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研究生：	吳博仁 Wu, Po-Jen
論文名稱：	探討異染色質於果蠅初級免疫反應中的角色 The role of heterochromatin in Drosophila innate immune response
指導教授：	顏賢章 Yan, Shian-Jang
學位類別：	碩士 Master
系所名稱：	醫學院 - 生理學研究所 Department of Physiology
論文出版年：	2015
畢業學年度：	103
語文別：	英文
論文頁數：	34
中文關鍵詞：	異染色質、初級免疫、綠膿桿菌
外文關鍵詞：	Heterochromatin, Innate immune, Pseudomonas aeruginosa
相關次數：	點閱：122 下載：0
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先天免疫系統在對抗入侵病原體的感染中扮演著重要角色。而表觀遺傳包含了染色質結構的改變，能區分成「寬鬆的」「活潑的」真染色質(Euchromatin) 與「緊密的」「沉默的」異染色質(Heterochromatin) 狀態，它們管理著生理上基因的表達。但是，我們對於Heterochromatin在調控先天免疫反應的分子和細胞層次機制仍然不清楚。在此論文中，我們利用果蠅作為模式生物系統，探討Heterochromatin的形成是否會調控有毒性的人類病原體「革蘭氏陰性綠膿桿菌(Pseudomonas aeruginosa) PA14」的抗菌反應和如何調控。Heterochromatin的提升有助於生物體在綠膿桿菌PA14感染下的存活，而Heterochromatin的減少則會導致其在感染後的存活時間縮短。而Heterochromatin的程度會與抗菌胜肽(Antimicrobial peptide) Drosomycin的活性有關。並且，我們發現Imd免疫訊息傳遞中重要的負調控分子Pirk，其基因的表達在Heterochromatin高的果蠅中會下降，而在heterochromatin低的果蠅中會上升。總結以上結果，我們認為Heterochromatin的形成能抑制pirk基因的表達進而促進先天的抗菌反應，並期待此研究能對於預防及治療人類的感染性疾病有所貢獻。

Innate immune system plays an important role against infections of invading pathogens. Epigenetic mechanisms involving the regulation of chromatin structures, which are divided into “loose” “active” euchromatin and “compact” “silent” heterochromatin states, govern gene expression of physiological systems. However, the molecular and cellular mechanisms by which heterochromatin regulates innate immune responses remain unclear. Here, using Drosophila as the model system, we determined whether and how heterochromatin formation mediates antimicrobial responses to the human pathogen, Pseudomonas aeruginosa PA14, which is Gram-negative and highly virulent. Upon PA14 infection, animals with increased heterochromatin levels survived longer, whereas decreasing heterochromatin exhibits shortened life span. Heterochromatin levels are associated with the activity of the antimicrobial peptide (AMP) gene Drosomycin. Moreover, we found that pirk, an important negative regulator of the Imd pathway, was downregulated in flies with more heterochromatin, and up-regulated in flies with less heterochromatin. Taken together, these data suggest that heterochromatin formation promotes innate antimicrobial responses via downregulating pirk. Our work may contribute to the prevention and treatment of human infectious diseases.

中文摘要	I
Abstract	II
致謝	III
Contents	IV
Figure index	VI
Introduction	1
1.	Drosophila as a powerful model system for innate immunity studies	1
2.	Epigenetics in innate immunity	2
3.	To investigate the role of heterochromatin in innate immune responses against Pseudomonas aeruginosa	3
Materials and Methods	5
1. Fly strains and crosses	5
2. Bacterial stocks and infection	6
3. Survival analysis	6
4. RNA expression analysis	7
5. Bacterial growth and quantification	7
Results	8
1.	Heterochromatin Protein 1 (HP1) promotes antimicrobial responses upon P. aeruginosa infection	8
2.	Heterochromatin formation enhances antimicrobial responses against bacterial infection via pirk down-regulation	11
3.	Fibrillarin regulated heterochromatin-mediated gene silencing, and promoted the survival upon P. aeruginosa PA14 infection...	13
Discussion 	15
Reference 	18
Figures 	23
 
Figure index

Figure 1. P. aeruginosa PA14 infection via needle pricking enhances host mortality during 3 to 5 days post-inoculation. .....23
Figure 2. Heterochromatin formation enhances Drosophila survival in response to bacterial infection. ..........24
Figure 3. Heterochromatin formation promotes antimicrobial responses against bacterial growth in Drosophila. .......26
Figure 4. Heterochromatin formation upregulates expression of Drosomycin in flies upon bacterial infection. ........27
Figure 5. Figure 5. HP1 silencing upregulates expression of pirk involved in the Imd pathway.  .............28
Figure 6. The model of HP1-mediated pirk gene expression in Imd signaling of Drosophila innate immune. ........29

Supplement
Figure S1. The larval lethal phenotype of Fibrillarin (Fib) homozygous mutants. ...........30
Figure S2. The quantification of phenotype of DX1 and 118E-10 PEV gene by eye pigment. ..........31
Figure S3. The quantification of phenotype of wm4 PEV gene by eye pigment. ...........32
Figure S4. The quantification of phenotype of SbV PEV gene by stubble bristles.  ............33
Figure S5. Heterochromatin regulate Drosophila anti-microbial responses against P. aeruginosa infection-induced mortality. .....34
                                    

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