粒線體為細胞內負責能量產生與調控細胞生長及活性的重要胞器。粒線體的形態透過不斷的融合與分裂達到動態平衡，許多研究指出粒線體的形態改變與其活性具有高度的關聯性。茶黃素為多酚類化合物，是紅茶中色素與抗氧化的主要成份，茶黃素的研究指出其對於生物具有抗老化、抑制腫瘤生長與降低血膽固醇等功能。然而，目前茶黃素對於老化細胞粒線體動態的影響所知甚少。因此，本篇論文透過酵母菌來研究茶黃素與粒線體之間的關係。本論文的研究中發現，在培養過程中加入茶黃素的衰老酵母菌擁有更多碎裂形態的粒線體。我發現最常存在於粒線體中的活性氧化物質並非造成衰老細胞粒線體碎裂的主因。將負責粒線體分裂的基因DNM1剔除掉後可以抑制衰老細胞與茶黃素所造成的碎裂形態。此外，我的研究結果也指出茶黃素能夠減緩細胞分裂的速度。透過檢測粒線體的呼吸能力也發現茶黃素能減緩老化細胞中粒線體功能的衰退。在本研究中，我的結果提供了更多資訊去了解茶黃素對衰老酵母菌中的粒線體的影響，將有助於進一步的了解粒線體動態調控機制。

Mitochondria are highly dynamic organelles. Their morphology were mainly determined by continuous fusion and fission. Many research studies indicated that aging process is associated with mitochondrial disintegration. Theaflavins are the major compounds extracted from black tea. Theaflavins dominated the pigment and fragrance. The benefits of theaflavins, such as antiaging, inhibition of tumor proliferation and blood cholesterol reduction have been suggested in previous studies. However, little is known about the effect of theaflavins on mitochondria in senescent yeasts, so I aim to clarify the effects of theaflavins on mitochondrial dynamics and activity. Previous work found that mitochondria networks turn into fragmented in senescent yeast cells. Surprisingly, my results demonstrated that treating theaflavins caused mitochondrial morphology more fragmented than untreated senescent cells. In addition, my results indicated that the fragmented mitochondria in senescent yeasts are not caused by reactive oxygen species. The fragmented mitochondria in senescent cells under theaflavins treatment derived from fission protein Dnm1. Furthermore, my experiment results showed that theaflavins increase mitochondrial respiratory capacity and reduced budding scars. Taken together, the results suggested that theaflavins may slow down cell cycle progression and aid to maintain proper mitochondrial activity. These information shed light on the underlying mechanisms of theaflavins effects on mitochondria in senescent yeasts.

1. Bandy, B., and Davison, A.J. (1990). Mitochondrial mutations may increase oxidative stress: implications for carcinogenesis and aging? Free Radic Biol Med 8, 523-539.
2. Barker, M.G., and Walmsley, R.M. (1999). Replicative ageing in the fission yeast Schizosaccharomyces pombe. Yeast 15, 1511-1518.
3. Bockler, S., Chelius, X., Hock, N., Klecker, T., Wolter, M., Weiss, M., Braun, R.J., and Westermann, B. (2017). Fusion, fission, and transport control asymmetric inheritance of mitochondria and protein aggregates. J Cell Biol 216, 2481-2498.
4. Bonawitz, N.D., Chatenay-Lapointe, M., Pan, Y., and Shadel, G.S. (2007). Reduced TOR signaling extends chronological life span via increased respiration and upregulation of mitochondrial gene expression. Cell Metab 5, 265-277.
5. Bonawitz, N.D., Rodeheffer, M.S., and Shadel, G.S. (2006). Defective mitochondrial gene expression results in reactive oxygen species-mediated inhibition of respiration and reduction of yeast life span. Mol Cell Biol 26, 4818-4829.
6. Bordt, E.A., Clerc, P., Roelofs, B.A., Saladino, A.J., Tretter, L., Adam-Vizi, V., Cherok, E., Khalil, A., Yadava, N., Ge, S.X., et al. (2017). The Putative Drp1 Inhibitor mdivi-1 Is a Reversible Mitochondrial Complex I Inhibitor that Modulates Reactive Oxygen Species. Dev Cell 40, 583-594 e586.
7. Burte, F., Carelli, V., Chinnery, P.F., and Yu-Wai-Man, P. (2015). Disturbed mitochondrial dynamics and neurodegenerative disorders. Nat Rev Neurol 11, 11-24.
8. Chance, B., and Williams, G.R. (1956). The respiratory chain and oxidative phosphorylation. Adv Enzymol Relat Subj Biochem 17, 65-134.
