請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/79048
完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 史有伶(Yu-Ling Shih) | |
dc.contributor.author | You-Lin Tsai | en |
dc.contributor.author | 蔡侑霖 | zh_TW |
dc.date.accessioned | 2021-07-11T15:39:35Z | - |
dc.date.available | 2023-08-21 | |
dc.date.copyright | 2018-08-21 | |
dc.date.issued | 2018 | |
dc.date.submitted | 2018-08-13 | |
dc.identifier.citation | 1. Xiong, Y. and K.L. Guan, Mechanistic insights into the regulation of metabolic enzymes by acetylation. J Cell Biol, 2012. 198(2): p. 155-64.
2. Bannister, A.J. and T. Kouzarides, Regulation of chromatin by histone modifications. Cell Res, 2011. 21(3): p. 381-95. 3. Choudhary, C., et al., Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science, 2009. 325(5942): p. 834-40. 4. Schilling, B., et al., Protein acetylation dynamics in response to carbon overflow in Escherichia coli. Mol Microbiol, 2015. 98(5): p. 847-63. 5. Castano-Cerezo, S., et al., Protein acetylation affects acetate metabolism, motility and acid stress response in Escherichia coli. Mol Syst Biol, 2014. 10: p. 762. 6. Hentchel, K.L. and J.C. Escalante-Semerena, Acylation of Biomolecules in Prokaryotes: a Widespread Strategy for the Control of Biological Function and Metabolic Stress. Microbiol Mol Biol Rev, 2015. 79(3): p. 321-46. 7. de Diego Puente, T., et al., The protein acetyltransferase PatZ from Escherichia coli is regulated by autoacetylation-induced oligomerization. Journal of Biological Chemistry, 2015. 290(38): p. 23077-23093. 8. Starai, V.J. and J.C. Escalante-Semerena, Identification of the protein acetyltransferase (Pat) enzyme that acetylates acetyl-CoA synthetase in Salmonella enterica. J Mol Biol, 2004. 340(5): p. 1005-12. 9. Liu, X.X., W.B. Liu, and B.C. Ye, Regulation of a Protein Acetyltransferase in Myxococcus xanthus by the Coenzyme NADP. J Bacteriol, 2015. 198(4): p. 623-32. 10. Weinert, B.T., et al., Acetyl-phosphate is a critical determinant of lysine acetylation in E. coli. Mol Cell, 2013. 51(2): p. 265-72. 11. Zhao, K., X. Chai, and R. Marmorstein, Structure and substrate binding properties of cobB, a Sir2 homolog protein deacetylase from Escherichia coli. J Mol Biol, 2004. 337(3): p. 731-41. 12. Tu, S., et al., YcgC represents a new protein deacetylase family in prokaryotes. Elife, 2015. 4: p. e05322. 13. Kremer, M., et al., Comment on 'YcgC represents a new protein deacetylase family in prokaryotes'. Elife, 2018. 7. 14. Kuhn, M.L., et al., Structural, kinetic and proteomic characterization of acetyl phosphate-dependent bacterial protein acetylation. PLoS One, 2014. 9(4): p. e94816. 15. Castano-Cerezo, S., et al., cAMP-CRP co-ordinates the expression of the protein acetylation pathway with central metabolism in Escherichia coli. Mol Microbiol, 2011. 82(5): p. 1110-28. 16. Thao, S., et al., Nepsilon-lysine acetylation of a bacterial transcription factor inhibits Its DNA-binding activity. PLoS One, 2010. 5(12): p. e15123. 17. Ma, Q. and T.K. Wood, Protein acetylation in prokaryotes increases stress resistance. Biochem Biophys Res Commun, 2011. 410(4): p. 846-51. 18. Liang, W., A. Malhotra, and M.P. Deutscher, Acetylation regulates the stability of a bacterial protein: growth stage-dependent modification of RNase R. Mol Cell, 2011. 44(1): p. 160-6. 