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Electro-ultrafiltration study on Aldrich humic substances with different molecular weights
No full textGoing Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe 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
Get PDF“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
Get PDFWe 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
Divergent Voltage-dependent Gating Properties of REag1 and REag2 Potassium Channels
No full text電位控制開關的鉀離子通道(voltage-gated potassium channel)是一種陽離子通道，可以感應膜電位的變化選擇性的讓鉀離子通透。大鼠的ether à gogo（rEag） 鉀離子通道是屬於EAG家族(EAG family)的一種，有兩個子類型分別為rEag1和rEag2。rEag主要分布在中樞神經系統當中，對於調控神經細胞的興奮性和神經物傳導物質的釋放扮演重要角色。和其他鉀離子通道比較，rEag不同的地方除了在選擇性濾孔（selective filter）的指紋(signature)為GFG之外，還包括Ｎ端有PAS domain(Per-Arnt-Srm)，C端有環核苷酸結合區（cyclic-nucleotide binding domain）。
Eag鉀離子通道的開啟速度(activation kinetic)會受到不同的前置過極化負電位(hyperpolarized prepulse)所影響，越hyperpolarized的電位除了會造成activation delay之外還會讓eag的activation kinetics變慢。和其他鉀離子通道比較，前置過極化負電位只會延緩鉀離子通道的開啟時間。Eag對前置過極化負電位特殊的反應暗示了eag在activation之前可能會經過一個以上rate-limiting close state的transition。除了前置過極化電位之外，鎂離子和pH值也會改變通道開啟的速度。
本論文的研究目的是探討rEag1和rEag2電位活化機制之差異為何，並進一步了解產生這些差異的潛在分子結構基礎。我們的假設是造成rEag1和rEag2有不相同的gating property的主因可能為兩者在C端的差異。實驗時使用雙電極膜電位箝制(two-electrode voltage clamp)的電生理紀錄方法，首先比較rEag1和rEag2的gating propetty。之後並利用PCR的方法配合適當的引子(primer)以及利用兩次PCR( first run and second run)的技術，將rEag1和rEag2上相對應的C端互相置換，來探討不同的C端片段與兩者電位活化機制差異的可能關係。本實驗一共選擇了六個不同片段長度的C端段落置換，總計得到12個突變(chimera)。之後將這些突變轉型（transform）到pcDNA3的vector上，經由轉錄後（transcription）將cRNA注射到Xenopus oocytes 內表達(express)，最後測試這十二個突變對於前置過極化電位的反應以及其他的電位活化特性。
我們實驗的結果發現，rEag1和rEag2兩者在gating kinetics上存有先天明顯的差異。另外經由分析chimeras gating property的差異，我們首先觀察到rEag的C端會影響rEag1和rEag2 voltage-dependent gating property，但是不會改變rEag1和rEag2 steady-state voltage dependence。Voltage-gated potassium channels are cation channels which upon membrane potential change, undergo significant comformational change, leading to the opening of potassium-selective pores. Rat ether à gogo（rEag）potassium channels, which belongs to EAG family, specifically express in the central nervous system and are thought to plays an important roles in the modulation neuron of excitability and neurontransmeter release. Unlike other potassium channels, the signature sequence in the selective filter of rEag potassium channel is GYG. Furthermore, all EAG channels contain the PAS (Per-Arnt-Srm) domain and cyclic-nucleotide binging domain in the amino (N) and carboxyl-(C) terminus, respectively.
The activation kinetics of rEag potassium channel is significantly slower in the presence of hyperpolarizing prepulses, which is also known as the non-superimposable Cole -Moore shift. This phenomenon is consistent with the idea that prior to pore opening, ion channel must go through several voltage-dependent conformation changes. In addition, Mg2+ and pH can also modulate the activation kinetics of rEag potassium channels. There are two rEag potassium channels subtypes: rEag1 and rEag2. The two isofoms share 70% indntity in amino acid sequence, which encompasses the six transmembrane segments. Their activation kinetics, however, displays distinct features.
The goal of this thesis is to characterize the divergence voltage-dependent activation of rEag1 and rEag2 K+ channels and to find out the potential underlying structural bases. We applied two-electrode voltage clamp (TEVC) technique to study different rEag1 and rEag2 K+ channels heterologously expressed in Xenopus oocytes. Moreover, N and C termini, have previously been suggested to may a play role in the modulation channel activation. Since the major difference in amino acid sequence between rEag1 and rEag2 K+ channels lies in the C terminals, we aim to test the hypothesis that C terminal may confer the unique channel activation feature of rEag channels by using PCR mutagenesis technique. A total of 12 chimeras, 6 in each rEag backbone, will we constructed and tested for their biophysical properties.
