定義新型前列腺癌致癌及轉移驅動因素

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姓名	荷娜(Nurul Hayati) 查詢紙本館藏	畢業系所	生命科學系
論文名稱	定義新型前列腺癌致癌及轉移驅動因素 (Define Novel Prostate Cancer Oncogenic and Metastasis Driver)
檔案	[Endnote RIS 格式] [Bibtex 格式] [相關文章] [文章引用] [完整記錄] [館藏目錄] 至系統瀏覽論文 (2028-6-25以後開放)
摘要(中)	前列腺癌（PCa）仍然是發病和死亡的一個重要原因，病患帶有癌轉移腫瘤與較差的存活率有關。我們研究的目標是確定新的治療策略並改善癌轉移患者的預後。採用多方面的方法，結合臨床基因體分析、in vitro 和in vivo 實驗，發現前列腺癌（PCa）中新的致癌驅動因素和轉移促進因素，我們專注於一個精氨酸 N-降解決定子途徑相關的基因，稱為WDYHV1 (NTAQ1)。我們透過慢病毒轉染在LNCaP 前列腺癌細胞株過度表現該基因，證明了該基因的致癌功能，例如刺激增殖、集落形成、3D 侵襲和跨孔遷移。並且，我們發現WDYHV1 (NTAQ1)促進在NSG 免疫缺陷小鼠上的異質腫瘤生長與癌轉移。轉錄體分析顯示有顯著差異的585 個基因表現，透過路徑分析表明，上調基因參與上皮間質轉化 (EMT) 和透過NF-κB 進行的TNF-α 訊號傳導。這些發現凸顯了 WDYHV1 可作為開發減輕轉移性前列腺癌的標靶療法的有前途的候選基因。我未來的研究將專注於透過驗證轉錄體分析的發現和與尋找精氨酸 N-降解決定子調節相關的 WDYHV 直接受質來闡明潛在致癌機制。
摘要(英)	Prostate cancer (PCa) remains a significant cause of morbidity and mortality, and the presence of metastasis is linked to poor survival rates. The goal of our study is to identify novel therapeutic strategies and improve outcomes for patients with metastasis. A multifaceted approach was employed combining clinical genomic analysis, in vitro assays and in vivo assays to uncover novel oncogenic drivers and metastasis-promoting factors in prostate cancer (PCa) focusing on a specific gene associated with the arginine N-degron pathway, called WDYHV1 (NTAQ1). Via overexpression of WDYHV1(NTAQ1) in LNCaP prostate cancer cell line using lentiviral transduction, we demonstrated its oncogenic functions including stimulation of proliferation, colony formation, 3D invasion, and transwell migration ability. Furthermore, we found that WDYHV1(NTAQ1) promotes in vivo xenograft tumor growth and metastasis in NSG immunodeficient mice utilizing the luciferase / dTomato expression as the reporter. Transcriptome profiling uncovered that 585 genes upregulated dramatically and pathway analysis of these up-regulated genes indicated the involvement of epithelial-mesenchymal transition (EMT) and TNF-alpha signaling via NF-κB. These findings highlight the WDYHV1 gene as a promising candidate for developing targeted therapies to mitigate metastatic prostate cancer. My future studies will focus on elucidating the underlying oncogenic mechanism from transcriptome analysis and looking for potential direct WDYHV substracts associated with arginine N-degron regulation.
