http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/151 Wed, 20 May 2026 05:12:14 GMT 2026-05-20T05:12:14Z http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/102076 標題: 黏蛋白型瞬時受體電位通道基因變異K370Q對流感病毒感染的影響及胞內運輸機轉分析; TRPML2 K370Q Mutation Disrupts Endolysosomal Trafficking and Modulates Influenza Virus Infection 作者: 林維瑄; WEI-SHUAN LIN 摘要: A型流感病毒（IAV）仍然是全球重要的健康議題，不僅引發季節性流行，還靠能導致大規模疫情。IAV為具包膜的病毒，擁有分節負鏈RNA基因組，主要透過網格蛋白介導的內吞作用（CME）進入宿主細胞。一旦內化，病毒會劫持宿主的內溶體-溶酶體系統，以促進自身的複製與釋放。病毒核糖核蛋白（vRNPs）仰賴內溶體運輸以有效進入細胞核，進可組裝新的病毒顆粒。內溶體-溶酶體系統由早期內溶體、回收內溶體、晚期內溶體及溶酶體組成，負責細胞內物質運輸，並在病毒感染過程中扮演了關鍵角色。該系統主要由Rab蛋白與離子通道所調控，其中包黏黏蛋白型瞬時受體電位通道2（TRPML2）。TRPML2主要而位於早期內體(early endosome)與回收內體(recycling endosome)以協助囊泡運輸。近期研究顯示，TRPML2在A型流感病毒感染中具有重要作用，不僅能促進病毒囊泡運輸，還有助於病毒高效逃離內體區室。值得注意的是，編碼TRPML2的基因MCOLN2的一種罕見等位基因變異被鑑定為功能喪失突變，導致病毒進入受阻。這些發現表明，TRPML2活性在內溶體動態調控中扮演關鍵角色，並可能成為調控感染的潛在標的。 在這項研究中，我們發現TRPML2 K370Q突變在A549細胞中減弱了A型流感病毒的複製，這個結果和不具活性的TRPML2 R310A突變體表現相似，進一步支持了 TRPML2在病毒感染過程中的角色。螢光成像結果顯示，TRPML2 K370Q與早期及晚期內體的共定位增加，但對於回收內體則沒有太大影響。此外，細胞在TRPML2專一性激活劑ML2-SA2的作用下可以逆轉TRPML2 K370Q與早期及晚期內體共而位增加的現象，這表明TRPML2的活性靠能會影響它在細胞中的位置。接著，透過活體即時螢光奈米域鈣成像我們發現K370Q的鈣釋放量低於野生型，暗示它的通道活性靠能受損，進而影響它在病毒運輸和感染過程中的作用。最後，在TRPML2活化劑ML2-SA1作用下，我們在表現K370Q的細胞中觀察到與野生型相當的A型流感病毒表現量，證實了K370Q較弱的活性是導致其無法增強流感病毒感染的主要原因。然而，K370Q如何具體影響流感病毒感染的機制，還需要進一步的研究支解釋。; Influenza A virus (IAV) remains a significant global health concern, causing seasonal epidemics and occasional pandemics. As an enveloped virus with a segmented negative-sense RNA genome, IAV primarily enters host cells via clathrin-mediated endocytosis (CME). Once internalized, the virus hijacks the host endolysosomal system to facilitate its replication and release. The viral ribonucleoproteins (vRNPs) depend on efficient endosomal trafficking for nuclear import and subsequent virion assembly. The endolysosomal system, composed of early endosomes, recycling endosomes, late endosomes, and lysosomes, is essential for intracellular cargo transport and viral infection. This highly dynamic network is regulated by Rab proteins and ion channels, including transient receptor potential mucolipin 2 (TRPML2). TRPML2, a member of the TRPML family, is primarily localized in early and recycling endosomes, where it facilitates vesicular trafficking. Recent studies have highlighted its role in IAV infection, showing that TRPML2 enhances viral vesicular trafficking and promotes efficient viral escape from endosomal compartments. Notably, a rare allelic variant of MCOLN2, the gene encoding TRPML2, has been identified as a loss-of-function mutation, impairing viral entry. These findings suggest that TRPML2 activity plays a crucial role in endosomal dynamics and may serve as a potential target for modulating IAV infection. In this study, we found that K370Q weakens IAV replication in A549 cells, similar to the inactive R310A mutant, reinforcing the role of TRPML2 in viral infection. Using fluorescence imaging, we observed that K370Q increase its colocalization with early and late endosomes, while having no significant impact on recycling endosomes. Further, ML2-SA2 treatment reversed the increased colocalization between K370Q and endosomes, suggesting that the activity of TRPML2 influence its intracellular localization. Subsequently, calcium imaging revealed less calcium release in cells expressing K370Q, implying a potential defect in channel activity, which may further result in disrupting its enhancement in viral trafficking and infection. Finally, upon treatment with the TRPML2 agonist