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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 蔡丰喬(Feng-Chiao Tsai) | |
| dc.contributor.author | Ting-Jen Tseng | en |
| dc.contributor.author | 曾亭禎 | zh_TW |
| dc.date.accessioned | 2021-07-11T15:10:20Z | - |
| dc.date.available | 2024-08-28 | |
| dc.date.copyright | 2019-08-28 | |
| dc.date.issued | 2019 | |
| dc.date.submitted | 2019-08-09 | |
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Journal of Biological Chemistry, 2017. 292(1): p. 351-360. 15. Yu, H.Y., et al., Deletion of STK40 Protein in Mice Causes Respiratory Failure and Death at Birth. Journal of Biological Chemistry, 2013. 288(8): p. 5342-5352. 16. Wang, L.N., et al., Deletion of Stk40 impairs definitive erythropoiesis in the mouse fetal liver. Cell Death & Disease, 2017. 8. 17. Ding, D., et al., Reticulocalbin-2 enhances hepatocellular carcinoma proliferation via modulating the EGFR-ERK pathway. Oncogene, 2017. 36(48): p. 6691-6700. 18. Etienne-Manneville, S., Adherens junctions during cell migration. Subcell Biochem, 2012. 60: p. 225-49. 19. Krause, M. and A. Gautreau, Steering cell migration: lamellipodium dynamics and the regulation of directional persistence. Nat Rev Mol Cell Biol, 2014. 15(9): p. 577-90. 20. Wozniak, M.A., et al., Focal adhesion regulation of cell behavior. Biochim Biophys Acta, 2004. 1692(2-3): p. 103-19. 21. Tojkander, S., G. Gateva, and P. Lappalainen, Actin stress fibers--assembly, dynamics and biological roles. J Cell Sci, 2012. 125(Pt 8): p. 1855-64. 22. Hartsock, A. and W.J. Nelson, Adherens and tight junctions: Structure, function and connections to the actin cytoskeleton. Biochimica Et Biophysica Acta-Biomembranes, 2008. 1778(3): p. 660-669. 23. Ehrlich, J.S., M.D.H. Hansen, and W.J. Nelson, Spatio-temporal regulation of Rac1 localization and lamellipodia dynamics during epithelial cell-cell adhesion. Developmental Cell, 2002. 3(2): p. 259-270. 24. Koukouritaki, S.B., A. Gravanis, and C. Stournaras, Tyrosine phosphorylation of focal adhesion kinase and paxillin regulates the signaling mechanism of the rapid nongenomic action of dexamethasone on actin cytoskeleton. Molecular Medicine, 1999. 5(11): p. 731-742. 25. Turner, C.E., Paxillin and focal adhesion signalling. Nature Cell Biology, 2000. 2(12): p. E231-E236. 26. Katoh, K., et al., Stress fiber organization regulated by MLCK and Rho-kinase in cultured human fibroblasts. American Journal of Physiology-Cell Physiology, 2001. 280(6): p. C1669-C1679. 27. Vicente-Manzanares, M. and A.R. Horwitz, Adhesion dynamics at a glance. Journal of Cell Science, 2011. 124(23): p. 3923-3927. 28. Amano, M., et al., Formation of actin stress fibers and focal adhesions enhanced by Rho-kinase. Science, 1997. 275(5304): p. 1308-1311. 29. Burridge, K. and C. Guilluy, Focal adhesions, stress fibers and mechanical tension. Experimental Cell Research, 2016. 343(1): p. 14-20. 30. Oakes, P.W., et al., Tension is required but not sufficient for focal adhesion maturation without a stress fiber template. Journal of Cell Biology, 2012. 