維持細胞生理功能需要嚴格調節細胞氧化還原平衡。然而，對酶和化學物質如何影響細胞內的氧化還原平衡的理解仍然有限。為了解決這個問題，我在此建立了穩定表達mito-roGFP2-Grx1、mito-roGFP2-Orp1、cyto-roGFP2-Grx1 或 cyto-roGFP2-Orp1生物傳感器的SH-SY5Y神經母細胞瘤細胞株，以檢測粒線體和細胞質中穀胱甘肽氧化還原能力和H2O2含量的變化。利用病毒轉染方法，我將這四種生物傳感器分別引入SH-SY5Y細胞中，並以共軛焦顯微鏡檢查了兩種mito-roGFP2在細胞中的表現情形。結果顯示以MitoView™633為指示標記，兩種mito-roGFP2確實在粒線體中表達。這些表達roGFP2的SH-SY5Y細胞株對 1 mM 二酰胺（一種氧化劑）或 5 mM 二硫蘇糖醇（一種還原劑）的處理非常敏感，證明它們能夠反映氧化還原變化。此roGFP2 生物傳感器對氧化劑-還原劑交換過程中的反應敏感，可用於活細胞成像以追踪即時氧化還原變化。以50-200 μM H2O2處理含有cyto-roGFP2-Orp1或mito-roGFP2-Orp1的細胞三小時，細胞內氧化程度隨著劑量增加而上升。以30-100 μM Pb(II)處理含有cyto-roGFP2-Orp1的細胞三小時也增加了氧化程度，但相同的處理不會改變粒線體的H2O2含量。總體而言，本論文的結果表明roGFP2生物傳感器是測量穀胱甘肽氧化還原能力和H2O2含量的可靠工具，可用於未來研究氧化還原相關問題的細胞效應。

Maintaining cellular physiological functions requires tight regulation of cellular redox balance. However, the understanding of how enzymes and chemicals affect cellular redox balance is still limited. To address this issue, here I established SH-SY5Y neuroblastoma cell lines stably expressing mito-roGFP2-Grx1, mito-roGFP2-Orp1, cyto-roGFP2-Grx1, or cyto-roGFP2-Orp1 biosensors to detect changes in glutathione redox capability and H2O2 levels in mitochondria and cytosol. By using a viral transduction protocol, I separately introduced the four biosensors into SH-SY5Y cells, and examined cellular expression pattern of the two mito-roGFP2 by confocal microscopy. The results showed that the two mito-roGFP2 were indeed expressed in mitochondria using MitoView™633 as an indicating marker. The roGFP2-expressing SH-SY5Y cell lines were very sensitive in response to the treatments of 1 mM diamide, an oxidant, or 5 mM dithiothreitol, a reducing agent, indicating that they are capable of reflecting redox changes. The roGFP2 biosensors were sensitive to oxidant-reductant exchanges and could be applied for live-cell imaging to track real-time redox alterations. Administering 50-200 μM H₂O₂ to mito-roGFP2-Orp1 or cyto-roGFP2-Orp1 containing cells for 3 h increased the oxidation degree in dose-dependent manners. Administering 30-100 μM Pb (II) to cyto-roGFP2-Orp1 containing cells for 3 h also increased the oxidation degree, but the same treatment did not alter the H₂O₂ levels in mitochondria. Overall, results of this thesis suggest that the roGFP2 biosensors are reliable tools for measuring glutathione redox capability and H2O2 levels that can be adapted for future studies on cellular effect of redox-related issues.

1.Abdal Dayem, A., Hossain, M. K., Lee, S. B., Kim, K., Saha, S. K., Yang, G. M., . . . Cho, S. G. (2017). The Role of Reactive Oxygen Species (ROS) in the Biological Activities of Metallic Nanoparticles. Int J Mol Sci, 18(1). doi:10.3390/ijms18010120

2.Aquilano, K., Baldelli, S., & Ciriolo, M. R. (2014). Glutathione: new roles in redox signaling for an old antioxidant. Front Pharmacol, 5, 196. doi:10.3389/fphar.2014.00196

3.Chai, Y. C., Ashraf, S. S., Rokutan, K., Johnston, R. B., Jr., & Thomas, J. A. (1994). S-thiolation of individual human neutrophil proteins including actin by stimulation of the respiratory burst: evidence against a role for glutathione disulfide. Arch Biochem Biophys, 310(1), 273-281. doi:10.1006/abbi.1994.1167

4.Cross, C. E., Halliwell, B., Borish, E. T., Pryor, W. A., Ames, B. N., Saul, R. L., . . . Harman, D. (1987). Oxygen radicals and human disease. Ann Intern Med, 107(4), 526-545. doi:10.7326/0003-4819-107-4-526

5.Di Marzo, N., Chisci, E., & Giovannoni, R. (2018). The Role of Hydrogen Peroxide in Redox-Dependent Signaling: Homeostatic and Pathological Responses in Mammalian Cells. Cells, 7(10). doi:10.3390/cells7100156

6.Dooley, C. T., Dore, T. M., Hanson, G. T., Jackson, W. C., Remington, S. J., & Tsien, R. Y. (2004). Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators. J Biol Chem, 279(21), 22284-22293. doi:10.1074/jbc.M312847200
7.Droge, W. (2002). Free radicals in the physiological control of cell function. Physiol Rev, 82(1), 47-95. doi:10.1152/physrev.00018.2001

8.Flora, G., Gupta, D., & Tiwari, A. (2012). Toxicity of lead: A review with recent updates. Interdiscip Toxicol, 5(2), 47-58. doi:10.2478/v10102-012-0009-2

