臺灣博碩士論文加值系統

目 錄
摘要……………………………………………………………….......................……….i
ABSTRACT……...……………………………………………………........................ii
目錄…………………………………………………………………….........................iv
圖目錄………………………………………………………………......................…….v
表目錄………………………………………………………………......................…...ix
縮寫列表…………………………………………………………......................………x
緒論………………………………………………………….....................….…………..1
材料方法……………………………………………………......................……………4
實驗結果………………………………………………………….....................……..13
討論……………………………………………………………….....................……...27
圖表…………………………………………………………….....................………...32
附表……………………………………………………….....................……………...94
參考文獻…………………………………………………………….....................…..99
附錄…………………………………………………………...................…………...103

圖 目 錄
Figure 1-1. rgef-1p::hsf-1::sl2::mCherry ; daf-16::gfp 突變株的螢光
照相結果......................................................................32
Figure 1-2. 神經過度表現HSF-1會促使腸道DAF-16進核…….....…….33
Figure 1-3. 神經過度表現HSF-1會活化DAF-16的活性……………..…..34
Figure 1-4. 神經過度表現HSF-1足以延長線蟲壽命…………….…….…..35
Figure 1-5. 神經過度表現HSF-1在基因插入特定染色體處理
（Integration）後仍可以延長線蟲壽命…..…………....……..36
Figure 1-6. 神經過度表現HSF-1增加線蟲的壓力抵抗能力………...…..37
Figure 2-1. unc-25p::hsf-1::sl2::YFP; daf-16::gfp 突變株表現的神經
細胞分佈.....................................................................38
Figure 2-2. unc-25p::hsf-1::sl2::YFP; daf-16::gfp 突變株的螢光照相
結果……......................................................................39
Figure 2-3. GABAergic神經過度表現HSF-1不會造成腸道DAF-16進
核...............................................................................40
Figure 2-4. unc-25p::hsf-1::sl2::YFP; daf-16::gfp突變株的腸道DAF-
16核質比定量結果……………………………..............…….......41
Figure 2-5. GABAergic神經過度表現HSF-1不會影響線蟲壽命….…..42
Figure 3-1. eat-4p::hsf-1::sl2::mCherry; daf-16::gfp 突變株表現的神
經細胞分佈…...…………………………………..........………….….43
Figure 3-2. eat-4p::hsf-1::sl2::mCherry; daf-16::gfp 突變株的螢光
照相結果.....................................................................44
Figure 3-3. Glutamatergic神經過度表現HSF-1不會造成腸道DAF-16
進核….....................................................................….45
Figure 3-4. eat-4p::hsf-1::sl2::mCherry; daf-16::gfp突變株的腸道
DAF-16核質比定量結果…………………………….....…………..46
Figure 3-5. Glutamatergic神經過度表現HSF-1不會影響線蟲壽命...47
Figure 4-1. tph-1p::hsf-1::sl2::mCherry; daf-16::gfp 突變株表現的神
經細胞分佈……………..………………………………........…...…...48
Figure 4-2. tph-1p::hsf-1::sl2::mCherry; daf-16::gfp 突變株的螢光
照相結果…...................................................................49
Figure 4-3. Serotonergic神經過度表現HSF-1不會造成腸道DAF-16進
核…….......................................................................…50
Figure 4-4. tph-1p::hsf-1::sl2::mCherry; daf-16::gfp突變株的腸道
DAF-16核質比定量結果………………………………….…...……51
Figure 4-5. Serotonergic神經過度表現HSF-1不會影響線蟲壽命.....52
Figure 5-1. tdc-1p::hsf-1::sl2::mCherry; daf-16::gfp 突變株表現的神
經細胞分佈…………………………….....……..........………………53
Figure 5-2. tdc-1p::hsf-1::sl2::mCherry; daf-16::gfp 突變株的螢光照
相結果.........................................................................54
Figure 5-3. Tyraminergic/ Octopaminergic神經過度表現HSF-1不會
造成腸道DAF-16進核………………………………………….......55
Figure 5-4. tdc-1p::hsf-1::sl2::mCherry; daf-16::gfp突變株的腸道
DAF-16核質比定量結果…………………………...…........…….56
Figure 5-5. Tyraminergic/ Octopaminergic神經過度表現HSF-1不會
影響線蟲壽命……….............……….....…………………………..57
Figure 6-1. dat-1p::hsf-1::sl2::mCherry; daf-16::gfp 突變株表現的神
經細胞分佈……………………………………................……...….58
Figure 6-2. dat-1p::hsf-1::sl2::mCherry; daf-16::gfp 突變株的螢光照
