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Biomedical Application of Activity Probe for Neuraminidase and Steroid Sulfatase
No full text以酵素的活性為基礎的而設計的小分子活性探針可以用來標示、濃縮、分離及區分蛋白質。它會選擇性的與酵素反應並共價鍵結於酵素上。活性探針的組成包含四個主要部分：辨識端、捕捉機制、連結鏈與發報端。在本研究中，活性探針6、11及12被用來測試標定流感病毒唾液酸酶及固醇硫酸酶。以活性探針12為基礎而設計的化合物17、18及19則用以抑制固醇硫酸酶的活性。
[第一部分]
爲了偵測及抑制流行性感冒病毒唾液酸酶，探針6的結構包含四個部分：唾液酸辨識端、quionoe methide捕捉機制、連結鏈及用以偵測分離的生物素發報端。一但唾液酸酶水解唾液酸，所產生的高活性quinone methide便會共價修飾酵素。在本研究中，我們以西方墨點法觀察到生物素標示的Arthrobacter ureafaciens唾液酸酶。針對病毒捕捉的實驗，探針藉由卵白素與生物素的交互作用固定於微孔盤上，並利用酵素免疫分析法證實可以成功地捕捉到流行性感冒病毒。合併反轉錄-聚合酶鏈反應與專一性的引子對可成功的區分被捕捉的流行性感冒病毒種類。本研究這個新的方法提供了一個機會可以快速利用抗體篩選流行性感冒病毒及發展更敏感快速的診斷方法。
[第二部分]
固醇硫酸酶藉由調控estrone sulfate的脫硫反應促進乳癌細胞吸收estrogen。人類固醇硫酸酶被發現在乳癌腫瘤上有表現量及活性增加的趨勢也因此成為藥物發展的標的。在本研究中，兩種固醇硫酸酶活性探針(探針11及12)及三種抑制劑(化合物17、18及19被用來標示及抑制固醇硫酸酶。標示的研究說明了探針11會專一性的與固醇硫酸酶反應。墨點分析也說明了探針11所標示的訊號強度與酵素的活性有關。更進一步的研究發現，探針12比探針11具有更高的特異性與較少的擴散反應發生。在抑制的研究方面，三價的酪氨酸硫酯化衍生物，化合物19，顯示有較化合物17和18更好的抑制能力。活細胞的實驗也證明了化合物19對倉鼠卵巢細胞具有適當的通透性及不具細胞毒性的特性。對於發展乳癌的治療固醇硫酸酶探針12及化合物19將會是一個有用的化學方法用於固醇硫酸酶的鑑別、抑制劑的設計及蛋白質體內標的物功能性狀態的決定。Depending on their enzymatic activities, the small molecular activity probe can be used to tag, enrich or isolate distinct sets of proteins. It only selectively reacts with enzymes that have a catalytic activity and covalent linkage with targeted enzyme. Activity probe contains four major components: a recognition head, a trapping device, a linker and a reporter group. The designed probe 6 was employed by this study to test its labeling effects on influenza virus neuraminidase (NA), whereas for the application of steroid sulfatase (STS), probes 11 and 12 were adopted. Compounds 17, 18 and 19, whose structures were designed on the basis of that of probe 12, were used for inhibiting STS activity.
Part I
For the detection and inhibition of influenza virus NA, the structure of probe 6 was designed to contains four fragments, a sialic acid for recognition, a latent quinone methide for trapping device, a linker and a biotin as reporter for detection and separation. Once a sialic acid is released by the hydrolysis of NA, the enzyme can be covalently modified by the resulting highly reactive quinone methide. In our study, the biotin labeled Arthrobacter ureafaciens NA were observed by Western blotting. For virus capturing experiment, the probe was be used to attach the microplates through avidin-biotin interactions and the captured virus were successfully demonstrated by the ELISA assay. To identify the captured virus, RT-PCR method combined with specific primer sets was future implemented. Overall, the novel approach adopted in this study offers opportunities for the rapid screening of antibodies against influenza virus and development of sensitive, rapid diagnostic methods.
