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作者(中文):黃健倫
作者(外文):Huang, Chien-Lun.
論文名稱(中文):TER94 在調控有絲分裂後細胞之細胞核大小以及結構上所扮演的角色
論文名稱(外文):Role of TER94 in Regulating nuclear size in post-mitotic cells
指導教授(中文):桑自剛
指導教授(外文):Sang, Tzu-Kang
口試委員(中文):張慧雲
郭崇涵
口試委員(外文):Chang, Hui-yun
Kuo, Tsung-Han
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
學號:105080593
出版年(民國):107
畢業學年度:106
語文別:英文
論文頁數:74
中文關鍵詞:細胞核自噬作用DNA修復DNA損害細胞週期有絲分裂後細胞
外文關鍵詞:nucleusp53DNA damage responseautophagycell cycle arrestpost-mitotic cell
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細胞核儲存著遺傳訊息並且在細胞中扮演重要的角色,細胞核大多維持固定結構,在一般情況下不會有太多的形態上的變化,除非細胞正在進行細胞分裂,在我們先前對於AAA(ATPase associated with diverse cellular activities)-ATPase , TER94的研究中,我們發現當在果蠅複眼感光細胞中表現其顯性抑制(dominant negative)的突變TER94K2A時,細胞核有異常變大的現象,而有趣的是,感光細胞是屬於分化完成且已不再進行細胞分裂的post-mitotic cells。此外,我們在這些變大的細胞中發現有許多被泛素化標定的蛋白質(ubiquitinated protein)堆積,顯示TER94可能與核蛋白的穩定有所關係。透過基因篩選,我們發現p53基因敲落可以回覆TER94K2A造成的細胞核增大現象,而當我們降低p53的拮抗基因mdm2/Corp的表現時,也得到的同樣的結果,顯示p53參與在此機制中。由於p53參與在DNA損壞時的修復反應(DNA damage repair),我們深入探討這是否與TER94K2A造成的細胞核增大有關。γ-H2AV是參與在辨識DNA雙股螺旋斷裂的蛋白,在免疫組織染色中,我們發現變大的細胞核中,p53與γ-H2AV的表現量皆有大幅的增加。此外,我們以過氧化氫分別餵食野生型以及表現TER94異形合子(heterozygous)的果蠅,造成氧化壓力並誘發DNA損壞,發現只帶有一半的TER94基因之果蠅表現較野生型多的γ-H2AV,顯示TER94在DNA修復機制上的重要性。p53也調控著細胞中自噬作用(autophagy)之進行,當基因敲落自噬作用相關之蛋白質時,TER94K2A造成的細胞核增大現象也會被部分的抑制。以上的實驗顯示,TER94在調控DNA修復機制與p53的穩定性上扮演重要的角色,當此機制失調時,可能影響p53下游的自噬作用,最後造成細胞核增大。
The nucleus houses genetic information to control essentially every aspect of eukaryotic cell activities. The structure of this organelle is rather stable in general unless the cell is undergoing the division. In our previous study of the type II AAA-ATPase, TER94, we found that expression of a dominant-negative mutant TER94K2A causes nuclear enlargement in fly photoreceptor cells (R cell) of which are at the end-dividing, post-mitotic state. Also, we found that the ubquitinated protein accumulated in the enlarged nucleus, suggesting that TER94 may play a role in nuclear protein quality control. Through the genetic screen, we identified p53 might play a crucial role in TER94K2A-induced nuclear expansion. p53 only accumulated in the enlarged nuclei but not in those with regular size. In agree with this, downregulation of the fly Mdm2, Corp, which could antagonize p53 activity, also suppressed the nuclear phenotype. Since p53 participates in DNA damage response (DDR), we asked if p53 is involving in TER94-associated nuclear size control. Indeed, we observed the increase of γ-H2AV, a DNA double-strand break marker, in TER94K2A-expressing nuclei. Moreover, γ-H2AV accumulation appeared more robust in TER94 heterozygous background after treating DNA damage agent hydrogen peroxide, indicating that TER94 shortfall might cause DDR defect. DNA damage could trigger autophagy signaling, and we found that down-regulation of autophagy-related protein could partially suppress TER94K2A-induced nuclear phenotype. Together, these results suggest that TER94 may be instrumental in regulating DNA damage response and p53 stability. Damage of this mechanism may affect downstream autophagy regulation, which eventually results in nuclear enlargement.
Abstract 1
中文摘要 4
致謝 6
Introduction 9
Materials and methods 14
Drosophila stocks and genetics 14
Immunohistochemistry 14
Hydrogen peroxide treatments 15
Results 16
TER94 dysfunction causes nuclear enlargement in postmitotic photoreceptor cells 16
The nuclear function of TER94 is instrumental in nuclear protein quality control 17
p53 participates in TER94K2A-induced nuclear phenotype 18
TER94 loss of function causes DNA damage repair defect 20
Autophagy regulation impairment caused by TER94K2A may result in nuclear enlargement 24
p53-induced cell-cycle arrest or cell-cycle re-entry may be important in TER94K2A-induced nuclear enlargement regulation 25
Discussion 27
The identity of ubiquitinated proteins 30
Downregulation of Corp also suppressed TER94K2A-induced nuclear enlargement 31
The regulation of lipid biosynthesis involves in TER94K2A-induced nuclear enlargement 32
Tables 61
References 64

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