19. Liang, W. and M.P. Deutscher, Post-translational modification of RNase R is regulated by stress-dependent reduction in the acetylating enzyme Pka (YfiQ). RNA, 2012. 18(1): p. 37-41. 20. de Diego Puente, T., et al., The Protein Acetyltransferase PatZ from Escherichia coli Is Regulated by Autoacetylation-induced Oligomerization. J Biol Chem, 2015. 290(38): p. 23077-93. 21. Margolin, W., FtsZ and the division of prokaryotic cells and organelles. Nat Rev Mol Cell Biol, 2005. 6(11): p. 862-71. 22. Erickson, H.P., FtsZ, a tubulin homologue in prokaryote cell division. Trends Cell Biol, 1997. 7(9): p. 362-7. 23. Hugonnet, J.E., et al., Factors essential for L,D-transpeptidase-mediated peptidoglycan cross-linking and beta-lactam resistance in Escherichia coli. Elife, 2016. 5. 24. Carballido-Lopez, R., The bacterial actin-like cytoskeleton. Microbiol Mol Biol Rev, 2006. 70(4): p. 888-909. 25. Cho, H., et al., Bacterial cell wall biogenesis is mediated by SEDS and PBP polymerase families functioning semi-autonomously. Nat Microbiol, 2016: p. 16172. 26. Ouzounov, N., et al., MreB Orientation Correlates with Cell Diameter in Escherichia coli. Biophys J, 2016. 111(5): p. 1035-43. 27. Dyer, N., Tubulin and its prokaryotic homologue FtsZ: a structural and functional comparison. Sci Prog, 2009. 92(Pt 2): p. 113-37. 28. Chien, A.C., N.S. Hill, and P.A. Levin, Cell size control in bacteria. Curr Biol, 2012. 22(9): p. R340-9. 29. Wu, L.J. and J. Errington, Nucleoid occlusion and bacterial cell division. Nat Rev Microbiol, 2011. 10(1): p. 8-12. 30. Tonthat, N.K., et al., Molecular mechanism by which the nucleoid occlusion factor, SlmA, keeps cytokinesis in check. EMBO J, 2011. 30(1): p. 154-64. 31. Pichoff, S. and J. Lutkenhaus, Escherichia coli division inhibitor MinCD blocks septation by preventing Z-ring formation. J Bacteriol, 2001. 183(22): p. 6630-5. 32. Lutkenhaus, J. and M. Sundaramoorthy, MinD and role of the deviant Walker A motif, dimerization and membrane binding in oscillation. Mol Microbiol, 2003. 48(2): p. 295-303. 33. Hu, Z., C. Saez, and J. Lutkenhaus, Recruitment of MinC, an inhibitor of Z-ring formation, to the membrane in Escherichia coli: role of MinD and MinE. J Bacteriol, 2003. 185(1): p. 196-203. 34. Taheri-Araghi, S., et al., Cell-size control and homeostasis in bacteria. Curr Biol, 2015. 25(3): p. 385-391. 35. Pierucci, O., Dimensions of Escherichia coli at various growth rates: model for envelope growth. J Bacteriol, 1978. 135(2): p. 559-74. 36. Harris, L.K. and J.A. Theriot, Relative Rates of Surface and Volume Synthesis Set Bacterial Cell Size. Cell, 2016. 165(6): p. 1479-1492. 37. Lee, H.L., et al., Quantitative Proteomics Analysis Reveals the Min System of Escherichia coli Modulates Reversible Protein Association with the Inner Membrane. Mol Cell Proteomics, 2016. 15(5): p. 1572-83. 38. Ducret, A., E.M. Quardokus, and Y.V. Brun, MicrobeJ, a tool for high throughput bacterial cell detection and quantitative analysis. Nat Microbiol, 2016. 1(7): p. 16077. 39. Froger, A. and J.E. Hall, Transformation of plasmid DNA into E. coli using the heat shock method. J Vis Exp, 2007(6): p. 253. 40. Datsenko, K.A. and B.L. Wanner, One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A, 2000. 97(12): p. 6640-5. 41. Karimova, G., et al., A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proc Natl Acad Sci U S A, 1998. 95(10): p. 5752-6. 