極度高眼壓造成組織缺血，而當缺血後再灌流，會產生大量活性氧分子及氧化性壓力，進而造成組織受損。本研究以 lysine-labeling-based two-dimensional difference gel electrophoresis (2D-DIGE) 合併 MALDI-TOF/TOF mass spectrometry (MALDI-TOF/TOF MS) 方法測定眼內缺血再灌流一小時後對角膜、結膜、葡萄膜、視網膜、鞏膜等組織蛋白質表現量之變化。結果發現因缺血再灌流傷害所造成的組織蛋白變化量以角膜 (cornea) 最多，其次是結膜 (conjunctiva) 、葡萄膜 (uvea) 與鞏膜 (sclera)，最後才為視網膜 (retina)。
　　隨後，我們往下延伸進行細胞實驗的機制性和訊號傳遞路徑探討，培視網膜細胞 Retinal Ganglion Cell, RGC-5/小樑網細胞 Human Trabecular Meshwork Cell, HTMC。已有文獻指出，氧化壓力誘導的TGF-β信號路徑與細胞外基質（Extracellular matrix, ECM）纖維化有關，造成組織損傷，並刺激細胞的抗氧化機制；另一方面，TGF-β也可作為神經保護蛋白，提升細胞耐受性。為了闡明TGF-β對RGC和HTMC的雙重潛在作用和調節機制，以過氧化氫不同濃度與作用時間誘發細胞產生氧化壓力。通過一系列細胞功能定性分析，包含MTT細胞活性測試、傷口癒合能力測定、細胞凋亡測定、細胞內ROS偵測、西方墨點法、細胞內GSH含量和粒線體活性測定法，我們闡明了RGC-5 細胞受氧化刺激誘導的損傷狀態。在H2O2刺激後，RGC中的TGF-β1和TGF-β2 表現量上升；細胞功能測定結果中，shTGF-β1 和 shTGF-β2會降低存活率，促進細胞凋亡和ROS積累。特別是TGF-β1 (5 ng/mL) 的上升促進了ALDH3A1 蛋白表達，並增加了抗氧化劑和神經保護途徑的活性。此外，TGF-β1和TGF-β2的抗氧化信號與HO-1和Nrf2的活化有關，ROS的累積導致HIF-1α表現量上升，進而引起線粒體損傷並導致神經病變。TGF-β1通過活化 Nrf2和HO-1信號平衡HIF-1α的表現量上升來保護RGC免受自由基引發的損傷，這暗示TGF-β1在視網膜神經保護相關療法中具有前瞻性作用。另一方面在HTMC中，與正常TM細胞相比下，H2O2作用的HTMC具有更不規則的肌動蛋白結構。rhTGF-β1 (1 ng/mL) 預處理的組別降低了細胞凋亡率、纖維化狀態和ROS累積，同時還提高了存活率。此外，就抗氧化信號而言，TGF-β1和TGF-β2與纖維化反應蛋白膠原 (collagen I) 和laminin的活化有關。簡而言之，我們的研究表示低濃度的TGF-β1 (1 ng/mL)使HTMCs免受自由基引起的p-p38 MAPK路徑和p-AKT信號的損害，從而提出了TGF-β1在HTMC氧化中的信號機制；而提高TGF-β1 濃度至 5 ng/mL則可能加劇損傷。
　　糖尿病的一個顯著病徵是高血糖，其被認為會誘發進行性神經變性疾病的糖尿病性視網膜病變。為了研究TGF-β在高血糖誘發的RGC-5和HTMC細胞損傷中的潛在作用和調控機制，我們以不同糖濃度細胞培養液來模擬糖尿病人體內高糖生理狀態。細胞功能實驗結果顯示，高血糖的RGC中TGF-β1/ 2表現量上升。降低TGF-β會增加高血糖症中ROS的積累，抑制細胞增殖速率並降低細胞內GSH含量。此外，TGF-β調控的抗氧化信號的增強與細胞內刺激反應蛋白的活化和抗氧化途徑有關，例如ALDH3A1, HO-1, Nrf2, HIF-1α。我們的結果表示，TGF-β通過促進抗氧化劑途徑的活化而使RGC免受高血糖觸發的損害，顯示糖尿病性視網膜病的潛在抗糖尿病療法。另一方面，依照實驗結果，高血糖培養下HTMCs中p-JNK，p-p38，p-AKT，TGF-β1和TGF-β2及SMAD表現量上升。細胞功能測定結果表明，在高血糖的HTMC中添加rhTGFβ-1 (1 ng/mL) 結果具有較高的增殖速率，較低的ROS和鈣離子濃度。另外，抗氧化劑信號傳導中的TGF-β1和TGF-β2與纖維化反應蛋白α-SMA，collagen I和laminin的活化有關。因此，當一定濃度的TGF-β會啟動細胞的抗氧化機制；但當產生過高濃度的 TGF-β 時會導致HTMC 纖維化，進而導致細胞受損、功能喪失。
　　此研究透過一系列分子細胞生物學的機制鑑定與蛋白質體學的分析，探討 TGF-β 在氧化壓力誘導 (過氧化氫 & 高糖濃度) 下，對細胞的功能性影響與引發之訊號傳遞路徑。

