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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 羅凱尹(Kai-Yin Lo) | |
| dc.contributor.author | Ching-Hsiang Hsu | en |
| dc.contributor.author | 許景翔 | zh_TW |
| dc.date.accessioned | 2023-03-19T23:28:55Z | - |
| dc.date.copyright | 2022-09-23 | |
| dc.date.issued | 2022 | |
| dc.date.submitted | 2022-09-22 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/85918 | - |
| dc.description.abstract | 細胞中核醣體蛋白的基因突變或是rDNA的複製數出現問題會導致核醣體組裝出現缺陷進而造成核醣體疾病。先前研究顯示，在核醣體基因突變時，會增加膜的壓力反應 (membrane stress response) ，並導致ROS的增加。從結果中發現核醣體蛋白基因缺失的菌株在menadione 或是過氧化氫中的生長相較於野生株都更為敏感。利用Amplex red試劑測試菌株中過氧化氫的濃度發現部分的對數期核醣體突變株中過氧化氫濃度有增加。接著觀察抗氧化酵素的表現，諸如過氧化氫酶及超氧岐化酶的活性，發現突變株在靜止期 (stationary-phase) 的過氧化氫酶活性會大量增加，但對數期 (log-phase) 中的過氧化氫酶活性則沒有影響；然而，對數期的核醣體突變株則多數具有較高的超氧岐化酶活性。以qPCR測試過氧化氫酶活性的提高是否因為在轉錄層面有增加，發現過氧化氫酶的基因 (Cytosolic catalase T, CTT1) 在多數對數期的核醣體突變株會大量表現，且轉錄因子Msn2、Msn4和Hog1能夠參與過氧化氫酶轉錄的調控。前人研究指出過氧化氫會透過fenton reaction在25S rRNA上進行裁切，使其能夠轉譯抗氧化相關基因以適應氧化壓力，結果顯示在核醣體基因缺失的菌株中，25S rRNA亦有被裁切的現象。以蔗糖梯度的方式分離轉譯和非轉譯中的mRNA，發現抗氧化酵素在突變株以及野生株在靜止期中的CTT1和Peroxisomal catalase A (CTA1) 轉譯效率都有明顯的提高，但兩者間的轉譯效率則沒有明顯差異，說明轉譯效率的提高並非核醣體突變造成的現象。另外也發現核醣體突變株的過氧化氫酶活性在抗壞血酸以及生育酚的處理中有不同反應。透過以上的數據，推測核醣體蛋白基因的缺失，會導致體內過氧化氫的增加，以轉錄的方式提高抗氧化酵素的活性。 | zh_TW |
| dc.description.abstract | Ribosomopathy is caused by defects in the assembly of ribosomes, which may be caused by mutations of biogenesis-related genes or incorrect copy numbers of rDNA. Previous studies have shown that when ribosomal protein (RP) genes are mutated, the membrane stress response and ROS generation increase. Our results showed that RP gene mutant strains were more sensitive to menadione or hydrogen peroxide than wild type. Our results also showed that the concentrations of hydrogen peroxide measured with Amplex red reagent in the RP mutants were increased in the log phase. We speculated that higher H2O2 might induce the activity of antioxidant enzymes, i.e., catalase and superoxide dismutase (SOD), which can consume ROS. The stationary-phase RP mutants had higher activity but remained unchanged in the log-phase cells. In contrast, most log-phase RP mutants expressed a higher SOD activity. Consistently, the transcription levels of cytosolic catalase genes (CTT1) were higher at the log phase in the RP mutants. We found the transcription factors related to stress response, Msn2, Msn4, and Hog1, were involved in the regulation. Previous studies have shown that H2O2 can cleave 25S rRNA by Fenton reaction to make ribosomes more capable of translating oxidative stress-related genes. Our results showed that 25S rRNA was also cleaved in RP mutants. Using a sucrose