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YAO, YUN-GIN
No full text為建立一方便、敏感、迅捷，能適用於實驗室及野外現場，對豬假性狂犬病血清診斷
試驗方法，並探討酵素結合免疫吸附測定法的本質與特性，以及診斷其他傳染性疾病
的可行性。
試比較下列各種病毒處理來做為ELISA 試驗之抗原製備。分別以⑴遠心沈澱⑵polye-
thylene glycol６○○○ ８％濃縮⑶硫酸銨濃縮半純化的豬假性狂犬病毒及⑷經細
胞消化液處理⑸界面活性劑Trixton x －１○○ １％在Tris－glycine 緩衝液粹取
病毒感染細胞所得之可溶性抗原等。結果發現以遠心沈澱法來純化病毒及Trixton x
－１○○粹取的可溶性抗原，做為ELISA 試驗的抗原，效果判訣容易，特異性及敏感
性均高。
試用各種不同的洗液：如PBS 緩衝液，PBS －Tween ２○，蒸餾水，蒸餾水－Tween
２○，生理食鹽水－Tween ２○，高鹽緩衝液－Tween ８○；各種不同的血清、酵素
標示抗體的稀釋液：如PBS 緩衝液，PBS －Tween ２○，PBS －○．５％BSA ，PBST
－○．５％BSA ，PBS －４％屬血清，PBS －４％兔血清；及各種試劑作用時間的長
短，都對結果的判定影響不大。唯有抗原與酵素標示抗體的製備是否精純優良，是決
定ELISA 試驗成功的關鍵。
以遠心沈澱法來純化病毒及應用Trixton x －１○○粹取的可溶性抗原，做為間接酵
素結合免疫吸附法的抗原，來測定豬假性狂犬病抗體，其結果與應用血清中和試驗測
定的結果大致相同。以遠心沈澱法半純化病毒做為抗原所測定８○支血清的結果，比
較兩者之符合性為７７／８○（９６％），特異性為４６／４８（９６％），敏感性
為３１／３２（９７％），以粹取的可溶性抗原測定８○支血清，與中和試驗比較，
兩者的符合性為７５／８○（９４％），特異性為４９／５○（９８％），敏感性為
２６／３○（８７％）。
應用可溶性抗原為ELISA
試驗的判訣，結果用肉眼即可判定，無須藉助機器判訣。故可供野外測試血清之應用
，同時在２小時內即可獲得可靠的結果。
本研究制定了對豬假性狂犬病血清抗體測定時，應用ELISA 之標準方法，此法不但可
在野外迅速應用，不須假藉儀器之判訣，且準確度、特異性及敏感性均高的測定方法
，值得現場推廣應用
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
Investigation on the Active Hypoglycemic Components of Siraitia Grosvenori by Glucose Uptake Assay in 3T3-L1 Adipocytes
No full text糖尿病是一種體內葡萄糖恆定失調的代謝性疾病，主要由於胰島素分泌或胰島素作用有缺陷，而影響血中葡萄糖進入肝臟、肌肉及脂肪等組織所引起的高血糖。脂肪與肌肉細胞皆為受胰島素調控來維持血糖恆定的主要組織。文獻指出，週邊組織攝入葡萄糖，對於血糖的恆定及清除餐後血糖是很重要的步驟。因此，評估細胞的葡萄糖攝取能力，在糖尿病相關研究中，扮演一個很基礎的角色。羅漢果的食用、藥用價值已有很悠長的歷史。除了甜味的特性之外，研究也發現羅漢果在體內與體外試驗中，均具有降血糖之活性。因此本次實驗目的為利用3T3-L1脂肪細胞之葡萄糖攝取試驗為篩選模式，找出羅漢果中可能具有降血糖活性之成分。結果顯示，羅漢果粗萃物能夠顯著提升脂肪細胞的葡萄糖攝取量，且具濃度效應。將粗萃物依極性區分後，發現以乙酸乙酯層的效果最佳。更進一步將乙酸乙酯層利用管柱層析分離後，在其次區分層中分離出S1、S2與S3；此三個樣品皆能顯著增加脂肪細胞的葡萄糖攝取能力。當單純以樣品處理時，S1具有最好的效果，然而與胰島素共同處理下，S1則降低了胰島素的活性。經由西方點墨法分析，發現S1本身可增加Akt磷酸化；S2的促進效果同時存在於單獨處理與有10 nM胰島素刺激的狀況。綜合以上結果，推測S1具有類似胰島素之作用，S2應有協同胰島素作用之活性。Diabetes mellitus is a metabolic disorder characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. Muscle and fat cells are both insulin-stimulated target tissues which regulate the blood glucose concentration. Glucose uptake in target tissues is a critical step in maintaining glucose homeostasis and clearing the postprandial glucose load. Therefore, evaluation of glucose uptake ability in cells plays a fundamental role in diabetes research. Siraitia grosvenori (SG), also called Luo-Han-Guo (LHG), is a fruit used in traditional Chinese medicine and also used as food additives. Besides its sweet taste, studies showed that LHG-extract exhibited an antidiabetic effect on both in vivo and in vitro trials. The aim of this study is to find the active compounds with hypoglycemic potential in LHG by glucose uptake assay in 3T3-L1 cells. LHG crude extract increased glucose uptake in a dose-dependent manner, and after solvent partition of crude extract, the ethyl acetate fraction showed the best glucose uptake activity. There were three components (S1, S2 and S3) separated from EA fraction by column chromatography, and all of them can significantly increase glucose uptake activity in 3T3-L1 adipocytes. Among them, S1 showed the best activity in basal glucose uptake. In combination with insulin, however, S1 slightly reduced insulin-stimulated glucose uptake. By western blot analysis, S1 enhanced the phosphorylation level of Akt. S2 was found to have a promotion effect on both basal and insulin-stimulated glucose uptake. In conclusion, S1 had an insulin-mimetic action and S2 showed synergistic effect with insulin in 3T3-L1 adipocytes