9.Go, Y. M., Roede, J. R., Walker, D. I., Duong, D. M., Seyfried, N. T., Orr, M., . . . Jones, D. P. (2013). Selective targeting of the cysteine proteome by thioredoxin and glutathione redox systems. Mol Cell Proteomics, 12(11), 3285-3296. doi:10.1074/mcp.M113.030437

10.Gutscher, M., Pauleau, A. L., Marty, L., Brach, T., Wabnitz, G. H., Samstag, Y., . . . Dick, T. P. (2008). Real-time imaging of the intracellular glutathione redox potential. Nat Methods, 5(6), 553-559. doi:10.1038/nmeth.1212

11.Gutscher, M., Sobotta, M. C., Wabnitz, G. H., Ballikaya, S., Meyer, A. J., Samstag, Y., & Dick, T. P. (2009). Proximity-based protein thiol oxidation by H2O2-scavenging peroxidases. J Biol Chem, 284(46), 31532-31540. doi:10.1074/jbc.M109.059246

12.Han, D., Williams, E., & Cadenas, E. (2001). Mitochondrial respiratory chain-dependent generation of superoxide anion and its release into the intermembrane space. Biochem J, 353(Pt 2), 411-416. doi:10.1042/0264-6021:3530411

13.Hanson, G. T., Aggeler, R., Oglesbee, D., Cannon, M., Capaldi, R. A., Tsien, R. Y., & Remington, S. J. (2004). Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators. J Biol Chem, 279(13), 13044-13053. doi:10.1074/jbc.M312846200

14.Holmstrom, K. M., & Finkel, T. (2014). Cellular mechanisms and physiological consequences of redox-dependent signalling. Nat Rev Mol Cell Biol, 15(6), 411-421. doi:10.1038/nrm3801

15.Kohlgruber, S., Upadhye, A., Dyballa-Rukes, N., McNamara, C. A., & Altschmied, J. (2017). Regulation of Transcription Factors by Reactive Oxygen Species and Nitric Oxide in Vascular Physiology and Pathology. Antioxid Redox Signal, 26(13), 679-699. doi:10.1089/ars.2016.6946

16.Le Gal, K., Schmidt, E. E., & Sayin, V. I. (2021). Cellular Redox Homeostasis. Antioxidants (Basel), 10(9). doi:10.3390/antiox10091377

17.Liou, G. Y., & Storz, P. (2010). Reactive oxygen species in cancer. Free Radic Res, 44(5), 479-496. doi:10.3109/10715761003667554

18.Meister, A. (1988). Glutathione metabolism and its selective modification. J Biol Chem, 263(33), 17205-17208. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/3053703

19.Meyer, A. J., & Dick, T. P. (2010). Fluorescent protein-based redox probes. Antioxid Redox Signal, 13(5), 621-650. doi:10.1089/ars.2009.2948

20.Morgan, M. J., & Liu, Z. G. (2011). Crosstalk of reactive oxygen species and NF-kappaB signaling. Cell Res, 21(1), 103-115. doi:10.1038/cr.2010.178

21.Murphy, M. P., Bayir, H., Belousov, V., Chang, C. J., Davies, K. J. A., Davies, M. J., . . . Halliwell, B. (2022). Guidelines for measuring reactive oxygen species and oxidative damage in cells and in vivo. Nat Metab, 4(6), 651-662. doi:10.1038/s42255-022-00591-z

22.Nguyen, G. T., Green, E. R., & Mecsas, J. (2017). Neutrophils to the ROScue: Mechanisms of NADPH Oxidase Activation and Bacterial Resistance. Front Cell Infect Microbiol, 7, 373. doi:10.3389/fcimb.2017.00373

23.Niemeyer, J., Scheuring, D., Oestreicher, J., Morgan, B., & Schroda, M. (2021). Real-time monitoring of subcellular H2O2 distribution in Chlamydomonas reinhardtii. Plant Cell, 33(9), 2935-2949. doi:10.1093/plcell/koab176

24.Ostergaard, H., Henriksen, A., Hansen, F. G., & Winther, J. R. (2001). Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein. EMBO J, 20(21), 5853-5862. doi:10.1093/emboj/20.21.5853

25.Pizzino, G., Irrera, N., Cucinotta, M., Pallio, G., Mannino, F., Arcoraci, V., . . . Bitto, A. (2017). Oxidative Stress: Harms and Benefits for Human Health. Oxid Med Cell Longev, 2017, 8416763. doi:10.1155/2017/8416763

26.Schrader, M., & Fahimi, H. D. (2006). Peroxisomes and oxidative stress. Biochim Biophys Acta, 1763(12), 1755-1766. doi:10.1016/j.bbamcr.2006.09.006

27.Sousa, C. A., & Soares, E. V. (2014). Mitochondria are the main source and one of the targets of Pb (lead)-induced oxidative stress in the yeast Saccharomyces cerevisiae. Appl Microbiol Biotechnol, 98(11), 5153-5160. doi:10.1007/s00253-014-5631-9

28.Ubezio, P., & Civoli, F. (1994). Flow cytometric detection of hydrogen peroxide production induced by doxorubicin in cancer cells. Free Radic Biol Med, 16(4), 509-516. doi:10.1016/0891-5849(94)90129-5

29.Valko, M., Leibfritz, D., Moncol, J., Cronin, M. T., Mazur, M., & Telser, J. (2007). Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol, 39(1), 44-84. doi:10.1016/j.biocel.2006.07.001

30.Zhang, J., Wang, X., Vikash, V., Ye, Q., Wu, D., Liu, Y., & Dong, W. (2016). ROS and ROS-Mediated Cellular Signaling. Oxid Med Cell Longev, 2016, 4350965. doi:10.1155/2016/4350965