臺灣博碩士論文加值系統

一氧化氮(NO)在生物體中扮演重要功能，哺乳類生物的NO與細胞訊息傳遞、免疫反應有關，而在細菌中NO則被發現具有保護免於氧化傷害之作用。另外在其他物種如真菌、藻類及原生動物等都發現可能具有一氧化氮合成酶(NOS)的活性。有學者指出雖然NOS的功能隨著生物演化，但其結構和作用機制並未有太大改變。從2006年Harris的實驗中，發現陰道滴蟲可能具有NOS的功能，但其基因體未被註解出NOS。因此，本研究目的為：(1)使用NCBI protein資料庫提供之所有物種NOS蛋白質序列及陰道滴蟲全部蛋白質序列，以生物資訊學方法比對，預測出陰道滴蟲NOS序列TV1及TV2，並經由實驗驗證其功能。(2) 了解生物資訊學方法之比對正確預測性，分別對陰道滴蟲已知蛋白比對進行內部驗證，以及對其他物種NOS比對進行外部驗證，證明本研究方法之可行性。研究方法使用跨物種之蛋白質序列與物種全序列列以Smith-Waterman演算法區域排比，設定選取分數大於100分，以進行後續次數比例判讀。本研究實驗結果發現TV1及TV2具有NOS活性反應，且經由內部驗證及外部驗證的方式可證明本研究分析方法之可行性，預期未來能發展出一套有效註解物種未知蛋白質的方式。

Nitric oxide (NO) plays an important role in the organism. The mammals’ NO is related to signal transduction in cells and immune response, while NO in bacteria is found to protect against oxidative damage. Previous studies have shown that nitric oxide synthase (NOS) may be widely found in many organisms, from bacteria to animals; also, a few in algae, protozoa, fungi and other organisms have found a similar sequence structure of NOS or perform NO activity. Though the biological evolution, its structure and mechanism are only slightly changed. The genome of Trichomonas vaginalis has been published in 2007, although it has not yet been annotated NOS-related genes. However, some research has demonstrated its ability to produce NO. So, we want to annotate the sequences of nitric oxide synthase in Trichomonas vaginalis. Data are downloaded from NCBI protein database. Total 120,757 records of T. vaginalis and 6,631 of NOS protein sequences are aligned by Smith-Waterman algorithm. We also designed two ways of internal and external validation to verify our method. Two sequences (Tv1, Tv2) identified as [Iron only hydrogenase large subunit, C-terminal domain containing protein] are annotated as NOS in T. vaginalis in our study, and it needs experiment validation in further research. In conclusion, we establish an effective way to annotate unknown protein in the organisms, and provide the researchers to find candidate sequences in interest for experiments.

目　　錄
目　　錄 I
表目錄 IV
圖目錄 VI
附錄目錄 XI
摘 要 1
Abstract 2
第一章　緒論 3
第一節 研究背景與重要性 3
第二節 研究動機 7
第三節 研究目的 11
第二章 文獻探討 12
第一節 一氧化氮合成酶 12
一、 結構與生化特性 12
二、 不同物種之一氧化氮合成酶 14
第二節 陰道滴蟲 21
一、 陰道滴蟲簡介 21
二、 陰道滴蟲感染宿主方式 21
三、 陰道滴蟲感染之流行病學特性 22
四、 陰道滴蟲基因體註解 23
第三節 陰道滴蟲與一氧化氮合成酶 25
第四節 NOS蛋白質活性測試方法 27
第三章 材料與方法 30
第一節 研究架構 30
第二節 研究假設 31
第三節 研究方法 32
一、 資料來源 32
二、 資料比對與分析方法 36
三、 實驗方法 39
第四章 結果 45
第一節 陰道滴蟲之一氧化氮合成酶預測 45
一、 序列比對結果 45
二、 實驗驗證結果 55
第二節 內部驗證結果 62
一、 陰道滴蟲「磷酸二脂磷酸β-葡萄糖基轉移酶」之驗證 62
二、 陰道滴蟲「葡萄糖激酶」之驗證 68
三、 陰道滴蟲「焦磷酸--果糖-6-磷酸磷酸基團轉移酶」之驗證 73
四、 陰道滴蟲「琥珀酸輔酶A連接酶」之驗證 83
第三節 外部驗證結果 92
一、 「多頭絨泡黏菌」一氧化氮合成酶之驗證 92
二、 「綠藻」一氧化氮合成酶之驗證 96
三、 「米麴黴」一氧化氮合成酶之驗證 100
四、 「利什曼原蟲」一氧化氮合成酶之驗證 104
五、 「梨形鞭毛蟲」一氧化氮合成酶之驗證 108
第五章、討論 112
第一節 陰道滴蟲之NOS序列比對結果 112