關鍵字(中)	★ 前列腺癌 ★ 轉移 ★ N-降解決定子途徑	關鍵字(英)	★ Prostate Cancer ★ metastasis ★ N-Degron pathway
論文目次	中文摘要 .................................................................................................................................... v Abstract ...................................................................................................................................... vi Acknowledgments ................................................................................................................... viii Table of Contents ...................................................................................................................... ix List of Figures ............................................................................................................................ xi Explanation of Symbols and Abbreviations ............................................................................. xii Chapter I. Introduction ............................................................................................................... 1 1-1 Epidemiology of Prostate Cancer (PCa) ................................................................. 1 1-2 Prostate Cancer Therapies ...................................................................................... 2 1-3 Molecular Biology of Prostate Cancer ................................................................... 4 1-4 N-terminal Glutamine Amidohydrolase (NTAQ) .................................................. 4 1-4-1 Protein Degradation ................................................................................................ 4 1-4-2 N-end Rule Pathways ............................................................................................. 6 1-4-3 WDYHV1: From PTM to Potential Cancers Implications ..................................... 7 1-5 Research Objectives ............................................................................................... 8 Chapter II. Materials and Methods ........................................................................................... 10 2-1 Generation of pHIV-dTomato-NSF-WDYHV1 ................................................... 10 2-1-1 Vector Design ....................................................................................................... 10 2-1-2 Construction of pHIV-dTomato-Luciferase Vector ............................................. 11 2-1-3 cDNA Synthesis ................................................................................................... 11 2-1-4 Polymerase Chain Reaction (PCR) ...................................................................... 12 2-1-5 Transfection and generation of stable cell lines ................................................... 13 2-2 Cell Culture .......................................................................................................... 14 2-3 Western Blotting Analysis and Antibodies .......................................................... 14 2-4 Cancer Cell Proliferation ...................................................................................... 15 2-5 Colony Formation ................................................................................................. 15 2-6 Cancer Cell Migration .......................................................................................... 15 2-7 Cancer Cell Invasion ............................................................................................ 16 2-8 Xenograft Experiment in NSG Mice .................................................................... 16 2-9 Experimental Metastasis Assays .......................................................................... 16 2-10 Transcriptome Analysis ........................................................................................ 17 x 2-10-1 RNA Extraction .................................................................................................... 17 2-10-2 Microarrays Analysis ............................................................................................ 17 2-11 Statistical Analysis ............................................................................................... 