Our date indicate that there is distinct divergence of gating property between rEag1 and rEag2. Besides, instead of steady state voltage dependence,we first report that C terminus will modulate rEag voltage-dependent gating property.目錄
誌謝 ……………………………………………………………………………………I
目錄……………………………………………………………………………………II
圖次……………………………………………………………………………………IV
中文摘要 ……………………………………………………………………………V
英文摘要 …………………………………………………………………………VII
第一章、導論 …………………………………………………………………1
I. EAG鉀離子通道介紹 ………………………………………………………2
A. 分類 ………………………………………………………………………… 2
B. EAG鉀離子通道家族功能 …………………………………………………2
C. EAG鉀離子通道的gating mechanism…………………………………… 3
II. Eag鉀離子通道的N端和C端 ………………………………………… 5
A. 一般結構 ……………………………………………………………… 6
B. N terminus………………………………………………………………… 6
C. C terminus………………………………………………………………… 7
III. 本實驗展望 …………………………………………………………… 9
A. rEag1和rEag2鉀離子通道的差異 …………………………………… 9
B. 實驗假說及實驗步驟 ……………………………………………… 10
第二章、材料與方法 ……………………………………………………………… 12
I. 分子生物技術 ……………………………………………………… 12
A. 置換GFP-rEag1和GFP-rEag2的C端位置，產生突變 ………… 12
B. 將突變從GFP vector換到pcrEag1或是pcrEag2上 ……………… 15
C. Gel extraction ………………………………………………………… 16
D. transformation，inoculation，miniprep …………………………… 16
E. RNA transcription ……………………………………………………… 17
II. 電生理記錄………………………………………………………………… 17
A. Xenopus laevis （南非爪蟾） oocyte取得和處理 ………………… 17
B. oocyte RNA injection ……………………………………………………18
C. 電生理記錄………………………………………………………………… 19
D. 電生理紀錄之分析………………………………………………………… 20
第三章、結果 ……………………………………………………………… 21
I. rEag1和rEag2鉀離子通道的voltage-dependent gating property 21
A. rEag1和rEag2鉀離子通道activation kinetics的異同性 ……… 23
B. rEag1和rEag2鉀離子通道deactivation kinetics的異同性……… 24
C. 前置過極化負電位對rEag1和rEag2鉀離子通道
activation kinetics的影響 …………………………………………26
II. 以rEag1為骨架的chimeras其電生理特性與rEag1鉀離子通道的差別29
A. steady-state voltage dependence………………………………………29
B. activation kinetics of chimeras………………………………………30
C. Deactivation kinetics of chimeras……………………………………30
D. 前置過極化負電位對chimeras的影響……………………………………31
III. 以rEag2為骨架的chimeras其電生理特性與rEag2鉀離子通道的差別32
第四章、討論 …………………………………………………………………………36
I. C端對於rEag1和rEag2 gating property異同性的影響………………37
A. Steady-state voltage dependence………………………………………37
B. Voltage dependent gating kinetics……………………………………39
II. C端在rEag1和rEag2 gating過程中扮演的角色………………………40
III. 相關的實驗討論 ………………………………………………………… 45
參考資料 ………………………………………………………………………… 7
The recognition region in SulA by ClpYQ protease from Escherichia coli
No full text大腸桿菌中ClpYQ蛋白酶為一種ATP依賴蛋白酶，由具有ATPase及unfoldase活性的ClpY，與具有peptidase活性的ClpQ所構成的雙單元體。這類蛋白酶在細胞中，可降解構形錯誤或是具危害性的蛋白質，以維持細胞正常生理作用，避免細胞受到危害。在ClpYQ蛋白酶中，ClpY會負責辨識基質，並水解ATP作為能量來源，將基質結構打開並傳送至ClpQ的活性區，以進行降解作用。然而，關於ClpYQ蛋白酶是如何選擇辨識基質，及後續降解作用的詳細機制，目前仍不清楚。SulA是一個細胞分裂的抑制物，當細胞暴露在逆境下時會產生SOS反應，誘導大量SulA蛋白表現，以避免受損的DNA傳到子代細胞。目前已知可分解SulA的蛋白酶為Lon及ClpYQ，其中又以Lon為主要負責分解的蛋白酶。之前有研究指出，Lon可以藉由辨認SulA之C-端末8個胺基酸，與之結合並將其降解，但是ClpYQ卻不能。對於ATP依賴蛋白酶來說，為避免不必要的降解，如何選擇辨認需要降解的基質是非常重要的。本實驗中，為確認ClpYQ蛋白酶辨認SulA蛋白的區域，建構SulA之C-端不同大小片段缺失的突變蛋白，以酵母菌雙雜交系統測試，各個SulA突變蛋白與ClpY之間交互作用的情形，發現ClpY辨識的區域可能位於C-端的高疏水性片段，C-端第20 - 30個胺基酸。