196(3): p. 363-374. 31. Pasapera, A.M., et al., Myosin II activity regulates vinculin recruitment to focal adhesions through FAK-mediated paxillin phosphorylation. Journal of Cell Biology, 2010. 188(6): p. 877-890. 32. Imamura, F., et al., Y-27632, an inhibitor of Rho-associated protein kinase, suppresses tumor cell invasion via regulation of focal adhesion and focal adhesion kinase. Japanese Journal of Cancer Research, 2000. 91(8): p. 811-816. 33. Sun, Z., S.S. Guo, and R. Fassler, Integrin-mediated mechanotransduction. J Cell Biol, 2016. 215(4): p. 445-456. 34. Fincham, V.J., et al., Active ERK/MAP kinase is targeted to newly forming cell-matrix adhesions by integrin engagement and v-Src. Embo Journal, 2000. 19(12): p. 2911-2923. 35. Ihermann-Hella, A., et al., Mitogen-activated protein kinase (MAPK) pathway regulates branching by remodeling epithelial cell adhesion. PLoS Genet, 2014. 10(3): p. e1004193. 36. Deng, M.Q., C.J. Williams, and R.M. Schultz, Role of MAP kinase and myosin light chain kinase in chromosome-induced development of mouse egg polarity. Developmental Biology, 2005. 278(2): p. 358-366. 37. Hu, J., et al., Stk40 deletion elevates c-JUN protein level and impairs mesoderm differentiation. J Biol Chem, 2019. 38. Roskoski, R., Jr., ERK1/2 MAP kinases: structure, function, and regulation. Pharmacol Res, 2012. 66(2): p. 105-43. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78658 | - |
| dc.description.abstract | 細胞遷移對於胚胎發育、血管新生和癌細胞轉移都佔有一席之地，近期研究找出一些和細胞遷移相關的基因，不過並不清楚這些基因之間是如何交互作用的，為了了解基因交互作用對細胞遷移之影響，我們使用了短髮夾RNA和小分子抑制劑的方式來建立雙擊篩選平台，釐清分子之間交互作用，在這個篩選平台中，令我們感到有趣的組合是STK40和MAPK的交互作用，先前研究便有指出敲弱STK40會改變細胞遷移，而在我們的結果中，STK40和MAPK雙重抑制時會顯著性降低細胞遷移，因此我們認為細胞遷移中STK40和MAPK之間是有交互作用的存在。
首先我們使用單一細胞遷移追蹤平台，進而得知STK40敲弱會使細胞速度和協調性減低的趨勢，在雙重抑制實驗中，發現到STK40會改變細胞骨架-黏著斑，於是我們便進一步探討，發現STK40會增加黏著斑的訊號，為了更了解這之間的機制，我們會有兩大方向: (1)了解STK40對於黏著斑影響 (2)還有研究STK40的功能，上述這些方向能讓我們知道STK40如何影響細胞遷移和MPAK之間的交互作用。 在這篇論文中我們證明了，(1)STK40會透過牽引力-黏著斑這條路徑調控細胞遷移 (2)STK40會和MAPK交互作用而影響黏著斑以及細胞遷移，目前我們發現到STK40可能是個接頭蛋白，改變p-ERK的位置，進而影響黏著斑以及細胞遷移，於是我們致力於尋找更多證據來闡述STK40是否為接頭蛋白。 | zh_TW |
| dc.description.abstract | Cell migration is important for embryonic development, angiogenesis and cancer metastasis. Recent study identified migration-related gene, but how they interact with each other remains elusive. To find out their interaction, we using “two-hit” screen to identify interaction by short hairpin RNA (shRNA) and small molecule inhibitor. The screen generated several candidate shRNA-inhibitor pairs, among which Serine-Threonine Kinase 40 (STK40)-MAPK specifically caught our attention. STK40 is a putative serine/threonine kinase. Previous study showed that STK40 knockdown (shSTK40) increased cell migration. However, our two-hit screen shSTK40 plus MAPK inhibitor (MAPKi) dramatically reduced cell migration, indicating that co-inhiition of STK40 and MAPK disrupted cell migration in a synergistic manner. Therefore, we hypothesize that STK40 interacts with MAPK pathway to regulate cell migration.