18 Chapter III. Results ................................................................................................................... 19 3-1 Identification of candidate gene for prostate cancer metastasis ........................... 19 3-2 WDYHV1 expression in prostate cancer cells ..................................................... 20 3-3 Western blot analysis of overexpressing WDYHV1 in LNCaP ........................... 20 3-4 WDYHV1 overexpression increased a proliferation rate in LNCaP .................... 21 3-5 WDYHV1 overexpression increases colony number and size ............................. 22 3-6 WDYHV1 overexpression promotes prostate cancer cell migration ................... 23 3-7 WDYHV1 overexpression promotes cancer cell invasion ................................... 23 3-8 WDYHV1 promotes tumor growth in-vivo .......................................................... 24 3-9 WDYHV1 promotes the metastasis ability .......................................................... 25 3-10 Transciptome analysis of WDYHV1 overexpression .......................................... 27 Chapter IV. Discussion ............................................................................................................. 29 4-1 General Discussion ............................................................................................... 29 4-2 Limitations and future works ................................................................................ 29 4-1-1 Gene knockdown of WDYHV1 ........................................................................... 29 4-1-2 Validating the mechanism based on transcriptome profiling ............................... 30 4-1-3 N-end Rule Degradation Pathway ........................................................................ 30 4-1-4 Potential Downstream Target ............................................................................... 31 Chapter V. Conclusion ............................................................................................................. 32 Bibliography ............................................................................................................................. 33 Appendix A. ............................................................................................................................. 41 Appendix B. .............................................................................................................................. 45
參考文獻	1. Alshalalfa, M., Seldon, C., Franco, I., Vince, R., Carmona, R., Punnen, S., Kaochar, S., Dess, R., Kishan, A., Spratt, D. E., Sharma, J., Dal Pra, A., Pollack, A., Abramowitz, M. C., & Mahal, B. A. (2022). Clinicogenomic characterization of prostate cancer liver metastases. Prostate Cancer and Prostatic Diseases, 25(2), 366–369. https://doi.org/10.1038/s41391-021-00486-2 2. Alvarez, A., Barisone, G. A., & Diaz, E. (2014). Focus formation: a cell-based assay to determine the oncogenic potential of a gene. Journal of Visualized Experiments : JoVE, 94. https://doi.org/10.3791/51742 3. ARCHS4. (2024). WDYHV1. https://maayanlab.cloud/archs4/gene/WDYHV1#tissueexpression 4. Asiedu, M. K., Thomas, C. F., Dong, J., Schulte, S. C., Khadka, P., Sun, Z., Kosari, F., Jen, J., Molina, J., Vasmatzis, G., Kuang, R., Aubry, M. C., Yang, P., & Wigle, D. A. (2018). Pathways impacted by genomic alterations in pulmonary carcinoid tumors. Clinical Cancer Research, 24(7), 1691–1704. https://doi.org/10.1158/1078-0432.CCR-17-0252 5. Aslan, M., Hsu, E. C., Liu, S., & Stoyanova, T. (2021). Quantifying the invasion and migration ability of cancer cells with a 3D Matrigel drop invasion assay. Biology Methods and Protocols, 6(1). https://doi.org/10.1093/biomethods/bpab014 6. Banerjee, P. P., Banerjee, S., Brown, T. R., & Zirkin, B. R. (2018). Androgen action in prostate function and disease. American Journal of Clinical and Experimental Urology, 6(2), 62–77. 