梨形鞭毛蟲(Giardia lamblia)是一種早期分化出來且廣泛分布於全球的腸內致病性寄生原蟲，通常是藉由飲用受汙染之水源而受到感染。梨形鞭毛蟲生活週期主要分為兩種，滋養體時期和囊體時期，在梨形鞭毛蟲進入宿主體內後是以滋養體型式生活，在腸道中酸鹼改變時會進行囊體化作用成為囊體。自噬作用是生物體內自我降解之作用，在生物體內細胞週期中發育、代謝的重要平衡的機制。ATG8在啤酒酵母中發現與形成自噬小體(autophagosome)有關，可作為自噬作用的標記，FYVE蛋白質在人類中發現可促進自噬小體形成，在人類研究中，FYVE蛋白質與人類自噬作用相關蛋白LC3有交互作用。我們先前發現Myeloid leukemia factor (MLF)蛋白質會出現於囊泡。在梨形鞭毛蟲資料庫中找到自噬小體(autophagosome) 形成所需之ATG8基因及在人類中具有調節囊泡運輸之FYVE domain相似之蛋白，稱之為FYVE蛋白質。
本研究想了解梨形鞭毛蟲自噬作用之相關機轉，過去發現梨形鞭毛蟲的ATG8和FYVE蛋白質會出現於囊泡中，這些囊泡與MLF (myeloid leukemia factor)所在的囊泡相同，MLF是一個未知功能的蛋白質，已知會出現在囊泡中，MLF大量表現時，梨形鞭毛蟲的囊壁蛋白質CWP1表現量會增加。而過去發現調整細胞週期及分化之CDK2蛋白質，將其CDK2 domain中49-1742胺基酸刪除，成為CDK2dm突變蛋白質，發現CDK2dm會出現於囊泡中，這些囊泡與MLF所在的囊泡相同。
為測試這些囊泡的特性，首先進行飢餓實驗分析，結果發現在標準株MLF蛋白質表現上升。接著加入具有抑制自噬作用之藥物。Chloroquine可抑制自噬小體(autophagosome)與溶酶體(lysosome)融合，使自噬小體形成增加。Nocodazole造成溶酶體損傷使其導致自噬小體與溶酶體融合能力下降，使自噬小體形成增加。MG132是抑制蛋白酶體，使壞的蛋白質增加，並使壞的蛋白質進入自噬作用增加。Wortmannin功能是抑制吞噬泡(phagophore)生成，抑制自噬作用。Dithiothreitol(DTT)會在ER干擾氧化蛋白的折疊，引發自噬作用關鍵蛋白LC3表現量上升。Puromycin(PU)、G418會導致梨形鞭毛蟲死亡。E.coli會引發細胞對抗外來物而產生的自噬作用(Xenophagy)，可用來測試梨形鞭毛蟲之Xenophagy。首先在野生型WB分別加入Chloroquine、Nocodazole、MG132、Wortmannin、DTT、G418、Puromycin(PU)藥物，及使用E.coli處理後進行免疫螢光分析。發現實驗組加入Chloroquine、Nocodazole、MG132、Wortmannin、DTT、G418、Puromycin及E.coli後，其含有MLF囊泡數目增加。接著進行西方墨點法，在WB加入Chloroquine、Nocodazole、MG132、Wortmannin、DTT、G418、Puromycin藥物及E.coli之實驗組進行Xenophagy實驗之實驗組，其MLF蛋白質表現量均有上升。而加入Chloroquine處理之實驗組也發現BIP蛋白質表現量上升。接著我們對ATG8蛋白質細胞株進行飢餓實驗分析，結果發現ATG8蛋白質表現量上升。加入Chloroquine、Nocodazole、MG132、DTT、G418、Ecoli實驗組並進行西方墨點法，發現其ATG8蛋白質表現量上升。藉由免疫沉澱分析發現ATG8蛋白質會與BIP蛋白質交互作用，推測其可能與蛋白質運輸有關。對人類LC3b蛋白質表現細胞株之pLC3b細胞株與控制組p5’Δ5N-pac細胞株相比MLF及CWP1表現量上升，且藉由免疫沉澱發現LC3b細胞株會與梨形鞭毛蟲MLF交互作用。接著對pFYVE細胞株進行飢餓實驗分析，結果發現FYVE蛋白質表現量上升。加入Chloroquine、Nocodazole、MG132、DTT、G418、Ecoli實驗組並進行西方墨點法，發現其FYVE蛋白質表現量上升。對FYVE蛋白質表現細胞株之pFYVE細胞株與控制組p5’Δ5N-pac細胞株和pFYVE m1細胞株比較進行西方墨點法，結果發現pFYVE細胞株之MLF及CWP1表現量上升，而突變株pFYVE m1細胞株與pFYVE細胞株相比MLF及CWP1表現量下降。也藉由免疫沉澱分析發現FYVE會與BIP及ATG8蛋白質交互作用。