ML2-SA1, cells expressing the K370Q variant exhibited influenza A virus expression levels comparable to those of wild-type cells. This finding supports that the reduced activity of K370Q is the primary reason for its inability to enhance viral infection. However, it still needs further investigation to clarify the underlying mechanism of how this mutant affects influenza viral infection. Wed, 01 Jan 2025 00:00:00 GMT http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/102076 2025-01-01T00:00:00Z http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76904 標題: 高脂飲食促進肺腺癌腫瘤生長機制探討; Investigation of Enhanced Lung Adenocarcinoma Progression Induced by High-Fat Diet 作者: Wei-Ming Chen; 陳薇名 摘要: 飲食習慣與癌症的發生明顯相關，據估計約30%的癌症病例與飲食有關。某些具抗癌效果的營養素被發現可抑制癌細胞生長的路徑，相反的部分脂質、低密度膽固醇及脂肪分泌的激素會促進癌細胞生長，於大腸癌、乳癌、前列腺癌等曾被報導，但在肺癌的探討相當有限且機制尚不明瞭。我們利用基因轉殖小鼠以藥物促使突變型表皮生長因子受體L858R表現作為肺腺癌動物模式，小鼠另外餵食高脂飲食及正常飲食以比較肺部腫瘤的生長情形，以及後續蛋白及基因的表現分析。結果顯示餵食高脂飲食的肺癌小鼠其腫瘤體積較大，Ki67的基因表現也顯著上升。利用RNA定序分析全基因表現發現高脂及正常飲食的肺腫瘤小鼠之基因表現主要差異在細胞分裂的功能，經qPCR確認ROS1基因表現在高脂飲食的肺腫瘤小鼠中顯著上升，且與腫瘤大小有高度正相關 (R=0.7058, p=0.0033)，然而ROS1蛋白磷酸化及其下游訊號無明顯差異。此外，FOXM1基因表現量亦在高脂飲食的肺腫瘤顯著上升，其功能已有促進腫瘤生長的報導。另一方面，在餵食野生種小鼠高脂飲食可使鼠蹊部白脂肪的TIMP1、pentraxin3和IL-6分泌量顯著上升，亦與RNA定序結果不謀而合。Kaplan-Meier 生存分析顯示高表現TIMP1或IL-6的肺腺癌患者其總存活期顯著下降。未來可用重組蛋白或抑制劑測試癌細胞的生長變化，以期能找出高脂飲食促使肺腫瘤生長的關鍵並進而應用於臨床。; Diet is attributed to about 30% of risk factors of cancers. Some nutrients with anti-tumor effect is also known for inhibition of signaling pathway related to tumor formation. In contrast, specific lipid uptake, low-density lipoprotein cholesterol and cytokine produced by adipose tissue can induce cancer cells proliferation and reported to correlate with several cancers. However, investigation of lung cancer and diet are limited and the mechanism is not clarified yet. We have bred inducible transgenic mice with spontaneous lung cancer formation driven by mutant EGFRL858R for animal model of lung adenocarcinoma. Lung tissues were harvested for hematoxylin and eosin (HE) stain, immunohistochemical (IHC) stain, western blotting and RNA for whole-genome transcriptomic analysis. Mice fed with high-fat diet and tumor induction (LC-HFD group) had larger tumor burden than regular-diet group (LC-RD) in observation of HE, IHC staining for Ki67 and lung weight. Gene set enrichment analysis of RNA sequencing revealed that the gene expression related to the function of cell division was more enhanced in LC-HFD than LC-RD. In addition, expression of ROS1 gene in LC-HFD was also increased significantly and highly correlated to tumor burden. However, there was no obvious phosphorylated ROS1 protein in western blotting and activation of downstream signals. The sequencing result also indicated that FOXM1 expression increased in LC-HFD, a transcription factor that has been known for promoting tumor growth. Tumor progression induced by high-fat diet may be triggered by FOXM1 rather than ROS1. On the other hand, TIMP1, pentraxin3, and IL-6 expression from inguinal white adipose tissue (iWAT) of high-fat diet treated wild-type mice were significantly upregulated, complying with the result of iWAT RNA sequencing. Kaplan-Meier plot showed high expression of TIMP1 or IL-6 was associated with lower overall survival in patients with lung adenocarcinoma. In order to find out the key leads high-fat diet promoting tumor progression, it is suggested to utilize recombinant protein or inhibitor for in vitro and in vivo assay to clarify the underlying mechanism in future. Wed, 01 Jan 2020 00:00:00 GMT http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76904 2020-01-01T00:00:00Z http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/38629 標題: 骨髓系增生疾病病人之端粒長度之研究：俱潛力之診斷標的; Study on Telomere Length in Patients with Myeloproliferative Neoplasms: A Potential Diagnostic Marker 作者: Tzu-Hsuan Chuang; 莊子璇 摘要: 骨髓系增生疾病(Myeloproliferative neoplasms, MPNs)是骨髓系幹細胞發生異常變化使得下游的血液細胞發生過度增生所造成的一種疾病，典型的骨髓增生性疾病病人可分為真性紅血球增多症、血小板增多症和原發性骨髓纖維症。