博碩士論文 100284001 詳細資訊




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姓名 廖弘玉(Hung-Yu Liao)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 三硝基甲苯之毒理機制及生物降解暨多氯乙烯汙染模場生物整治
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摘要(中) 三硝基甲苯常被使用在軍事或名間爆破用途,已經有許多研究證明三硝基甲苯會殘留在土地或地下水中,由於三硝基甲苯具有毒性,而其毒性機制尚未澄清,本研究第一部分,使用人類肝癌細胞HepG2和Hep3B,證明三硝基甲苯會誘導自由基產生,造成DNA的損傷,如果長期有此壓力存在可能導致癌症,進一步我們發現在高濃度的三硝基甲苯培養條件下會導致細胞內質網壓力及粒線體的破壞,造成細胞死亡;第二部分,由於上述我們證實三硝基甲苯對細胞會造成毒性,因此清除環境中三硝基甲苯的污染刻不容緩,我們於台灣南部受三硝基甲苯汙染之廠址篩選出一株可降解三硝基甲苯之本土菌株Citrobacter sp.,我們發現Citrobacter sp. 可以將三硝基甲苯轉化成4-amino-2,6-dinitrotoluene (4-ADNT)和2-amino-4,6-dinitrotoluene (2-ADNT),並且我們利用轉錄體學結和蛋白質體學調查Citrobacter sp.在降解三硝基甲苯過程中的分子機制,發現在降解過程中中心代謝機制受到抑制,並且抵抗環境壓力相關基因(例如: chaperones, transport-related proteins, and membrane proteins)表現量增加,我們推測這些基因與Citrobacter sp. 的去毒性有關,最後我們純化降解三硝基甲苯的關鍵蛋白NemA,證實此蛋白質確實為降解三硝基甲苯之關鍵酵素;第三部分,我們將整治研究實際於一多氯乙烯污染場址施作,多氯乙烯是一種難以用化學或物理法去除的環境汙染物,我們於台南永康區一處多氯乙烯汙染模場,添加slow polycolloid releasing substrate (SPRS),進行生物刺激法整治並在整治過程中利用次世代定序取代傳統變性析度凝膠電泳分析調查環境菌相變化,結果證實SPRS確實可刺激降解多氯乙烯菌株生長並增加降解。
摘要(英) There have been several reports of widespread contamination of soil and groundwater resulting from explosives, such as 2,4,6- trinitrotoluene (TNT). The accumulation of TNT also presents potentially hazardous effects to both humans and animals. In chapter I, we examined TNT-induced apoptosis via ROS dependent mitochondrial dysfunction and ER stress in HepG2 and Hep3B cells. Chapter II, the bacterial strain Citrobacter sp. was isolated from 2,4,6-trinitrotoluene (TNT) contaminated soil. We investigated the transcriptomic and proteomic responses of Citrobacter sp. to TNT by comparing profiles at 0 h and 12 h to understand how Citrobacter sp. can survive and transform under TNT stress. Chapter III, Tetrachloroethene (PCE) and trichloroethene (TCE) are pollutants found in large quantities in industrial areas around the world. The slow polycolloid releasing substrate (SPRS) has been developed to continuously provide biodegradable substrates for the enhancement of TCE reductive dechlorination. We investigated microbial groundwater community before and after SPRS addition and proved that can be stimulated the dechlorinating.
