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系統識別號 U0026-2102201812353400
論文名稱(中文) 秀麗隱桿線蟲之RIOK-1激酶為p38 MAPK先天免疫路徑之免疫抑制分子
論文名稱(英文) RIOK-1 is a suppressor of the p38 MAPK innate immune pathway in Caenorhabditis elegans
校院名稱 成功大學
系所名稱(中) 基礎醫學研究所
系所名稱(英) Institute of Basic Medical Sciences
學年度 107
學期 1
出版年 107
研究生(中文) 陳怡偉
研究生(英文) Yi-Wei Chen
學號 S58024088
學位類別 博士
語文別 英文
論文頁數 93頁
口試委員 口試委員-高承源
口試委員-許鴻猷
召集委員-蔡佩珍
指導教授-陳昌熙
指導教授-柯文謙
口試委員-陳柏齡
中文關鍵字 riok-1激酶  p38 MAPK訊息傳遞路徑  轉錄因子skn-1  先天免疫力  恆定狀態  免疫抑制分子  達卡產氣單胞菌  秀麗隱桿線蟲 
英文關鍵字 riok-1  p38 MAPK  skn-1  innate immunity  homeostasis  immune suppressor  Aeromonas dhakensis  Caenorhabditis elegans 
學科別分類
中文摘要 先天免疫力的恆定狀態是後生動物抗感染防禦機制與免疫調節之關鍵。研究已證實先天免疫功能異常亢進對個體實為傷害。包含秀麗隱桿線蟲在內,後生動物具有之p38 MAPK先天免疫調控路徑扮演抵抗諸多感染的關鍵角色,然而現今對於參與調控維持免疫力恆定狀態所需之負調控因子仍所知甚寡。在以線蟲激酶資料庫為研究標的進行RNA干擾的實驗中,我們發現RIOK-1激酶是p38 MAPK免疫路徑未知的抑制分子,抑制riok-1激酶的表現竟使得線蟲對於達卡產氣單胞菌感染具有抵抗力。藉由使用riok-1 激酶轉殖基因線蟲株與即時定量聚合酶連鎖反應,我們也發現線蟲之riok-1激酶表現量會隨著感染達卡產氣單胞菌而增加。在使用遺傳學上位基因調控分析方法下,我們也判斷出riok-1激酶坐落於p38 MAPK訊息傳遞路徑上游,而且藉由抑制riok-1激酶竟可促進活化p38 MAPK訊息傳遞路。據此可推知riok-1激酶是線蟲先天免疫調控路徑之負調控因子。同樣藉由遺傳學上位基因調控分析方法,我們進一步釐清riok-1激酶同樣坐落於p38 MAPK訊息傳遞路徑下游之轉錄因子skn-1之上游,且轉錄因子skn-1又具有活化riok-1激酶能力,與p38 MAPK訊息傳遞路徑及riok-1激酶形塑出一反饋迴路。總結本研究,我們證實線蟲之riok-1激酶是一新發現的免疫抑制分子,以及存在於p38 MAPK訊息傳遞路徑、轉錄因子skn-1與riok-1激酶之間的反饋迴路模式。
英文摘要 The homeostasis of innate immunity is critical for the primary defense mechanism against infection and regulating immune response in metazoans. It has been proven that aberrant up-regulation of innate immune signaling pathways can be detrimental to the host. The p38 MAPK/PMK-1 innate immune signaling pathway has been demonstrated to play essential roles in cellular defenses against numerous infections in metazoans, including Caenorhabditis elegans. However, the negative regulators that maintain the homeostasis of this important innate immune pathway remain largely understudied. By screening a focused kinome RNAi library of C. elegans, we identified a human RIO kinase homolog RIOK-1 as a novel suppressor of the p38 MAPK/PMK-1 signal pathway. We showed that the suppression of riok-1 confers resistance to Aeromonas dhakensis infection in C. elegans. Using riok-1 reporter worms and qRT-PCR, we found the expression levels of riok-1 were significantly up-regulated in A. dhakensis infected worms. With genetic epistasis analysis of many immune pathways in C. elegans, we also discovered that riok-1 acts on the upstream of the p38 MAPK/pmk-1genetic pathway. Moreover, the suppression of riok-1 enhanced the activity of p38 MAPK, suggesting that riok-1 is a negative regulator of this innate immune pathway in C. elegans. The epistatic results also put riok-1 upstream of skn-1, which encodes a p38 MAPK downstream transcription factor, participates as an upstream activator to riok-1 and serves as a feedback loop to the p38 MAPK pathway during A. dhakensis infection. In conclusion, we proposed riok-1 is a novel innate immune suppressor and a negative feedback loop model involving p38 MAPK cascade, SKN-1, and RIOK-1 in C. elegans.
