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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 王致恬 教授(Chih-Tien Wang) | |
| dc.contributor.author | Ting-Yu Wo | en |
| dc.contributor.author | 吳亭諭 | zh_TW |
| dc.date.accessioned | 2021-06-15T12:41:58Z | - |
| dc.date.available | 2021-08-03 | |
| dc.date.copyright | 2016-08-03 | |
| dc.date.issued | 2016 | |
| dc.date.submitted | 2016-07-26 | |
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(1998). Cysteine-string proteins regulate exocytosis of insulin independent from transmembrane ion fluxes. FEBS Lett, 437(3), 267-272. Zhang, J., & Allen, M. D. (2007). FRET-based biosensors for protein kinases: illuminating the kinome. Mol Biosyst, 3(11), 759-765. Zhang, J., Hupfeld, C. J., Taylor, S. S., Olefsky, J. M., & Tsien, R. Y. (2005). Insulin disrupts beta-adrenergic signalling to protein kinase A in adipocytes. Nature, 437(7058), 569-573. Zhang, J., Ma, Y., Taylor, S. S., & Tsien, R. Y. (2001). Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering. Proc Natl Acad Sci U S A, 98(26), 14997-15002. Zinsmaier, K. E., Hofbauer, A., Heimbeck, G., Pflugfelder, G. O., Buchner, S., & Buchner, E. (1990). A cysteine-string protein is expressed in retina and brain of Drosophila. J Neurogenet, 7(1), 15-29. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/50467 | - |
| dc.description.abstract | 發育中的神經系統在有感覺經驗之前，模式化自發性作用又稱做視網膜波對於建立功能性的神經網路很重要。第二期視網膜波發生於囓齒類動物出生後一周間，被發現可以促使一些先天性免疫蛋白的表現，其中包含了第一型主要組織相容性複合體 (major histocompatibility complex class I; MHCI)，MHCI與突觸的修剪以及視神經節細胞 (RGCs) 軸突投射到腦區所導致的雙眼專一性投射分層，造成雙眼視野的隔離。這時期的視網膜波是由突觸前的星狀無軸突細胞 (SACs) 釋放神經傳導物質到鄰近的SACs或是RGCs，造成像波一樣的傳遞，因此有獨特的空間及時間傳播特性。伴隨著視網膜波的產生，視網膜神經節細胞層上會有大量的鈣離子流入。目前還不知道這些受到視網膜波調控的免疫基因表現主要是透過視網膜的空間還是時間特性。在先前的研究中發現半胱胺酸串鍊蛋白(CSP) 在突觸前神經元細胞內的磷酸化修飾可以增加神經傳導物質的釋放以及視網膜波的頻率，並且不會影響到視網膜波的空間傳播特性。因此，透過改變突觸前神經元內的CSP磷酸化程度可以用來研究視網膜波時間特性對於這些免疫基因表現的影響。在本篇研究中，我們探討了發育中大鼠視網膜內，視網膜波的時間特性如何調控活性依賴型的基因(activity-dependent gene)表現。我們將CSP 專一性的表現到SAC中，並且發現當CSP過量表現於SAC時相對於控制組會使得這些先天性免疫蛋白的表現增加了，但是若將無法被磷酸化的CSP突變株過量表現到SAC就無法增加這些先天性免疫基因的表現，除此之外，我們利用追蹤MHCI上游轉錄因子，磷酸化CREB (pCREB)的表現量，發現CSP-WT表現在SAC中會使突觸後神經元的pCREB增加，而突變株與對照組沒有顯著差異，因此，我們得知在SAC內，CSP的磷酸化可以增加視網膜波的頻率並且調控先天性免疫基因在視網膜中的表現。這些結果代表視網膜波的時間特性對於發育中大鼠視網膜內的活性依賴型基因表現扮演重要的角色。 | zh_TW |
| dc.description.abstract | Prior to sensory experience, patterned spontaneous activity (termed retinal waves in the developing visual system) is essential for establishing functional neural circuits. In rodents, retinal waves during the first postnatal week up-regulate the expression of innate immune proteins, such as histocompatibility complex class I (MHCI), leading to synaptic refinement and eye-specific segregation of retinogeniculate projection. These waves are initiated by presynaptic starburst amacrine cells (SACs) releasing neurotransmitters to neighboring SACs or retinal ganglion cells (RGCs), displaying unique spatial and temporal patterns, with wave-like propagation of correlated firings and Ca2+ transients in the RGC layer. However, which pattern (spatial or temporal) of retinal waves is important for this activity-dependent gene expression remains unknown. We previously found that phosphorylation of cysteine string protein-α (CSPα) in presynaptic cells increases neurotransmitter release and wave frequency, without altering the spatial correlation of retinal waves. Thus, the effects by altering the CSPα phosphorylated state in wave-initiating cells can reflect how the wave temporal pattern regulates activity-dependent gene expression. In this study, we determine how the temporal pattern of retinal waves regulates activity-dependent gene expression in the developing rat retina. By utilizing the cell type-specific molecular perturbation, we found that overexpression of wild-type CSPα in SACs increased the expression of innate immune proteins compared to control, but overexpression of the CSPα phosphodeficient mutant (CSPα-S10A) in SACs did not. In addition, CSP-WT in SACs also increased the expression of the upstream transcription factor, pCREB, in postsynaptic cells compared to control or CSPα-S10A. Therefore, through increasing wave frequency, phosphorylation of CSPα in SACs regulates the retinal expression of innate immune genes. These results suggest that the temporal pattern of retinal waves is important for activity-dependent gene expression in the developing rat retina. | en |