對於梨形鞭毛蟲MLF及相關自噬作用基因有更深入了解後，要釐清人類的MLF2與梨形鞭毛蟲之MLF作用是否相似。將人類hMLF2基因轉染入梨形鞭毛蟲之中形成phMLF2細胞株，並與控制組p5’Δ5N-pac細胞株和梨形鞭毛蟲pMLFHA細胞株比較進行西方墨點法結果phMLF2細胞株及pMLFHA細胞株之梨形鞭毛蟲MLF及CWP1表現量上升。進行飢餓實驗分析，結果發現hMLF2蛋白質表現量上升。將hMLF2蛋白質細胞株加入Chloroquine、Nocodazole、MG132、G418、DTT、Ecoli實驗組並進行西方墨點法，發現其hMLF2蛋白質表現量上升。也藉由免疫沉澱分析發現hMLF2會與梨形鞭毛蟲MLF蛋白質交互作用。
下一步我們針對pCDK2dm細胞株加入Chloroquine進行免疫螢光分析，在實驗組同樣發現含有CDK2dm蛋白質之囊泡數目也大量增加。pCDK2dm細胞株加入Chloroquine實驗組進行西方墨點法後CDK2dm、MLF、BIP皆有上升情況。而在野生型WB蟲株中與梨形鞭毛蟲囊體化形成有關聯之CWP1蛋白質受到Chloroquine處理後表現量下降，認為是因為Chloroquine阻止囊泡進入溶酶體之中阻斷自噬作用，而影響梨形鞭毛蟲發育及囊體化形成。
為了釐清MLF蛋白質對於CDK2dm蛋白質之影響，送入含有表現帶有HA tag之MLF基因的質體pMLFHA進入pCDK2dm細胞株中，希望經由大量表現表現細胞MLF蛋白質，觀察對CDK2dm的影響，並分別送入H2O和empty vector質體作為控制組。我們經由免疫螢光分析發現送入pMLFHA表現質體之pCDK2dm細胞株中，相較於只送入H2O及empty vector質體，送入MLFHA表現質體之pCDK2dm細胞蟲株，其囊泡數目增加。進行西方墨點法後發現送入MLFHA表現質體後其CDK2dm和MLF上升而CWP1下降。說明MLF的大量表現會促使CDK2dm囊泡增加，但增加的CDK2dm囊泡造成囊體化蛋白下降。
我的發現瞭解了MLF、ATG8、FYVE在自噬作用相關藥物處理會增加表現，及所在囊泡數目增加。以突變蛋白質CDK2dm為模式，也了解到這些囊泡會運輸突變蛋白質。

關鍵字：梨形鞭毛蟲、自噬作用、ATG8、FYVE domain、MLF、Chloroquine

Giardia lamblia is an early-branching and widely distributed intestinal protozoan parasites. People get infected by drinking the contaminated water. G. lamblia have two stages in the life cycle: a binucleate trophozoite and a quadrinucleate cyst. G. lamblia trophozoite parasitizies in small intestine and encysts by PH value changes. Autophagy is a self-degradative process of organisms that is an important balance of cell cycle in development and metabolism. In the previous reference, ATG8 was associated with the autophagosomes formation in Saccharomyces cerevisiae, and can be used as markers of autophagy. FYVE protein promotes autophagosomes formation in human and interacts with human autophagy-related protein-LC3 .We found localization of myeloid leukemia factor (MLF) in the vesicles. We’ve discovered autophagosome maker protein LC3/ATG8 related protein and FYVE protein that may transport vesicles in the Giardia by search genome database.