相結果….....................................................................59
Figure 6-3. Dopaminergic神經過度表現HSF-1不會造成腸道DAF-16
進核…......................................................................…60
Figure 6-4. dat-1p::hsf-1::sl2::mCherry; daf-16::gfp突變株的腸道
DAF-16核質比定量結果……………………....…………………..61
Figure 6-5. Dopaminergic神經過度表現HSF-1不會延長線蟲壽命….62
Figure 7-1. unc-17p::hsf-1::sl2::mCherry; daf-16::gfp 突變株表現的
神經細胞分佈……………………………………………….....………..63
Figure 7-2. Cholinergic神經過度表現HSF-1不會造成腸道DAF-16進
核……..........................................................................64
Figure 7-3. unc-17p::hsf-1::sl2::mCherry; daf-16::gfp突變株的腸道
DAF-16核質比定量結果……………………………………………..65
Figure 7-4. Cholinergic神經過度表現HSF-1不會影響線蟲的壓力抵抗
能力….........................................................................66
Figure 7-5. Cholinergic神經過度表現HSF-1會延長線蟲壽命….....….67
Figure 7-6. acr-2p::hsf-1::sl2::CFP; daf-16::gfp 突變株表現的神經細
胞分佈…......................................................................68
Figure 7-7. acr-2p::hsf-1::sl2::CFP; daf-16::gfp 突變株能有效表現
CFP的基因片段…….........………………………………………..…69
Figure 7-8. acr-2p::hsf-1::sl2::CFP; daf-16::gfp 突變株的螢光照相結
果…….......................................................................….70
Figure 7-9. Cholinergic神經過度表現HSF-1（acr-2p::hsf-1 O.E.）不
會造成腸道DAF-16進核………………………………………..……71
Figure 7-10. acr-2p::hsf-1::sl2::CFP; daf-16::gfp突變株的腸道DAF-
16核質比定量結果……………………………………………….….72
Figure 7-11. Cholinergic神經過度表現HSF-1（acr-2p::hsf-1 O.E.）足
以延長線蟲壽命..……………………………………….…………....73
Figure 8. 神經過度表現HSF-1需要DAF-16來延長線蟲壽命….…………74
Figure 9. Cholinergic神經過度表現HSF-1足以延長線蟲壽命並且需要
DAF-16的活化…………………………………………………………....75
Figure 10-1. unc-17p::hsf-1 O.E.; eat-4p::hsf-1 O.E.; tph-1p::hsf-1
O.E.; tdc-1p::hsf-1 O.E.; dat-1p::hsf-1 O.E.; unc-
25p::hsf-1 O.E.; daf-16::gfp 突變株表現的神經細胞分
佈……………........................………………………………………76
Figure 10-2. 分泌神經傳導物質的神經中過度表現HSF-1會造成腸道
DAF-16明顯進核…..…………………………………………………77
Figure 11-1. 神經增強hsf-1 RNAi knockdown會縮短線蟲壽命……....78
Figure 11-2. hsf-1 RNAi能有效降低hsf-1 mRNA的表現量…………..…79
Figure 12. GABAergic神經hsf-1 knockdown不會影響線蟲壽命…....80
Figure 13. Glutamatergic神經hsf-1 knockdown不會影響線蟲壽命..81
Figure 14. Dopaminergic神經hsf-1 knockdown不會影響線蟲壽命..82
Figure 15. Cholinergic神經hsf-1 knockdown不會影響線蟲壽命…...83
Figure 16-1. Serotonergic 神經RNAi knockdown突變株表現的神經細
胞分佈.......................................................................84
Figure 16-2. Serotonergic神經hsf-1 knockdown不會影響線蟲壽
命..............................................................................85
Figure 17-1. Tyraminergic/ Octopaminergic 神經RNAi knockdown突
變株表現的神經細胞分佈………………..............…………....86
Figure 17-2. Tyraminergic/ Octopaminergic神經hsf-1 knockdown不
會影響線蟲壽命………….....……………………………………....87
Figure 18-1. 神經RNAi knockdown 突變株給予rab-3 RNAi後的照相結
果…...........................................................................88
Figure 18-2. 神經RNAi knockdown突變株具有RNAi knockdown的效
果……........................................................................89
Figure 18-3. 神經hsf-1 RNAi knockdown不會影響線蟲壽命…………90
Figure 19. 調控腸道DAF-16進核與長壽的機制為兩條獨立的路徑.…..91
Figure 20. 神經增強RNAi knockdown突變株會影響肌肉組織的功
能.................................................................................92
Figure 21. 腸道hsf-1 RNAi knockdown會縮短線蟲壽命…………...…..93

表 目 錄
Table 1. 神經細胞分類與相對應的特定神經啟動子………….…..…........94
Table 2. 過度表現HSF-1於氧化壓力及壽命分析的實驗記錄……........95
Table 3. HSF-1、DAF-16 Knockdown於壽命分析的實驗記錄…....…97

1. Lithgow, G.J. and G.A. Walker, Stress resistance as a determinate of C. elegans lifespan. Mechanisms of ageing and development, 2002. 123(7): p. 765-771.