　　Glaucoma is a group of eye diseases that can cause vision loss and optical nerve damage. The ischemia–reperfusion (IR) injury model combined with the proteomic analysis approach of two-dimensional difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to monitor the protein expression alterations in two groups of specimens ( IR / control group). Based on the ratio of protein expression alterations, the alterations in the ocular tissues were in the following order: the cornea, conjunctiva, uvea, sclera, and retina.
　　Furthermore, oxidative stress generated by reactive oxygen species (ROS) plays a critical role in the pathogenesis of glaucoma and may induce retinal ganglion cell (RGC) and trabecular meshwork cell (HTMC) damage. It is known that the TGF-β signaling pathway is an important component of oxidative stress-induced damage related to ECM fibrosis and activates cell antioxidative mechanisms. To elucidate the dual potential roles and regulatory mechanisms of TGF-β in effects on RGC-5 and HTMCs, we established an in vitro oxidative model using hydrogen peroxide (H2O2) sufficiency. By a series of cell functional qualitative analysis, we illustrated the RGC-5 cell state in oxidative stress-induced injury. Cell functional assays resulted that knockdown of TGF-β1 & TGF-β2 reduced survival rate whereas enhanced apoptosis and accumulation of ROS. Especially TGF-β1 upregulation promoted the protein expression of ALDH3A1 and increased the activity of antioxidant and neuroprotection pathways. In summary, our results demonstrated that TGF-β1 preserves RGC-5 from free radicals-mediated injury by upregulating the activation of Nrf2 expression and HO-1 signaling balance HIF-1α upregulation, implying a prospective role of TGF-β1 in retinal neuroprotection-related therapies. Cell functional assays showed that HTMCs with H2O2 exposure duration had a more irregular actin architecture compared to normal TM cells, and data with rhTGF-β1 (1 ng/mL) pretreatment enhanced survival. Furthermore, TGF-β1 in terms of antioxidant signaling were related to the activation of collagen I and laminin, which are fibrosis-response proteins. Succinctly, our study demonstrated that low concentrations of TGF-β1 (1ng/mL) preserves HTMCs from free radical-mediated injury by p-p38 MAPK level and p-AKT signaling balance, presenting a signaling transduction mechanism of TGF-β1 in HTMC oxidative stress-related therapies.
　　A characteristic of diabetes mellitus is hyperglycemia, which is considered with an emphasis on the diabetic retinopathy of progressive neurodegenerative disease. To investigate the potential roles and regulatory mechanisms of TGF-β in hyperglycemia-triggered damage of RGC-5 and HTMCs in vitro, functional experiments showed that the knockdown of TGF-β enhanced the accumulation of ROS, in hyperglycemia. Furthermore, the results showed that the TGF-β-mediated enhancement of antioxidant signaling was correlated with the activation of stress response proteins and the antioxidant pathway, such as ALDH3A1, HO-1, Nrf2, and HIF-1α. Summarizing, our results demonstrated that TGF-β keeps RGC-5 from hyperglycemia-triggered harm, suggesting a potential anti-diabetic therapy. Results of protein expression showed that p-JNK, p-p38, p-AKT, TGF-β1 & TGF-β2 and its related SMAD family were upregulated in HTMCs after hyperglycemia. Cell functional assays resulted that HTMCs with rhTGFβ-1 (1 ng/mL) and hyperglycemia have higher proliferation rate, lower ROS and calcium level. Additionally, TGF-β1 & TGF-β2 on antioxidant signaling was related to activation of α-SMA, collagen I, and laminin, which are fibrosis-response proteins. 　
　　Mechanistic and proteomic analyses in ocular cells explored the characteristics and effects of TGF-β on RGC-5 and HTMC cells, while further investigated the signal transduction pathway induced by oxidative stress.