9. Comfort, A. (1956). The biology of ageing. Lancet 271, 772-778.
10. De Vecchis, D., Cavellini, L., Baaden, M., Henin, J., Cohen, M.M., and Taly, A. (2017). A membrane-inserted structural model of the yeast mitofusin Fzo1. Sci Rep 7, 10217.
11. Desler, C., Hansen, T.L., Frederiksen, J.B., Marcker, M.L., Singh, K.K., and Juel Rasmussen, L. (2012). Is There a Link between Mitochondrial Reserve Respiratory Capacity and Aging? J Aging Res 2012, 192503.
12. Dhawan, J., and Laxman, S. (2015). Decoding the stem cell quiescence cycle--lessons from yeast for regenerative biology. J Cell Sci 128, 4467-4474.
13. Endo, T., Yamamoto, H., and Esaki, M. (2003). Functional cooperation and separation of translocators in protein import into mitochondria, the double-membrane bounded organelles. J Cell Sci 116, 3259-3267.
14. Frank, S., Gaume, B., Bergmann-Leitner, E.S., Leitner, W.W., Robert, E.G., Catez, F., Smith, C.L., and Youle, R.J. (2001). The role of dynamin-related protein 1, a mediator of mitochondrial fission, in apoptosis. Dev Cell 1, 515-525.
15. Friedman, J.R., and Nunnari, J. (2014). Mitochondrial form and function. Nature 505, 335-343.
16. Fu, G., Wang, H., Cai, Y., Zhao, H., and Fu, W. (2018). Theaflavin alleviates inflammatory response and brain injury induced by cerebral hemorrhage via inhibiting the nuclear transcription factor kappa beta-related pathway in rats. Drug Des Devel Ther 12, 1609-1619.
17. Gems, D. (2015). The aging-disease false dichotomy: understanding senescence as pathology. Front Genet 6, 212.
18. Halder, J., and Bhaduri, A.N. (1998). Protective role of black tea against oxidative damage of human red blood cells. Biochem Biophys Res Commun 244, 903-907.
19. Handee, W., Li, X., Hall, K.W., Deng, X., Li, P., Benning, C., Williams, B.L., and Kuo, M.H. (2016). An Energy-Independent Pro-longevity Function of Triacylglycerol in Yeast. PLoS Genet 12, e1005878.
20. Harman, D. (1956). Aging: a theory based on free radical and radiation chemistry. J Gerontol 11, 298-300.
21. Harman, D. (1981). The aging process. Proc Natl Acad Sci U S A 78, 7124-7128.
22. Hatch, A.L., Gurel, P.S., and Higgs, H.N. (2014). Novel roles for actin in mitochondrial fission. J Cell Sci 127, 4549-4560.
23. Heden, T.D., Neufer, P.D., and Funai, K. (2016). Looking Beyond Structure: Membrane Phospholipids of Skeletal Muscle Mitochondria. Trends Endocrinol Metab 27, 553-562.
24. Hermann, G.J., Thatcher, J.W., Mills, J.P., Hales, K.G., Fuller, M.T., Nunnari, J., and Shaw, J.M. (1998). Mitochondrial fusion in yeast requires the transmembrane GTPase Fzo1p. J Cell Biol 143, 359-373.
25. Hoppins, S., Lackner, L., and Nunnari, J. (2007). The machines that divide and fuse mitochondria. Annu Rev Biochem 76, 751-780.
26. Imran, A., Arshad, M.U., Arshad, M.S., Imran, M., Saeed, F., and Sohaib, M. (2018). Lipid peroxidation diminishing perspective of isolated theaflavins and thearubigins from black tea in arginine induced renal malfunctional rats. Lipids Health Dis 17, 157.
27. Jensen, R.E., Hobbs, A.E., Cerveny, K.L., and Sesaki, H. (2000). Yeast mitochondrial dynamics: fusion, division, segregation, and shape. Microsc Res Tech 51, 573-583.
28. Jiang, J.C., Stumpferl, S.W., Tiwari, A., Qin, Q., Rodriguez-Quinones, J.F., and Jazwinski, S.M. (2016). Identification of the Target of the Retrograde Response that Mediates Replicative Lifespan Extension in Saccharomyces cerevisiae. Genetics 204, 659-673.
29. Kaeberlein, M., Kirkland, K.T., Fields, S., and Kennedy, B.K. (2004). Sir2-independent life span extension by calorie restriction in yeast. PLoS Biol 2, E296.
30. Kaeberlein, M., McVey, M., and Guarente, L. (1999). The SIR2/3/4 complex and SIR2 alone promote longevity in Saccharomyces cerevisiae by two different mechanisms. Genes Dev 13, 2570-2580.
31. Khan, S.M., Sobti, R.C., and Kataria, L. (2005). Pesticide-induced alteration in mice hepato-oxidative status and protective effects of black tea extract. Clin Chim Acta 358, 131-138.
