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研究生:林宥成
研究生(外文):Yu-Cheng Lin
論文名稱:以新發展之微生物泳動分析演算法揭示 Haloarcula marismortui 之光趨性
論文名稱(外文):Phototaxis of Haloarcula marismortui Revealed through a Novel Microbial Motion Analysis Algorithm
指導教授:楊啓伸
指導教授(外文):Chii-Shen Yang
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:微生物與生化學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:英文
論文頁數:105
中文關鍵詞:Haloarcula marismortui光趨性微生物泳動分析演算法
外文關鍵詞:Haloarcula marismortuiphototaxismicrobial motion analysis algorithm
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過去微生物學家認為Haloarcula marismortui 不會泳動,然而近年的研究證明了H. marismortui 鞭毛的存在,顯示其具有泳動能力。本研究觀察H. marismortui 的活菌泳動現象,並以ImageJ 追蹤其泳動軌跡 (swimming trajectory)。由於其軌跡相當雜噪 (noisy),因此難以適用於前人所建立的泳動分析演算法 (motion analysis algorithm)。在此我提出 “window vector” 的概念,以Microscope Excel-VBA 程式語言建立一個微生物泳動分析演算法 (microbial motion analysis algorithm),具有以下功能:(1) 不需以實驗結果個別化估算分析參數 (empirical parameter customization),而用冪次率關係 (power-law relationship) 自動分辨細胞為泳動或非泳動;(2) 減低布朗運動對泳動軌跡造成的雜訊 (noise),因此提升泳動折返 (swim reversal) 判斷的準確性。藉此演算法,本研究證明了HmSRI 和HmSRII 這兩個近來被確認的感光視紫質 (sensory rhodopsin) 的生理功能,分別是趨光性 (photoattractant) 和避光性 (photorepellent) 反應。H. marismortui 是除了 Halobacterium salinarum 外,唯一被確認具有光趨性 (phototaxis) 的古生菌。

Haloarcula marismortui has been described to be nonmotile prior to the recent identification of flagellar filaments, suggesting the motile nature of H. marismortui. Here we observed the locomotion of freshly cultured H. marismortui cells and tracked the swimming trajectories via ImageJ. Trajectories of H. marismortui are intrinsically noisy, posing difficulties in motion analysis with previously established algorithms. By introducing the concept of ‘‘window vector,’’ a Microsoft Excel-VBA-implemented microbial motion analysis algorithm reported here was able to (1) discriminate nonswimming objects from swimming cells without empirical customization by applying a power-law relationship and (2) reduce the noise caused by Brownian motion, thus enhancing the accuracy of swim reversal identification. Based on this motion analysis algorithm, two recently identified sensory rhodopsins, HmSRI and HmSRII, were shown to mediate photoattractant and photorepellent responses, respectively, revealing the phototactic activity of H. marismortui, the only archaeon showing such phenomenon other than Halobacterium salinarum.

口試委員會審定書…i
謝誌…ii
Contents…iii
List of Figures …vi
List of Tables…viii
中文摘要…ix
Abstract…x
Chapter 1 Introduction…1
1.1 Haloarcula marismortui, a halophilic archaeon from the Dead Sea…1
1.2 Genome sequencing of H. marismortui implied a phototactic nature…4
1.3 Mechanism of swim-reversal motility – a biased random walk…7
1.4 Brief introduction of microbial rhodopsins…9
1.5 Biophysical properties of the six rhodopsins in H. marismortui…14
1.6 Predictions for the physiologic functions of the three sensory rhodopsins,
HmSRI, HmSRII and HmSRM…17
1.7 Main questions and framework of this study…18
Chapter 2 Materials and Methods…22
2.1 Motile cell culture and sample preparation…22
2.1.1 Preparation of Halomedium…22
2.1.2 Preparation of swarm plate…22
2.1.3 Motile cell culture…22
2.2 Flagella stain…23
2.3 Hardware…25
2.4 Software…28
2.4.1 Image processing with ImageJ…29
2.4.2 Particle tracking…35
2.4.3 The concept of “window vector”…38
2.4.4 Trajectory discrimination using the power law…42
2.4.5 Mathematical descriptions of the power law…45
2.4.6 User manual of the Excel-VBA program…49
2.4.7 An overview of the motion analysis software system…53
2.5 Verification of the motion analysis system…56
Chapter 3 Results…57
3.1 The swimming pattern and autonomous reversal rate of freshly cultured
H. marismortui cells in the dark…57
3.2 Pulse illumination experiments demonstrate HmSRII mediates photorepellent
responses…60
3.3 Continuous illumination experiments demonstrate HmSRI and HmSRII
mediate photoattractant and photorepellent responses, respectively…62
3.4 The photorepellent signal of HmSRII was antagonized by HmSRI…63
3.5 HmSRM does not mediate phototactic responses…65
3.5.1 Activation of HmSRM did not elicit distinct phototactic effect…66
3.5.2 Simultaneous activation of HmSRM and HmSRI / HmSRII did not
elicit distinct phototactic effect…69
3.5.3 The signaling kinetics of HmSRII was not affected by HmSRM…71
3.6 Conclusions…73
Chapter 4 Discussion…74
4.1 Advantages of the concept of window vector in noise reduction…74
4.2 Estimation of the optimal window width…74
4.3 Advantage of using power law for trajectory discrimination…75
4.4 Prerequisites for accurate trajectory discrimination…76
4.5 Potential methods to reveal the physiologic function of HmSRM…77
4.6 Future perspectives…77
Reference…79
Appendices…85
Appendix 1. Thesis defense PowerPoint slides…85
Appendix 2. Thesis defense questions and answers…97
Appendix 3. Poster…105


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