Table of Contents

中文摘要 I
Abstract III
Table of Contents V
List of Figures and Tables XIV
Chapter 1　INTRODUCTION 1
1.1 Overview of oxidative stress on ocular cells 1
1.2 Proteomic analysis of various rat ocular tissues after ischemia–reperfusion injury and possible relevance to acute glaucoma 5
1.3 TGF-β1 signaling protects retinal ganglion cells from oxidative stress via modulation of the HO-1/Nrf2 pathway 9
1.4 Characterization of TGF-β by induced oxidative stress in human trabecular meshwork cells 11
1.5 The role of transforming growth factor-beta in retinal ganglion cells with hyperglycemia and oxidative stress 14
1.6 Effects of hyperglycemia on the TGF-β pathway in trabecular meshwork cells 16
Chapter 2　MATERIALS and METHODS 19
2.1 Chemicals, reagents and antibodies 19
2.2 Animals 20
2.2.1 IR Injury Model 20
2.3 Proteomic analysis 21
2.3.1 2D-DIGE and Gel Image Analysis 21
2.3.2 Protein Staining 22
2.3.3 In-Gel Digestion 23
2.3.4 Protein Identification by MALDI-TOF MS 23
2.4 Cell lines and cell cultures 24
2.4.1 H2O2 sufficiency 25
2.4.2 Hyperglycemia cell culture 25
2.4.3 shTGF-β1/2 knockdown RGC-5 & HTMC establishment 26
2.4.4 siRNA knockdown and transfection 26
2.5 Immunoblotting analysis 27
2.6 Immunofluorescence 29
2.7 In vitro functional assays 29
2.7.1 Cell viability assay 29
2.7.2 Proliferation Assay 30
2.7.3 Wound healing ability assay 30
2.7.4 Detection of ROS increase 30
2.7.5 Intracellular calcium-level measurement 31
2.7.6 Apoptosis assay by flow cytometry analysis 31
2.7.7 Measurement of intracellular GSH content 32
2.7.8 High-resolution respirometry 32
2.8 Cell treatment with recombinant human TGF-β protein1 (rhTGF-β1) 33
2.9 Statistical analysis 33
Chapter 3　RESULTS 34
3.1 Proteomic Analysis of Various Rat Ocular Tissues after Ischemia–Reperfusion Injury and Possible Relevance to Acute Glaucoma 34
3.1.1 Analysis of Various Ocular Tissues Including Cornea, Conjunctiva, Uvea, Retina, and Sclera by 2D-DIGE and MALDI-TOF 34
3.1.2 Validation of Characterized Ocular Retinal Proteins through Immunoblotting 41
3.2 TGF-β1 signaling protects retinal ganglion cells from oxidative stress via modulation of the HO-1/Nrf2 pathway 42
3.2.1 Cell viability at different H2O2 concentrations and exposure durations in RGCs 42
3.2.2 Low levels (50 μM) of H2O2 promote the cell ability of proliferation, migration, and wound healing 44
3.2.3 Effects of H2O2 on cell apoptotic status and increased iROS levels on RGCs 46
3.2.4 GSH content of RGCs under H2O2 (1 mM) stimulation 48
3.2.5 Immunoblot analysis of RGCs exposed to H2O2 49
3.2.6 Immunofluorescence analysis of RGCs under H2O2 (1 mM) stimulation 50
3.2.7 Cell viability and iROS accumulation in RGCs under H2O2 (1 mM) stimulation with/without N-acetyl-L-cysteine (NAC, 5mM) 51
3.2.8 Cell viability, apoptosis analysis and ROS production in shTGF-β1/2 knockdown RGCs 53
3.2.9 Immunoblot analysis of shTGF-β1/2 knockdown RGCs with hydrogen peroxide (1 mM) 55
3.2.10 Oxygen consumption rate (OCR) of RGC-5 shTGF-β1/2 cells with hydrogen peroxide treatment 56
3.2.11 Cell viability, apoptosis analysis and ROS accumulation in RGC-5 cells treated with recombinant TGF-β1 protein (5 ng/ml) with or without hydrogen peroxide (1 mM) 57
3.2.12 Immunoblot analysis of activation of downstream substrates in RGC-5 cells treated with recombinant TGF-β1 protein (5 ng/ml) with or without hydrogen peroxide (1 mM) 59
3.3 Characterization of TGF-β by induced oxidative stress in human trabecular meshwork cells 60
3.3.1 Impairments by H2O2 of HTMCs 60
3.3.2 Immunoblot analysis and identification of H2O2-induced oxidative stress response signaling pathways 63