2. Kenyon, C., et al., A C. elegans mutant that lives twice as long as wild type. Nature, 1993. 366(6454): p. 461.
3. Bluher, M., B.B. Kahn, and C.R. Kahn, Extended longevity in mice lacking the insulin receptor in adipose tissue. Science, 2003. 299(5606): p. 572-574.
4. Tatar, M., et al., A mutant Drosophila insulin receptor homolog that extends life-span and impairs neuroendocrine function. Science, 2001. 292(5514): p. 107-110.
5. Suh, Y., et al., Functionally significant insulin-like growth factor I receptor mutations in centenarians. Proceedings of the National Academy of Sciences, 2008. 105(9): p. 3438-3442.
6. Kimura, K.D., et al., daf-2, an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditis elegans. Science, 1997. 277(5328): p. 942-946.
7. Pierce, S.B., et al., Regulation of DAF-2 receptor signaling by human insulin and ins-1, a member of the unusually large and diverse C. elegans insulin gene family. Genes & development, 2001. 15(6): p. 672-686.
8. Malone, E.A., T. Inoue, and J.H. Thomas, Genetic Analysis of the Roles of daf28 and age-1 in Regulating Caenorhabditis elegans Dauer Formation. Genetics, 1996. 143(3): p. 1193-1205.
9. Carter, C.S., M.M. Ramsey, and W.E. Sonntag, A critical analysis of the role of growth hormone and IGF-1 in aging and lifespan. TRENDS in Genetics, 2002. 18(6): p. 295-301.
10. Paradis, S. and G. Ruvkun, Caenorhabditis elegans Akt/PKB transduces insulin receptor-like signals from AGE-1 PI3 kinase to the DAF-16 transcription factor. Genes & development, 1998. 12(16): p. 2488-2498.
11. Apfeld, J. and C. Kenyon, Cell nonautonomy of C. elegans daf-2 function in the regulation of diapause and life span. Cell, 1998. 95(2): p. 199-210.
12. Murphy, C.T., et al., Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans. Nature, 2003. 424(6946): p. 277.
13. Kenyon, C.J., The genetics of ageing. Nature, 2010. 464(7288): p. 504.
14. Libina, N., J.R. Berman, and C. Kenyon, Tissue-specific activities of C. elegans DAF-16 in the regulation of lifespan. Cell, 2003. 115(4): p. 489-502.
15. Hartl, F.U., Molecular chaperones in cellular protein folding. Nature, 1996. 381(6583): p. 571.
16. Jolly, C. and R.I. Morimoto, Role of the heat shock response and molecular chaperones in oncogenesis and cell death. Journal of the National Cancer Institute, 2000. 92(19): p. 1564-1572.
17. Garigan, D., et al., Genetic analysis of tissue aging in Caenorhabditis elegans: a role for heat-shock factor and bacterial proliferation. Genetics, 2002. 161(3): p. 1101-1112.
18. Pirkkala, L., P. Nykanen, and L. Sistonen, Roles of the heat shock transcription factors in regulation of the heat shock response and beyond. The FASEB Journal, 2001. 15(7): p. 1118-1131.
19. Prahlad, V., T. Cornelius, and R.I. Morimoto, Regulation of the cellular heat shock response in Caenorhabditis elegans by thermosensory neurons. Science, 2008. 320(5877): p. 811-814.
20. Finkel, T. and N.J. Holbrook, Oxidants, oxidative stress and the biology of ageing. Nature, 2000. 408(6809): p. 239.
21. Hsu, A.-L., C.T. Murphy, and C. Kenyon, Regulation of aging and age-related disease by DAF-16 and heat-shock factor. Science, 2003. 300(5622): p. 1142-1145.
22. Douglas, P.M., et al., Heterotypic signals from neural HSF-1 separate thermotolerance from longevity. Cell reports, 2015. 12(7): p. 1196-1204.
23. Baird, N.A., et al., HSF-1–mediated cytoskeletal integrity determines thermotolerance and life span. Science, 2014. 346(6207): p. 360-363.
24. Brenner, S., The genetics of Caenorhabditis elegans. Genetics, 1974. 77(1): p. 71-94.
25. Chiang, W.-C., et al., HSF-1 regulators DDL-1/2 link insulin-like signaling to heat-shock responses and modulation of longevity. Cell, 2012. 148(1-2): p. 322-334.
26. Lascano, R., et al., Paraquat: an oxidative stress inducer, in Herbicides-Properties, Synthesis and Control of Weeds. 2012, InTech.
27. Serrano-Saiz, E., et al., A neurotransmitter atlas of the Caenorhabditis elegans male nervous system reveals sexually dimorphic neurotransmitter usage. Genetics, 2017. 206(3): p. 1251-1269.
