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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 董馨蓮(Shin-Lian Doong) | |
| dc.contributor.author | Yi-Han How | en |
| dc.contributor.author | 侯以涵 | zh_TW |
| dc.date.accessioned | 2021-06-16T17:40:52Z | - |
| dc.date.available | 2018-09-24 | |
| dc.date.copyright | 2013-09-24 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-08-14 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64326 | - |
| dc.description.abstract | CBM複合體(CARMA1-BCL10-MALT1)參與在NF-κB活化訊息傳遞中。其中MALT1除了做為鷹架(scaffold)的功能外，也是paracaspase蛋白酶。此外有研究指出ABC-DLBCL淋巴瘤之存活和增生是極度依賴 MALT1的切割活性，所以抑制MALT1活性可作為治療的標靶。本論文第一部分的實驗目的為利用先前實驗室建立的試管中MALT1受質切割系統，來尋找MALT1的活性抑制劑。
首先利用細菌表現並純化His-tag MALT1及BCL10，在系統中確認MALT1可以切割BCL10，而catalytic diad (H415-C464)突變則失去切割活性。另外使用Ac-LRSR-AMC螢光胜肽當做受質，以切割後的發射螢光值來計算MALT1切割活性。在這個系統中， z-VRPR-fmk(已報導的MALT1抑制劑)對MALT1之IC50約12 nM，但z-VAD-fmk (caspase抑制劑) IC50約2 μM。而zVRPR-fmk也能對trypsin造成抑制，且IC50與對MALT1相當(約16 nM)。接著對65個化學物進行篩選，以抑制50%活性為初步門檻，篩選出三個候選藥物IC50約為20 ng/μl。 未來可繼續利用此in vitro系統大量篩選抑制劑，並進一步在in vivo系統中如ABC-DLBCL細胞株進行抑制試驗。 本論文第二部份利用酵母菌來探討MALT1是否具有與YCA1相似的功能。出芽酵母菌具有與人類唯一的paracaspase MALT1序列相似的metacaspase YCA1，已被發現會參與在酵母菌的死亡機制中，也發現能扮演清除細胞內protein aggregates的角色。 首先在H2O2或緩慢加熱刺激後，可觀察到BY4742 Δyca1突變株比野生種的生存率高。然而大量表現YCA1的Δyca1突變株在H2O2處理後生存率卻無降低。將野生種之YCA1 caspase-like domain (CLD)取代為MALT1的CLD對耐受性也無影響。另外，以不同selection marker製備的Δyca1突變株皆不具有比野生種高的耐受性。而BY4741背景之酵母菌也無法證實YCA1參與在酵母菌的死亡機制。 此外BY4742及BY4741背景的Δyca1突變株也觀察不到過去文獻指出的multi-vacuole細胞所佔比例高於野生種2.5倍的現象，starvation刺激後也無法區分差異。因此本部份由於無法重覆YCA1之功能，而無法更進一步探討MALT1。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2021-06-16T17:40:52Z (GMT). No. of bitstreams: 1 ntu-101-R99445103-1.pdf: 9088310 bytes, checksum: b5d044a0b551f79c1794e78845e75c6b (MD5) Previous issue date: 2012 | en |
| dc.description.tableofcontents | 誌謝 i
中文摘要 ii ABSTRACT iv 目 錄 vii [第一部分] 1 第一章、序論 1 MALT1 (Mucosa-Associated-Lymphoid-Tissue Lymphoma-Translocation Gene 1) 1 MALT1在NF-κB活化路徑中扮演鷹架蛋白(scaffold protein)角色 2 MALT1具有蛋白酶活性(proteolytic activity) 3 MALT1蛋白酶活性影響NF-κB的活性 4 目前已被發現的MALT1受質 5 MALT1能成為臨床上治療之標靶 7 第二章、研究目的 9 第三章、材料與方法 10 本實驗使用之菌株 10 本實驗使用之質體 10 製備勝任細胞 (Preparation of competent cells) 10 細菌轉形 (Bacteria transformation) 10 小量質體製備 (Mini plasmid preparation) 11 His-tag MALT1 蛋白質的純化 11 His-tag BCL10蛋白質的純化 12 蛋白質定量 (Protein quantification analysis) 12 蛋白質電泳 (SDS-polyacrylamide gel electrophoresis) 12 西方墨點法 (Western