First, we conducted single cell migration assays and find out shSTK40 decreased cell motility and coordination. In double knockdown experiments, we discovered that STK40 control cell migration via cell-matrix adhesion (Paxillin). To find out its mechanism, we (1) examined how STK40 affect focal adhesion (FA) and (2) studied STK40 function. In this master thesis, we discovered that (1)STK40 regulate cell migration via traction force-focal adhesion pathway and (2)STK40-MAPK interaction affect focal adhesion and cell migration. In our recent data showed that STK40 may be a adaptor protein. STK40 alter p-ERK localization to regulate focal adhesion and cell migration. Thus, we need more evidence to find out STK40 function. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-11T15:10:20Z (GMT). No. of bitstreams: 1 ntu-108-R06443008-1.pdf: 8956590 bytes, checksum: 289c58df4c24a8f6c651415be2b34ba7 (MD5) Previous issue date: 2019 | en |
| dc.description.tableofcontents | 致謝 i
中文摘要 ii ABSTRACT iii 目錄 iv 圖目錄 x 表目錄 xii 第一章 導論 1 1. 細胞遷移 (cell migration) 1 2. 細胞遷移相關基因(gene) 1 3. 尋找cell-migration genes的交互溝通 (cross-talk) 2 4. 雙擊篩選平台找出STK40-MAPK交互作用 3 5. 絲氨酸/蘇氨酸激酶40 (serine-threonine kinase 40, STK40) 4 6. STK40和絲裂原活化蛋白激酶 (Mitogen-Activated Protein Kinase, MAPK)的交互作用 (interaction) 5 7. 研究目的: 研究STK40和MAPK interaction對於cell migration的影響 5 第二章 材料與方法 7 1. 細胞株和細胞培養 7 2. 材料 7 3. 細胞遷移實驗 (Cell Migration Assay) 7 3-1 細胞前置處理 7 3-2 活細胞影像攝影(Live Cell Imaging) 7 3-3 影像分析 8 4. Short-hairpin RNA(shRNA)基因靜默 (Gene knockdown)質體 (Plasmid) 8 5. 過度表現質體 (Overexpression Plasmid) 8 6. Lentivirus包裝與感染 (Infection) 8 7. 蛋白質萃取 (Protein Extraction)與西方墨點法(Western Blot) 9 7-1 細胞蛋白質萃取 9 7-2 蛋白質定量與樣品配置 9 7-3 膠體製備與電泳 9 7-4 蛋白質轉漬 (Transfer)和封阻 (Blocking) 10 7-5 一級抗體與二級抗體免疫結合 10 7-6 ECL呈色與定量分析 10 7-7 Western blot buffer成分配方 10 8. 細胞免疫螢光染色 (Immunofluorescent Assay, IF) 10 9. 黏著斑動態實驗 (Focal Adhesion Dynamic Assay) 11 10. 流式細胞儀 11 11. 蛋白質陣列 (Protein array) 11 12. 蛋白質核質分離 (Nuclear and cytosol fraction) 12 第三章 結果 13 1. STK40對cell migration之影響 13 1-1 以cell migration assay觀察STK40對於細胞的變化 13 1-2 STK40在HUVEC中對cell migration之影響 13 1-3 STK40在SAS中對cell migration之影響 13 2. STK40對cell migration結構之影響 14 2-1 利用雙重抑制 (Double inhibition)研究STK40對cell migration之影響 14 2-2 STK40和Focal adhesion有關連性 14 3. STK40對Focal adhesion之影響 15 3-1 shSTK40會使Focal adhesion signal增強 15 3-2 STK40對Focal adhesion影響為普遍現象 (universal phenomenon) 16 4. STK40對於Focal adhesion dynamic之影響 16 5. STK40對stress fiber之影響 17 6. STK40對牽引力 (traction force)之影響 18 6-1 STK40會改變traction force 18 6-2 Traction force inhibitor無法消弭shSTK40對Focal adhesion的效果 18 6-3 STK40對p-MLC的protein表現量並沒有影響 19 7. STK40在細胞之位置 19 7-1 市售抗體無法偵測內生性 (endogenous)的STK40蛋白表現 19 7-2 STK40 domain對其localization之影響 19 8. STK40 localization對Focal adhesion之影響 20 8-1 Cytosol-STK40能rescue shSTK40對Focal adhesion之影響 20 8-2 沒有EYFP的干擾，STK40都可以resccue shSTK40的效果 21 9. STK40是否為kinase 21 9-1 無法找出STK40的substrate 21 9-2 STK40 kinase domain對Focal adhesion之影響 22 10. STK40-MAPK