Part II
Steroid sulfatase (STS) facilitates the estrogen uptake of breast cancer cells by mediating the desulfation of estrone sulfate. Human steroid sulfatase has become a target for drug development due to its increased expression and activity in breast carcinoma. In this study, two activity probes (probes 11 and 12) and three inhibitors (compounds 17, 18, and 19) were used for STS labeling and inhibition. Regarding STS labeling, probe 11 was shown to specifically react with STS. The dot signal intensity of probe 11 was illustrated by dot blot analysis to be correlated with enzymatic activity. Compared with probe 11, probe 12 demonstrated that higher specificity and less cross-reactivities. For STS inactivation study, compound 19, trivalent tyrosine sulfate ester derivative, revealed better inhibition ability than compounds 17 and 18. For compound 19, moderate cell permeability and no cytotoxicity for CHO cells were demonstrated in live cell assay. As a result, for the development of breast cancer treatment, probe 12 and compound 19 may be two effective chemical devices in STS identification, STS inhibition, and functional state determination in complex proteome.口試委員會審定書 i
誌謝 ii
中文摘要 iii
Abstract v
Abbreviations x
List of Figures xiii
List of Tables xv
Chapter 1 Biomedical Application of Activity Probe for Influenza Virus Neuraminidase
1.1 Introduction 1
1.1.1 Influenza virus ……………………………………………... 1
1.1.2 NA-targeted inhibitors ………………………………………. 4
1.1.3 Activity probe for NA labeling………………………………... 7
1.1.4 Aim and experimental approaches …………………………… 10
1.2 Materials and Methods 12
1.2.1 Materials …………………………………………………... 12
1.2.2 NA probe labeling ……………………………….................. 13
1.2.3 Western blot analysis ………………………………………... 13
1.2.4 NA activity assay …………………………………………… 14
1.2.5 Virus particle ELISA assay …………………………………... 14
1.2.6 Purification of RNA from influenza virus and RT-PCR amplification .......................................................................... 15
1.2.7 Agarose gel electrophoresis ………………………………….. 16
1.3 Results 17
1.3.1 NA labeling by probe 6 ………………………….................... 17
1.3.2 Effect of probe 6 inhibition ………………………………….. 18
1.3.3 Virus captured by immobilized probe 6 ……………….............. 21
1.3.4 Detection of influenza virus after probe capturing experiment …… 23
1.3.5 Typing and subtyping of influenza virus after probe capturing ….... 25
1.4 Discussion 27
1.4.1 Labeling and inactivation of NA by probe 6……………............. 27
1.4.2 Capturing influenza virus by immobilized probe 6 ……………... 28
1.5 Conclusion 32
Chapter 2 Development of an Activity Probe and Inhibitors for Steroid Sulfatase
2.1 Introduction 33
2.1.1 Reversible sulfation pathway and steroid sulfatase (STS) ……….. 33
2.1.2 Steroid sulfatase (STS) in health and disease …………………... 39
2.1.3 Activity probes and inhibitors for STS ………………………... 42
2.1.4 Aim and experimental approaches …………………………… 46
2.2 Materials and Methods 47
2.2.1 Materials …………………………………………………... 47
2.2.2 Cell culture and transfection …………………………………. 48
2.2.3 Cell fractionation …………………………………………… 49
2.2.4 STS activity assay …………………………………………. 49
2.2.5 STS probe labeling ………………………………………….. 50
2.2.6 Western blot ………………………………………………... 50
2.2.7 Dot blot ……………………………………………………. 51
2.2.8 Cell viability assay ………………………………………….. 51
2.3 Results 52
2.3.1 Human STS Transfection ……………………………………. 52
2.3.2 Labeling of STS by probe 11 ………………………………… 53
2.3.3 Dot blot analysis of probe 11 in labeling reaction ……………… 55
2.3.4 Labeling of STS by activity probe 12 …………………………. 57
2.3.5 Inactivation by STS inhibitors …………………....................... 59
2.3.6 Cytotoxicity studyof compound 19 ………………………….... 61
2.4 Discussion 63
2.4.1 Effect of STS labeling by activity probes 11 and 12 …………….. 63
2.4.2 Inactivation by STS inhibitors………………………………… 65
2.5 Conclusion 67
References 68
Curriculum Vitae 7
Nitrophenylboronic Acids as Highly Chemoselective Probes To Detect Hydrogen Peroxide in Foods and Agricultural Products
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Going 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
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
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