gradient to separate the translated and non-translated mRNAs, it was also found that the translation efficiency of CTT1 and CTA1 were significantly improved both in wild type and RP mutants in the stationary phase but no differences were observed in between. In addition, the treatment of antioxidants, i.e., ascorbic acid and tocopherol, caused different responses to the catalase activities in RP mutants. Our results showed that the RP mutants would increase H2O2 in vivo, which in turn elevated the expression of antioxidant enzymes mainly via transcription. | en |
| dc.description.provenance | Made available in DSpace on 2023-03-19T23:28:55Z (GMT). No. of bitstreams: 1 U0001-1909202214303700.pdf: 5035846 bytes, checksum: dee9453ed05123e2556f36c03546c23c (MD5) Previous issue date: 2022 | en |
| dc.description.tableofcontents | 致謝 i 摘要 ii Abstract iii 目錄 v 圖目錄 vii 表目錄 viii 第壹章、 文獻回顧 1 一、核醣體 (ribosome) 1 二、核醣體疾病 (Ribosomopathies) 1 三、 核醣體疾病的表徵 3 四、 活性氧物質 (Reactive oxygen species, ROS) 5 i. ROS產生方式 : 5 ii. ROS攻擊的標的 6 iii. 抗氧化系統介紹 7 五、 核醣體蛋白同源類似物(paralog) 12 第貳章、 研究動機 14 第參章、 材料方法 15 (一)菌株、質體 (Strains and plasmid) 15 (二) 生長測試 (Spot assay) 15 (三) 過氧化氫酶活性染色分析 (Catalase zymography) 15 (四) 超氧歧化酶活性染色分析 (Superoxide dismutase zymography) 16 (五) qPCR基因表達分析: 16 (六) 北方墨點法 (Northern blot) 18 (七) 過氧化氫濃度測試 19 (八) 轉譯mRNAs的比例分析 (Analysis of the ratio of translating mRNAs) 19 (九) 氫氧化鈉破菌 (Post-alkaline extraction from yeast) 21 (十) 西方墨點法 (Western blot) 21 (十一) 流式細胞儀 (Flow cytometry) 22 第肆章、 結果 24 一、核醣體突變株對於氧化壓力敏感 24 二、核醣體突變株體內的過氧化氫較野生株高 26 三、核醣體突變株產生較高的抗氧化酵素活性 28 四、核醣體突變株的粒線體膜電位異常 30 五、添加不同抗氧化劑後對過氧化氫酶的活性有不同的影響 32 六、對數期時，部分核醣體突變株在抗氧化酵素的轉錄較野生株高 33 七、在特定轉錄因子缺失時過氧化氫酶無法正常表現 34 八、轉譯相關因子的調控會改變過氧化氫酶活性 35 九、H2O2可促進25S核醣體RNA的位點特異性切割且在靜止期更為明顯 36 十、抗氧化酵素的轉譯在靜止期中效率較高 37 第伍章、 討論 38 第陸章、 結論 44 | |
| dc.language.iso | zh-TW | |
| dc.title | 啤酒酵母中核醣體蛋白基因缺失對抗氧化壓力的影響 | zh_TW |
| dc.title | Impact of the mutations of ribosomal protein genes on the oxidative stress in Saccharomyces cerevisiae | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 110-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 洪傳揚(Chwan-Yang Hong),張麗冠(Li-Kwan Chang),鄭梅君(Mei-Chun Cheng) | |
| dc.subject.keyword | 核醣體,核醣體疾病,活性氧物質,過氧化氫酶,超氧歧化酶, | zh_TW |
| dc.subject.keyword | ribosome,ribosomopathy,reactive oxygen species,catalase,superoxide dismutase, | en |
| dc.relation.page | 78 | |
| dc.identifier.doi | 10.6342/NTU202203568 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2022-09-23 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 農業化學研究所 | zh_TW |
| dc.date.embargo-lift | 2022-09-23 | - |
| 顯示於系所單位： | 農業化學系 | |
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