第二節 陰道滴蟲之NOS功能驗證 128
第三節 內部驗證結果探討 130
第四節 外部驗證結果探討 134
第五節 反面結果驗證 140
第六節 序列比對結果判定標準 142
第六章、結論 145
參考文獻 146
附錄 150

邱怡嘉, 魏夢麗, 呂椿棠, 陳政道, 呂秀英, Chiu, Y. C., . . . Lu, H. Y. 表現序列標幟(EST)功能註解分析的新流程
A New Procedure of Functional Annotation and Analysis in Expressed Sequence Tag (EST). 49-64.
莊樹諄. (2005). 大海撈針-尋找基因的方法. 科學發展 396.
劉秀鈴. (2012). 於NCBI資料庫中以Smith-Waterman演算法比對出陰道滴蟲之目標基因並加以驗證–以一氧化氮合成酶為例. (碩士), 國防醫學院, 台北市.
Alderton, W. K., Cooper, C. E., & Knowles, R. G. (2001). Nitric oxide synthases: structure, function and inhibition. Biochemical Journal, 357(3), 593-615.
Almeida, B., Buttner, S., Ohlmeier, S., Silva, A., Mesquita, A., Sampaio-Marques, B., . . . Ludovico, P. (2007). NO-mediated apoptosis in yeast. Journal of Cell Science, 120(18), 3279-3288. doi:10.1242/jcs.010926
Andreakis, N., D'Aniello, S., Albalat, R., Patti, F. P., Garcia-Fernandez, J., Procaccini, G., . . . Palumbo, A. (2011). Evolution of the nitric oxide synthase family in metazoans. Mol Biol Evol, 28(1), 163-179. doi:10.1093/molbev/msq179
Basu, N. K., Kole, L., Ghosh, A., & Das, P. K. (1997). Isolation of a nitric oxide synthase from the protozoan parasite, Leishmania donovani. Fems Microbiology Letters, 156(1), 43-47. doi:DOI 10.1111/j.1574-6968.1997.tb12703.x
Carlton, J. M., Hirt, R. P., Silva, J. C., Delcher, A. L., Schatz, M., Zhao, Q., . . . Johnson, P. J. (2007). Draft genome sequence of the sexually transmitted pathogen Trichomonas vaginalis. Science, 315(5809), 207-212. doi:10.1126/science.1132894
Cheng, W. H., Huang, K. Y., Huang, P. J., Hsu, J. H., Fang, Y. K., Chiu, C. H., & Tang, P. (2015). Nitric oxide maintains cell survival of Trichomonas vaginalis upon iron depletion. Parasit Vectors, 8, 393. doi:10.1186/s13071-015-1000-5
Correa-Aragunde, N., Foresi, N., & Lamattina, L. (2013). Structure diversity of nitric oxide synthases (NOS): the emergence of new forms in photosynthetic organisms. Frontiers in Plant Science, 4, 232. doi:10.3389/fpls.2013.00232
Davis, A., Dasgupta, A., Goddard-Eckrich, D., & El-Bassel, N. (2016). Trichomonas vaginalis and Human Immunodeficiency Virus Coinfection Among Women Under Community Supervision: A Call for Expanded T. vaginalis Screening. Sex Transm Dis, 43(10), 617-622. doi:10.1097/OLQ.0000000000000503
Foresi, N., Correa-Aragunde, N., Parisi, G., Calo, G., Salerno, G., & Lamattina, L. (2010). Characterization of a Nitric Oxide Synthase from the Plant Kingdom: NO Generation from the Green Alga Ostreococcus tauri Is Light Irradiance and Growth Phase Dependent. Plant Cell, 22(11), 3816-3830. doi:10.1105/tpc.109.073510
Foresi, N., Correa-Aragunde, N., Santolini, J., & Lamattina, L. (2016). Analysis of the Expression and Activity of Nitric Oxide Synthase from Marine Photosynthetic Microorganisms. Methods Mol Biol, 1424, 149-162. doi:10.1007/978-1-4939-3600-7_13
