1,720,978 research outputs found
Evaluation on the Feasibility of Using RNA Interference for Inhibition of Hepatitis B Virus Replication
No full textRNA interference (RNAi) 為一普遍存在於動物及植物細胞中的機制，由雙股RNA與其序列上有互補的目標mRNA結合之後，造成目標mRNA的分解。在哺乳動物細胞，可經由外送約21個核苷酸的小片段雙股RNA (siRNA)至細胞中而引發RNAi的產生。在本實驗中，我們試圖評估以siRNA作為治療B型肝炎病毒(HBV)感染的可行性。
在本實驗的第一部份，我們想瞭解是否能利用siRNA在肝癌細胞株中作為控制基因表現的工具。首先針對綠色螢光蛋白質(GFP)的基因序列設計了兩個siRNA，利用pSUPER載體表達siRNA，發現能夠在不同的肝癌細胞株(Hep G2, Huh 7, PLC/PRF5 and Hep 3B)中有效並具特異性地降低GFP的表現。接下來針對HBV基因體我們建構了六個表達siRNA的質體(命名為pSUPER/HBx1, pSUPER/HBx2, pSUPER/HBx3, pSUPER/TP, pSUPER/RT, pSUPER/ε)，將這些表達質體各別與pHBV48共同轉染至HepG2細胞中，以北方墨點法分析這些siRNA表達質體抑制pHBV48轉錄本表現的效果。六個pSUPER質體均能表現siRNA。然而，針對HBV基因體上相當具保守性、且與encapsidation有關的序列所設計的pSUPER/ε無法抑制HBV轉錄本的表現。pSUPER/TP，pSUPER/RT及pSUPER/HBx3則證明了在共同轉染的系統中能夠有效並特異地將HBV轉錄本分解。
HepG 2 2.2.15為一能夠持續穩定表現HBV轉錄本及產生HBV病毒顆粒的細胞株，實驗的最後一部份，我們以HepG 2 2.2.15細胞為模式模擬慢性肝炎的患者，觀察siRNA是否能對於已存在於細胞中的HBV複製造成影響。我們採用兩種方法企圖提高細胞的轉染效率，其一是將pSUPER/TP(Hyg)，pSUPER/RT(Hyg)及pSUPER/HBx3(Hyg)各別轉染至HepG 2 2.2.15細胞後，經過Hygromycin篩選後所建立的pooled clones，雖然這些pooled clones能夠表現siRNA，但對於HBV轉錄本並沒有明顯的抑制效果。此外我們建構以lentiviral vector為骨架表達siRNA的質體，利用病毒感染的方式將質體送入HepG 2 2.2.15細胞中，發現可以將轉染效率提高至80~ 90 %以上，然而對於HBV轉錄本只有約30~ 40 %的抑制效果。綜合上述結果，對於siRNA是否為治療慢性肝炎一個良好的工具還存有不確定性，相關的問題及解決方法將會在內文中詳加討論。RNA interference (RNAi) is a process by which double-stranded RNA (dsRNA) directs sequence-specific degradation of messenger RNA in animal and plant cells. In mammalian cells, RNAi can be triggered by 21-nucleotide duplexes of small interfering RNA (siRNA). In this study, we would like to evaluate the potential of using siRNAs for inhibition of hepatitis B virus (HBV) replication.
In the first part of the study, we investigated the feasibility of using siRNAs for controlling gene expression in hepatoma cell lines. Two anti-GFP siRNAs expressed by pSUPER vectors were able to efficiently and specifically knockdown GFP expression in several hepatoma cell lines (Hep G2, Huh 7, PLC/PRF5 and Hep 3B). Next, six siRNA expression vectors (designated as pSUPER/HBx1, pSUPER/HBx2, pSUPER/HBx3, pSUPER/TP, pSUPER/RT, pSUPER/ε) were constructed. Northern blot analysis was performed to evaluate the efficacy of degradation of HBV-specific transcripts after co-transfection of individual siRNA-expressing construct with pHBV-48 in Hep G2 cells. All six constructs expressed the siRNAs of interest. However, the effects on the HBV-specific transcripts varied. , pSUPER/εtargeting the conserved encapsidation sequences, failed to exert its effect. pSUPER/HBx3, pSUPER/TP, and pSUPER/RT were demonstrated to knockdown the HBV-specific transcripts specifically and efficiently in the co-transfection system.
