DC 欄位 | 值 | 語言 |
dc.contributor | 李芳仁 | zh-TW |
dc.contributor | Lee, Fang-Jen | en |
dc.contributor | 臺灣大學:分子醫學研究所 | zh-TW |
dc.contributor.author | 陳吳姍 | zh-TW |
dc.contributor.author | Chen, Wu-Shan | en |
dc.creator | 陳吳姍 | zh-TW |
dc.creator | Chen, Wu-Shan | en |
dc.date | 2009 | en |
dc.date.accessioned | 2010-05-04T07:09:13Z | - |
dc.date.accessioned | 2018-07-09T01:18:47Z | - |
dc.date.available | 2010-05-04T07:09:13Z | - |
dc.date.available | 2018-07-09T01:18:47Z | - |
dc.date.issued | 2009 | - |
dc.identifier.other | U0001-1308200911401900 | en |
dc.identifier.uri | http://ntur.lib.ntu.edu.tw//handle/246246/178722 | - |
dc.description.abstract | 人類腺嘌呤核苷二磷酸核糖化相似因子4D (ARL4D) 屬於小分子GTPase腺嘌呤核苷二磷酸核糖化因子(ARF)家族,為一隨著發育而受到調控的蛋白質。我們實驗室觀察到ARL4D非活性突變蛋白質(ARL4DT35N) 在COS-7細胞株中具有許多不同的分布型態,包含座落於內小體(endosome)、粒線體(mitochondria),或是形成聚集體(aggresome)。座落於粒線體的外生性ARL4DT35N會造成粒線體膜電位(mitochondria membrane potential, MMP)消失。為了瞭解ARL4DT35N的功能,進行了酵母菌雙雜交篩檢(yeast two-hybrid screening),以ARL4DT35N為誘餌篩選小鼠第七天胚胎期的cDNA library。PS4A被選擇來進行後續的研究。VPS4A的片段(a.a. 286-437)和ARL4DT35N之間具專一且直接的交互作用,且此作用受到GTP/GDP鍵結狀態的影響。當外生性的VPS4A和ARL4DT35N在HeLa細胞株中共同表現時,會抑制VPS4A造成的細胞質分裂(cytokinesis),此現象與外生性的VPS4A顯性抑制突變蛋白質(VPS4AK173Q)表現時相似。除此之外,以小干擾RNA(siRNA)技術調降(knockdown) ARL4D蛋白質表達,會減低VPS4AK173Q所造成的細胞質分裂抑制現象。綜合以上實驗,推論ARL4DT35N可能藉由影響VPS4A的功能,進一步影響細胞質分裂。進一步研究ARL4D的生理功能,建立了ARL4D基因剔除小鼠。ARL4D基因剔除小鼠可正常地生長與繁殖。藉由蘇木素伊紅染色〈hematoxylin and eosin stain〉,發現雄性ARL4D基因剔除小鼠的肝臟中有許多小空泡,是初期的肝功能衰落現象;而另一雌性ARL4D基因剔除小鼠則在肝臟中觀察到嚴重的發炎現象。血清生化的分析資料則顯示,谷草轉氨酶〈GOT〉、谷丙轉氨酶〈GPT〉以及肌酸激酵素酶〈CPK〉三種酵素活性,ARL4D基因剔除小鼠都高於野生型與中間型。且雄性小鼠的差異較雌性小鼠明顯。由於現有樣本數不足,明確的結論仍需更多證據支持。 | zh-TW |
dc.description.abstract | ARL 4D is a regulated developmentally protein which belongs to ADP-ribosylation factor (ARF) small GTP binding protein family. In previous researches, our lab observed the heterogeneous distributions of constitutively inactive form of ARL4D (ARL4DT35N) at endosomes, mitochondria, and aggresome-like compartments in COS-7 cells. Indeed, ARL4DT35N overexpression diminished mitochondria membrane potential. To characterize function of ARL4DT35N, yeast two-hybrid screening was performed to identify the interacting partner in the mouse embryonic 7-day library.n interacting protein vacuolar protein sorting factor 4A (VPS4A) was chosen for further study. VPS4A (a.a. 286-437) interacts with ARL4DT35N specifically and directly in a GDP-dependent manner. ARL4DT35N and VPS4A co-overexpression in HeLa cells could enhance the cytokinesis inhibition induced by VPS4A. This increase was similar to dominant-negative VPS4AK173Q overexpression. In addition, ARL4D knockdown by siRNA would reduce cytokinesis inhibition induced by VPS4AK173Q. Together, these data suggested ARL4DT35N might affect the function of VPS4A, and subsequently affect the cytokinesis.RL4D knockout mice have been generated to investigate the physiological function of ARL4D. ARL4D knockout mice were viable and fertile. However, there were many vacuoles in the liver