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
Identification of Aberrant Type I Chain Elongation and Branching on Lactotetrasylceramides from Colon Carcinoma by Mass Spectrometry Analysis
No full text在癌症生物學的研究當中，常發現由不正常醣化結構所構成的癌相關抗原，即癌細胞常會表現出一般正常細胞中不會表現的特殊醣質結構。一般而言，以 Galb1-4GlcNAc，亦即 LacNAc，所聚合組成的第二型醣鍊為人體內較常表現的醣結構，通常可再線性延伸並形成分支狀。相對而言，構成第一型醣鍊的 Galb1-3GlcNAc 則通常不會再延伸。90年代初期，在大腸直腸癌細胞株 Colo205 細胞膜上第一次發現具有延伸性的第一型醣鍊位於醣脂質上，搭配其不同程度的岩藻醣化 (fucosylation) 形成二聚體的Leb/a-Lea 特殊抗原。
本論文的研究目標開始於應用一些已經確定結構的醣質標準品，以及表現在 Colo205 醣脂質上的第一型醣鍊及其衍生物為樣品，去探討並建立一綜合高撞擊能量與低撞擊能量串聯式質譜去分析、定序及區分第一型以及第二型醣鍊的方法。接下來，利用此互輔性的質譜分析法，搭配酵素、高效能液相層析分離以及化學反應，鑑定出 Colo205 醣脂質上由三個 Lewis 單位聚合組成的醣鍊，含延伸性和分支狀的純第一型醣鍊，及第一型與第二型不同排列複合組成的醣鍊。
由於第一型醣鍊的延伸性醣脂質為一罕見結構並與癌化細胞相關，遂從已知的人類醣轉移酵素中，藉由體外酵素合成的方式來分析何種酵素可能具有分支第一型醣鍊的能力。實驗結果顯示已知的三種分支合成酵素 b6-N-acetyl-glucosaminyltransferases (IGnTs) 皆含有此活性，其中以 IGnT2 的活性最高，並為 Colo205 細胞內表現量較為顯著的酵素。配合質譜分析鑑定其合成結構，可進而確定其分支點位置。此外，藉由分析此酵素合成產物，得以再度應證已建立的串聯式質譜分析方法的確能有效分析、辨識第一型以及第二型醣鍊。搭配醣轉移酵素的應用，亦可有效合成的各式第一型以及第二型醣鍊結構，形成多種抗原組合以探討抗體對不同結構的辨識能力，進而定出其相對應之抗原結構。
總言之，本論文的主要研究成果在於建立並有效應用高敏感度串聯式質譜分析，搭配高效能液相層析以及化學和酵素的反應，成功的鑑定表現於大腸癌細胞醣脂質上罕見的第一型延伸性醣鍊，並探討其相關生合成酵素，遂再以生化應用之角度辨別最小抗原決定結構。Tumor associated antigens arising from aberrant glycosylation is a well recognized phenomenon in cancer biology. Among the more notable structural features is the rare occurrence of extended type 1 chain on the lactosylceramides of the human colonic adenocarcinoma cell line, Colo205, concomitant with multiple fucosylation to give multimeric Lewisb/a terminal epitopes. While a dimeric Leb/a-Lea determinant has been well characterized previously, a trimeric stretch of type 1 units, -3Galb1-3GlcNAcb1-, and its fucosylated variants have not been unequivocally demonstrated. In a broader context of developing facile mass spectrometry (MS) methodologies for the sequencing of linear and branched poly-N-acetyl-lactosamine (polyLacNAc), this thesis demonstrates that the type 1 unit can be not only linearly extended to three or more tandem repeats, but also branched at C6 of 3-linked Gal in a manner similar to its type 2 polyLacNAc counterparts. A combination of chemical and enzymatic approaches, HPLC purification, and both low and high energy CID tandem mass spectrometry (MS/MS) analyses showed that the glycan chains carried on the lactosylceramides of Colo205 comprise a complex mixture of type 1 and 2 hybrids as well as those exclusively of extended type 1 chain. Complementary fragmentation characteristics were established to allow discrimination of linkage specific branching versus linear extension.
