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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 黃火鍊(Fore-Lien Huang) | |
dc.contributor.author | Ai-Pei Chi | en |
dc.contributor.author | 紀艾佩 | zh_TW |
dc.date.accessioned | 2021-06-08T06:02:01Z | - |
dc.date.copyright | 2007-07-30 | |
dc.date.issued | 2007 | |
dc.date.submitted | 2007-07-25 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/25089 | - |
dc.description.abstract | Cas (p130Cas, Crk-associated substrate)為分子量130千道爾頓的焦點黏附蛋白,當其酪胺酸磷酸化時可以執行訊息傳遞功能因而可以調節細胞骨架的重組及參與細胞遷移,然而目前對Cas在發育過程中所扮演腳色並不是很清楚,為了瞭解Cas在發育上的功能我們選擇使用果蠅遺傳系統來研究,優點在於果蠅基因體中只含有唯一的同源基因可以便於分析哺乳動物Cas複雜的功能。
全株原位雜交與增強子捕捉試驗的結果顯示在果蠅胚胎發育過程中p130cas的表現集中在相當於哺乳動物中樞神經系統的腹部神經索,並且發現在三齡幼蟲時期p130cas大量的在腦部表現。為了瞭解p130cas的功能以及更進一步的分析p130cas在神經發育過程所扮演的角色,我建立一系列可以執行RNA干擾技術的轉殖果蠅,結果顯示在果蠅的神經中降低p130cas的表現會造成腹部神經索中原來排列整齊的commissure axons神經束偶爾會出現不正常的融合; 若將p130cas專一地在果蠅眼睛中降低則會造成果蠅視神經突觸目標辨識與突觸導向的缺陷; 而如果局部的運動神經系統中降低p130cas的表現會造成在神經肌肉接點的神經突觸有澎大的現象; 此外在果蠅的感覺器官前細胞以及原神經細胞叢,降低p130cas的表現則會導致剛毛發育的缺陷。這些p130cas RNAi所產生的表型和一些參與Rho GTPase的效應蛋白以及調控Arp2/3 (actin-related protein)活性的Wasp/wave複合體的突變所產生的表型類似。綜合這些發現,我推測p130cas在果蠅發育過程中主要的功能在於調節細胞骨架進而影響神經發育。 除此之外,我也發現在發育中的翅膀降低p130cas的表現會造成翅膀以及翅脈不正常的發育,而這樣的性狀與已知其他的焦點黏附蛋白突變果蠅類似。因此由種種得結果推測p130cas應該是在不同的組織、不同的發育階段,藉由與不同的蛋白交互作用來調節各種發育過程。 | zh_TW |
dc.description.abstract | Cas (p130Cas, Crk-associated substrate) is a tyrosine-phosphorylated 130 kilodalton docking protein that is required for the regulation of actin cytoskeleton organization and cell migration in mammalian fibroblasts. However, the role of p130cas during development is poorly understood. To investigate the function of p130cas during development, I have identified the single Drosophila ortholog of mammalian Cas family.
In situ hybridization and enhancer trap techniques revealed that p130cas is concentrated in the ventral nerve cord during embryogenesis and is enriched in the brain in third instar larvae. I have therefore generated several p130cas RNA interference (RNAi) transgenic lines to determine the possible function of p130cas during neurogenesis. Knockdown of p130cas expression in developing neurons resulted in partial fusion in the commissures axon fascicles of CNS, photoreceptor (R cell) axon targeting and guidance defects, and bulged synapse boutons at the neuromuscular junction. Moreover, reduced expression of p130cas in sensory organ precursor cells and proneural clusters resulted in bristle development defects. These phenotypes are similar to those of several effector mutants of Rho GTPase and mutants of regulatory proteins of Wasp/Wave complex. Taken together, these results suggest that the major functional roles of p130cas in Drosophila might be involved in the regulation of cytoskeleton reorganization via a Rho GTPase signaling pathway, and therefore affecting the appropriate neuron wiring and targeting. Furthermore, I also found that reduced expression of p130cas in the developing wing often resulted in abnormal wing vein formation and blistered wings. These phenotypes are similar to mutants involved in focal adhesion formation. In summary, my results suggest that p130cas is likely to interact with multiple targets in a variety of developing processes by virtue of its modular protein binding domains. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T06:02:01Z (GMT). No. of bitstreams: 1 ntu-96-R94b43022-1.pdf: 5583273 bytes, checksum: 0791efaefa6085e728de2d94f79ac53e (MD5) Previous issue date: 2007 | en |
dc.description.tableofcontents | 中文摘要…………………………………………………………….…………….……iii