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| dc.description.tableofcontents | Contents
國立臺灣大學碩士學位論文口試委員會審定書 I 致謝 II 中文摘要 III Abstract IV Abbreviations VI Contents X Chapter I Introduction 1 1.1 The nervous system and neural development 1 1.2 Structure and development of retinas 1 1.3 Retinal waves 2 1.4 PKA activity during retinal waves 5 1.5 Ca2+-dependent exocytosis and synaptic transmission during stage II retinal waves 6 1.6 Cysteine string protein (CSP) 8 1.7 Innate immune proteins and circuit refinement 10 1.7-1 Histocompatibility complex (MHC) 11 1.7-2 Complement proteins 13 1.8 Specific aims 14 Chapter II Material and Methods 17 2.1 Animals 17 2.2 Plasmid construction and subcloning 17 2.2-1 A kinase activity reporter (AKAR3) 17 2.2-2 HA tag 21 2.3 Site-directed mutagenesis 23 2.4 Retinal dissection 24 2.5 Retinal explant culture 25 2.6 Transient transfection 26 2.7 RNA extraction 27 2.8 Reverse transcriptase-quantitative real-time PCR (RT-qPCR) 28 2.9 Pharmacological treatments for RT-qPCR 29 2.10 Immunofluorescence staining for whole mount retinas 30 2.11 Immunofluorescent staining for dissociated retinal cells 31 2.12 Calcium imaging 33 Chapter III Results 35 3.1 Pharmacological treatments that regulated stage II retinal waves can alter the expression of innate immune genes 35 3.2 CSP-WT and CSP-S10A in SACs can regulate the temporal properties of retinal waves 37 3.3 The immunoreactivity levels of pPKA substrates in SACs is declined by CSP-S10A in SACs 39 3.4 CREB and pCREB are expressed in the whole mount retina and partially colocalize to SACs or RGCs 40 3.5 pCREB immunoreactivity levels in SACs are declined by CSP-S10A in SACs 41 3.6 The pCREB immunoreactivity levels in RGCs are increased by CSP-WT in SACs 42 3.7 The population of SACs or RGCs containing pCREB is not changed after transfection 43 3.8 CSP phosphorylation in SACs regulates the expression of innate immune genes by regulating the temporal properties of retinal waves 44 Chapter IV Discussion 47 4.1 The impact of CSP phosphorylation on immune gene expression by temporal properties of stage II retinal waves 48 4.2 Expression of MHCI, C1qa and C1qb is increased by CSP-WT in SACs 51 4.3 The limitation of whole retina RT-qPCR 51 4.4 Whether the spatial properties of retinal waves can regulate the immune gene expression 52 4.5 The signaling pathway triggers pCREB expression 53 4.6 Expression of immune genes modulated by the wave temporal properties may affect synaptic pruning and eye-specific segregation 54 Chapter V Conclusion 55 References 56 List of Figures Figure 1. The structure of the retina 61 Figure 2. The spatial and temporal properties of retinal waves 63 Figure 3. Three stages of retinal waves initiated by different mechanisms 64 Figure 4. The eye-specific segregation and synaptic pruning 65 Figure 5. Pharmacology agents regulate retinal waves 66 Figure 6. CSP phosphorylation regulates exocytosis and the temporal properties of retinal waves of retinal waves 68 Figure 7. Synaptic pruning regulated by immune proteins 70 Figure 8. Pharmacology treatments that change stage II retinal wave properties can regulate Syt IV gene expression 72 Figure 9. Muscimol up-regulates MHCI and PirB gene expression 73 Figure 10. Pharmacological treatments regulate complement protein expression 74 Figure 11. The CSP mRNA level is increased by FSK 75 Figure 12. Transient transfection of CSP-WT and CSP-S10A successfully increases CSP gene expression in whole retinas 76 Figure 13. Transfection of CSP-WT and CSP-S10A successfully elevates the CSP