Identification of these novel structures further prompted an investigation into the candidate glycosyltransferases that may be responsible for mediating their aberrant synthesis. Based on in vitro enzymatic synthesis systems, linear extended type 1 and 2 chains and their hybrids were first synthesized which serve as authentic standards to validate MS/MS fragmentation pattern established; as well as substrates for further enzymatic (a-fucosylation and to assay for the specific activities of various I branching enzyme, (b6-N-acetyl-glucosaminyltransferases (IGnT or(b6GnT). The former allows a reconstitution of authentic oligomeric Lewis X and A antigens to be used in conjunction with other analytical methods to define the specificity of a monoclonal antibody which apparently recognizes only the GSLs from Colo205 of a size larger than those carrying a dimeric Lewis A epitopes. Variably fucosylated hybrid type 1 and 2 chains with at least an internal Lewis X epitope were established as being the most likely carrier of the epitope recognized. The latter led to a successful demonstration that extended type 1 and the hybrid chains can be branched in a manner similar to type 2 polyLacNAc chains by all three available human IGnTs albeit at a lower reactivity. IGnT2 is the only transcript expressed at any significant level in Colo205 and the most active in branching an extended type 1 chain with the branched Gal position critically established by further MS/MS analysis of the synthesized products.
In short, this work has unambiguously identified the presence of linear and branched extended type 1 chains on the GSLs of a colonic adenocarcinoma cell line based on advanced MS/MS analysis. With the following up in vitro enzymatic synthesis studies coupled with further MS/MS analysis of the products made, the implicated glycosyltransferases were shown to be capable of making the novel tumor-associated structures and the derived synthetic glycan epitopes were further used successfully to define the specificity of a monoclonal antibody raised against the tumor-associated antigens on GSLs, as well as to validate the MS/MS methodologies developed.中文摘要 1
Abstract 3
Chapter 1 Introduction 5
1.1 Aberrant glycosylation in cancer 5
1.2 Implications of polyLacNAc, extended type I chain and the underlying glycosyltransferase 7
1.3 Glycomics and identification of tumor associated antigens on GSLs 10
1.4 Strategies in glycosylation analysis 12
1.5 Current advances in mass spectrometry for mapping and sequencing glycans 14
1.6 Specific aims 20
Chapter 2 Materials and methods 23
2.1 Cell lines 23
2.2 Glycolipids extraction and fractionation 23
2.3 Glycan release from GSLs 24
2.4 Chemical and enzymatic treatment 24
2.5 Fractionation and purification of trimeric LacNAc glycans 25
2.6 Sodium hydroxide permethylation 25
2.7 GSL staining and immunoblotting on HPTLC 25
2.8 MS and MS/MS analysis 26
Chapter 3 Results 28
3.1. The overall profile and characteristic patterns of GSLs from Colo205 28
3.2 MALDI-MS/MS fragmentation pattern for distinguishing type 1 and type 2 LacNAc, as established from standards 31
3.3 Structural characterization and identification of trimeric type 1 chain 39
3.4 In vitro synthesis of linear and branched type 1 chains and their structure characterization 52
3.5 Delineation of the specificity of a mAb putatively recognizing the trimeric type 1 and/or 2 chains on the GSLs from Colo205 58
Chapter 4 Discussion 68
4.1 Application of mass spectrometry in novel structure identification 68
4.2 Occurrence of linear and branched extended type 1 chains 69
4.3 Glycosyltransferases implicated in the biosynthesis of linear and branched extended type 1 chains 72
4.4 Functional implications of extended type 1 chain or poly-Lea 74
4.5 Concluding Remarks 76
Chapter 5 References 7
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
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