7. Basse, P., Hokland, P., Heron, I., & Hokland, M. (1988). Fate of tumor cells injected into left ventricle of heart in BALB/c mice: Role of natural killer cells. Journal of the National Cancer Institute, 80(9). https://doi.org/10.1093/jnci/80.9.657 34 8. Belkahla, S., Nahvi, I., Biswas, S., Nahvi, I., & Ben Amor, N. (2022). Advances and development of prostate cancer, treatment, and strategies: A systemic review. Frontiers in Cell and Developmental Biology, 10(September), 1–11. https://doi.org/10.3389/fcell.2022.991330 9. Borley, N., & Feneley, M. R. (2009). Prostate cancer: Diagnosis and staging. Asian Journal of Andrology, 11(1), 74–80. https://doi.org/10.1038/aja.2008.19 10. Castel, P. (2022). Defective protein degradation in genetic disorders. Biochimica et Biophysica Acta - Molecular Basis of Disease, 1868(5). https://doi.org/10.1016/j.bbadis.2022.166366 11. Cheng, L., Montironi, R., Bostwick, D. G., Lopez-Beltran, A., & Berney, D. M. (2012). Staging of prostate cancer. Histopathology, 60(1), 87–117. https://doi.org/10.1111/j.1365-2559.2011.04025.x 12. Cooper, G. (2000). The Cell A Molecular Approach. 2nd edition. Sunderland (MA) Sinauer Associates. In Biochemical Education. 13. Cornford, P., Tilki, D., Bergh, Van Den, R.C.N., Briers, E. et. al. (2024, April). EAU-EANM-ESTRO-ESUR-ISUP-SIOG Guidelines on Prostate Cancer. https://d56bochluxqnz.cloudfront.net/documents/pocket-guidelines/EAUEANM- ESTRO-ESUR-ISUP-SIOG-Pocket-on-Prostate-Cancer-2024_2024-04- 16-125527_rzmb.pdf 14. Dana, H., Chalbatani, G. M., Mahmoodzadeh, H., Karimloo, R., Rezaiean, O., Moradzadeh, A., Mehmandoost, N., Moazzen, F., Mazraeh, A., Marmari, V., Ebrahimi, M., Rashno, M. M., Abadi, S. J., & Gharagouzlo, E. (2017). Molecular Mechanisms and Biological Functions of siRNA. International Journal of Biomedical Science : IJBS, 13(2), 48–57. 15. de Haan, K., Zhang, Y., Zuckerman, J. E., Liu, T., Sisk, A. E., Diaz, M. F. P., Jen, K. Y., Nobori, A., Liou, S., Zhang, S., Riahi, R., Rivenson, Y., Wallace, W. D., & Ozcan, A. (2021). Deep learning-based transformation of H&E stained tissues into special stains. Nature Communications, 12(1). https://doi.org/10.1038/s41467-021-25221-2 35 16. Desarnaud, F., Geck, P., Parkin, C., Carpinito, G., & Makarovskiy, A. N. (2011). Gene expression profiling of the androgen independent prostate cancer cells demonstrates complex mechanisms mediating resistance to docetaxel. Cancer Biology and Therapy, 11(2). https://doi.org/10.4161/cbt.11.2.13750 17. Detlefsen, A. J., Paulukinas, R. D., & Mesaros, C. (2023). Chapter Thirteen - High sensitivity LC-MS methods for quantitation of hydroxy- and keto-androgens. In T. M. Penning (Ed.), Steroid Biochemistry (Vol. 689, pp. 355–376). Academic Press. https://doi.org/https://doi.org/10.1016/bs.mie.2023.04.009 18. Ferlay J, E. M. L. F. L. M. C. M. M. L. P. M. Z. A. S. I. B. F. (2024). Global Cancer Observatory: Cancer Today. Lyon, France: International Agency for Research on Cancer. 19. Gandaglia, G., Abdollah, F., Schiffmann, J., Trudeau, V., Shariat, S. F., Kim, S. P., Perrotte, P., Montorsi, F., Briganti, A., Trinh, Q.-D., Karakiewicz, P. I., & Sun, M. (2014). Distribution of metastatic sites in patients with prostate cancer: A population-based analysis. The Prostate, 74(2), 210–216. https://doi.org/10.1002/pros.22742 20. Glick, B. R., & Patten, C. L. (2017). Molecular biotechnology : principles and applications of recombinant DNA / Bernard R. Glick, Cheryl L. Patten. Molecular Biotechnology : Principles and Applications of Recombinant DNA. 21. Harshitha, R., & Arunraj, D. R. (2021). Real-time quantitative PCR: A tool for absolute and relative quantification. Biochemistry and Molecular Biology Education, 49(5), 800–812. https://doi.org/https://doi.org/10.1002/bmb.21552 22. Heiden, M. G. Vander, Cantley, L. C., & Thompson, C. B. (2009). Understanding the Warburg Effect: The Metabolic Requirements of Cell Proliferation. Science, 324(5930), 1029–1033. https://doi.org/10.1126/science.1160809 23. Heo, A. J., Kim, S. Bin, Kwon, Y. T., & Ji, C. H. (2023). The N-degron pathway: From basic science to therapeutic applications. In Biochimica et Biophysica Acta - Gene Regulatory Mechanisms (Vol. 1866, Issue 2). https://doi.org/10.1016/j.bbagrm.2023.194934 36 24. Hershko, A., & Ciechanover, A. (1998). The ubiquitin system. In Annual Review of Biochemistry (Vol. 67). https://doi.org/10.1146/annurev.biochem.67.1.425 25. Jang, I. K., & Gu, H. (2003). Negative regulation of