immunoreactivity level in SACs and regulates Ca2+ transient frequency 78 Figure 14. The immunoreactivity level of PKA substrates is decreased in SACs by overexpressing CSP-S10A 81 Figure 15. The relative distribution of total CREB or pCREB in the SACs of whole-mount retinas 84 Figure 16. The relative distribution of total CREB or pCREB in the RGCs of whole-mount retinas 86 Figure 17. Total CREB immunoreactivity level is not changed in SACs after transfection 87 Figure 18. CSP-S10A significantly downregulates the pCREB immunoreactivity level in SACs 89 Figure 19. The immunoreactivity level of total CREB is not changed in RGCs after transfection 91 Figure 20. CSP-WT significantly increases pCREB expression in RGCs 93 Figure 21. The population of pCREB-positive SACs is not changed by the CSP phosphorylation level in SACs 95 Figure 22. The population of pCREB-positive RGCs is not changed by the CSP phosphorylation level in SACs 97 Figure 23. CSP phosphorylation in SACs does not change Syt IV gene expression 99 Figure 24. MHCI gene expression is increased by CSP-WT in SACs 100 Figure 25. Expression of C1qa and C1qb genes is upregulated by CSP-WT in SACs 101 Figure 26. The working model from this study 103 List of Tables Table 1. Primers used for plasmid DNA construction 104 Table 2. Primers used for site-directed mutagenesis 105 Table 3. Primers used for RT-qPCR 106 Table 4. The primary antibodies used in the study 107 Table 5. The secondary antibodies used in the study 108 Table 6. Expression of immune genes influenced by pharmacological reagents 109 Table 7. Expression of immune genes influenced by changing the wave temporal properties 110 Appendix 111 AKAR (A kinase activity reporter) 112 Fluorescence resonance energy transfer (FRET) imaging 114 Figure 1. Expression of Syt IV after 2, 4, 8, 16, or 24 hr following application of pharmacological reagents. 116 Figure 2. Wave-associated Ca2+ transients cannot be affected after co-transfection. 117 | |
| dc.language.iso | en | |
| dc.subject | 先天免疫基因 | zh_TW |
| dc.subject | 視網膜波 | zh_TW |
| dc.subject | 星狀無軸突細胞 | zh_TW |
| dc.subject | 半胱胺酸串鍊蛋白 | zh_TW |
| dc.subject | 視網膜神經節細胞 | zh_TW |
| dc.subject | 第一型組織相容性複合體 | zh_TW |
| dc.subject | 視網膜波 | zh_TW |
| dc.subject | 星狀無軸突細胞 | zh_TW |
| dc.subject | 半胱胺酸串鍊蛋白 | zh_TW |
| dc.subject | 先天免疫基因 | zh_TW |
| dc.subject | 視網膜神經節細胞 | zh_TW |
| dc.subject | 第一型組織相容性複合體 | zh_TW |
| dc.subject | Retinal ganglion cells | en |
| dc.subject | Retinal waves | en |
| dc.subject | Histocompatibility complex class I | en |
| dc.subject | Retinal ganglion cells | en |
| dc.subject | Histocompatibility complex class I | en |
| dc.subject | Innate immune genes | en |
| dc.subject | Cysteine string protein | en |
| dc.subject | Starburst amacrine cells | en |
| dc.subject | Retinal waves | en |
| dc.subject | Starburst amacrine cells | en |
| dc.subject | Cysteine string protein | en |
| dc.subject | Innate immune genes | en |
| dc.title | 在星狀無軸突細胞中的半胱胺酸串鍊蛋白磷酸化對發育中大鼠視網膜的基因表現的影響 | zh_TW |
| dc.title | Phosphorylation of cysteine string protein-α in starburst amacrine cells modulates gene expression in the developing rat retina | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 104-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 焦傳金 教授(Chuan-Chin Chiao),盧主欽 助理教授(Juu-Chin Lu),徐立中 副教授(Li-Chung Hsu) | |
| dc.subject.keyword | 視網膜波,星狀無軸突細胞,半胱胺酸串鍊蛋白,先天免疫基因,視網膜神經節細胞,第一型組織相容性複合體, | zh_TW |
| dc.subject.keyword | Retinal waves,Starburst amacrine cells,Cysteine string protein,Innate immune genes,Retinal ganglion cells,Histocompatibility complex class I, | en |
| dc.relation.page | 119 | |
| dc.identifier.doi | 10.6342/NTU201601431 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2016-07-27 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 分子與細胞生物學研究所 | zh_TW |
| 顯示於系所單位： | 分子與細胞生物學研究所 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-105-1.pdf 未授權公開取用 | 6.57 MB | Adobe PDF |
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