This research will determine G. lamblia autophagy related mechanism. We observed ATG8 and FYVE proteins both located in MLF vesicles. MLF over expression can induce CWP1 protein level. In earlier experiments, we found the G. lamblia cell cycle relate CDK2 protein is cytosolic, but CDK2dm with a deletion of 49-1742a.a localized in MLF contained vesicles.
To test the characteristics of these vesicles, we performed the starvation analysis, and treated PBS to cause starvation response in Giardia wild type WB strain. We found increased levels of MLF protein expression. We tested various autophagy inhibitors. Chloroquine inhibits the fusion of autophagosomes with lysosomes, and causes increased autophagosome formation. Nocodazole causes lysosomal damage, and decreases fusion ability of autophagosomes with lysosomes, resulting in an increase in autophagosome formation. MG132 is a proteasome inhibitor, that increases damage proteins and autophagy formation. Wortmannin is phagophore inhibitor and can inhibit autophagy formation. Dithiothreitol (DTT) can interfere the folding of proteins in ER, and induce expression of autophagosme maker protein-LC3. Puromycin (PU) and G418 will cause Giardia death. E. coli can induce autophagy (Xenophagy) in mammal cell, and can be used to test Xenophagy in Giardia. First, we treated chloroquine, nocodazole, MG132, wortmannin, DTT, G418, puromycin (PU) , and E. coli in the Giardia wild type WB cells. We found increased numbers of MLF vesicles and increased MLF protein expression by chloroquine, nocodazole, MG132, wortmannin, DTT, G418, puromycin and E. coli treatment. The expression of BIP protein was also increased in the chloroquine treatment experimental group. Then we tested the starvation effect in pATG8 experession strain. Increased numbers of ATG8 vesicles and increased ATG8 protein expression was found. We treated chloroquine, nocodazole, MG132, DTT, G418, and E. coli in the pATG8 expression strain, and found increased numbers of ATG8 vesicles and increased ATG8 protein expression. Using immunoprecipitation methods, we found that ATG8 proteins interacted with BIP. We transfected human LC3b gene in Giardia and created Giardia’s human LC3b expression strain-pLC3b. We found pLC3b interacted with G. lamblia MLF protein. The results showed that human LC3b was similar to G. lamblia ATG8 protein.
Next we tested the starvation effect in pFYVE experession strain. Increased numbers of FYVE vesicles and increased FYVE protein expression was found. We treated chloroquine, nocodazole, MG132, DTT, G418, and E. coli in the pFYVE expression strain, and found increased numbers of FYVE vesicles and increased FYVE protein expression. We created Giardia’s mutation strain-pFYVEm1, and found that pFYVEml expression strain, expressed lower level of CWP1 protein than pFYVE expression strain.
Using immunoprecipitation methods, we found that FYVE proteins interacted with BIP, and FYVE protein also interacted with ATG8 protein.
MLF protein may be an autophagy related protein in Giardia. We also wanted to know the function of human MLF2 protein in Giardia. We transfected human MLF2 gene in Giardia and created Giardia’s human MLF2 expression strain-phMLF2. We tested the starvation effect in phMLF2 experession strain. Increased numbers of hMLF2 and increased hMLF2 protein expression was found. We treated chloroquine, nocodazole, MG132, DTT, G418, and E. coli in the phMLF2 expression strain, and found increased numbers of hMLF2 vesicles and increased hMLF2 protein expression. Using immunoprecipitation methods, we found hMLF2 proteins interacted with G. lamblia MLF protein.
We treat chloroquine in the pCDK2dm expression strain, and found increased numbers of CDK2dm vesicles and increased CDK2dm protein expression. Next, we transfected pMLFHA plasmid in the CDK2dm expression strain. And found increased level of MLF and CDK2dm proteins and number of those vesicles, but reduced CWP1 protein expression.