關鍵字(中) ★ 生物降解 關鍵字(英) ★ Biodegradation
論文目次 Table of Contents
致謝 i
中文摘要 ii
ABSTRACT iii
Chapter I-2,4,6-Trinitrotoluene Induces Apoptosis via ROS-Regulated Mitochondrial Dysfunction and Endoplasmic Reticulum Stress in HepG2 and Hep3B Cells 1
ABSTRACT 2
INTRODUCTION 3
MATERIALS AND METHODS 5
RESULTS 11
DISCUSSION 16
Chapter II-Biodegradation of 2, 4, 6-Trinitrotoluene by Citrobacter sp. 20
ABSTRACT 21
INTRODUCTION 23
MATERIALS AND METHODS 25
RESULTS 32
DISCUSSION 38
Chapter Ⅲ-The Change of Microbial Community from Chlorinated Solvent-Contaminated Groundwater after Biostimulation using the Metagenome Analysis 44
ABSTRACT 45
INTRODUCTION 46
MATERIALS AND METHODS 49
RESULTS AND DISCUSSION 54
REFERENCES 59
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List of Figures
Figure 1. TNT-induced cytotoxicity, DNA damage and apoptosis in HepG2 cells in a dose- and-time dependent manner... 70
Figure 2. TNT-induced ER stress in HepG2 cells. 72
Figure 3. TNT triggered the mitochondrial dysfunction and apoptosis pathways. 73
Figure 4. TNT induced the generation of ROS. 74
Figure 5. ROS-dependent ER stress was involved in the TNT-mediated ROS production. 75
Figure 6. Effects of CHOP siRNA on TNT-induced ER stress in HepG2 cells. 76
Figure 7. The schematic representation of proposed mechanisms according to TNT-induced apoptosis in HepG2 cells. 77
Figure 8. Growth curve of Citrobacter sp.in TNT-NFG medium. 78
Figure 9. TNT biodegradation by Citrobacter sp.. 79
Figure 10. Representative two-dimensional gel electrophoresis (2-DE) gel images (pH 4-7) of whole cell lysates of Citrobacter sp. 80
Figure 11. Identification of differential abundances of proteins of Citrobacter sp. in TNT-NFG medium at different time. 81
Figure 12. Functional classification of 42 protein spots 82
Figure 13. Proteins with significant differential abundances sorted by COG categories. 83
Figure 14. Venn diagram showing differentially expressed genes and proteins of Citrobacter sp. 84
Figure 15. Plasmid drawing of pET21b-NemA and SDS-PAGE of His6-NemA. 85
Figure 16. Time course of contamination levels of PCE,TCE,DCE,VC and ethene in monitoring well. 86
Figure 17. The graph showed the most abundant genera according to the RDP classification for V1-V3 region, in 16S rRNA sequences. 87
Figure 18. Metagenome reads were mapped on genomes of bacteria using LCA classification approach. 88
Figure 19. The predicted enzymes were mapped on the TCE biodegradation pathways
89

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List of Supplementary Figures
Supplementary Figure S1. ROS-dependent ER stress was involved in the TNT-mediated ROS production.... 97
Supplementary Figure S2. HepG2 cells were transfected with CHOP siRNA for 24h, and analysis by western blot. 98
Supplementary Figure S3. Differentially expressed gene profile, according to clusters of orthologous groups of proteins (COG) analysis.. 99
Supplementary Figure S4. The predicted genes were mapped on the glycolysis/gluconeogenesis pathways. 100
Supplementary Figure S5. The predicted genes were mapped on the oxidative phosphorylation pathways. 101
Supplementary Figure S6. Site map showing the groundwater flow direction 102
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List of Tables
Table 1. Draft genome assembly statistics of Citrobacter sp.... 90
Table 2. Summary of reads generation. 90
Table 3. Functional categorization of differentially expressed proteins of Citrobacter sp. in TNT-NFG medium at different time.. 91
Table 4. A list of common differentially expressed genes between transcriptome and proteome.. 94
Table 5. Geochemical characteristics of groundwater in monitoring wells. 95
Table 6. Mapping of assembled reads on xenobiotic degradation pathways present in KEGG database. 96
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List of Supplementary Tables
Supplementary Table S1. Primer list... 104
Supplementary Table S4. Comparison of RNA-seq results by quantitative PCR on differentially expressed genes. 105
Supplementary Table S5. Differential Pathway detected in TNT (30 μM). 106
Supplementary Table S6. Differential Pathway detected in TNT (80 μM). 108
Supplementary Table S8. Primer list 111
Supplementary Table S10. Comparison of RNA-seq results by quantitative PCR on differentially expressed genes. 112
Supplementary Table S14. The most abundant genera according to the RDP classification approach. 113
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指導教授 陳師慶、喻秋華(Ssu-Ching Chen Chiou-Hwa Yuh) 審核日期 2017-9-21
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