3.3.3 Immunofluorescence analysis of H2O2 (0.5 mM)-treated HTMC 66
3.3.4 Effects of rhTGF-β1 protein on oxidative stress response on HTMC
68
3.3.5 Immunoblot analysis and identification of rhTGF-β1 protein in oxidative stress pathways 71
3.3.6 Immunofluorescence analysis of rhTGF-β1 protein-treated HTMCs with or without H2O2 exposure (0.5 mM for 1 h) 74
3.3.7 Effects of shTGF-β1/2 knockdown in HTMCs with H2O2 exposure
76
3.3.8 Immunoblot analysis and identification of shTGF-β1/2 knockdown HTMCs in oxidative stress pathways 79
3.3.9 Effects of sip38 MAPK knockdown in HTMCs with H2O2 exposure
82
3.3.10 Immunoblot analysis and identification of sip38 MAPK knockdown HTMCs in oxidative stress pathways 84
3.4 The Role of Transforming Growth Factor-Beta in Retinal Ganglion Cells with Hyperglycemia and Oxidative Stress 87
3.4.1 Effects of Hyperglycemia on RGCs 87
3.4.2 Immunoblot Analysis of RGCs with Hyperglycemia 89
3.4.3 Immunofluorescence Analysis of RGCs with Hyperglycemia. 91
3.4.4 Effects in TGF-β1/2 Knockdown RGCs with Hyperglycemia 93
3.4.5 Immunoblot Analysis of Activation of Downstream Substrates in TGF-β1/2 Knockdown RGC-5 cells Treated with Hyperglycemia. 95
3.4.6 Effects in RGCs with Hyperglycemia with or w/o rhTGF-β1 Protein (5 ng/mL). 97
3.4.7 Immunoblot Analysis in RGCs with Hyperglycemia with or w/o Recombinant TGF-β1 Protein (5 ng/mL). 100
3.4.8 Effects in RGCs with Hyperglycemia with or without Hydrogen Peroxide for 1 h. 102
3.4.9 Immunoblot Analysis in RGCs with Hyperglycemia with or without Hydrogen Peroxide (1 mM) for 1 h. 104
3.5 Effects of hyperglycemia on TGF-β pathway in trabecular meshwork cells 107
3.5.1 Hyperglycemia effects on HTMCs　 107
3.5.2 Immunoblot analysis of hyperglycemia effects on HTMCs 110
3.5.3 Immunofluorescence analysis of the effects of hyperglycemia on HTMCs　　. 112
3.5.4 Hyperglycemia effects on TGF-β1/2 knockdown HTMCs 114
3.5.5 Immunoblot analysis to determine the effects of hyperglycemia on TGF-β1/2 knockdown HTMCs. 117
3.5.6 Hyperglycemia effects of on HTMCs with or without rhTGF-β1 protein (1 and 5 ng/ml). 120
3.5.7 Immunoblot analysis to determine the effects of hyperglycemia on HTMCs with or without recombinant TGF-β1 protein (1 ng/ml). 123
3.5.8 Hydrogen peroxide-induced oxidative stress and effects of hyperglycemia on HTMCs. 126
3.5.9 Immunoblot analysis to determine the effects of hyperglycemia on HTMCs with or without H2O2 (0.5 mM) for 1 h. 129
Chapter 4　DISCUSSION 132
4.1 Proteomic Analysis of Various Rat Ocular Tissues after Ischemia–Reperfusion Injury and Possible Relevance to Acute Glaucoma 132
4.2 TGF-β1 signaling protects retinal ganglion cells from oxidative stress via modulation of the HO-1/Nrf2 pathway 136
4.3 Characterization of TGF-β by induced oxidative stress in human trabecular meshwork cells 141
4.4 The Role of Transforming Growth Factor-Beta in Retinal Ganglion Cells with Hyperglycemia and Oxidative Stress 145
4.5 Effects of hyperglycemia on the TGF-β pathway in trabecular meshwork cells 150
Chapter 5　 CONCLUSION 155
5.1 Proteomic Analysis of Various Rat Ocular Tissues after Ischemia–Reperfusion Injury and Possible Relevance to Acute Glaucoma 155
5.2 TGF-β1 signaling protects retinal ganglion cells from oxidative stress via modulation of the HO-1/Nrf2 pathway 155
5.3 Characterization of TGF-β by induced oxidative stress in human trabecular meshwork cells 156
5.4 The Role of Transforming Growth Factor-Beta in Retinal Ganglion Cells with Hyperglycemia and Oxidative Stress 156
5.5 Effects of hyperglycemia on the TGF-β pathway in trabecular meshwork cells 157
Chapter 6　 REFERENCES 158

