：…………………………………………….	7
2.7	抽取粒線體：………………………………………………..	8
2.8	分離粒線體基質：…………..………………………………..10
2.9	NADH / NAD+ 量測…………………..………………...……11
第三章	實驗結果	13
3.1	型態學：…………………………………………………….	13
3.2	NAD+、NADH濃度與型態關係之統計：………………….	16
第四章	討論	19
第五章	附錄：	21
第六章	常用藥品	25
第七章	REFERENCE 	27

圖表目錄
圖 a-ADH3-pYES2 BJ2168	14
圖 b-ADH3-pYES2 BY4742	14
圖 c-∆ADH3-pYES2 BY4742(∆YLL001W)	14
圖 d,圖 e分別為ADH3-pYES2 BJ2168、ADH3-pYES2 BY4742、∆ADH3-pYES2 BY4742表現形態	15
圖 f-為型態對細胞質之[NAD+] / Total [NAD/NADH]作分析	17
圖 g-為三種型態對粒線體內[NAD+] / Total [NAD/NADH]作分析	17
圖 h-為三種型態對細胞質之[NADH]/ [NAD+]作分析	18
圖 i-為三種型態對粒線體內[NADH] / [NAD+]作分析	18


表格 i- 統計母體表格	21
表格 ii-  NADH標準曲線	22
表格 iii-   SC-ura Plate	25
表格 iv-   YPD Plate	25
表格 v-   YPD medium	26
表格 vi-   Phosphate-buffered saline（PBS）	26

方程式 1 -  細胞質[NADH]/[NAD+] 對型態之Kruskal-wallis rank sum test	22
方程式 2-   粒線體[NADH]/[NAD+] 對型態之Kruskal-wallis rank sum test	22
方程式 3-   粒線體[NADH]/[NAD+] 對型態之anova test	23
方程式 4-   細胞質[NADH]/[NAD+] 對型態之anova test	23
方程式 5-  細胞質 [NAD+] / total 對型態之Kruskal-wallis rank sum test	23
方程式 6-   粒線體 [NAD+] / total 對型態之Kruskal-wallis rank sum test	23
方程式 7-   粒線體 [NAD+] / total 對型態之Kruskal-wallis rank sum test	24
方程式 8-   細胞質 [NAD+] / total 對型態之anova test	24

[1]	K. Mao and D. J. Klionsky, "Mitochondrial fission facilitates mitophagy in Saccharomyces cerevisiae," Autophagy, vol. 9, no. 11, pp. 1900-1, Nov 1 2013.
[2]	A. M. van der Bliek, Q. Shen, and S. Kawajiri, "Mechanisms of mitochondrial fission and fusion," Cold Spring Harb Perspect Biol, vol. 5, no. 6, Jun 1 2013.
[3]	古佳弘, "過度表現 ADH3p 對啤酒酵母粒線體形態上的影響," 淡江大學生命科學研究所碩士班學位論文, pp. 1-32, 2010.
[4]	陳宏仁, "(I) 啤酒酵母菌 YNR029Cp 的表現和純化及甲基化活性之研究 (II) 過度表現啤酒酵母菌 ADH3p 對其粒線體形態影響," 淡江大學化學學系碩士班學位論文, pp. 1-69, 2011.
[5]	林建利, "ADH3p 的表現在酵母菌 BY4742 和 ΔADH3 (BY4741) 中對粒線體型態的影響," 淡江大學化學學系碩士班學位論文, pp. 1-51, 2014.
[6]	邱明諄, "啤酒酵母菌醇類去氫酶 3 與 4 之選殖, 過度表現, 純化及動力學分析," 淡江大學生命科學研究所碩士班學位論文, pp. 1-83, 2005.
[7]	B. M. Bakker, C. Bro, P. Kötter, M. A. Luttik, J. P. Van Dijken, and J. T. Pronk, "The mitochondrial alcohol dehydrogenase Adh3p is involved in a redox shuttle in Saccharomyces cerevisiae," Journal of bacteriology, vol. 182, no. 17, pp. 4730-4737, 2000.
[8]	H. Kitagaki, Y. Araki, K. Funato, and H. Shimoi, "Ethanol-induced death in yeast exhibits features of apoptosis mediated by mitochondrial fission pathway," FEBS Lett, vol. 581, no. 16, pp. 2935-42, Jun 26 2007.
[9]	V. Palermo, C. Falcone, and C. Mazzoni, "Apoptosis and aging in mitochondrial morphology mutants ofS. cerevisiae," Folia Microbiologica, vol. 52, no. 5, pp. 479-483, 2007.
[10]	E. T. Young and D. Pilgrim, "Isolation and DNA sequence of ADH3, a nuclear gene encoding the mitochondrial isozyme of alcohol dehydrogenase in Saccharomyces cerevisiae," Molecular and Cellular Biology, vol. 5, no. 11, pp. 3024-3034, 1985.
[11]	D. Pilgrim and E. T. Young, "Primary structure requirements for correct sorting of the yeast mitochondrial protein ADH III to the yeast mitochondrial matrix space," Molecular and cellular biology, vol. 7, no. 1, pp. 294-304, 1987.
[12]	R. Hauser, M. Pypaert, T. Hausler, E. K. Horn, and A. Schneider, "In vitro import of proteins into mitochondria of Trypanosoma brucei and Leishmania tarentolae," Journal of Cell Science, vol. 109, no. 2, pp. 517-523, 1996.