於此區域內再建構點突變蛋白，分析不同性質的胺基酸對於ClpY交互作用的影響，結果顯示當點突變取代為親水性胺基酸時，會降低SulA蛋白與ClpY之間的交互作用。之後測試各個SulA突變蛋白的活性表現，及被ClpYQ蛋白酶降解之情形，結果顯示SulA之C-端第20 - 45個胺基酸的區域，對於其抑制細胞分裂的活性表現是重要的，且對於ClpYQ蛋白酶的降解作用也會造成影響，因此ClpYQ蛋白酶應可藉由SulA蛋白之活性表現與否，來辨別其是否需要降解。ClpYQ is an ATP-dependent protease from Escherichia coli and a two component complex composed of ClpY, which is an ATPase and unfoldase, and ClpQ peptidase. Degradation of denatured or damaged proteins by this proteases helps protect the normal cell growth from the harmful effects of these proteins. The ClpY is thought to recognize protein substrates, denature them, and translocate the unfolded polypeptide into the catalytic cavity of the ClpQ for degradation. However, little information is available on the recognition of substrates for ClpYQ and on the mechanism by which they were selected, unfolded, and translocated by ClpY to the interior of the ClpQ. SulA, induced in the SOS response, is a cell division inhibitor and prevents the distribution of damaged DNA into daughter cells during DNA repair processes. SulA can be degraded by ATP-dependent proteases such as Lon and ClpYQ, and the degradation in vivo seems to be predominantly by Lon, while ClpYQ appears to act as a backup for Lon. It was reported previously that the region of C-terminal eight amino acid residues of SulA was essential for interaction with Lon but not with ClpYQ. To avoid unnecessary degradation of cellular proteins, substrate selection by ATP-dependent proteases is tightly regulated; therefore, it is interesting to investigate the recognition region of SulA by ClpYQ. In this study, the deletion mutants of SulA with regard to C-terminus were constructed and the interaction with ClpY was analyzed in yeast two-hybrid system. The results showed that ClpY recognized the hydrophobic region of SulA, C20 - 30 aa, and the recognition was also likely to rely on hydrophobic interaction following the observation that the binding activity decreased if the substituted residue was polar. The C-terminal region of SulA, C20 - 45 aa, seemed to be important for its activity with an inhibition of cell division, and the region is necessary for the degradation by ClpYQ. Therefore, ClpYQ protease would be able to distinguish whether SulA is to be degraded by the activity of inhibition.目錄試委員會審定書 i謝 ii要 iiibstract iv錄 vi目錄 viii目錄 ix圖目錄 x、前言 1、ATP依賴蛋白酶 1、ClpYQ蛋白酶 2、ClpYQ複合體結構 4、ClpYQ蛋白酶之基質 5、細胞分裂抑制物SulA 7、蛋白酶對基質SulA辨認之相關研究 9、研究動機與目的 12、材料與方法 13、實驗材料 13一) 菌株與質體 13二) 藥品與試劑 14三) 器材設備 15四) 分析軟體 15、實驗方法 16一) 一般實驗方法 16二) 菌體的建構 20三) 突變基因建構 25四) 酵母菌雙雜交系統分析 28五) 大腸桿菌選殖基因表現系統 33六) 西方墨點分析 (Western Blotting) 35、實驗結果 41、確認SulA蛋白被ClpY辨識之區域 41一) 建構SulA蛋白C-端不同大小片段缺失之突變 41二) 以酵母菌雙雜交系統測試各個SulA突變蛋白與ClpY之交互作用 41、SulA蛋白被辨識區域之特性 43一) SulA蛋白C-端序列特性分析 43二) 於SulA蛋白被辨識區域中建構不同胺基酸性質之點突變 43三) 利用酵母菌雙雜交系統測試SulA點突變蛋白與ClpY之交互作用 44、不同蛋白酶對於SulA蛋白辨識區域之差異 45一) Lon蛋白酶與SulA缺失突變蛋白間之交互作用 45二) ClpY I domain上兩個loop對於SulA蛋白辨識之影響 46、SulA突變蛋白之活性表現及受ClpYQ蛋白酶降解之現象 47一) C-端末20個胺基酸缺失之SulA突變蛋白 48二) C-端二級結構部份缺失之SulA突變蛋白 50三) SulA不同性質胺基酸之點突變蛋白 51、討論 53、SulA蛋白被ClpY辨識之區域及其特性 53、不同蛋白酶對於辨認相同基質之差異 54、SulA突變蛋白之活性表現與FtsZ的相關性 55、ClpYQ蛋白酶對於SulA突變蛋白之降解情形 57、質體SulA的表現會誘導染色體之SulA表現 58、結論 60、參考文獻 61目錄一、本論文所使用的菌株與噬菌體 67二、本論文所使用的質體 68三、本論文所使用的引子對 69四、在酵母菌雙雜交系統中SulA突變蛋白與ClpY之交互作用情形 71五、SulA突變蛋白之活性測試及受ClpYQ蛋白酶降解之情形 72目錄一、大腸桿菌之SulA結構圖 73二、以酵母菌雙雜交系統分析Sul
