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Nachweis, Lokalisation und Funktion von Polyphosphat im Endothel
No full textAnorganisches Polyphosphat (PolyP) ist ein Polymer bestehend aus wenigen bis hin zu einigen hunderten Orthophosphaten, welche durch energiereiche Phosphoanhydridbindungen verbunden sind. Beim Menschen spielt PolyP eine wichtige Rolle bei der Gerinnung, indem bei der Aktivierung von Thrombozyten an deren Zelloberfläche PolyP präsentiert wird und den Gerinnungsfaktor XII (FXII) dadurch aktiviert.
Auch andere humane Zellen besitzen PolyP, jedoch ist die Lokalisation und Funktion bei vielen Zelltypen nicht erforscht. Ziel dieser Arbeit war es endogenes PolyP in Endothelzellen (EZs) nachzuweisen und subzelluläre Lokalisation sowie Funktionen von PolyP zu analysieren.
Methodisch erfolgte zunächst die Detektion von endogenem PolyP in humaner Endothelzelllinie mittels des Malachite Green Assays. Ergänzend wurde hierzu PolyP in murinen und humanen Endothelzelllinien sowie Human Umbilical Vein Endothelial Cells (HUVECs) nach der neu überarbeiteten Methode der PolyP-Extraktion in der Urea-PAGE analysiert. Der Nachweis erfolgte hierbei jeweils durch den spezifischen Verdau des PolyPs durch die Exopolyphosphatase aus E. coli (PPX). Der mikroskopische in situ Nachweis der (sub-) zellulären Lokalisation wurde mit der PolyP-Sonde PPX_∆1,2 durchgeführt. Die PolyP-Sonde ist eine gentechnisch modifizierte Exopolyphosphatase und bindet an PolyP, hydrolysiert dieses jedoch nicht. Die Spezifität der Färbung wurde bestätigt durch eine kompetitive Hemmung von PPX_∆1,2 mittels synthetischem PolyP P70. Anhand konfokaler Fluoreszenzmikroskopie konnte weiter gezeigt werden, dass PolyP vor allem in den Mitochondrien der EZs lokalisiert ist, was die Verknüpfung mit ATP aufzeigen könnte. Des Weiteren wurde eine Ko-Lokalisation von PolyP und Nucleoli-ähnlichen Strukturen aufgezeigt. Dieses könnte auf eine mögliche Rolle von PolyP bei der Genexpression hinweisen. Außerdem wurde PPX_∆1,2 mutmaßlich in den Weibel-Palade-Körperchen (WPBs) von HUVECs angereichert, welche möglicherweise neben Von-Willebrand-Faktor (VWF) auch als Speicher für PolyP dienen könnten und somit auch die Hämostase beeinflussen.
Xenotropic and Polytropic Retrovirus Receptor 1 (XPR1) ist der einzig bekannte Phosphatexporter in Metazoa und reguliert als Transmembranprotein auch den Gehalt von PolyP in Thrombozyten. Deshalb wurde eine siRNA-vermittelte Downregulation von XPR1 durchgeführt, um den endogenen Gehalt an PolyP zu modulieren und die Auswirkungen in EZs zu untersuchen. Wie erwartet akkumuliert PolyP nach dem Verlust von XPR1 auch in EZs und ein deutlicher Anstieg der mRNA der Proliferationsmarker CCNB1, CCNB2, MKI67 sowie MYBL2 wurde beobachtet. Dies kann ein Hinweis auf eine mögliche Funktion von PolyP bei der Proliferation sein. Außerdem war die Morphologie der Mitochondrien beeinträchtigt und der Gehalt an VWF intrazellulär gemindert.
Hinsichtlich der Ziele dieser Arbeit konnte PolyP vor allem in den Mitochondrien der EZs nachgewiesen werden sowie mutmaßlich in Nucleoli-ähnlichen Strukturen und WPBs der HUVECs. Zudem besteht ein Zusammenhang zwischen XPR1 der HUVECs und dem PolyP-Gehalt der Zellen sowie der Proliferation. Weitere Funktionen von PolyP im Endothel müssen durch weitere Versuchsreihen analysiert werden.Inorganic polyphosphate (PolyP) is a polymer consisting of a few to several hundred orthophosphates linked by energetic phosphoanhydride bonds. In humans, polyP plays an important role in coagulation. Activated platelets presenting their polyp at their cell surface and thereby activating clotting factor XII (FXII).
Other human cells also possess polyP, but its localization and function in most other cell types has not been explored. The aim of this work was to detect endogenous polyP in endothelial cells (EZs) and to analyze subcellular localization and functions of polyP.
Methodically, endogenous polyP was first detected in human endothelial cell line using the Malachite Green assay. In addition, polyP was analyzed in murine and human endothelial cell lines as well as human umbilical vein endothelial cells (HUVECs) using the newly revised method of polyP extraction in urea-PAGE. In each case, detection was performed by specific digestion of polyP by exopolyphosphatase from E. coli (PPX). Microscopic in situ detection of (sub-) cellular localization was performed using the PPX_∆1,2 polyP-probe. The polyP-probe is a genetically modified exopolyphosphatase and binds to polyP but does not hydrolyze it. Specificity of staining was confirmed by competitive inhibition of PPX_∆1,2 using synthetic polyP P70. Using confocal fluorescence microscopy, it was further shown that polyP is mainly localized in the mitochondria of EZs, which could reveal the linkage with ATP. Furthermore, co-localization of polyP and nucleoli-like structures was revealed. This could indicate a possible role of polyP in gene expression. In addition, PPX_∆1,2 was putatively enriched in the Weibel-Palade-bodies (WPB s) of HUVECs, which could possibly serve as a store for polyP in addition to von-Willebrand-factor (VWF), thus also affecting hemostasis.
Xenotropic and Polytropic Retrovirus Receptor 1 (XPR1) is the only known phosphate exporter in Metazoa and as a transmembrane protein, also regulates the level of polyP in platelets. Therefore, siRNA-mediated downregulation of XPR1 was performed to modulate the endogenous level of polyP and to study its effects in EZs. As expected, polyP also accumulated in EZs after loss of XPR1, and a marked increase in mRNA of the proliferation markers CCNB1, CCNB2, MKI67, as well as MYBL2 was observed. This may indicate a possible function of polyP in proliferation. In addition, mitochondrial morphology was impaired and VWF content was decreased intracellularly.
Regarding the aims of this work, polyP was detected mainly in the mitochondria of EZs and putatively in nucleoli-like structures and WPBs of HUVECs. In addition, there is a correlation between XPR1 of HUVECs and polyP content of cells and proliferation. Additional functions of polyP in the endothelium need to be analyzed by further series of experiments
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
EVL regulates VEGF receptor‐2 internalization and signaling in developmental angiogenesis
No full textDeutsche Forschungsgemeinschaft https://doi.org/10.13039/50110000165
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