42. Kaleta, C., et al., Metabolic costs of amino acid and protein production in Escherichia coli. Biotechnol J, 2013. 8(9): p. 1105-14. 43. Shih, Y.L., et al., Division site placement in E.coli: mutations that prevent formation of the MinE ring lead to loss of the normal midcell arrest of growth of polar MinD membrane domains. EMBO J, 2002. 21(13): p. 3347-57. 44. Lima, B.P., et al., Involvement of protein acetylation in glucose-induced transcription of a stress-responsive promoter. Mol Microbiol, 2011. 81(5): p. 1190-204. 45. Zhang, Q., et al., Reversible lysine acetylation is involved in DNA replication initiation by regulating activities of initiator DnaA in Escherichia coli. Sci Rep, 2016. 6: p. 30837. 46. Venkat, S., et al., Biochemical Characterization of the Lysine Acetylation of Tyrosyl-tRNA Synthetase in Escherichia coli. Chembiochem, 2017. 18(19): p. 1928-1934. 47. Bi, J., et al., Modulation of Central Carbon Metabolism by Acetylation of Isocitrate Lyase in Mycobacterium tuberculosis. Sci Rep, 2017. 7: p. 44826. 48. Park, K.T., et al., The Min oscillator uses MinD-dependent conformational changes in MinE to spatially regulate cytokinesis. Cell, 2011. 146(3): p. 396-407. 49. Wu, W., et al., Determination of the structure of the MinD-ATP complex reveals the orientation of MinD on the membrane and the relative location of the binding sites for MinE and MinC. Mol Microbiol, 2011. 79(6): p. 1515-28. 50. Bi, E.F. and J. Lutkenhaus, FtsZ ring structure associated with division in Escherichia coli. Nature, 1991. 354(6349): p. 161-4. 51. Carabetta, V.J., et al., Temporal Regulation of the Bacillus subtilis Acetylome and Evidence for a Role of MreB Acetylation in Cell Wall Growth. mSystems, 2016. 1(3). 52. Ko, S., et al., Chylopericardium Secondary to Lymphangiomyoma - A case report. Korean J Thorac Cardiovasc Surg, 2011. 44(5): p. 377-9. 53. Shih, Y.L., I. Kawagishi, and L. Rothfield, The MreB and Min cytoskeletal-like systems play independent roles in prokaryotic polar differentiation. Mol Microbiol, 2005. 58(4): p. 917-28. 54. Wu, F., et al., Symmetry and scale orient Min protein patterns in shaped bacterial sculptures. Nat Nanotechnol, 2015. 10(8): p. 719-26. 55. de Boer, P.A., R.E. Crossley, and L.I. Rothfield, A division inhibitor and a topological specificity factor coded for by the minicell locus determine proper placement of the division septum in E. coli. Cell, 1989. 56(4): p. 641-9. 56. Shih, Y.L., et al., The N-terminal amphipathic helix of the topological specificity factor MinE is associated with shaping membrane curvature. PLoS One, 2011. 6(6): p. e21425. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/79048 | - |
dc.description.abstract | 乙醯化修飾是細胞中常見且重要的蛋白質轉譯後修飾,可改變蛋白質結構及功能,用以調節細胞生理。細菌中的蛋白質乙醯化修飾可根據機制分為兩類,其中主要的乙醯化修飾來源,是由帶有高能量的乙醯磷酸 (Acetyl-phosphate) 所進行的非酵素催化乙醯化,以及由蛋白質乙醯基轉移酶 (Protein lysine acetyltransferase, Pka) 所催化的專一性乙醯化反應。Pka本身具有高度保留的第一型Gcn5相關乙醯轉移酶 (Gcn5-related N-acetyltransferase, GNAT) 功能性區域,能夠精準的將乙醯基自乙醯輔酶A (Acetyl CoA) 轉移至目標蛋白賴氨酸上的ε胺基上。另一方面,實驗室先前的研究發現,大腸桿菌中的Pka會和細胞分裂相關小細胞蛋白產生直接的交互作用;而Min系統可以協助細菌將分裂的位置放置到中央,使得細菌可以在長軸中心進行分裂,產生兩個等同大小的子細胞。基於蛋白質乙醯化修飾與細胞分裂的重要性,本篇論文主旨在探討大腸桿菌內Pka利用乙醯化修飾影響細胞分裂的機制,以及整體的乙醯化修飾路徑對細菌生長的影響。