32. Kraujalyte, V., Pelvan, E., and Alasalvar, C. (2016). Volatile compounds and sensory characteristics of various instant teas produced from black tea. Food Chem 194, 864-872.
33. Kuilman, T., Michaloglou, C., Mooi, W.J., and Peeper, D.S. (2010). The essence of senescence. Genes Dev 24, 2463-2479.
34. Kuroda, Y., and Hara, Y. (1999). Antimutagenic and anticarcinogenic activity of tea polyphenols. Mutat Res 436, 69-97.
35. Lakowski, B., and Hekimi, S. (1996). Determination of life-span in Caenorhabditis elegans by four clock genes. Science 272, 1010-1013.
36. Lane, N., and Martin, W. (2010). The energetics of genome complexity. Nature 467, 929-934.
37. Li, S., Lo, C.Y., Pan, M.H., Lai, C.S., and Ho, C.T. (2013). Black tea: chemical analysis and stability. Food Funct 4, 10-18.
38. Lin, J.K., Chen, P.C., Ho, C.T., and Lin-Shiau, S.Y. (2000). Inhibition of xanthine oxidase and suppression of intracellular reactive oxygen species in HL-60 cells by theaflavin-3,3'-digallate, (-)-epigallocatechin-3-gallate, and propyl gallate. J Agric Food Chem 48, 2736-2743.
39. Liu, S., Lu, H., Zhao, Q., He, Y., Niu, J., Debnath, A.K., Wu, S., and Jiang, S. (2005). Theaflavin derivatives in black tea and catechin derivatives in green tea inhibit HIV-1 entry by targeting gp41. Biochim Biophys Acta 1723, 270-281.
40. Longo, V.D., Antebi, A., Bartke, A., Barzilai, N., Brown-Borg, H.M., Caruso, C., Curiel, T.J., de Cabo, R., Franceschi, C., Gems, D., et al. (2015). Interventions to Slow Aging in Humans: Are We Ready? Aging Cell 14, 497-510.
41. Longo, V.D., and Fabrizio, P. (2012). Chronological aging in Saccharomyces cerevisiae. Subcell Biochem 57, 101-121.
42. Longo, V.D., Shadel, G.S., Kaeberlein, M., and Kennedy, B. (2012). Replicative and chronological aging in Saccharomyces cerevisiae. Cell Metab 16, 18-31.
43. Lu, J., Ho, C.T., Ghai, G., and Chen, K.Y. (2000). Differential effects of theaflavin monogallates on cell growth, apoptosis, and Cox-2 gene expression in cancerous versus normal cells. Cancer Res 60, 6465-6471.
44. Ludovico, P., Sansonetty, F., and Côrte-Real, M. (2001). Assessment of mitochondrial membrane potential in yeast cell populations by flow cytometry. Microbiology 147, 3335-3343.
45. Nunnari, J., and Suomalainen, A. (2012). Mitochondria: in sickness and in health. Cell 148, 1145-1159.
46. Okunuki, K. (1965). [Structure and function of mitochondria. Introduction]. Tanpakushitsu Kakusan Koso 10, 1241-1242.
47. Oliveira, A.V., Vilaca, R., Santos, C.N., Costa, V., and Menezes, R. (2017). Exploring the power of yeast to model aging and age-related neurodegenerative disorders. Biogerontology 18, 3-34.
48. Osman, C., Noriega, T.R., Okreglak, V., Fung, J.C., and Walter, P. (2015). Integrity of the yeast mitochondrial genome, but not its distribution and inheritance, relies on mitochondrial fission and fusion. Proc Natl Acad Sci U S A 112, E947-956.
49. Otsuga, D., Keegan, B.R., Brisch, E., Thatcher, J.W., Hermann, G.J., Bleazard, W., and Shaw, J.M. (1998). The dynamin-related GTPase, Dnm1p, controls mitochondrial morphology in yeast. J Cell Biol 143, 333-349.
50. Payne, B.A., and Chinnery, P.F. (2015). Mitochondrial dysfunction in aging: Much progress but many unresolved questions. Biochim Biophys Acta 1847, 1347-1353.
51. Polymenis, M., and Kennedy, B.K. (2012). Chronological and replicative lifespan in yeast: do they meet in the middle? Cell Cycle 11, 3531-3532.
52. Powell, C.D., Quain, D.E., and Smart, K.A. (2003). The impact of brewing yeast cell age on fermentation performance, attenuation and flocculation. FEMS Yeast Res 3, 149-157.
53. Rockstein, M., and Brandt, K.F. (1963). Enzyme changes in flight muscle correlated with aging and flight ability in the male housefly. Science 139, 1049-1051.
54. Rolland, F., Winderickx, J., and Thevelein, J.M. (2002). Glucose-sensing and -signalling mechanisms in yeast. FEMS Yeast Res 2, 183-201.