論文目次 目錄
中文摘要-----------iv
英文摘要-----------v
論文本文
Introduction
1. Caenorhabditis elegans -------1
2. Aeromonas dhakensis -------1
3. Kinome- kinase database of C. elegans -----2
4. Innate immunity of C. elegans ------2
5. RIOK-1 ---------4
6. Homeostasis --------4
Materials and Methods
1. Bacteria and C. elegans strain -----6
2. Screen of RNAi kinome library -----6
3. Lifespan assay of C. elegans with RNAi ----7
4. Lifespan assay of C. elegans with infection -----7
5. Measurement of gene expression ------8
6. Construction of the riok-1 reporter ------9
7. Tissue-specific RNAi -------9
8. p-p38 western blot --------9
9. Statistical analysis -------10
Results
1. RNAi screening of C. elegans kinome. -----11
2. Suppressing expression of riok-1 confers resistance against A. dhakensis infection in C. elegans. ------11
3. Expression level of riok-1 is increased under A. dhakensis infection in C. elegans. -------13
4. Knockdown of riok-1 in intestine and neurons conferred resistance to A. dhakensis in C. elegans. ------13
5. Progeny deficiency is not associated with resistance to A. dhakensis infection mediated by suppressing riok-1. ----14
6. RIOK-1 is a suppresser of the p38 MAPK immune pathway. -14
7. vhp-1 is not involved in the RIOK-1-supressed p38 MAPK signaling pathway upon A. dhakensis infection. ----15
8. RIOK-1 feeds back negatively to the p38 MAPK pathway through transcriptional factor skn-1 after A. dhakensis infection in C. elegans. ---------15
9. RIOK-1 deficiency in C. elegans increases the susceptibility to different gram-negative bacterial infections. -----17
Conclusion ----------18
Discussion
1. Kinases regulate immunity- activation and suppression --19
2. Homeostasis of innate immunity-----19
3. Potential mechanisms how riok-1 suppressed p38 MAPK pathway-20
4. The sites of action of riok-1 in innate immunity----21
5. The kinase activity of RIOK-1 in A. dhakensis infection--22
6. The up-regulation of riok-1 is a strategy to enhance the infection by pathogen---------22
7. The possible mechanism how A. dhakensis activates riok-1--23
8. The potential of riok-1 as a therapeutic target in infection--23
References ----------25
Tables
Table 1. The results of kinome RNAi screen. ----32
Table 2. Hit numbers of the kinome RNAi screen. ----41
Figures
Figure 1. Flowchart showing the screening of the RNAi kinome library in C. elegans. -------42
Figure 2. Loss of riok-1 confers resistance to Aeromonas dhakensis infection in C. elegans. -------43
Figure 3. The locations of riok-1 genetic tools. ----44
Figure 4. Expression levels of riok-1 isoforms by qRT-PCR. --45
Figure 5. Complementation of riok-1 returns the sensitivity to A. dhakensis infection. --------46
Figure 6. Lifespan of riok-1 over-expressed worms. ----47
Figure 7. Expression levels of riok-1 transcriptional reporter. ---48
Figure 8. Quantification of Figure 7. -----49
Figure 9. Expression levels of riok-1 translational reporter. --50
Figure 10. Quantification of Figure 9. ------51
Figure 11. riok-1 expression levels as determined by qRT-PCR. --52
Figure 12. Expression pattern of riok-1 in riok-1 transcriptional reporter.-53
Figure 13. Expression pattern of riok-1 in riok-1 translational reporter. -54
Figure 14. The tissue specificity of tissue-specific RNAi worm strains. -55
Figure 15. The sites of action of riok-1 in A. dhakensis infection. --56
Figure 16. Progeny deficiency is not associated with A. dhakensis resistance mediated by riok-1deletion in C. elegans. ---57
Figure 17. Screen of the well-known immune pathways epistatic to riok-1. ----------58
Figure 18. riok-1 lays on the upstream to p38 MAPK/pmk-1. --59
Figure 19. riok-1 is a suppressor laying on the upstream of p38 MAPK pathway. -------60
Figure 20. riok-1 suppresses the phosphorylation of p38 MAPK/pmk-1. –61
Figure 21. The expression levels of downstream genes of p38 MAPK pathway. --------62
Figure 22. C. elegans tyrosine-protein phosphatase vhp-1 is not involved in the p38 MAPK- RIOK-1 signaling pathway upon A. dhakensis infection. -------63
Figure 23. The increase in riok-1 is skn-1-dependent in C. elegans with Aeromonas dhakensis infection. -----64
Figure 24. riok-1 is an upstream suppressor of p38 MAPK/pmk-1 downstream transcription factor Nrf/skn-1. ---65
Figure 25. The expression level of riok-1 in p38 gain-of-function mutant worms nsy-1(ums8). ------66
Figure 26. The expression level of riok-1 in p38 MAPK/pmk-1 mutant worms. ---------67
Figure 27. Suppressed expression of riok-1 increases the resistance to pathogens in C. elegans. ------68
Figure 28. Model of a p38 MAPK, SKN-1, and RIOK-1 feedback loop upon infection in C. elegans. -----69
Figure 29. Enlargement of the neuron part in Figure 23A-D. ---70
附錄
簡歷----------71
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