28. Ruvinsky, I. and G. Ruvkun, Functional tests of enhancer conservation between distantly related species. Development, 2003. 130(21): p. 5133-5142.
29. Serrano-Saiz, E., et al., Modular control of glutamatergic neuronal identity in C. elegans by distinct homeodomain proteins. Cell, 2013. 155(3): p. 659-673.
30. Chang, A.J., et al., A distributed chemosensory circuit for oxygen preference in C. elegans. PLoS biology, 2006. 4(9): p. e274.
31. Sze, J.Y., et al., The C. elegans POU-domain transcription factor UNC-86 regulates the tph-1 tryptophan hydroxylase gene and neurite outgrowth in specific serotonergic neurons. Development, 2002. 129(16): p. 3901-3911.
32. Pirri, J.K., D. Rayes, and M.J. Alkema, A change in the ion selectivity of ligand-gated ion channels provides a mechanism to switch behavior. PLoS biology, 2015. 13(9): p. e1002238.
33. Doitsidou, M., et al., A Caenorhabditis elegans Zinc Finger Transcription Factor, ztf-6, Required for the Specification of a Dopamine Neuron-Producing Lineage. G3: Genes, Genomes, Genetics, 2018. 8(1): p. 17-26.
34. Bessa, C., P. Maciel, and A.J. Rodrigues, Using C. elegans to decipher the cellular and molecular mechanisms underlying neurodevelopmental disorders. Molecular neurobiology, 2013. 48(3): p. 465-489.
35. Jospin, M., et al., A neuronal acetylcholine receptor regulates the balance of muscle excitation and inhibition in Caenorhabditis elegans. PLoS biology, 2009. 7(12): p. e1000265.
36. Pereira, L., et al., A cellular and regulatory map of the cholinergic nervous system of C. elegans. Elife, 2015. 4: p. e12432.
37. Firnhaber, C. and M. Hammarlund, Neuron-specific feeding RNAi in C. elegans and its use in a screen for essential genes required for GABA neuron function. PLoS genetics, 2013. 9(11): p. e1003921.
38. Akay, A., P. Sarkies, and E.A. Miska, E. coli OxyS non-coding RNA does not trigger RNAi in C. elegans. Scientific reports, 2015. 5: p. 9597.
39. Tabara, H., et al., The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell, 1999. 99(2): p. 123-132.
40. Nonet, M.L., et al., Caenorhabditis elegans rab-3 mutant synapses exhibit impaired function and are partially depleted of vesicles. Journal of Neuroscience, 1997. 17(21): p. 8061-8073.
41. Henderson, S.T. and T.E. Johnson, daf-16 integrates developmental and environmental inputs to mediate aging in the nematode Caenorhabditis elegans. Current Biology, 2001. 11(24): p. 1975-1980.
42. Kondo, M., et al., The p38 signal transduction pathway participates in the oxidative stress-mediated translocation of DAF-16 to Caenorhabditis elegans nuclei. Mechanisms of ageing and development, 2005. 126(6-7): p. 642-647.
43. Kondo, M., et al., Effect of oxidative stress on translocation of DAF-16 in oxygen-sensitive mutants, mev-1 and gas-1 of Caenorhabditis elegans. Mechanisms of ageing and development, 2005. 126(6-7): p. 637-641.
44. Sun, X., W.-D. Chen, and Y.-D. Wang, DAF-16/FOXO transcription factor in aging and longevity. Frontiers in pharmacology, 2017. 8: p. 548.
45. Chen, A.T.Y., et al., Effects of C aenorhabditis elegans sgk‐1 mutations on lifespan, stress resistance, and DAF‐16/F ox O regulation. Aging cell, 2013. 12(5): p. 932-940.
46. Wang, Y. and H.A. Tissenbaum, Overlapping and distinct functions for a Caenorhabditis elegans SIR2 and DAF-16/FOXO. Mechanisms of ageing and development, 2006. 127(1): p. 48-56.
47. Takahashi, Y., et al., Asymmetric arginine dimethylation determines life span in C. elegans by regulating forkhead transcription factor DAF-16. Cell metabolism, 2011. 13(5): p. 505-516.
48. Bansal, A., et al., Transcriptional regulation of Caenorhabditis elegans FOXO/DAF-16 modulates lifespan. Longevity & healthspan, 2014. 3(1): p. 5.
49. Tatum, M.C., et al., Neuronal serotonin release triggers the heat shock response in C. elegans in the absence of temperature increase. Current Biology, 2015. 25(2): p. 163-174.
50. Berendzen, K.M., et al., Neuroendocrine coordination of mitochondrial stress signaling and proteostasis. Cell, 2016. 166(6): p. 1553-1563. e10.