We provide evidence that the MLF、ATG8 and FYVE protein expression increased and numbers of vesicles by autophagy inhibitors trement. Using a mutant protein model CDK2dm, we also know that vesicles may transport mutant protein.


Key words: Giardia lamblia, Autophagy, ATG8, FYVE domain, MLF, Chloroquine

目錄(CONTENTS)
誌謝 I
中文摘要 IV
ABSTRACT V
目錄(CONTENTS) VIII
第一章 前言(Introduction) 1
1.1 梨形鞭毛蟲簡介 1
1.2 自噬作用 2
1.3 FYVE 蛋白質 3
1.4 MLF蛋白質 3
1.5 CDKs(Cyclin dependent kinases) 4
1.6 細胞壓力實驗 5
1.7 研究動機 6
第二章 材料與方法(Materials and Methods) 7
2.1 梨形鞭毛蟲滋養體時期以及囊體化時期之培養(Giardia lamblia culture) 7
2.2 轉殖質體之建構(Plasmid construction) 7
2.2.1 5’Δ5N-pac 7
2.2.2 pATG8 7
2.2.3 pmATG8N 8
2.2.4 pFYVE 8
2.2.5 pFYVE-m1 9
2.2.6 phMLF-2 9
2.2.7 pMLF-HA 10
2.2.8 pLC3b 10
2.3 質體的轉型與萃取(Transformation and extraction) 10
2.3.1 質體的轉型 10
2.3.2 質體的萃取 11
2.4 梨形鞭毛蟲的轉染與選殖(Transfection and Selection) 11
2.5 重組蛋白質之表現與純化(Expression and Purification of Recombinant) 11
2.5.1 蛋白表現量測試 12
2.5.2 重組ATG8蛋白質表現所使用之質體建構 12
2.5.3 ATG8重組蛋白質之表現與純化 13
2.6 分子篩管柱層析法(Gel filtration) 13
2.7 磷脂酶D處理實驗(Phospholipase D ; PLD) 14
2.8 Imaris | 3D and 4D Real-Time Interactive Data Visualization 14
2.9 免疫螢光染色(Immunofluorescence assay；IFA) 15
2.10 西方墨點法與Coomassie blue染色(Weatern blot and Coomassie blue stain) 15
2.11 反轉錄聚合酶鍊式反應(RT-PCR) 16
2.12 即時定量反轉錄聚合酶鍊式反應(quantitative RT-PCR；qRT-PCR) 17
2.13 囊體計數(Cyst count) 18
2.14 免疫共沉澱法(Co-immunoprecipitation Assay；Co-IP) 19
2.15 ATG8、FYVE基因剔除實驗 (ATG8 and FYVE genes KO by Cas9 System) 20
第三章 實驗結果(Result) 21
3.1 梨形鞭毛蟲MLF蛋白質與自噬作用相關研究 21
3.2 梨形鞭毛蟲ATG8蛋白質之鑑定 23
3.3 梨形鞭毛蟲FYVE蛋白質序列之分析命名為FYVE 26
3.4 梨形鞭毛蟲FYVE蛋白質之鑑定 28
3.5 比較梨形鞭毛蟲MLF蛋白質與人類MLF2之功能 31
3.6 梨形鞭毛蟲MLF蛋白質在損壞蛋白質中扮演角 34
第四章 討論(Discussion) 36
4.1 梨形鞭毛蟲標準株MLF蛋白質出現在囊泡中 36
4.2 梨形鞭毛蟲ATG8功能釐清 36
4.3 梨形鞭毛蟲之FYVE蛋白質分析 37
4.4 人類MLF2轉染入梨形變毛蟲與梨形鞭毛蟲MLF蛋白質相似 38
4.5 梨形鞭毛蟲飢餓情況下自噬作用相關蛋白質上升 39
4.6 梨形鞭毛蟲會有Xenophagy之現象 40
4.7 CDK2dm突變株自噬作用的影響 40
4.8 ATG8/FYVE K.O之細胞株，製備困難 41
附圖(Figures) 42
圖一 ..... 42
圖二 ..... 56
圖三 ..... 73
圖四 ..... 76
圖五 ..... 92
圖六 ..... 109
附圖 ..... 116
Reference 117

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