blotting analysis) 13 銀染色 (Silver staining) 13 In vitro BCL10切割試驗 14 In vitro發射螢光胜肽Ac-LRSR-AMC切割試驗及MALT1抑制劑篩選 14 第四章、結果 15 His-tag MALT1與His-tag BCL10蛋白質的純化 15 MALT1可以在In vitro切割 BCL10 15 利用In vitro發射螢光胜肽切割試驗測試抑制劑對MALT1的影響 16 利用In vitro發射螢光胜肽切割試驗篩選MALT1可能的抑制劑 17 第五章、討論 18 第六章、圖表 21 圖一、His-tag MALT1蛋白質的表現與純化 21 圖二、BCL10蛋白質的表現與純化 22 圖三、在In vitro切割系統中，純化的MALT1可以對BCL10進行切割 23 圖四、利用In vitro發射螢光胜肽切割試驗測試抑制劑對MALT1的影響 24 圖五、利用In vitro發射螢光胜肽切割試驗篩選MALT1可能的抑制劑 25 附圖一、MALT1 受質專一性口袋結構能辨識z-VRPR-fmk抑制劑 26 [第二部分] 27 第壹章、序論 27 YCA1 (Yeast Caspase 1) 27 YCA1的功能 27 利用酵母菌來研究其他物種之metacaspase的功能 30 YCA1的酵素活性 31 第貳章、研究目的 32 第參章、材料與方法 33 本實驗使用之菌株 33 本實驗使用之質體 33 本實驗使用之引子 34 構築表現質體 34 酵母菌的轉型 (Yeast transformation) 35 酵母菌中表現蛋白質 35 TCA沉澱法萃取酵母菌總蛋白質 36 酵母菌genomic DNA之抽取法 36 酵母菌突變株之製備 36 酵母菌對H2O2耐受性試驗 38 緩慢加熱對酵母菌生存率試驗(Heat ramp cell death assay) 38 南方墨點法(Southern blotting) 39 FM 4-64酵母菌液胞染色 40 第肆章、結果 41 第伍章、討論 47 第陸章、圖表 51 圖一、 以PCR確認BY4742 Δyca1突變株 51 圖二、 H2O2處理BY4742野生種及突變株 52 圖三、 緩慢加熱(Heat ramp)處理BY4742野生種及突變株 53 圖四、 非誘導情況下，帶有pYES2-MALT1質體之BY4742野生種及Δyca1突變株在H2O2處理下的生存率 54 圖五、 在BY4742 Δyca1突變株表現質體pYES2-HA-YCA1及pYES2-MALT1 55 圖六、 利用基因重組將YCA1 caspase-like domain (CLD)取代成MALT1 CLD 56 圖七、 以南方墨點法再次確認染色體基因重組之BY4742突變株 57 圖八、 染色體基因重組的MALT1-CLD、MALT1-C464A-CLD BY4742突變株可以表現YCA1 prodomain-MALT1 caspase-like domain融合蛋白 58 圖九、 BY4742各種基因重組突變株在H2O2或加熱處理後之耐受性 59 圖十、 BY4741野生種及突變株之耐受性試驗 60 圖十一、 緩慢加熱(Heat ramp)處理BY4741野生種及突變株 61 圖十二、 BY4741表現質體pYES2-HA-YCA1及pYES2-MALT1 62 圖十三、 以不同selection marker製備YCA1基因刪除突變株 64 圖十四、 緩慢加熱處理YCA1基因刪除突變株 65 圖十五、 以H2O2處理YCA1基因刪除突變株 66 圖十六、 對酵母菌進行FM4-64螢光染色並觀察液胞結構 67 圖十七、 利用nitrogen starvation誘導酵母菌自噬作用並觀察液胞結構 68 附圖一、限制酶截切圖譜 (供南方墨點法使用)。 69 參考文獻 70 | |
| dc.language.iso | zh-TW | |
| dc.title | (一)利用試管中切割系統篩選MALT1之抑制劑
(二)利用酵母菌尋找MALT1之新功能 | zh_TW |
| dc.title | (I)In vitro cleavage assay for screening of MALT1 inhibitors
(II)Investigation on MALT1 functions in Saccharomyces cerevisiae | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 鄧述諄(Shu-Chun Teng),詹世鵬(Shih-Peng Chan) | |
| dc.subject.keyword | CBM複合體,MALT1,蛋白酶,抑制劑,酵母菌,YCA1, | zh_TW |
| dc.subject.keyword | CBM complex,MALT1,protease inhibitor,yeast,YCA1, | en |
| dc.relation.page | 76 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2012-08-15 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 微生物學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
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