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe 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
The proteins needed for indole induced biofilm formation and their functions in E. coli DH5α
No full textABSTRACT
之前我們已經以二維膠片的分析技術發現當大腸桿菌的重要病原菌O157被處以抗生素枯草桿菌素時會經由EvgS/EvgA雙蛋白質系統引起Tryptophanase基因的表現，並進而分解Tryptophan引起Indole的產生，接著引起生物膜的形成以及一系列相關蛋白質的表達。
在這篇論文中我們以被跳躍子基因破壞的大腸桿菌DH5α突變株作為研究材料，進一步研究哪些基因為由Indole引起大腸桿菌形成生物膜所必須，結果發現當yhiP、cyaA、trkA、phr、fimE突變時大腸桿菌無法為Indole引起生物膜的形成。經由互補實驗我們也進一步證實這些基因確實為Indole引起大腸桿菌形成生物膜所必須。我們也利用報導基因分析再進一步證實Indole確實能引起這些基因的表現，其中又以yhiP、cyaA活性增加至200% 以上最為明顯﹔另外經由互補實驗和報導基因分析我們也部分的闡明了Indole與這些基因上游的轉錄調控子之間的關聯﹔以及各種常見抗生素對這些基因表現的影響。除此之外，在這篇論文中也以butanol等實驗材料分析了這些基因對大腸桿菌形成生物膜以對抗逆境的貢獻。
另外由於我們之前發現了在大腸桿菌中蛋白質B0363能為autoinducer (indole)引發表現且為金黃色葡萄球菌IcaA的類似物，因為它被預測是被分泌至細胞膜外側的部分水溶性蛋白質較易於分析，所以在這篇論文中我們也表達純化了這個蛋白質並做了相關的活性分析並且得到了高純度且具有活性的蛋白質。ABSTRACT
By using the two-dimensional gel method, we already discovered that when E. coli O157 was exposed to bacitracin, the expression of tryptophanase gene was induced through the EvgS/EvgA two-component system. Tryptophanase then converted tryptophan to indole,which caused biofilm formation as well as a series of related protein expression.
In this study, we screened the E. coli DH5α transposon library mutants to find out which genes are crucial for indole induced biofilm formation. These results show that when yhiP, cyaA, trkA, phr, and fimE genes are disrupted by transposon, indole can not induce these mutant strains to form biofilm. By using complementary assays, we confirmed that these genes are truly necessary for Indole induced biofilm formation in E. coli DH5α. Besides, we also used the reporter gene analysis method to further confirm that indole can cause these genes expressions with the promoter activity of yhiP, cyaA increased up to 200%. Moreover, we also clarified the relationship between indole and expressions of some transcription factor genes upstream to these genes, as well as the influences of each kind of common antibiotic to the expressions of these genes. In addition, we also used other experiment material such as butanol to test if expressions of these genes also contribute to the resistence to the adverse circumstance when E. coli formed biofilm.
Besides, the expression of B0363 can be induced byindole in E.coli. B0363 is an analogous protein of IcaA of staphylococcus, which is secreted to outside of cell after transcription. I also expressed and purified this protein successfully and made sure it has catalytic activity
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