TCR signaling and T-cell activation by selective protein degradation. Current Opinion in Immunology, 15(3), 315–320. https://doi.org/10.1016/S0952-7915(03)00048-7 26. Kawahata, I., & Fukunaga, K. (2020). Degradation of tyrosine hydroxylase by the ubiquitin-proteasome system in the pathogenesis of Parkinson’s disease and dopa-responsive dystonia. International Journal of Molecular Sciences, 21(11). https://doi.org/10.3390/ijms21113779 27. Li, Y., Li, S., & Wu, H. (2022). Ubiquitination-Proteasome System (UPS) and Autophagy Two Main Protein Degradation Machineries in Response to Cell Stress. In Cells (Vol. 11, Issue 5). https://doi.org/10.3390/cells11050851 28. Liberti, M. V, & Locasale, J. W. (2016). The Warburg Effect: How Does it Benefit Cancer Cells? Trends in Biochemical Sciences, 41(3), 211–218. https://doi.org/10.1016/j.tibs.2015.12.001 29. Magi, B., & Liberatori, S. (2005). Immunoblotting techniques. Methods in Molecular Biology (Clifton, N.J.), 295, 227–254. https://doi.org/10.1385/1- 59259-873-0:227 30. Mamiatis, T., Fritsch, E. F., Sambrook, J., & Engel, J. (1985). Molecular cloning– A laboratory manual. New York: Cold Spring Harbor Laboratory. 1982, 545 S., 42 $. Acta Biotechnologica, 5(1), 104. https://doi.org/https://doi.org/10.1002/abio.370050118 31. Mašić, S., Pezelj, I., & Krušlin, B. (2019). Prostate-Spesific Antigen (PSA) Values in Patients with Low- and High-Risk Prostatic Adenocarcinoma. Acta Clinica Croatica, 58(Suppl 2), 12–15. https://doi.org/10.20471/acc.2019.58.s2.02 32. Monies, D., Abouelhoda, M., AlSayed, M., Alhassnan, Z., Alotaibi, M., Kayyali, H., Al-Owain, M., Shah, A., Rahbeeni, Z., Al-Muhaizea, M. A., Alzaidan, H. I., Cupler, E., Bohlega, S., Faqeih, E., Faden, M., Alyounes, B., Jaroudi, D., Goljan, 37 E., Elbardisy, H., … Alkuraya, F. S. (2017). The landscape of genetic diseases in Saudi Arabia based on the first 1000 diagnostic panels and exomes. Human Genetics, 136(8). https://doi.org/10.1007/s00439-017-1821-8 33. Moore, C. B., Guthrie, E. H., Huang, M. T. H., & Taxman, D. J. (2010). Short hairpin RNA (shRNA): design, delivery, and assessment of gene knockdown. Methods in Molecular Biology (Clifton, N.J.), 629. https://doi.org/10.1007/978- 1-60761-657-3_10 34. Müller, M. M. (2018). Post-Translational Modifications of Protein Backbones: Unique Functions, Mechanisms, and Challenges. In Biochemistry (Vol. 57, Issue 2). https://doi.org/10.1021/acs.biochem.7b00861 35. Ni, X., Wei, Y., Li, X., Pan, J., Fang, B., Zhang, T., Lu, Y., Ye, D., & Zhu, Y. (2024). From biology to the clinic — exploring liver metastasis in prostate cancer. Nature Reviews Urology. https://doi.org/10.1038/s41585-024-00875-x 36. Nolsøe, A. B., Jensen, C. F. S., Østergren, P. B., & Fode, M. (2021). Neglected side effects to curative prostate cancer treatments. International Journal of Impotence Research, 33(4), 428–438. https://doi.org/10.1038/s41443-020- 00386-4 37. Oh, J. H., Hyun, J. Y., Chen, S. J., & Varshavsky, A. (2020). Five enzymes of the Arg/N-degron pathway form a targeting complex: The concept of superchanneling. Proceedings of the National Academy of Sciences of the United States of America, 117(20), 10778–10788. https://doi.org/10.1073/pnas.2003043117 38. Park, M. S., Bitto, E., Kim, K. R., Bingman, C. A., Miller, M. D., Kim, H.-J., Han, B. W., & Phillips, G. N. J. (2014). Crystal structure of human protein N-terminal glutamine amidohydrolase, an initial component of the N-end rule pathway. PloS One, 9(10), e111142. https://doi.org/10.1371/journal.pone.0111142 39. Paudel, R. R., Lu, D., Roy Chowdhury, S., Monroy, E. Y., & Wang, J. (2023). Targeted Protein Degradation via Lysosomes. In Biochemistry (Vol. 62, Issue 3). https://doi.org/10.1021/acs.biochem.2c00310 38 40. Peng, C., & Gao, F. (2014). Protein localization analysis of essential genes in prokaryotes. Scientific Reports, 4. https://doi.org/10.1038/srep06001 41. Piatkov, K. I., Colnaghi, L., Békés, M., Varshavsky, A., & Huang, T. T. (2012). The auto-generated fragment of the Usp1 deubiquitylase is a physiological substrate of the N-end rule pathway. Molecular Cell, 48(6), 926– 933. https://doi.org/10.1016/j.molcel.2012.10.012 42. Pohl, C., & Dikic, I. (2019). Cellular quality control by the ubiquitin-proteasome system and autophagy. Science, 366(6467), 818–822. https://doi.org/10.1126/science.aax3769 43. Potocki, P. M., & Wysocki, P. J. (2022). Evolution of prostate cancer therapy. Part 1. Oncology in Clinical Practice, 18(3), 177–188. https://journals.viamedica.pl/oncology_in_clinical_practice/article/view/OCP.20 21.0001 44. Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A., & Zhang, F. (2013). Genome engineering using the CRISPR-Cas9 system. Nature Protocols, 8(11), 2281–2308. https://doi.org/10.1038/nprot.2013.143 45. Rawla, P. (2019). Epidemiology of Prostate Cancer. World Journal of Oncology, 10(2), 63–89. https://doi.org/10.14740/wjon1191 46. Ray, D., Cuneo, K. C., Rehemtulla, A., Lawrence, T. S., & Nyati, M. K. (2015). Inducing oncoprotein degradation to improve targeted cancer therapy. In Neoplasia (United States) (Vol. 17, Issue 9). https://doi.org/10.1016/j.neo.2015.08.008 47. Rea, D., Del Vecchio, V., Palma, G., Barbieri, A., Falco, M., Luciano, A., De Biase, D., Perdonà, S., Facchini, G., & Arra, C. (2016). Mouse Models in Prostate Cancer Translational Research: From Xenograft to PDX. BioMed Research International, 2016. https://doi.org/10.1155/2016/9750795 48. Resources, A. of G. (2024). NTAQ1. https://doi.org/https://www.ncbi.nlm.nih.gov/gene/55093 39 49. Ruffalo, M., & Bar-Joseph, Z. (2019). Protein interaction disruption in cancer. BMC Cancer, 19(1). https://doi.org/10.1186/s12885-019-5532-5 50. Singer, E. A., Kaushal, A., Turkbey, B., Couvillon, A., Pinto, P. A., & Parnes, H. L. (2012). Active surveillance for prostate cancer: past, present and future. Current Opinion in Oncology, 24(3), 243–250. https://doi.org/10.1097/CCO.0b013e3283527f99 51. Sung, H., Ferlay, J., Siegel, R. L., Laversanne, M., Soerjomataram, I., Jemal, A., & Bray, F. (2021). Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA: A Cancer Journal for Clinicians, 71(3), 209–249. https://doi.org/10.3322/caac.21660 52. The Human Protein Atlas. (2024). WDYHV1. https://v15.proteinatlas.org/ENSG00000156795-WDYHV1/subcellular 53. Townsend, M. H., Ence, Z. E., Cox, T. P., Lattin, J. E., Burrup, W., Boyer, M. K., Piccolo, S. R., Robison, R. A., & O’Neill, K. L. (2020). Evaluation of the upregulation and surface expression of hypoxanthine guanine phosphoribosyltransferase in acute lymphoblastic leukemia and Burkitt’s B cell lymphoma. Cancer Cell International, 20(1). https://doi.org/10.1186/s12935- 020-01457-8 54. UniProt. (2024). Q96HA8 · NTAQ1_HUMAN. https://www.uniprot.org/uniprotkb/Q96HA8/entry#names_and_taxonomy 55. Van, V., & Smith, A. T. (2020). ATE1-Mediated Post-Translational Arginylation Is an Essential Regulator of Eukaryotic Cellular Homeostasis. In ACS Chemical Biology (Vol. 15, Issue 12, pp. 3073–3085). American Chemical Society. https://doi.org/10.1021/acschembio.0c00677 56. Varshavsky, A. (2019). N-degron and C-degron pathways of protein degradation. Proceedings of the National Academy of Sciences, 116(2), 358–366. https://doi.org/10.1073/pnas.1816596116 40 57. Vu, T. T. M., Mitchell, D. C., Gygi, S. P., & Varshavsky, A. (n.d.). The Arg/Ndegron pathway targets transcription factors and regulates specific genes. https://doi.org/10.1073/pnas.2020124117/-/DCSupplemental 58. Wasim, S. , P. J. , N. S. , and K. J. (2023). Prostate Cancer Patients. Cancers , 15(5615), 1–27. https://doi.org/https://doi.org/10.3390/ cancers15235615 59. You, M., Xie, Z., Zhang, N., Zhang, Y., Xiao, D., Liu, S., Zhuang, W., Li, L., & Tao, Y. (2023). Signaling pathways in cancer metabolism: mechanisms and therapeutic targets. Signal Transduction and Targeted Therapy, 8(1), 196. https://doi.org/10.1038/s41392-023-01442-3 60. Zhang, S., Shen, T., & Zeng, Y. (2023). Epigenetic Modifications in Prostate Cancer Metastasis and Microenvironment. Cancers, 15(8). https://doi.org/10.3390/cancers15082243 61. Zubair, H., & Ahmad, A. (2017). Chapter 1 - Cancer Metastasis: An Introduction. In A. Ahmad (Ed.), Introduction to Cancer Metastasis (pp. 3–12). Academic Press. https://doi.org/https://doi.org/10.1016/B978-0-12-804003-4.00001-3
指導教授	許恩旗范世榮(En-Chi Hsu Shih-Jung Fan)	審核日期	2024-7-26
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