近年來有研究發現在細胞中負責保護染色體末端的端粒，其長度在骨髓增生性疾病病人中有明顯縮短的趨勢。我們藉由一對能結合至端粒重複序列的引子與一對能結合至單一基因(single copy gene)的引子，利用即時監控聚合酶連鎖反應的原理來測量各檢體的相對端粒長度--相對端粒長度(Relative telomere length)正比於端粒產物量(T)比上單一基因產物量(S)之比例(T/S ratio)。 我們分析63 位被測得有高於正常人血球數目的病人其端粒長度並與255 位測有正常血球數目的人做比較，再將此端粒長度的分析結果與其他骨髓增生性疾病突變檢測結果綜合評估。因為在正常人群組中，端粒長度會受個人的性別與年齡影響，因此這些因素在病人中亦被分別分析。 在63 位被測得有高於正常人血球數目的病人中有35 位為JAK2 V617F 陽性的病人。其中27 位V617F 陽性病人的端粒長度較同齡正常人有明顯縮短之現象。此外此端粒長度縮短的現象只有在病人的多核細胞檢體中被發現，在其單核細胞檢體中則否。而在不同性別的V617F 陽性病人比較中，女性病人與男性病人的端粒長度則沒有顯著的差異。在63 位被測得有高於正常人血球數目的病人中剩下的28 位為JAK2 V617F 陰性的病人，藉由甲基特異化聚合酶連鎖反應 (Methylation-specific PCR assay)的結果我們將V617F 陰性的病人分為在JAK2的抑制子SOCS3 promoter 有部份甲基化及無甲基化組。而在SOCS3 promoter有部份甲基化的四位病人中，都可發現其端粒長度都有較同齡正常人下降的表現。 因此實驗認為端粒長度分析可作為檢測JAK2V617F 陰性骨髓增生性疾病中於JAK2 的抑制子SOCS3 promoter 有部份甲基化的病人之另一項標的。; Myeloproliferative neoplasms (MPNs) are caused by several disorders of myeloid clonal stem cells and these diseases are characterized by chronic proliferation of hematopoietic precursors. There are three major types of classic myeloproliferative neoplasms: polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF), and JAK2V617F is the most common biomarker to screen the patients with classic MPNs. Recent studies have reported that telomeres, which are complexes of tandem repeats and able to protect the chromosomes against homologous recombination and non-homologous end joining during cell division, showed significant shortening in patients with MPNs. In this study, the quantitative-PCR assay measured the relative telomere length of 63 studied patients with abnormal increased leukocyte count, or increased platelet count, or increased hemoglobin level; and then the results of the patients was compared with 255 normal controls which have normal complete blood counts (the relative telomere length was the ratio of telomere product to single-copy gene product, T/S). Due to the telomere length was affected by different genders and ages in the normal control cohort, the comparisons of the patients between different genders and ages were processed separately. In the JAK2V617F-positive patient cohort, the results of paired comparisons showed that telomere lengths were remarkably shorter in 27 of 35 patients (27/35) than the age-matched normal controls. The telomere length shortening was observed mainly in the granulocytes but not in the mononuclear cells. Furthermore, no significant differences of telomere shortening were observed between female and maleJAK2V617F-positive patients. In addition, the mutation burden of JAK2V617F displayed no obvious correlation with telomere length shortening in the JAK2V617F-positive patient cohort. In the JAK2V617F-negative patients, abnormal SOCS3 promoter methylation pattern was found in four patients who had significantly shorter telomere lengths than their age-matched normal controls. SOCS3 promoter methylation has been reported among the patients with PMF in the previous reports, so these four SOCS3-positive patients with shorter telomere lengths could have primary myelofibrosis. In conclusion, this