在此研究中,我分析了一系列大腸桿菌的乙醯化和去乙醯化的相關酵素基因剔除菌株,發現不同乙醯化突變菌株在生長的對數期與靜止期早期的細胞大小出現明顯差異,因而推論乙醯化修飾可能參與大腸桿菌的細胞大小調節,也加強Pka研究的重要性。為了探討Pka調節細胞分裂的機制,我運用細菌雙雜合系統發現了Pka與細胞分裂蛋白MinC、MinD、MinE、FtsZ、與MreB之間具有直接交互作用關係,代表Pka可能透過與細胞分裂蛋白之間的交互做用調節細胞大小。此外,透過螢光顯微鏡的實驗,觀察到細胞分裂進程上游的關鍵步驟,亦即將FtsZ環定為到細胞中點的能力,會在將pka基因被剔除的菌株中減低產生偏移。而Pka於野生型中會於細胞兩端的形成點狀聚集,而在minCDE基因剔除的背景下Pka均勻散佈於細胞質中。這些細胞內定位的表型,均支持Pka與細胞分裂蛋白之間有密不可分的關聯性。為了進一步了解是否細胞分裂蛋白受Pka產生的乙醯化修飾調節,我以細胞外乙醯化實驗,配合質譜方式鑑定出MinE 蛋白的賴胺酸52為Pka的乙醯化位置。總結目前結果,大腸桿菌的Pka可與細胞分裂蛋白產生交互做用,相互影響並改變Pka與細胞分裂蛋白於細胞內的定位,可能得以調節細胞大小。雖然目前無法確認細胞分裂蛋白乙醯化後對生長分裂的影響,但初步結果已顯示MinE 具有Pka的專一性乙醯化賴氨酸。未來將再進一步進行實驗分析。 | zh_TW |
dc.description.abstract | Acetylation is a prevalent post-translational modification of proteins in Escherichia coli that regulates cell physiology through modulating protein structure and function. In addition to non-enzymatic acetylation activity involving the high-energy acetyl donor acetyl-phosphate, the protein acetylation can be catalyzed by protein lysine acetyltransferase, Pka. Pka contains a highly conserved, type I Gcn5-related N-acetyltransferases (GNAT) domain at the C-terminus that transfers the acetyl group from acetyl-CoA to the ε-amine of the lysine residue. Interestingly, Pka was identified as an interacting partner of a cell division machinery, the Min system, suggesting that Pka may be involved in regulating cell division. Therefore, this study aims at investigating the interplay between Pka and cell division by asking whether the enzymatic activity of Pka is involved, or Pka possesses moonlighting activity to regulate the process of cell division.
To understand the relationships between acetylation and cell division in E. coli, a series of mutants that carried deletions in genes involved in the acetylation and deacetylation cycles was examined for cell growth and morphology. The results showed that there were significant size variations between the mutants and the wild type strain at different growth phases, indicating that the acetylation activity may be involved in the cell size regulation. Among them, the Δpka mutant became smaller proportionally in both width and length at the exponential and late exponential phase when cultured at 30℃. To probe for the mechanism regulating the cell size, the bacterial cytoskeletal proteins FtsZ and MreB, that are involved in coordinating the bacterial growth and division, were found to directly interact with Pka in the bacterial two-hybrid assays. Coincided with the findings, placement of the FtsZ ring at the midcell zone became less robust in the Δpka, and polar localization of GFP-Pka was lost in the absence of minCDE. All evidence suggested that Pka may directly interact with the cell division machineries, including the Min system, FtsZ, and MreB to modulate the cell size. To address whether the acetylation activity of Pka on the cell division proteins could influence the cell division proteins, I used the in vitro acetylation assay in combination with the mass spectrometry analysis to identify lysine 52 of MinE was the specific acetylation site of Pka. Taken together, we conclude that the acetylation network serves as a novel mechanism to regulate cell size, growth and division in E. coli. Whether Pka could directly modulate the functions of the cell division proteins, or regulate cell size via modulating cell metabolism will be investigate. | en |
dc.description.provenance | Made available in DSpace on 2021-07-11T15:39:35Z (GMT). No. of bitstreams: 1 ntu-107-R05b46018-1.pdf: 5720482 bytes, checksum: 55e9b65879ca2d472a8ac06846f6fb86 (MD5) Previous issue date: 2018 | en |
