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研究生:王煜涵
研究生(外文):Wang, Yu-Han
論文名稱:以酵母菌探討粒線體形態調控以及細胞老化之關聯性
論文名稱(外文):Cellular Aging and Mitochondrial Dynamics in Saccharomyces cerevisiae
指導教授:張壯榮
指導教授(外文):Chang, Chuang-Rung
口試委員:汪宏達鄭子豪張壯榮
口試日期:2011-7-14
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:英文
論文頁數:56
中文關鍵詞:粒線體動態平衡神經退化性疾病老化酵母菌
外文關鍵詞:Mitochondrial dynamicsNeurodegenerative diseasesAgingYeast
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The mitochondrial dynamics are consisted of at least two distinct pathways: mitochondria fusion and fission. Emerging evidences indicate that mitochondrial dynamics plays a critical role in neurodegenerative diseases. The prevalence of neurodegenerative disorders is closely associated with age; however little is known about the correlation between mitochondria dynamics and aging. Therefore, the aim of thesis is to understand the mitochondrial dynamics in aged cells. I adopted an approach to enrich yeast aged cells system in our experiments. By examining these cells, I found that aged cells have more fragmented mitochondria. In addition, higher mRNA expression levels of mitochondria fission genes FIS1 and DNM1 were found in these aged cells. Furthermore, the exponential phase growth rate of DNM1 deletion strain was similar as wild type in my experiments. These results indicated that aging may activate conventional mitochondria fission pathway and cause fragmentation. Our results also demonstrated that budding yeast can be used as a model organism to study mitochondrial dynamics and cellular aging.
粒線體融合與分裂的動態調控影響其於細胞中的功能,而在老化所導致的神經退化性疾病中,已經有很多研究顯示與粒線體的功能喪失及形態調控有直接關聯。故而本篇論文是從細胞老化的角度切入,釐清粒線體融合、分裂之動態平衡與老化的關係。在本篇研究中,採用了釀酒酵母(Saccharomyces cerevisiae)此種被廣泛利用的模式生物來作為本研究之對象,以修飾過的生物素(Biotin)標記在細胞表面,利用其與卵白素(Streptavidin)之高度親和力,再利用其與已鍵結上卵白素的微鐵珠 (micro-magnetic beads)作用,即可使用強力磁鐵達到分離已標記之原始細胞的目的。藉由此技術,可以將出芽生長分裂代數較多的酵母菌從培養的族群中分離出來,再利用已送入含有可標記粒線體綠螢光蛋白(Green-Fluorescent Protein)之質體,便可以觀察酵母菌因粒線體融合與分裂平衡的傾向所導致的不同粒線體形態。在細胞老化的層次上,可發現生長代數較多之野生種酵母菌顯現出較多粒線體碎裂的特徵,此現象在基因剔除粒線體分裂機制上必要的FIS1 以及DNM1 菌株中即不復出現;在mRNA 的表現量而言,FIS1 以及DNM1基因在分裂代數較多之酵母菌的樣品裡表現量也明顯提高,證實粒線體的動態調控機制參與了老化細胞裡粒線體分裂多於融合的過程。我們的實驗驗證了以酵母菌為模式生物來研究細胞層次老化的過程是可行的。而我們的結果更指出粒線體動態平衡的調控機制
與細胞老化是有相關的。
CONTENTS

口試委員會審定書 #
誌謝 i
中文摘要 iii
ABSTRACT iv
CONTENTS v
LIST OF FIGURES viii
LIST OF TABLES ix
Chapter 1 Introduction 1
1.1 The mechanisms of mitochondrial dynamics 1
1.2 Mitochondrial dynamics in neurons 4
1.3 Monitoring mitochondrial dynamics 5
1.4 The unique feature of Saccharomyces cerevisiae cell dividing 6
1.5 Specific aim 7
Chapter 2 Materials and Methods 9
2.1 Special reagents & chemicals 9
2.2 Microbial strains 9
2.3 Experimental protocols 9
2.3.1 E. coli transformation & plasmid mini-preparation 9
2.3.2 Yeast transformation 10
2.3.3 Growth rate measurement 11
2.3.4 Cell sorting 11
2.3.5 Mitochondrial morphology classifications 12
2.3.6 Budding scar staining 12
2.3.7 Yeast RNA extraction 13
2.3.8 Reverse-transcription polymerase chain reaction 13
2.3.9 Yeast protein extraction 14
2.3.10 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting 14
Chapter 3 Results 16
3.1 The validation of aged cell sorting 16
3.1.1 Determination of the growth rate of strains and the re-culture time 16
3.1.2 Verifying aged cell enrichment by budding scar numbers 17
3.2 The determination of mitochondrial morphology 18
3.2.1 The classification of mitochondrial morphology 18
3.2.2 The aged WT yeast showed more fragmented mitochondria 18
3.2.3 The fission increasing index of mitochondrial morphology measurement 19
3.2.4 The increased mitochondria fission in aging cells was diminished in fission gene deletion strains 20
3.2.5 Δfzo1, a mitochondrial fusion related gene deletion strain demonstrated slightly fission increasing in the early culture 21
3.2.6 Srv2 gene knockout also showed the same tendency with WT yeast in mitochondrial fission increasing 21
3.3 The mRNA expression levels of mitochondrial fission related genes rise in W303 1a 22
Chapter 4 Conclusions & Discussion 23
4.1 Fragmented mitochondria are enriched in sorted aged yeast cells 23
4.2 Discussion 23
4.2.1 The distinct results of other studies 23
4.2.2 The cause or the consequence? 25
4.2.3 Perspectives of the future research 26
REFERENCES 51


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