55. Roy, M., Reddy, P.H., Iijima, M., and Sesaki, H. (2015). Mitochondrial division and fusion in metabolism. Curr Opin Cell Biol 33, 111-118.
56. Sauerwald, J., Jores, T., Eisenberg-Bord, M., Chuartzman, S.G., Schuldiner, M., and Rapaport, D. (2015). Genome-Wide Screens in Saccharomyces cerevisiae Highlight a Role for Cardiolipin in Biogenesis of Mitochondrial Outer Membrane Multispan Proteins. Mol Cell Biol 35, 3200-3211.
57. Schrempp, S.G., and van der Laan, M. (2015). Get Ready for Fusion: Insights into Mgm1-Mediated Membrane Remodeling. J Mol Biol 427, 2595-2598.
58. Sharma, V., and Rao, L.J. (2009). A thought on the biological activities of black tea. Crit Rev Food Sci Nutr 49, 379-404.
59. Shiraki, M., Hara, Y., Osawa, T., Kumon, H., Nakayama, T., and Kawakishi, S. (1994). Antioxidative and antimutagenic effects of theaflavins from black tea. Mutat Res 323, 29-34.
60. Smeal, T., Claus, J., Kennedy, B., Cole, F., and Guarente, L. (1996). Loss of transcriptional silencing causes sterility in old mother cells of S. cerevisiae. Cell 84, 633-642.
61. Stadtman, E.R., Van Remmen, H., Richardson, A., Wehr, N.B., and Levine, R.L. (2005). Methionine oxidation and aging. Biochim Biophys Acta 1703, 135-140.
62. Tan, Q., Peng, L., Huang, Y., Huang, W., Bai, W., Shi, L., Li, X., and Chen, T. (2018). Structure-activity Relationship Analysis on Antioxidant and Anticancer Actions of Theaflavins on Human Colon Cancer Cells. J Agric Food Chem.
63. Toussaint, M., Levasseur, G., Gervais-Bird, J., Wellinger, R.J., Elela, S.A., and Conconi, A. (2006). A high-throughput method to measure the sensitivity of yeast cells to genotoxic agents in liquid cultures. Mutat Res 606, 92-105.
64. Toyama, E.Q., Herzig, S., Courchet, J., Lewis, T.L., Jr., Loson, O.C., Hellberg, K., Young, N.P., Chen, H., Polleux, F., Chan, D.C., et al. (2016). Metabolism. AMP-activated protein kinase mediates mitochondrial fission in response to energy stress. Science 351, 275-281.
65. van Deursen, J.M. (2014). The role of senescent cells in ageing. Nature 509, 439-446.
66. Vinson, J.A., and Zhang, J. (2005). Black and green teas equally inhibit diabetic cataracts in a streptozotocin-induced rat model of diabetes. J Agric Food Chem 53, 3710-3713.
67. Wallace, D.C., and Fan, W. (2009). The pathophysiology of mitochondrial disease as modeled in the mouse. Genes Dev 23, 1714-1736.
68. Wang, I.H., Chen, H.Y., Wang, Y.H., Chang, K.W., Chen, Y.C., and Chang, C.R. (2014). Resveratrol modulates mitochondria dynamics in replicative senescent yeast cells. PLoS One 9, e104345.
69. Westermann, B. (2010). Mitochondrial fusion and fission in cell life and death. Nat Rev Mol Cell Biol 11, 872-884.
70. Wong, E.D., Wagner, J.A., Gorsich, S.W., McCaffery, J.M., Shaw, J.M., and Nunnari, J. (2000). The dynamin-related GTPase, Mgm1p, is an intermembrane space protein required for maintenance of fusion competent mitochondria. J Cell Biol 151, 341-352.
71. Wu, S., Zhou, F., Zhang, Z., and Xing, D. (2011). Mitochondrial oxidative stress causes mitochondrial fragmentation via differential modulation of mitochondrial fission-fusion proteins. FEBS J 278, 941-954.
72. You, Y., Yuan, X., Lee, H.J., Huang, W., Jin, W., and Zhan, J. (2015). Mulberry and mulberry wine extract increase the number of mitochondria during brown adipogenesis. Food Funct 6, 401-408.
Youle, R.J., and van der Bliek, A.M. (2012). Mitochondrial fission, fusion, and stress. Science 337, 1062-1065.
73. Zorova, L.D., Popkov, V.A., Plotnikov, E.Y., Silachev, D.N., Pevzner, I.B., Jankauskas, S.S., Babenko, V.A., Zorov, S.D., Balakireva, A.V., Juhaszova, M., et al. (2018). Mitochondrial membrane potential. Anal Biochem 552, 50-59.