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Erweiterung der zellbasierten Calcium-Imaging-Methode im eukaryotischen zellfreien Proteinsynthese-System für die transient-receptor-potential (TRP) - Ionenkanäle
No full textDie Fluoreszenz-Calcium-Imaging-Methode wird auch heute noch als gängige Methode verwendet, vor allem wegen der geringeren Kosten für das Wirkstoffscreening in der pharmazeutischen Forschung, wobei Ionenkanäle sowie einige der G-Protein gekoppelte Rezeptoren (GPCRs) die Mehrzahl der Wirkstoffziele ansprechen. Die zellfreie Synthese eukaryotischer Proteine hat nicht die Nachteile, die bei der Überexpression dieser ionenpermeablen Proteine in Zellen auftreten können, wie z. B. Zelltoxizität, geringere Proteinexpression und die Beseitigung der exprimierten Proteine aufgrund veränderter Domänen sowie die zeitaufwändige Pflege von Zelllinien. Die Synthese von Ionenkanälen in zellfreien Proteinsyntheseplattformen für das künftige Wirkstoffscreening ist noch in der Grundlagenforschung. Obwohl die Fluoreszenz-Calcium-Imaging-Methode in zellbasierten Assays weit verbreitet ist, wurde diese Methode bisher noch nicht in zellfreien Proteinexpressionssystemen verwendet. Insgesamt ist die neue Anwendung der Calcium-Imaging-Methode in eukaryontischen zellfreien Systemen eine Voraussetzung für die schnelle pharmakologische Analyse von Wirkstoffen. Das erste Ziel dieser wissenschaftlichen Arbeit bestand darin, die grundlegenden Prinzipien der Calcium-Imaging-Methode zur Untersuchung von Ionenkanälen in zellbasierten Systemen zu untersuchen. Hierfür wurden zwei Tumorzelllinien des Auges verwendet, und zwar benigne Pterygiumzellen und maligne Aderhautmelanom 92.1 Zellen. In diesen Studien wurde die Interaktion zwischen den nativ überexprimierten transient-receptor-potential-Ionenkanälen (TRPs) wie TRP Vanilliod 1 (TRPV1) (Capsaicinrezeptor) und TRP Melastatin 8 (TRPM8) (Mentholrezeptor) in diesen Tumorzellen nach Zugabe von verschiedenen Medikamenten und Hormonen untersucht. Das zweite Ziel dieser Arbeit war es, den Calcium-Mechanismus von GPCRs in den Zellen zu untersuchen. Zu diesem Zweck wurde Mas, ein GPCR und Angiotensin (1-7) -Hormonrezeptor, aus dem renin-angiotensin-aldosteron-system (RAAS) in der Human Embryonic Kidney-293 (HEK293) Zelllinie überexprimiert. In dieser Studie wurden insbesondere die Aktivierung klassischer GPCR-Signalwege wie Phospholipase C und Proteinkinase C durch Angiotensin-(1-7) über Mas und die Beteiligung von TRP-Kanälen nachgewiesen. Die zellbasierte-Calcium-Imaging-Methode für chemische Calcium-Indikatoren ließ sich aufgrund der Anwesenheit einer großen Menge cytosolischer Carboxylesterasen gut anwenden. Carboxylesterase ist das wichtigste Enzym in der Calcium Imaging Methode, das die Verarbeitung chemischen Calcium-Farbstoffe behandelt. Dieses Enzym fehlt jedoch in Mikrosomen, die als Basismembran für die Integration synthetisierter Ionenkanäle in eukaryontischen zellfreien Systemen verwendet werden. Das dritte Ziel dieser Forschungsarbeit war die Umsetzung der zellbasierten Calcium-Imaging Methode und der Calcium-Signalwege in zellfreie Systeme. Hier wurde die zellfrei synthetisierte Carboxylesterase in Mikrosomen von Spodoptera frugiperda (Sf21) als praktikables Calcium-Imaging-Werkzeug etabliert, um sowohl native ionenpermeable Proteine als auch zellfrei-synthetisierte Ionenkanäle zu untersuchen. Die Enzymaktivität der zellfrei-synthetisierten Carboxylesterase in Mikrosomen wurde durch Esterase-Assays und den Calcium-Fluoreszenzfarbstoff Fluo-5N Acetoxymethylester (Fluo-5N AM) Belastungstests nachgewiesen. Das Calcium-Imaging der nativ vorhandenen Ca2+-ATPase des sarkoplasmatischen/endoplasmatischen Retikulums (SERCA) und der Ryanodin-Rezeptoren (RyR) in den Mikrosomen sowie der zell-frei exprimierten TRP-Ionenkanäle wurden mit dem Fura-5N-AM- Fluoreszenzfarbstoff in mit Carboxylesterase vorsynthetisierten Mikrosomen nachgewiesen.
