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| ???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
|---|---|---|
| dc.contributor.advisor | 詹智強(Chih-Chiang Chan) | |
| dc.contributor.author | Jhih-Gang Jhang | en |
| dc.contributor.author | 張之綱 | zh_TW |
| dc.date.accessioned | 2021-05-19T17:42:33Z | - |
| dc.date.available | 2024-03-11 | |
| dc.date.available | 2021-05-19T17:42:33Z | - |
| dc.date.copyright | 2019-03-11 | |
| dc.date.issued | 2019 | |
| dc.date.submitted | 2019-02-12 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/7381 | - |
| dc.description.abstract | Infertile crescent (Ifc)具有酵素催化之功能，在果蠅的神經脂質(sphingolipid)從頭合成路徑(de novo pathway)中負責將二羥基神經醯胺(dihydroceramide)催化成神經醯胺(ceramide)。先前實驗室利用作用於眼睛的翻轉酶(eyeless-flippase, eyFLP)在果蠅眼睛產生ifc-KO同型合子的細胞(ifc-KO/ifc-KO)，發現果蠅眼睛失去ifc會出現光依賴性(light-dependent)神經退化。我們進一步利用ey3.5-FLP和repo-FLP分別只在視神經和神經膠細胞失去ifc，發現兩者皆會在視神經電位紀錄(electroretinogram)中出現神經功能退化，但只有在視神經細胞失去ifc才會看到其形態有明顯退化。在果蠅眼睛失去ifc的情況下，利用repo-ifc-mCherry在神經膠細胞中表現ifc-mCherry可以減緩光依賴性神經退化，從此結果可知神經膠細胞中的Ifc可能參與部分神經功能維持。另外，我們將Ifc上可能負責酵素催化功能之胺基酸序列突變，過表達(overexpress)此突變的ifc(∆C3)-mCherry則無法減緩果蠅眼睛失去ifc造成之神經退化，推論Ifc的酵素活性對神經維持功能有其重要性。
Ifc可將二羥基神經醯胺催化成神經醯胺，而文獻指出神經醯胺含量的多寡可能會影響胞外體分泌數量，因此我們探討ifc是否會調控胞外體分泌。在果蠅幼蟲眼碟(eye disc)實驗，以mCD63蛋白標定胞外體(exosome)，發現過表達ifc(WT)-mCherry會增加胞外mCD63 puncta數量，而失去ifc則會減少胞外mCD63 puncta數量；在果蠅成蟲眼睛中，利用持續活化的Rab5 (Rab5CA)標定多囊泡體(multivesicular body, MVB)，發現果蠅眼睛失去ifc會減少胞外體生合成，因此，推論ifc可能透過調控胞外體生合成來影響胞外體的分泌數量。ESCRT蛋白(Endosomal Sorting Complex Required for Transport)被認為是調控胞外體生合成的重要角色，所以我們探討ESCRT蛋白是否參與ifc所調控的胞外體生合成。在ESCRT蛋白突變(HrsD28/+或Vps25A3/+)的遺傳背景之下，同時過表達GFP-mCD63和ifc(WT)-mCherry，發現Hrs蛋白突變(HrsD28/+)會減少過表達ifc(WT)-mCherry所增加的胞外體分泌數量，代表Ifc可能會和Hrs蛋白一起調控胞外體生合成。 有研究指出Rab4會在網狀紅血球(reticulocyte)成熟時，和釋放的胞外體結合，先前實驗室也發現Ifc會和Rab4在細胞外共位，因此我們探討Ifc執行功能時是否需要Rab4協助。不管在Rab4KO的遺傳背景下過表達ifc(WT)-mCherry或在ifc-KO的遺傳背景下過表達YFP-Rab4，都可以減緩原本的光依賴性神經退化，所以我們認為ifc和Rab4對光依賴性神經退化的調控屬於兩條獨立的調控路徑且具有互補(complementary)特性。 | zh_TW |
| dc.description.abstract | infertile crescent (ifc) encodes proteins involved in converting dihydroceramide to ceramide. Previously we have found that light stimulation led to light-dependent neurodegeneration in ifc-KO photoreceptors. To distinguish the impact on light-dependent neurodegeneration between neurons and glia, we further used ey3.5-FLP and repo-FLP to generate ifc-KO/ifc-KO cells in neurons and glia respectively. We found that knockout of ifc in neurons or glia resulted in functional degeneration as indicated by electroretinogram (ERG), but only knockout of ifc in neurons caused significant morphological degeneration. Also, we observed that expressing ifc-mCherry in glia (repo-ifc-mCherry) could rescue the degenerating ifc-KO photoreceptors, indicating that Ifc in glia partially involved in neuronal maintenance. Furthermore, we found that overexpressing ifc(∆C3)-mCherry, catalytically mutant ifc, in the eye could not rescue the degenerating ifc-KO photoreceptors. As a result, we thought that the catalytic domain of Ifc may play an important role in neuronal maintenance.