Abstract………………………………………………………………..……………….iv Table of content………………………………………………………..……………….vi List of figures……………………………………………………………...…………….x Introduction……………………………………………………………..…………..…..1 I. p130cas has several protein-protein interaction domains and functions as an adaptor protein……………………………………………………………………..………..…....1 II. Cas family has three members with distinct functional roles…………...……………3 III. Biological functions of adaptor protein p130cas……………………………….…......5 1. Regulation of the actin cytoskeleton and cell migration…………..……….………5 2. Cell survival and apoptosis………………………………..……………..……..…..6 3. Mitosis and cell proliferation…………………………………..………..……….....7 4. Cell transformation and metastasis…………………………………..……..……....8 IV. p130cas participates in nervous system development in mammalian……..….……..10 V. Drosophila as a genetic and cellular model for studies on nervous system................10 1. Drosophila visual system………………………………………………………...11 2. The motor system of the Drosophila melanogaster larvae………………………12 VI. Regulators of actin polymerization are involved in the nervous system.……….….13 Material & Methods……………………………………………………………….….17 I. Molecular cloning………………… …...………………..……………………..17 1. pBSK+-p130cas-B-N ter…………………...………………………………….17 2. pUAST-p130cas-B …...………………………………………………………17 3. pWIZ-p130cas-IR……….……………………………………………………18 4. pAc5.1-p130cas-B…….………………………………………………………18 5. pAc5.1-p130cas-A…… ………………………………………………………19 6. pPRO EX HTa-p130cas C-terminal……………..……………………………..20 II. Drosophila genetics…………………………..…………………………...……20 III. Cell culture of S2R+ cells…………………………………..……………..……21 IV. Whole mount in situ hybridization of Drosophila embryos and imaginal discs.21 V. Antibody production…………………….……………………………………..23 VI. Immunolabeling…………………….…………………………………………..23 VII. Immunofluoresence staining of S2R+ cells……………………………………..25 VIII. The p{GawB} Enhancer trap technique………………………………………..26 Results………………………………………………………………………………….27 1. Multiple alignment of Human, Rat, and Drosophila p130cas………..……….27 2. p130cas was enriched in the nerve system during development…………...…28 3. Generation of p130cas RNAi (RNA interference) transgenic flies……..…….30 4. RNAi knockdown of p130cas in pan-neuron of Drosophila resulted in abnormal ventral nerve cord formation………………………..……………..…….32 5. Requirement of p130cas for R cell guidance and targeting in eye……….…..33 6. RNAi knockdown of p130cas in Drosophila motor neurons resulted in bulged synapse boutons at the neuromuscular junction (NMJs)………………….….…….34 7. p130cas participated in sensory organ development and wing patternin…......36 8. p130cas localized at the leading edge in S2R+ cell…………………………...37 Discussion……………………………………………………………………………...39 1. Drosophila p130cas is a good genetic tool to study the complicated functions of Cas family………………………………………………........………………….39 2. p130cas is highly expressed in neurons during development………………...40 3. p130cas is required for the guidance of photoreceptor axons……………...…40 4. The phenotypes of p130cas RNAi are similar to those of several of Rho GTPase effector mutants…………………………..……...…………..…………….41 5. Drosophila p130cas doesn’t associate with Dock in vitro……………......…..44 6. p130cas hypomorphic mutant P{GawB}NP4466……………….....…………45 7. Restricted expression level of p130cas proteins in a variety of developing processes…………………………………………………………………...……….45 References……………………………………………………………………...………47 Figures…………………………………………………………………………..……..58 | |
dc.language.iso | en | |
dc.title | p130cas參與果蠅發育過程之功能分析 | zh_TW |
dc.title | Functional analysis of p130cas in Drosophila development | en |
dc.type | Thesis | |
dc.date.schoolyear | 95-2 | |
dc.description.degree | 碩士 | |
dc.contributor.coadvisor | 陳光超(Guang-Chao Chen) | |
dc.contributor.oralexamcommittee | 李明亭(Ming-Ting Lee) | |
dc.subject.keyword | 果蠅,發育,130千道爾頓焦點黏附蛋白, | zh_TW |
dc.subject.keyword | p130cas,Drosophila,development, | en |
dc.relation.page | 57 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2007-07-27 | |
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 |
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