Zusammenfassend lässt sich sagen, dass das Prinzip der zellbasierten Calcium-Imaging -Methode vielversprechend an das eukaryotische zellfreie Sf21-System angepasst werden konnte, um Ionenkanäle zu analysieren. Nach entsprechender Forschung könnte die etablierte Methode in Zukunft auch auf andere Membranproteine ausgeweitet werden. Dies umfasst die Untersuchung anderer zell-frei exprimierte GPCRs oder anderer Ionenkanäle wie Kalium-, Natrium- und Chlorid-Ionenkanäle.Fluorescence calcium imaging is still used today used as the common method, mainly due to its cheaper costs for drug screening in pharmaceutical research encompassing both ion channels, and part of G- Protein Coupled Receptors (GPCRs) in the major share of drug targets. Eukaryotic cell-free protein translation overcomes several drawbacks that might occur for overexpression of these ion-permeable proteins in the cells such as cell toxicity, poor protein expression, and deletion due to modified protein domain and time-consuming cell line maintenance. Expressing ion channels in the cell-free protein synthesis platform for future drug screening processes is still ongoing basic research and so far, no calcium imaging technique for cell-free expressed ion channels is available. Taken together, the novel application of the fluorescence calcium imaging method in eukaryotic cell-free systems is a prerequisite for rapid pharmacological drug investigations, which are not feasible using conventional call-based approaches. The aim of this thesis is to investigate the common calcium signaling pathways relevant to drug research via ion channels and GPCRs in cell-based systems. Thereby, the basic mechanism of the cell-based calcium imaging for chemical calcium indicators is studied and then translate its principle to the cell-free protein synthesis platforms. In order to study the cell-based calcium imaging and calcium pathways, two types of eye tumor cell-based models, namely benign pterygial tumor cells and malign uveal melanoma 92.1 cells were used. Specifically, the interplay between the natively expressed Transient Receptor Potential Channels (TRPs) like TRP-Vanilloid 1 (TRPV1) (Capsaicin receptor) and TRP-Melastatin 8 (TRPM8) (Menthol receptor) in these tumor cells was investigated by application of various drugs and hormones. The second aim of this thesis was to investigate the cell-based calcium mechanism of GPCRs. For this, overexpressed Mas, a GPCR and Angiotensin-(1-7) hormone receptor, from the Renin Angiotensin Aldosterone System (RAAS) in the Human Embryonic Kidney (HEK293) cells was used. Particularly, the activation of classical GPCR pathways like Phospholipase C and Proteinkinase C via Ang-(1-7) through Mas and the involvement of TRPs was proven in this study. The third aim of this thesis is to translate the cell-based calcium imaging principle to cell-free systems. Cell-based calcium imaging for chemical calcium indicators could be performed with all ease due to the presence of a large amount of cytosolic carboxylesterase, the crucial enzyme which handles the chemical calcium dyes. But this enzyme is absent in microsomes. Microsomes are used as a backbone membrane to integrate the synthesized ion channels in a eukaryotic cell-free system. Cell-free synthesized carboxylesterase in Spodoptera frugiperda (Sf21) microsomes is established as a viable calcium imaging tool to study both natively present ion permeable proteins and cell-free synthesized ion channels. The enzyme activity of the carboxylesterase in microsomes was confirmed by esterase assays and fluorescent calcium dye Fluo-5N Acetoxymethylester (Fluo-5N AM) loading assays. Fluorescent calcium imaging of natively present SERCA (Sarco Endoplasmic Reticulum Calcium ATPase) and ryanodine channels in the microsomes and cell-free expressed TRPV1 channel was demonstrated using Fluo-5N AM fluorescent dye in carboxylesterase pre-synthesized microsomes. With adequate research in future, the established method could be promisingly extended to study other cell-free expressed GPCRs or other ion channels like potassium, sodium, and chloride ion channels
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
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
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
Author Under Sail The Imagination of Jack London, 1893-1902
No full textIn Author Under Sail, Jay Williams offers the first complete literary biography of Jack London as a professional writer engaged in the labor of writing. It examines the authorial imagination in London's work, the use of imagination in both his fiction and nonfiction, and the ways he defined imagination in the creative process in his business dealings with his publishers, editors, and agents. In this first volume of a two-volume biography, Williams traverses the years 1893 to 1902, from London's "Story of a Typhoon" to The People of the Abyss. The Jack London who emerges in the pages of Author Under Sail is a writer whose partnership with publishers, most notably his productive alliance with George Brett of Macmillan, was one of the most formative in American literary history. London pioneered many author models during the heyday of realism and naturalism, blurring the boundaries of these popular genres by focusing on absorption and theatricality and the representation of the seen and unseen. London created an impassioned, sincere, and extremely personal realism unlike that of other American writers of the time. Author Under Sail is a literary tour de force that reveals the full range of London as writer, creative citizen, and entrepreneur at the same time it sheds light on the maverick side of machine-age literature.Intro -- Title Page -- Copyright Page -- Dedication -- Contents -- Acknowledgments -- Introduction -- 1. Spirit Truth -- 2. From Absorption to Theatricality and Back Again -- 3. "I Will Build a New Present" -- 4. Sons as Authors -- 5. Fathers as Publishers -- 6. The Daughter as Author -- 7. Lovers as Authors -- 8. At Sea with the Family -- 9. Yellow News, Yellow Stories -- 10. The Return Home -- Notes -- Bibliography -- Index -- About Jay WilliamsIn Author Under Sail, Jay Williams offers the first complete literary biography of Jack London as a professional writer engaged in the labor of writing. It examines the authorial imagination in London's work, the use of imagination in both his fiction and nonfiction, and the ways he defined imagination in the creative process in his business dealings with his publishers, editors, and agents. In this first volume of a two-volume biography, Williams traverses the years 1893 to 1902, from London's "Story of a Typhoon" to The People of the Abyss. The Jack London who emerges in the pages of Author Under Sail is a writer whose partnership with publishers, most notably his productive alliance with George Brett of Macmillan, was one of the most formative in American literary history. London pioneered many author models during the heyday of realism and naturalism, blurring the boundaries of these popular genres by focusing on absorption and theatricality and the representation of the seen and unseen. London created an impassioned, sincere, and extremely personal realism unlike that of other American writers of the time. Author Under Sail is a literary tour de force that reveals the full range of London as writer, creative citizen, and entrepreneur at the same time it sheds light on the maverick side of machine-age literature.Description based on publisher supplied metadata and other sources.Electronic reproduction. Ann Arbor, Michigan : ProQuest Ebook Central, YYYY. Available via World Wide Web. Access may be limited to ProQuest Ebook Central affiliated libraries
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