Golderer, G., Werner, E. R., Leitner, S., Grobner, P., & Werner-Felmayer, G. (2001). Nitric oxide synthase is induced in sporulation of Physarum polycephalum. Genes & Development, 15(10), 1299-1309. doi:DOI 10.1101/gad.890501
Gorren, A. C. F., & Mayer, B. (2007). Nitric-oxide synthase: A cytochrome P450 family foster child. Biochimica Et Biophysica Acta-General Subjects, 1770(3), 432-445. doi:10.1016/j.bbagen.2006.08.019
Gotoh, O., Morita, M., & Nelson, D. R. (2014). Assessment and refinement of eukaryotic gene structure prediction with gene-structure-aware multiple protein sequence alignment. BMC Bioinformatics, 15, 189. doi:10.1186/1471-2105-15-189
Gould, S. B., Woehle, C., Kusdian, G., Landan, G., Tachezy, J., Zimorski, V., & Martin, W. F. (2013). Deep sequencing of Trichomonas vaginalis during the early infection of vaginal epithelial cells and amoeboid transition. Int J Parasitol, 43(9), 707-719. doi:10.1016/j.ijpara.2013.04.002
Harp, D. F., & Chowdhury, I. (2011). Trichomoniasis: evaluation to execution. Eur J Obstet Gynecol Reprod Biol, 157(1), 3-9. doi:10.1016/j.ejogrb.2011.02.024
Harris, K. M., Goldberg, B., Biagini, G. A., & Lloyd, D. (2006). Trichomonas vaginalis and Giardia intestinalis produce nitric oxide and display NO-synthase activity. J Eukaryot Microbiol, 53 Suppl 1, S182-183. doi:10.1111/j.1550-7408.2006.00224.x
Hirt, R. P., & Sherrard, J. (2015). Trichomonas vaginalis origins, molecular pathobiology and clinical considerations. Curr Opin Infect Dis, 28(1), 72-79. doi:10.1097/QCO.0000000000000128
Holden, J. K., Li, H. Y., Jing, Q., Kang, S., Richo, J., Silverman, R. B., & Poulos, T. L. (2013). Structural and biological studies on bacterial nitric oxide synthase inhibitors. Proceedings of the National Academy of Sciences of the United States of America, 110(45), 18127-18131. doi:10.1073/pnas.1314080110
Huang, K. Y., Chen, Y. Y., Fang, Y. K., Cheng, W. H., Cheng, C. C., Chen, Y. C., . . . Tang, P. (2014). Adaptive responses to glucose restriction enhance cell survival, antioxidant capability, and autophagy of the protozoan parasite Trichomonas vaginalis. Biochim Biophys Acta, 1840(1), 53-64. doi:10.1016/j.bbagen.2013.08.008
Jeandroz, S., Wipf, D., Stuehr, D. J., Lamattina, L., Melkonian, M., Tian, Z., . . . Wendehenne, D. (2016). Occurrence, structure, and evolution of nitric oxide synthase-like proteins in the plant kingdom. Sci Signal, 9(417), re2. doi:10.1126/scisignal.aad4403
Johnston, V. J., & Mabey, D. C. (2008). Global epidemiology and control of Trichomonas vaginalis. Curr Opin Infect Dis, 21(1), 56-64. doi:10.1097/QCO.0b013e3282f3d999