Appendix 172
Publications 200
 
List of Figures and Tables

Chapter 1　INTRODUCTION
Figure 1-1. The main concept of this research 3
Figure 1-2. The amino acid sequences of TGF-β1, TGF-β2, and TGF-β3 4
Figure 1-3. Schematic diagram of IR model and the anatomy of the rat’s eye 7
Figure 1-4. Schematic illustrations showing the ischemia/reperfusion injury eye model (ischemia for one hour and reperfusion for one hour), followed by 2D-DIGE (two-dimensional difference gel electrophoresis) and MALDI-TOF/TOF mass spectrometry based proteomic analysis has been performed 8

Chapter 2　MATERIALS and METHODS
Table 2-1. Antibodies information 28

Chapter 3　RESULTS
Figure 3-1. Comparison of proteomics between the ischemia–reperfusion (IR) injury and control groups (Ctrl) in cornea. 35
Figure 3-2. Comparison of proteomics between the IR injury and control groups in conjunctiva. 36
Figure 3-3. Comparison of proteomics between the IR injury and control groups in uvea. 37
Figure 3-4. Comparison of proteomics between the IR injury and control groups in sclera. 38
Figure 3-5. Comparison of proteomics between the IR injury and control groups in retina. 39
Figure 3-6. Percentage of total cellular proteins identified by 2D-DIGE/matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for (A) cornea, (B) conjunctiva, (C) uvea, (D) sclera, and (E) retina according to their biological functions. 40
Table 3-1. Order of protein expression alterations (i.e., cornea, conjunctiva, uvea, sclera, and retina) according to the ratio of protein expression alterations. 41
Figure 3-7. Representative immunoblotting analysis for selected differentially expressed protein identified by proteomic analysis between control groups and ischemia-reperfusion groups 42
Figure 3-8. Cell viability with different concentrations and exposure duration of H2O2 in RGC-5 cells. 43
Figure 3-9. Wound healing ability induced by low concentration H2O2 on RGC-5 cells. 45
Figure 3-10. Effects of H2O2 on cell apoptotic status and increasing iROS levels in RGC-5 cells. 47
Figure 3-11. GSH content of RGC-5 cells under H2O2 (1 mM) stimulation. 48
Figure 3-12. Immunoblot analysis of antioxidation pathway-associated proteins (Nrf2, Keap1, HIF-1α, ALDH3A1, HO-1, TGF-β1, TGF-β2, LDH, Lamin B1, and p53) under 1mM H2O2 treatment for different exposure times (5, 30, 60 min). (n = 3). 49
Figure 3-13. Immunofluorescence analysis of RGCs under H2O2 (1 mM) stimulation.
50
Figure 3-14. Cell viability and iROS accumulation in RGC-5 cells under H2O2 (1 mM) stimulation with/without NAC (N-acetyl-L-cysteine) (5 mM). 52
Figure 3-15. Effects of TGF-β1/2 knockdown on H2O2-induced oxidative damage.
54
Figure 3-16. Immunoblot analysis of antioxidation pathway-associated proteins (Nrf2, Keap1, HIF-1α, ALDH3A1, HO-1, TGF-β1, TGF-β2, LDH, Lamin B1 and p53) under 1mM H2O2 treatment in TGF-β1/2 knockdown RGC-5 cells (n = 3). 55
Figure 3-17. Oxygen consumption rate (OCR) of RGC-5 shTGFβ1/2 cells with H2O2.
56
Figure 3-18. Effects of rhTGF-β1 on H2O2-induced oxidative damage. 58
Figure 3-19. Immunoblot analysis of activation of downstream substrates for RGC-5 cells supplemented with recombinant TGF-β1 protein (5 ng/ml) for 24 h with or without H2O2 (1 mM) 60
Figure 3-20. Impairments by H2O2 of HTMCs. H2O2 treatment decreased cell viability and increased ROS along with Ca2+ levels in dose- and time-dependent manners. 61
Figure 3-21. Immunoblot analysis and identification of H2O2-induced oxidative stress response signaling pathways. 64
Figure 3-22. Immunofluorescence analysis of H2O2 (1 mM)-treated HTMCs. 67
Figure 3-23. Effects of rhTGF-β1 protein during oxidative stress response of HTMCs.
69
Figure 3-24. Immunoblot analysis and identification of rhTGF-β1 protein in oxidative stress pathways. 72
Figure 3-25. Immunofluorescence analysis of rhTGF-β1 protein-treated HTMCs with or without H2O2 exposure (0.5 mM for 1 h). 75
Figure 3-26. Effects of shTGF-β1 & shTGF-β2 knockdown HTMCs with H2O2 exposure. 77
Figure 3-27. Immunoblot analysis and identification of shTGF-β1/2 knockdown HTMCs in oxidative stress pathways. 80
Figure 3-28. Effects on sip38 MAPK knockdown HTMCs through H2O2 exposure.
83
Figure 3-29. Immunoblot analysis and identification of sip38 MAPK knockdown HTMCs in oxidative stress pathways. 85
Figure 3-30. Effects of hyperglycemia on retinal ganglion cells (RGCs). 88
Figure 3-31. Immunoblot analysis of RGCs with hyperglycemia. 90
Figure 3-32. Immunofluorescence analysis of the activation of stress fiber and actin dots formation with 5.5 mM, 25 mM, 50 mM, and 100 mM glucose supplemented medium. 92
Figure 3-33. Effects in TGF-β1/2 knockdown RGCs with hyperglycemia 94
Figure 3-34. Immunoblot analysis of activation of downstream substrates in TGF-β1/2 knockdown RGC-5 cells with hyperglycemia 96
Figure 3-35. Effects in RGCs with hyperglycemia with or without (w/o) recombinant TGF-β1 protein (5 ng/mL) 99
Figure 3-36. Immunoblot analysis in RGCs with hyperglycemia with or w/o recombinant TGF-β1 protein (5 ng/mL) 101
Figure 3-37. Effects in RGCs with hyperglycemia with or w/o hydrogen peroxide for 1 h 103
Figure 3-38. Immunoblot analysis in RGCs with hyperglycemia with or w/o hydrogen peroxide (1 mM) for 1 h 105
Figure 3-39. Hyperglycemic effects on human trabecular meshwork cells (HTMCs)
108
Figure 3-40. Immunoblot analysis of hyperglycemia effects on HTMCs 111
Figure 3-41. Immunofluorescence analysis of the effects of hyperglycemia on HTMCs 113
Figure 3-42. Effects of high glucose levels on TGF-β1/2 knockdown HTMCs 115
Figure 3-43. Immunoblot analysis to determine hyperglycemic effects on TGF-β1/2 knockdown HTMCs 118
Figure 3-44. Hyperglycemia effects on HTMCs with or without rhTGF-β1 protein (1 & 5 ng/mL) 121
Figure 3-45. Immunoblot analysis of hyperglycemia effects on HTMCs with or without recombinant TGF-β1 protein (1 ng/mL) 124
Figure 3-46. Hydrogen peroxide-induced oxidative stress in HTMCs treated with high glucose concentrations 127
Figure 3-47. Immunoblot analysis of the effects of hyperglycemia on HTMCs with or without hydrogen peroxide (0.5 mM) treatment for 1 h 130