dc.description.tableofcontents | Table of Contents
誌謝 i 中文摘要 iii Abstract v Table of Contents vii List of Tables xi List of Figures xii Abbreviation xiv 1.1 Protein acetylation in bacteria 1 1.2 Protein lysine acetyltransferase 3 1.3 Cell division and size control in E. coli 5 1.3.1 Cell growth and division in E. coli 5 1.3.2 The cell division site placement in E. coli 6 1.3.3 Size control 7 1.4 Objectives 8 Chapter 2. Materials and Methods 10 2.1 Materials: 10 2.1.1 List of chemicals, reagents, and medium 10 2.1.2 Culture medium 16 2.1.3 Antibiotics 17 2.1.4 Carbon source 17 2.2 Buffer 18 2.3 Stock solution 23 2.3.1 DNA experiment 23 2.3.2 Protein experiment 25 2.3.3 Microscope experiment 32 2.4 Bacterial strains 33 2.4.1 Growth condition 33 2.4.2 Storage 33 2.5 DNA techniques 34 2.5.1 Plasmid extraction 34 2.5.2 Polymerase chain reaction (PCR) 35 2.5.3 Restriction enzymes digestion 35 2.5.4 Purification of the PCR products 36 2.5.5 DNA electrophoresis 36 2.5.6 Extraction of DNA from agarose gel 37 2.6 Gene cloning 38 2.6.1 Transformation 38 2.7 Strain construction 40 2.7.1 λ red recombination 40 2.8 Protein techniques 41 2.8.1 Sodium dodecyl sulfate polyacrylamide gel electrophoresis 41 2.8.2 Western blotting 44 2.8.3 Quantify the signal on gels or blots 45 2.9 Protein overproduction and purification 45 2.9.1 Strains 45 2.9.2 Overproduction 45 2.9.3 Cell disruption 46 2.9.4 Ni-NTA affinity purification 46 2.10 Protein-protein interaction assays 47 2.10.1 Bacteria two-hybrid assay 47 2.11 Analysis of the acetylation pattern 48 2.12 Acetylation assay 49 2.12.1 Liposome preparation 49 2.12.2 the in vitro Acetylation reaction 49 2.12.3 Liquid chromatography–mass spectrometry/mass spectrometry (LC-MS/MS) analysis 50 2.13 Microscopy 52 2.13.1 Microscope 52 2.13.2 Cell culture condition 53 2.13.3 Quantification of the cell size 53 2.13.4 Subcellular localization of FtsZ and MreB 55 2.13.5 Statistical methods 56 Chapter 3. Results 57 3.1 Relationship between acetylation and cell size control 57 3.1.1 Construction of the acetylation and de-acetylation mutants 57 3.1.2 Growth of the mutants 57 3.1.3 Acetylation patterns of the wild-type and mutant strains 61 3.1.4 Effect of glucose concentration on the cell size regulation 62 3.2 Interplay between Pka and cell division proteins 64 3.2.1 Subcellular localization of Pka 64 3.2.2 Interaction between Pka and cell division proteins 65 3.3 Acetylation activity of Pka on the cell division proteins 67 3.3.1 Acetylation activity of Pka 67 3.3.2 Acetylation of Acs by Pka 69 3.3.3 Acetylation of Acs by Pka with the presence of liposomes 69 3.3.4 Acetylation of MinE by Pka 70 3.4. Dissecting the interacting domains of Pka with the Min proteins 71 Chapter 4. Discussion 73 4.1 Pka and cell size regulation 73 4.2 Acetylation activity of Pka on the cell division proteins 75 4.3 Interplay between Pka and cell division proteins 78 References: 82 List of Tables Table 1. Strain list 90 Table 2. Plasmid list 92 Table 3. Primer list 94 Table 4. Instrument list 96 Table 5. Plasmid combinations used in the bacterial two-hybrid assay 98 List of Figures Fig. 1-1. Protein lysine acetylation in bacteria 99 Fig. 1-2. Protein structures of Pka and its GNAT domain 100 Fig. 1-3. Illustration of the pole-to-pole oscillation