Lastly, Hep G2 2.2.15 cell line, stably expressing HBV transcripts and producing HBV viral particles, was utilized to examine the effect of siRNAs on an established-infection system. Two approaches were utilized to overcome the poor transfection efficiency problem. One approach was enrichment of cells with the siRNA expression cassette through selection. Though siRNAs of interest were demonstrated to be expressed in selected Hep G2 2.2.15 cells, they showed limiting effects on HBV-specific transcripts. The other approach was to deliver the siRNA expression cassette using HIV-based lentiviral vectors. The transduction efficiency could be reached by 80 to 90 %, however, only 30 to 40 % of HBV-specific transcripts were knocked down. Taken together, our results raised an uncertainty in utilizing siRNAs for treatment of chronic HBV-carriers. Problems and solutions were discussed in the text.英文摘要 I
中文摘要 II
目錄 III
序論 1
本論文研究目的 16
材料與方法 17
一. 質體構築 (Plasmid construction) 18
二. 細胞培養 (Cell culture) 18
三. 細胞轉染 (Transfection) 20
四. 建立穩定表現pSUPER/TP(Hyg)、pSUPER/RT(Hyg)及pSUPER/HBx3 (Hyg)的細胞株 (Establishment of stable clones expressing pSUPER/TP (Hyg), pSUPER/RT(Hyg) and pSUPER/HBx3 (Hyg) ) 20
五. 細胞轉型 (Transduction) 21
六. 製備勝任細胞 (Preparation of competent cell) 21
七. 細菌轉型 (Transformation) 22
八. 小量質體製備 (Mini-plasmid preparation) 22
九. 大量質體製備 (Maxi-plasmid preparation) 23
十. Sodium dodecylsulfate polyacrylamide gel electrophoresis
(SDS-PAGE) 24
十一. 西方墨點法分析 (Western blot analysis) 25
十二. 北方墨點法分析 (Northern blot analysis) 25
十三. 偵測21個核苷酸的小片段RNA (Detection of 21-nt siRNA) 27
十四. 試管內轉錄反應 (in vitro transcription) 28
結果 29
討論 36
結果圖表 41
參考文獻 6
Stability of Bingham Fluid Flowing Down Along an Inclined Plane
No full text近年來台灣土石流災情頻傳，災害範圍程度也較過去擴大及增加，研究土石流已成為刻不容緩一項課題。泥流是其中一種流動速度較快、顆粒較細的流體分類。目前已在泥流中，觀察到間歇性的湧浪。而若在泥流流動過程中發生滾波等不穩定現象，對防治設施、監測設備的位置、形狀、設計強度均有不同的規劃設計。本研究所作之不穩定性分析，目的是對泥流更加了解，以期減輕泥流所造成之損害。
本文從基本流況開始--穩態(steady)、均勻流(uniform)，再給一底床微小變化的擾動，探討經過一段時間、空間發展後，觀察擾動現象成長與否，探討流況的穩定性。之後再針對參數加以研究分析。
本研究採用修正過之賓漢流體(雙黏滯性)，所選定之底床擾動變化，第一種是週期變化，研究證明這種擾動對流體而言，仍是穩定狀況。另外，由參數討論中，發現 (特徵降伏應力)和 (坡度之效應)會影響振幅大小。而 也與向下游傳遞之波數相關，即降伏應力越大，其波數會越大。降伏應力之影響不可忽略之；第二種是緩升或緩降坡，也由解得知，只要斜率不為零，經過一段時間後，擾動會逐漸增加，為不穩定之狀況。摘要............................................................................................................一
目錄............................................................................................................二
第一章 緒論.............................................................................................. 1
1.1 前言.............................................................................................. 1
1.2 前人研究...................................................................................... 2
1.2.1 牛頓流體的滾波現象及穩定性分析之研究........................ 2
1.2.2 非牛頓流體本構關係之研究................................................ 3
1.2.3 非牛頓流體的滾波現象及不穩定性分析............................ 5
1.3 研究目的與方法.......................................................................... 6
1.4 論文架構...................................................................................... 8
第二章 基本理論介紹與推導..................................................................10
2.1泥流之數學表示式.......................................................................10
2.1.1賓漢本構關係式.................................................................13
2.1.2強剪層與弱剪層.................................................................14
2.1.2.1賓漢本構關係式.......................................................14
2.1.2.2交界面邊界條件.......................................................15
2.1.2.3控制方程式...............................................................17
2.2穩態均勻流...................................................................................20
2.2.1討論Bingham本構關係......................................................21
2.2.2邊界條件.............................................................................22
2.2.3解穩態均勻流之流況.........................................................25
2.2.4小結.....................................................................................28
第三章 無因次化與尺度分析.................................................................30
3.1無因次化......................................................................................30
3.1.1討論各參數之尺度.............................................................30
3.1.2各式無因次化.....................................................................31
3.1.2.1控制方程式...............................................................31
3.1.2.2邊界條件...................................................................38
3.1.2.3穩態均勻流解...........................................................42
3.2尺度分析.......................................................................................44
3.2.1比較各無因次參數.............................................................44
3.2.2尺度分析結果代回各方程式.............................................45
3.2.2.1控制方程式...............................................................45
3.2.2.2邊界條件...................................................................47
3.2.2.3穩態均勻流解...........................................................48
3.3確認零階解即穩態均勻流之解...................................................49
3.3.1零階方程式與邊界條件.....................................................49
3.3.2解零階解.............................................................................51
第四章 微擾法之應用..............................................................................53
4.1微擾法...........................................................................................53
4.1.1微擾法展開.........................................................................53
4.1.2控制方程式.........................................................................55
4.1.3邊界條件.............................................................................56
4.2一階方程式及邊界條件...............................................................60
4.2.1一階方程式.........................................................................60