Chapter 4　DISCUSSION
Figure 4-1. A hypothetical model detailing the role of TGF-β in H2O2-stimulated oxidative stress of RGCs 136
Figure 4-2. A hypothetical model detailing the role of the TGF-β-related oxidative stress pathway in HTMC 141
Figure 4-3. A hypothetical model detailing the role of TGF-β in hyperglycemia (RGC-5) 145
Figure 4-4. A hypothetical model detailing the effects of hyperglycemia on the TGF-β pathway in trabecular meshwork cells 150

Appendix
Table S1. Alphabetical list of identified differentially expressed cornea proteins between the IR injury and control groups after 2D-DIGE coupled with MALDI-TOF mass spectrometry analysis. 173
Table S2. Alphabetical list of identified differentially expressed conjunctiva proteins between the IR injury and control groups after 2D-DIGE coupled with MALDI-TOF mass spectrometry analysis 184
Table S3. Alphabetical list of identified differentially expressed uvea proteins between the IR injury and control groups after 2D-DIGE coupled with MALDI-TOF mass spectrometry analysis. 189
Table S4. Alphabetical list of identified differentially expressed sclera proteins between the IR injury and control groups after 2D-DIGE coupled with MALDI-TOF mass spectrometry analysis. 196
Table S5. Alphabetical list of identified differentially expressed retina proteins between the IR injury and control groups after 2D-DIGE coupled with MALDI-TOF mass spectrometry analysis. 197

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