section of male ARL4D knockout mice by hematoxylin and eosin staining, representing primary degeneration of liver function; severe inflammation was also observed in liver of a female ARL4D knockout mouse. Data of blood chemistry analysis indicated the enzyme activities of GOT, GPT, and CPK were higher in ARL4D knockout mice than wild-type and heterozygous mice. This increase was significant in male mice. More samples need to be examined before giving the final conclusion. | en |
dc.description.tableofcontents | 目錄able of Contents士學位論文口試委員會審定書 i謝 ii文摘要 iiibstract ivbbreviations vi. Introduction 11.1. Small GTPases of Ras Superfamily 11.2. ADP Ribosylation Factor (Arf) Family 21.3. ARL4DT35N and Mitochondria 51.4. ESCRT Complexes 61.5. VPS4 7. Materials and Methods 92.1. Cell Culture 92.2. Construction of Expression Plasmids 92.3. Transient Transfection 92.4. Western Blotting 102.5. Indirect Immunofluorescence Staining and Microscopy 102.6. Yeast Two‐Hybrid Screen and Interaction Assay 112.7. Bacterial Recombinant Protein Preparation 122.8. GST Pull Down Assay 132.9. Co-Immunoprecipitation 132.10. Gene Knockdown by Small Interfering RNA 132.11. Counting Cytokinesis-Inhibited Cells by Direct Visualization 142.12. ARL4D Knockout Mice Establishment 14. Results 173.1. Identification of Putative ARL4DT35N‐Interacting Proteins 173.2. VPS4A Specifically Interacts with ARL4DT35N 173.3. Specific ARL4DT35N-Interacting Region Mapping within VPS4A 183.4. VPS4A (a.a. 286-437) Preferentially Bbinds to ARL4DT35N in vitro and in vivo 183.5. Subcellular Localization of Exogenous VPS4A and VPS4AK173Q 193.6. ARL4DT35N Overexpression Enhanced the Cytokinesis Inhibition Induced by VPS4A 203.7. ARL4D Knockdown Would Reduce Cytokinesis Inhibition Induced by VPS4AK173Q 203.8. Phenotypic Function Study of ARL4D Knockout (ARL4D-/-) Mice 21. Discussion 23. Tables 27. Figures 30. Supplementary Figures 45. References 52 | en |
dc.format | application/pdf | en |
dc.format.extent | 5506943 bytes | - |
dc.format.mimetype | application/pdf | - |
dc.language | en | en |
dc.language.iso | en_US | - |
dc.subject | 人類腺嘌呤核苷二磷酸核糖化相似因子4D | zh-TW |
dc.subject | 腺嘌呤核苷二磷酸核糖化因子 | zh-TW |
dc.subject | 酵母菌雙雜交篩檢 | zh-TW |
dc.subject | 細胞質分裂 | zh-TW |
dc.subject | 基因剔除小鼠 | zh-TW |
dc.subject | ARL4D | en |
dc.subject | ARF | en |
dc.subject | yeast twouhybrid screening | en |
dc.subject | cytokinesis | en |
dc.subject | knockout mice | en |
dc.title | 人類腺嘌呤核苷二磷酸核糖化相似因子4D (ARL4D)與其結合蛋白 VPS4A 之特性探討以及ARL4D基因剔除小鼠之建立與表現型功能研究 | zh-TW |
dc.title | Identification and Characterization of ARL4D Interacting Protein VPS4A and Establishment and Phenotypic Function Study of ARL4D Knockout Mice | en |
dc.identifier.uri.fulltext | http://ntur.lib.ntu.edu.tw/bitstream/246246/178722/1/ntu-98-R95448005-1.pdf | - |
item.languageiso639-1 | en_US | - |
item.fulltext | with fulltext | - |
item.grantfulltext | open | - |
顯示於: | 分子醫學研究所
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