4.2.2一階邊界條件.....................................................................61
4.3整理一階方程式及邊界條件.......................................................62
4.3.1控制方程式.........................................................................62
4.3.2邊界條件.............................................................................63
4.4解一階方程式...............................................................................64
4.4.1列出一階方程式及邊界條件.............................................64
4.4.2解一階方程式.....................................................................65
4.5討論 與 之關係式....................................................................68
4.5.1強剪弱剪層交界面之邊界條件.........................................68
4.5.1.1一階...........................................................................68
4.5.1.2二階...........................................................................69
4.5.1.3三階...........................................................................70
4.5.2弱剪層x-動量關係式.........................................................72
4.5.3與空氣交界面之邊界條件.................................................72
4.5.4小結.....................................................................................73
第五章 不穩定性分析..............................................................................75
5.1底床擾動為週期擾動...................................................................75
5.1.1 穩定性分析........................................................................75
5.1.2 討論流深一階變化之解與各參數之間關係....................76
5.1.2.1整理流深關係式.......................................................76
5.1.2.2討論 與 之關係.....................................................83
5.1.2.3討論 ........................................................................83
5.1.2.4討論 ........................................................................84
5.1.3與相關文獻之比較.............................................................86
5.2底床擾動為微小線性變化，即一緩升坡或緩降坡....................87
5.2.1穩定性分析.........................................................................87
第六章 結論與建議..................................................................................90
6.1結論...............................................................................................90
6.2建議...............................................................................................90
附錄............................................................................................................92
參考文獻....................................................................................................96
圖目錄
圖1-1不同本構關係式之剪應力-剪切率關係示意圖............................ 5
圖1-2修正後賓漢本構關係式之剪應力-剪切率關係示意圖................ 7
圖1-3 架構流程圖.................................................................................... 9
圖2-1 探討之問題縱剖面示意圖............................................................11
圖2-2 控制方程式示意圖........................................................................12
圖2-3 邊界條件示意圖............................................................................17
圖2-4 穩態均勻流之水平流速示意圖....................................................29
圖5-1 底床為週期擾動之流深解示意圖................................................78
(A=1, =1,k=100= , =1, =0.3, =0.8)
圖5-2 和時間對一階流深之影響的關係圖........................................79
(A=1, =1,k=1= , =1, =1)
圖5-3 底床為週期擾動之解示意圖........................................................79
(A=1, =1,k=10= , =1, =0.3, =1000)
圖5-4 底床、強剪層厚度及流深一階變化關係圖..................................80
(A=1, =1,k=1= , =1, =0.8, =0.8)
圖5-5 底床、強剪層厚度及流深一階變化關係圖..................................81
(A=1, =1,k=1= , =1, =1, =0.8)
圖5-6 底床、強剪層厚度及流深一階變化關係圖..................................81
(A=1, =1,k=1= , =1, =0, =0.8)
圖5-7 底床、強剪層厚度及流深一階變化關係圖..................................82
(A=1, =1,k=1= , =1, =0.8, =0.6)
圖5-8 底床擾動變化與表面變化及強剪層厚度變化之關係圖............82
( k=1= , =1, =0.8, =1)
圖5-9 特徵降伏應力和振幅之關係圖....................................................83
圖5-10 特徵降伏應力和 (damping)之關係圖.....................................84
圖5-11 beta與振幅之關係圖(beta<1) .....................................................85
圖5-12 beta與P(振幅大小)之關係圖(beta>1) ........................................85
圖5-13 beta與 (damping)之關係圖.......................................................86
表目錄
表3-1無因次參數之尺度.........................................................................45
表A-1 各尺度與各無因次參數之關係...................................................94
表A-2 相關實驗與現場之文獻中各尺度列表.......................................9
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
Discuss the glamour of 7-word rhythmic suite poems in the period of Du Fu Kuizhou
No full textIn addition to the author, the formation of the style of literary works also involves the work, the world, and the reader. The four qualities influence each other to form an unique personal style. Moreover, \ue2Chenyu Duncuo\ue2is considered as the basic style of Du Fu\ue2s poetry. However, we have different interpretations about it and are ambiguous to really appreciate its true implication.
Therefore, this article will discuss from three aspects. First of all, through the introduction of Du Fu's life, the explanations of the influence of the world on the author and work will be discussed. Secondly, the historical readers\ue2 understanding to" Chenyu Duncuo " will be analyzed and how readers understand the work and the author will also be explained. Finally, according to Roman Ingarden, literatures are categorized into the followings: the stratum of linguistic sound form, the stratum of meaning units, the stratum of represented objects, and the stratum of schematized aspects. The four stratums will be the basic structure to analyze the three articles: Zhu Jiang, Yonghuai Guji, and Qiu Xing in the period of Du Fu Kuizhou so that the readers could taste the deep meaning and aesthetics Du conveyed as well as his characteristics. By concatenating the four parts of author, world, work and reader, it helps readers nowadays better understand the style of Du\ue2s poetry ,\ue2Chenyu Duncuo\ue2
- …