interaction對cell migration之影響 22 11. STK40對MAPK pathway之影響 23 11-1 STK40會些微改變MAPK pathway磷酸化 (phosphorylation) 23 11-2 STK40 overexpression無法rescue shSTK40對MAPK signal的現象 23 12. STK40和MAPK交互作用對MAPK磷酸化之影響 24 12-1 MAPKi無法消除shSTK40對MAPK磷酸化影響 24 12-2 shSTK40會對MAPKi更為敏感 (sensitivity) 24 13. STK40和MAPK交互作用對Focal adhesion影響 24 14. STK40和traction force對MAPK之影響 25 15. STK40不影響之現象 25 15-1 STK40不影響細胞週期(cell cycle) 25 15-2 STK40不影響Focal adhesion signaling pathway 26 15-3 STK40不影響上皮-間質轉換(Epithelial-Mesenchymal Transition, EMT) 26 第四章 討論 27 1. STK40透過Focal adhesion調節cell migration 27 2. STK40位置之重要性 27 3. STK40和MAPK之間的關聯性 28 4. STK40可能是adaptor protein 28 5. 未來方向 29 第五章 圖片 31 第六章 表格 59 第七章 參考文獻 63 第八章 MATLAB scripts 66 1. Cell migration assay scripts 66 1-1 z_calcnuclei 66 1-2 z_plot_cell_traces_all 68 1-3 z_getcellparameters 70 1-4 Calc_shRNA_speed 75 2. Focal adhesion Assay scripts 77 2-1 save_fluoref_cor_img 77 2-2 FA_background 77 2-3 YFP_background 78 2-4 FA_identification 78 2-5 FA_identification_YFP 83 3. Stress Fiber Assay scripts 87 3-1 save_fluoref_cor_img 87 3-2 phalloidin_idetification 88 4. p-MLC Assay scripts 91 4-1 save_fluoref_cor_img 91 4-2 p-MLC_idetification 92 5. Focal Adhesion Dynamic Assay scripts 95 5-1 save_fluoref_cor_img 95 5-2 FA_background 95 5-3 FAdynamic_identification 96 5-4 FA_align 100 5-5 z_plot_original_traces 101 第九章 Plasmid Map 106 1. STK40-EYFP-N1 107 2. eYFP-C2-STK40 110 3. STK40-EYFP-N1-NES 113 4. D1~21-STK40-EYFP-N1 116 5. D 1~21-STK40-EYFP-N1-NLS 119 6. pLAS2w.eYFP.Pbsd 122 7. pLAS2W.-Pbsd.-STK40-P2A-EYFP 125 8. pLAS2w.Pbsd.-STK40-EYFP 129 9. pLAS2w.Pbsd.- D 1~21-STK40-EYFP-NLS 133 10. pLAS2w.Pbsd.-STK40-EYFP-NES 137 11. pLAS2w.Pbsd.-STK40-NLS-P2A-EYFP 141 12. pLAS2w.Pbsd.-STK40-NES-P2A-EYFP 145 13. pLAS2W.Pbsd.-mcherry 149 14. pLAS2W.Pbsd.-STK40-P2A-mcherry 152 15. pLAS2W.Pbsd.-STK40- D Kinase domain-EYFP 156 16. pLAS2W.Pbsd.-STK40- D Kinase domain-P2A-EYFP 160 17. pLAS2w.Pbsd.-STK40-Kinase domain-P2A-EYFP 164 18. pLAS2w.Pbsd.-3flag-STK40 (Head) 168 19. pLAS2w.Pbsd.-STK40-339-3flag (Middle) 172 20. pLAS2w.Pbsd.-STK40-3flag (Tail) 176 | |
| dc.language.iso | zh-TW | |
| dc.title | 探討絲氨酸／蘇氨酸蛋白激酶40與絲裂原活化蛋白激酶的交互作用對細胞遷移之影響 | zh_TW |
| dc.title | Untangling Serine-Threonine Kinase 40 (STK40)- Mitogen activated protein kinase (MAPK) Interaction in Cell Migration | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 107-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 郭津岑(Jean-Cheng Kuo),林耿慧(Keng-hui Lin),陳青周(Ching-Chow Chen) | |
| dc.subject.keyword | STK40,MAPK,細胞遷移,黏著斑,接頭蛋白, | zh_TW |
| dc.subject.keyword | STK40,MAPK,cell migration,focal adhesion,adaptor protein, | en |
| dc.relation.page | 183 | |
| dc.identifier.doi | 10.6342/NTU201902264 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2019-08-12 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 藥理學研究所 | zh_TW |
| dc.date.embargo-lift | 2024-08-28 | - |
| 顯示於系所單位： | 藥理學科所 | |
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