Overexpressing ifc(WT)-mCherry in the eye disc increased the secretion of mCD63 (exosome marker) while knockout of ifc decreased the number of mCD63 puncta in distant regions. Furthermore, the colocalization of GFP-mCD63 and Rab5CA (enlarged endosome) showed that the biogenesis of exosome is reduced within the multivesicular body (MVB) of ifc-KO photoreceptors. Altogether, we proposed that ifc may mediate the secretion of exosomes at least in regulating exosome biogenesis. Also, cooverexpressing ifc(WT)-mCherry and GFP-mCD63 in ESCRT (Endosomal Sorting Complex Required for Transport) mutant background (HrsD28/+) reduced the secretion of mCD63, indicating that Ifc may synergize with Hrs to regulate exosome biogenesis. Whether overexpressing ifc(WT)-mCherry in Rab4KO genetic background or overexpressing YFP-Rab4 in ifc-KO photoreceptors rescued functional degeneration. As a result, we thought that ifc and Rab4 may act as two indepedentent pathways to regulate light-dependent neurodegeneration and compensate each other. | en |
| dc.description.provenance | Made available in DSpace on 2021-05-19T17:42:33Z (GMT). No. of bitstreams: 1 ntu-108-R05441001-1.pdf: 3339405 bytes, checksum: 01c1550279dd919bf8d8fa0b01431b9c (MD5) Previous issue date: 2019 | en |
| dc.description.tableofcontents | 中文摘要 ii
英文摘要 iv 目錄 vi 第一章 實驗背景 1 1.1 ifc的功能與神經脂質合成 2 1.2 果蠅眼睛與ifc-KO造成之光依賴性神經退化 3 1.3 ifc的細胞非自主調控和胞外體標記 4 1.4 胞外體維持神經功能之重要性 6 1.5 胞外體生合成調控 7 1.6 小結 8 第二章 實驗材料與方法 10 2.1 果蠅株及培養方法 11 2.2 果蠅食物培養基製備 13 2.3 群組分析(Clonal analysis) 14 2.4 免疫螢光染色與共軛焦顯微鏡(IHC & Confocal microscope) 14 2.5 免疫螢光染色抗體清單 15 2.6 視神經電位紀錄(Electroretinogram) 16 2.7 QuickChange聚合酶連鎖反應(QuickChange PCR) 17 第三章 實驗結果 20 3.1 剔除視神經細胞中的ifc造成視神經在形態和功能上的光依賴性神經退 21 3.2 剔除神經膠細胞中的ifc造成視神經在功能上出現光依賴性神經退化 21 3.3 在神經膠細胞中表現ifc-mCherry減緩果蠅眼睛失去ifc造成之光依賴性神經退化 22 3.4 突變Ifc上可能負責酵素催化功能之胺基酸序列降低Ifc-mCherry減緩光依賴性神經退化的能力 23 3.5 過表達ifc(WT)-mCherry造成胞外mCD63 puncta數量增加，其調控可能與Ifc的酵素活性有關 25 3.6 Hrs蛋白突變(HrsD28/+)使過表達ifc(WT)-mCherry所增加的胞外mCD63 