Karnkowska, A., Vacek, V., Zubacova, Z., Treitli, S. C., Petrzelkova, R., Eme, L., . . . Hampl, V. (2016). A Eukaryote without a Mitochondrial Organelle. Current Biology, 26(10), 1274-1284. doi:10.1016/j.cub.2016.03.053
Kumar, A., Castellano, I., Patti, F. P., Palumbo, A., & Buia, M. C. (2015). Nitric oxide in marine photosynthetic organisms. Nitric Oxide, 47, 34-39. doi:10.1016/j.niox.2015.03.001
Leitsch, D. (2016). Recent Advances in the Trichomonas vaginalis Field. F1000Res, 5. doi:10.12688/f1000research.7594.1
Loshchinina, E. A., & Nikitina, V. E. (2016). Role of the NO synthase system in response to abiotic stress factors for basidiomycetes Lentinula edodes and Grifola frondosa. Microbiology, 85(2), 165-171. doi:10.1134/S0026261716020120
Marcos, A. T., Ramos, M. S., Marcos, J. F., Carmona, L., Strauss, J., & Canovas, D. (2016). Nitric oxide synthesis by nitrate reductase is regulated during development in Aspergillus. Molecular Microbiology, 99(1), 15-33. doi:10.1111/mmi.13211
Martin, W., & Mentel, M. (2010). The origin of mitochondria. Nature Education, 3(9), 58.
Mercer, F., Diala, F. G., Chen, Y. P., Molgora, B. M., Ng, S. H., & Johnson, P. J. (2016). Leukocyte Lysis and Cytokine Induction by the Human Sexually Transmitted Parasite Trichomonas vaginalis. PLoS Negl Trop Dis, 10(8), e0004913. doi:10.1371/journal.pntd.0004913
Meyer, I. M., & Durbin, R. (2004). Gene structure conservation aids similarity based gene prediction. Nucleic Acids Res, 32(2), 776-783. doi:10.1093/nar/gkh211
Morada, M., Manzur, M., Lam, B., Tan, C., Tachezy, J., Rappelli, P., . . . Yarlett, N. (2010). Arginine metabolism in Trichomonas vaginalis infected with Mycoplasma hominis. Microbiology-Sgm, 156, 3734-3743. doi:10.1099/mic.0.042192-0
Nahrevanian, H. (2009). Involvement of nitric oxide and its up/down stream molecules in the immunity against parasitic infections. Braz J Infect Dis, 13(6), 440-448.
Newman, L., Rowley, J., Vander Hoorn, S., Wijesooriya, N. S., Unemo, M., Low, N., . . . Temmerman, M. (2015). Global Estimates of the Prevalence and Incidence of Four Curable Sexually Transmitted Infections in 2012 Based on Systematic Review and Global Reporting. PLoS One, 10(12), e0143304. doi:10.1371/journal.pone.0143304
Nishimura, A., Kawahara, N., & Takagi, H. (2013). The flavoprotein Tah18-dependent NO synthesis confers high-temperature stress tolerance on yeast cells. Biochemical and Biophysical Research Communications, 430(1), 137-143. doi:10.1016/j.bbrc.2012.11.023
Paveto, C., Pereira, C., Espinosa, J., Montagna, A. E., Farber, M., Esteva, M., . . . Torres, H. N. (1995). The Nitric-Oxide Transduction Pathway in Trypanosoma-Cruzi. Journal of Biological Chemistry, 270(28), 16576-16579.