of the Min proteins 101 Fig. 2-1. Cycle of Polymerase chain reaction 102 Fig. 2-2. Flow chart of λ red recombination 103 Fig. 2-3. Equation of SA/V calculation 104 Fig. 3-1. Confirmation of the mutants, Δacs, ΔackA, ΔackAΔpta, ΔcobBΔycgC and ΔpkaΔminCDE 105 Fig. 3-2. Cell size measurements of the acetylation and deacetylation mutants 107 Fig. 3-3. Analysis of the cell width and cell length in different growth phase 109 Fig. 3-4. Analysis of the SA/V ratio in different growth phase 111 Fig. 3-5. Cell size measurements of the acetylation and deacetylation mutants 113 Fig. 3-6. Summary of the morphology comparison between the mutants iandthe wild-type strain. 114 Fig. 3-7. Acetylation patterns of different mutants by Western blotting analyses 115 Fig. 3-8. Cell size measurements of the wild-type (WT) and the Δpka strains when grown in M9 medium supplemented with different concentration of glucose (0.416, 0.208, 0%) and different carbon source (0.54% acetate) 117 Fig. 3-9. Analyses of the SA/V ratio of wild-type and Δpka with 0.416%, 0.208%, 0% glucose and 0.52% sodium acetate.. 119 Fig. 3-10. Acetylation patterns of the wild-type (WT) and the Δpka strains when grown in M9 medium supplemented with different concentration of glucose (0.416, 0.208, 0%) and different carbon source (0.54% acetate). 120 Fig. 3-11. Subcellular localization of Pka in WT and Δmin mutant.. 121 Fig. 3-12. Interaction between Pka and MinC, MinD, and MinE, FtsZ, and MreB using the bacterial two-hybrid assays. 122 Fig. 3-13. Localization of MreB in the wild-type and Δpka strains. 123 Fig. 3-14. Localization of FtsZ in the wild-type and Δpka strains. 124 Fig. 3-15. Prediction of the acetylation sites on the target proteins of Pka. 125 Fig. 3-16. Purification of Pka. 126 Fig. 3-17. Purification of Acs. 127 Fig. 3-18. Purification of MinE. 129 Fig. 3-19. in vitro acetylation assay demonstrating acetylation of Acs by Pka. 130 Fig. 3-20. in vitro acetylation assay was unable to distinguish acetylated MinE by Pka. 131 Fig. 3-21. Identification of the acetylation sites on MinE. 132 Fig. 3-22. The MS2 spectra of LC-MS/MS were used to confirm the acetylated lysine on purified MinE. 133 Fig. 3-23. Designation of the Pka domains that were used in this study.. 134 Fig. 3-24. Interaction between the Pka domains and the Min proteins using the bacterial two-hybrid assays. 135 Fig. 3-25. Sequence alignment of MreB from E. coli and B. subtilis 137 | |
dc.language.iso | zh-TW | |
dc.title | 賴氨酸乙醯基轉移酶對大腸桿菌細胞分裂和大小的調控 | zh_TW |
dc.title | Roles of Protein Lysine Acetyltransferase
in Cell Division and Size Control in Escherichia coli | en |
dc.type | Thesis | |
dc.date.schoolyear | 106-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 吳世雄(Shih-Hsiung Wu),賴爾?(Lai, Erh-Min),鄧述諄 | |
dc.subject.keyword | 乙醯化反應,蛋白質乙醯轉移?,細胞尺寸,細胞分裂,小細胞系統, | zh_TW |
dc.subject.keyword | acetylation,protein lysine acetyltransferase,cell size,cell division,the Min system, | en |
dc.relation.page | 137 | |
dc.identifier.doi | 10.6342/NTU201802425 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2018-08-14 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科學研究所 | zh_TW |
dc.date.embargo-lift | 2023-08-21 | - |
顯示於系所單位: | 生化科學研究所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-107-R05b46018-1.pdf 目前未授權公開取用 | 5.59 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。