puncta數量減少 26 3.7 失去ifc減少胞外mCD63 puncta數量 27 3.8 失去ifc造成多囊泡體中的腔內囊泡數量有減少趨勢 28 3.9 失去ifc造成多囊泡體生合成減少 28 3.10 果蠅眼睛失去ifc和全身性失去Rab4造成的光依賴性神經退化屬於累加效應 29 3.11 ifc和Rab4對光依賴性神經退化的調控具有互補特性 29 第四章 實驗討論 31 4.1 在視神經或神經膠細胞中失去ifc對光依賴性神經退化的影響 32 4.2 Ifc(∆CAT)-mCherry是否真的破壞其酵素活性 32 4.3 胞外體標記選擇 33 4.4 脂質組成對胞外體的影響 33 4.5 Ifc可能和ESCRT蛋白一起調控胞外體分泌 34 4.6 ifc和Rab4互補調控在生理上的意義 35 第五章 未來實驗方向 36 5.1 探討Ifc和ESCRT蛋白一起調控胞外體分泌的可能機制 37 5.2 探討DEGS1-KO細胞分泌的胞外體數量和組成 37 5.3 探討失去Rab4是否會增加胞外體分泌 38 第六章 實驗圖表 39 Fig. 1 剔除視神經細胞中的ifc造成視神經在形態和功能上的光依賴性神經退化 40 Fig. 2 剔除神經膠細胞中的ifc造成視神經在功能上出現光依賴性神經退化 41 Fig. 3 在神經膠細胞中表現ifc-mCherry減緩果蠅眼睛失去ifc造成之光依賴性神經退化 42 Fig. 4 突變Ifc上可能負責酵素催化功能之胺基酸序列降低Ifc-mCherry減緩光依賴性神經退化的能力 43 Fig. 5 過表達ifc(WT)-mCherry造成胞外mCD63 puncta數量增加，其調控可能與Ifc的酵素活性有關 44 Fig. 6 Hrs蛋白突變(HrsD28/+)使過表達ifc(WT)-mCherry所增加的胞外mCD63 puncta數量減少 45 Fig. 7 失去ifc減少胞外mCD63 puncta數量 46 Fig. 8 失去ifc造成多囊泡體中的腔內囊泡數量有減少趨勢 47 Fig. 9 失去ifc造成多囊泡體生合成減少 48 Fig. 10 果蠅眼睛失去ifc和全身性失去Rab4造成的光依賴性神經退化屬於累加效應 49 Fig. 11 ifc和Rab4對光依賴性神經退化的調控具有互補特性 50 Supplementary Fig. 1 repo啟動子作用範圍不包含神經細胞 51 Supplementary Fig. 2 Ifc(WT)-mCherry和Ifc(∆C3)-mCherry的蛋白表現量無顯著差異 52 第七章 參考文獻 53 | |
| dc.language.iso | zh-TW | |
| dc.title | infertile crescent (ifc)調控胞外體分泌以維持神經功能之研究 | zh_TW |
| dc.title | Investigating the role of infertile crescent (ifc) in regulating exosome secretion for neuronal maintenance | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 107-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 李芳仁,李秀香,王昭雯 | |
| dc.subject.keyword | 果蠅,胞外體,神經退化, | zh_TW |
| dc.subject.keyword | Drosophila,exosome,neurodegeneration, | en |
| dc.relation.page | 64 | |
| dc.identifier.doi | 10.6342/NTU201900402 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2019-02-12 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 生理學研究所 | zh_TW |
| Appears in Collections: | 生理學科所 | |
Files in This Item:
| File | Size | Format | |
|---|---|---|---|
| ntu-108-1.pdf Until 2024-03-11 | 3.26 MB | Adobe PDF |
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