Pengkit, A., Jeon, S. S., Son, S. J., Shin, J. H., Baik, K. Y., Choi, E. H., & Park, G. (2016). Identification and functional analysis of endogenous nitric oxide in a filamentous fungus. Sci Rep, 6, 30037. doi:10.1038/srep30037
Poole, D. N., & McClelland, R. S. (2013). Global epidemiology of Trichomonas vaginalis. Sex Transm Infect, 89(6), 418-422. doi:10.1136/sextrans-2013-051075
Promponas, V. J., Iliopoulos, I., & Ouzounis, C. A. (2015). Annotation inconsistencies beyond sequence similarity-based function prediction - phylogeny and genome structure. Standards in Genomic Sciences, 10. doi:ARTN 108
10.1186/s40793-015-0101-2
Rafferty, S. (2011). Nitric oxide synthases of bacteria and other unicellular organisms. Open Nitric Oxide J, 3, 25-32.
Rosano, G. L., & Ceccarelli, E. A. (2014). Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol, 5, 172. doi:10.3389/fmicb.2014.00172
Samalova, M., Johnson, J., Illes, M., Kelly, S., Fricker, M., & Gurr, S. (2013). Nitric oxide generated by the rice blast fungus Magnaporthe oryzae drives plant infection. New Phytologist, 197(1), 207-222. doi:10.1111/j.1469-8137.2012.04368.x
Sanger, F., Nicklen, S., & Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A, 74(12), 5463-5467.
Sarti, P., Fiori, P. L., Forte, E., Rappelli, P., Teixeira, M., Mastronicola, D., . . . Brunori, M. (2004). Trichomonas vaginalis degrades nitric oxide and expresses a flavorubredoxin-like protein: a new pathogenic mechanism? Cell Mol Life Sci, 61(5), 618-623. doi:10.1007/s00018-003-3413-8
Schnoes, A. M., Brown, S. D., Dodevski, I., & Babbitt, P. C. (2009). Annotation Error in Public Databases: Misannotation of Molecular Function in Enzyme Superfamilies. Plos Computational Biology, 5(12). doi:ARTN e1000605
10.1371/journal.pcbi.1000605
Stadelmann, B., Hanevik, K., Andersson, M. K., Bruserud, O., & Svard, S. G. (2013). The role of arginine and arginine-metabolizing enzymes during Giardia - host cell interactions in vitro. BMC Microbiol, 13, 256. doi:10.1186/1471-2180-13-256
Surdel, M. C., Dutter, B. F., Sulikowski, G. A., & Skaar, E. P. (2016). Bacterial Nitric Oxide Synthase Is Required for the Staphylococcus aureus Response to Heme Stress. Acs Infectious Diseases, 2(8), 572-578. doi:10.1021/acsinfecdis.6b00081
Wang, B. S., Xi-Shengb; Zhang,Sheng-Huaa. (2005). 一氧化氮合酶的结构及其催化机理
Structure of Nitric Oxide Synthase and Its Catalytic Mechanism. Chinese Journal of Organic Chemistry, 25(3), 342-345.
Wetterstrand, K. (2016). DNA Sequencing Costs: Data from the NHGRI Genome Sequencing Program (GSP). Retrieved from https://www.genome.gov/sequencingcostsdata/
WHO. (2012). Global incidence and prevalence of selected sexually transmitted infections– 2008. Retrieved from http://www.who.int/reproductivehealth/publications/rtis/stisestimates/en/
Yamasaki, H., Watanabe, N. S., Sakihama, Y., & Cohen, M. F. (2016). An Overview of Methods in Plant Nitric Oxide (NO) Research: Why Do We Always Need to Use Multiple Methods? Methods Mol Biol, 1424, 1-14. doi:10.1007/978-1-4939-3600-7_1
Yandell, M., & Ence, D. (2012). A beginner's guide to eukaryotic genome annotation. Nat Rev Genet, 13(5), 329-342. doi:10.1038/nrg3174
Yarlett, N., & Bacchi, C. J. (1994). Parasite Polyamine Metabolism - Targets for Chemotherapy. Biochemical Society Transactions, 22(4), 875-879.
Zhu, Z., Davidson, K. T., Brittingham, A., Wakefield, M. R., Bai, Q., Xiao, H., & Fang, Y. (2016). Trichomonas vaginalis: a possible foe to prostate cancer. Med Oncol, 33(10), 115. doi:10.1007/s12032-016-0832-y