data suggested that telomere length analyses may support the diagnoses in patients with SOCS3 promoter methylation but without JAK2V617F. Sat, 01 Jan 2011 00:00:00 GMT http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/38629 2011-01-01T00:00:00Z http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/9480 標題: 香豆素衍生物BPRHIV001藉由Akt途徑抑制第一型人類免疫缺乏病毒Tat蛋白質調節之轉錄活性; Inhibition of HIV-1 Tat-mediated transcription by a coumarin derivative BPRHIV001 through Akt pathway 作者: Pi-Han Lin; 林必涵 摘要: 第一型人類免疫缺乏病毒(Human immunodeficiency virus type 1, HIV-1)的Tat調節蛋白質是病毒轉錄RNA所必需。因此，Tat功能受影響時，病毒的複製將會被抑制。先前我們設計一個可偵測Tat功能的細胞篩選平台，來篩選抑制HIV-1的新興化合物。經此平台篩選了292個香豆素衍生物，其中BPRHIV001在奈莫耳濃度（nanomolar concentration）即具有抑制Tat轉錄活化的活性。進一步實驗發現，BPRHIV001不是透過影響Tat的生成以及Tat轉錄活化相關蛋白質複合物- Tat/P-TEFb組成，來抑制其對RNA第二型聚合酶（RNAPII）的活化作用。因此我們想探究BPRHIV001是否透過調控Tat轉譯後的修飾來影響其功能。結果發現，加入BPRHIV001後，細胞內可以乙醯化Tat並且幫助其離開TAR RNA的p300蛋白質量明顯減少，但mRNA量並沒有受到影響。因為磷酸化的Akt可穩定p300蛋白質，減緩其衰減，我們接著發現Akt及其活化分子PDPK1的磷酸化都在BPRHIV001的存在下降低許多。我們進行蛋白質嵌合分析(Docking analysis)來探討BPRHIV001與PDPK1之間的關係，結果顯示BPRHIV001可能藉由變構效應(allosteric effect)與PDPK1交互作用進而抑制PDPK1的磷酸化使其活性降低。最後in vitro實驗發現，BPRHIV001可有效抑制HIV-1的複製，其50%有效濃度（50% inhibitory concentration；IC50）為1.3 nM。BPRHIV001也可與現行使用的反轉錄酶抑制劑azidothymidine (AZT) 以及efavirenz (EFV)有強烈的加成效應。 綜觀以上結果，BPRHIV001可能透過干擾PI3K/Akt 途徑，來抑制HIV-1 Tat蛋白質所調控的轉錄活化活性。由於其與現行使用的抗病毒藥物具有良好的加成作用，我們認為BPRHIV001可望發展成為抗HIV-1感染的新興治療藥物的前導化合物。; The HIV-1-encoded RNA-binding protein Tat is known to play an essential role in viral gene expression. In the search of novel compounds in inhibiting Tat transactivity, BPRHIV001 was identified after screening of 292 coumarin-derivatives. BPRHIV001 was shown to significantly inhibit Tat-mediated transactivation at nanomolar range. BPRHIV001 is likely to exert its effects at the stage after initiation of RNAPII elongation since Tat protein expression and the assembly of Tat/P-TEFb complex which is essential for processivity of RNAPII elongation complex, remained unchanged. Next, the level of p300 was determined since acetylation of Tat by p300/CBP could facilitate the dissociation of Tat from TAR and subsequent recycling of Tat. The p300 protein level was reduced in the presence of BPRHIV001, while the p300 mRNA level was unaffected. The reduced p300 protein level was possibly resulted from reduced stability of p300 since the level of phosphorylated Akt, which was shown to be closely related with p300 stability, decreased in the presence of BPRHIV001. A concordant reduction of phosphorylated PDPK1, a well-known Akt activator was also observed. Furthermore, the docking analysis revealed that the reduced PDPK1 phosphorylation is likely resulted from the allosteric effect of interaction between BPRHIV001 and PDPK1. Finally, BPRHIV001 was shown to effectively inhibit HIV-1 replication in vitro and the 50% inhibitory concentration (IC50) of BPRHIV001 against HIV-1 was 1.3 nM. BPRHIV001 also had strong synergistic effect with current reverse transcriptase inhibitor azidothymidine (AZT) and efavirenz (EFV). Above all, BPRHIV001 was shown to have strong inhibitory effect on HIV-1 Tat-mediated transactivation, possibly through repressing the PI3K/Akt pathway.With strong synergistic effects with current reverse transcriptase inhibitors, BPRHIV001 has the potential to become a promising lead compound for the development of novel therapeutic agent against HIV-1 infection. Sat, 01 Jan 2011 00:00:00 GMT http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/9480 2011-01-01T00:00:00Z