Identifizierung Ergothionein-haltiger Zellen im Zebrabärbling Danio rerio

Ergothionein ist eine natürlich vorkommende Aminosäure mit antioxidativen Eigenschaften in vitro. Der Mensch und andere Vertebraten synthetisieren Ergothionein nicht selbst, sondern können es nur über die Nahrung aufnehmen. Die physiologische Funktion von Ergothionein ist unbekannt. Cytoplasmamembranen sind für das hydrophile Ergothionein undurchlässig. Der Transport von Ergothionein in das Cytosol wird von einem hochspezifischen Transporter in der Plasmamembran, dem Ergothionein-Transporter, durchgeführt. Der Ergothionein-Transporter stellt den bis jetzt einzigen Biomarker für eine mögliche Ergothionein-Akkumulation/Aktivität dar. Beim Menschen bewirkt dieser Transporter (Gensymbol SLC22A4) eine Anreicherung von Ergothionein z. B. in Vorläuferzellen der Erythrozyten, in Monozyten und daher vermutlich auch in Makrophagen, die aus Monozyten hervorgehen. Monozyten und Makrophagen sind an chronischen Entzündungsprozessen beteiligt. Dies deutet auf eine Rolle von Ergothionein im Immunsystem hin, wofür auch spricht, dass das Auftreten der chronischen Entzündungserkrankungen Morbus Crohn, Colitis ulcerosa und rheumatoide Arthritis mit Varianten des Transportergens in Verbindung gebracht wurden. Ergothionein könnte daher möglicherweise in der Zukunft als neuer Wirkstoff bei der Prophylaxe oder bei der Therapie dieser Krankheiten zum Einsatz kommen. Das Ziel dieser Doktorarbeit war es, den Zebrabärbling Danio rerio als Modellorganismus für die Untersuchung der physiologischen Funktion von Ergothionein zu etablieren. Hierfür ist es essentiell, die exakten Funktionsorte von Ergothionein zu bestimmen. Daher sollte hier ein Werkzeug zur Identifizierung Ergothionein-haltiger Zellen mit Einzelzellauflösung im Zebrabärbling entwickelt werden. Zu diesem Zweck wurde die Herstellung einer transgenen Reporterlinie angestrebt, in der die Expression des grün-fluoreszierenden Proteins eGFP (enhanced Green Fluorescent Protein) unter Promotor-Kontrolle des Ergothionein-Transporters steht. Trotz umfassender Bemühungen war es jedoch nicht möglich, eine Zebrabärblinglinie mit einem eGFP-Expressionsprofil, das dem des Transportergens (slc22a4) entspricht, zu etablieren. Möglicherweise wird die slc22a4-Transkription von weit entfernt liegenden cis-regulatorischen Elementen beeinflusst, die hier nicht identifiziert werden konnten. Als Alternative wurde endogener Ergothionein-Transporter immunhistochemisch nachgewiesen. Hierzu wurden polyklonale Antikörper hergestellt, deren Spezifität mittels heterologer Expression gezeigt werden konnte. Der Slc22a4 Transporter konnte mit diesen Antikörpern in den Bürstensäumen von Darm und Niere nachgewiesen werden, in denen Slc22a4 vermutlich die Aufgabe der Ergothionein-Absorption und Ergothionein-Retention übernimmt. Zudem konnten in der Retina einzelne Zellschichten als Wirkungsorte von Ergothionein plausibel gemacht werden. Demgegenüber konnte der Transporter in Haut und Gehirn nicht immunhistochemisch nachgewiesen werden, obwohl sowohl Ergothionein als auch slc22a4-mRNS in beiden Organen in hohem Maße vorhanden sind. Es wäre denkbar, dass die Epitope des Slc22a4 in der Haut und im Gehirn maskiert sind. Als weitere Methode zur Lokalisierung des Slc22a4 Transporters wurde RT-PCR eingesetzt. Hierbei konnten Transkripte in peripheren Blutzellen des Zebrabärblings nachgewiesen werden. Die genaue Identität der slc22a4-positiven Zellen kann nun immunhistochemisch festgestellt werden. Falls wie beim Menschen der Ergothionein-Transporter in Monozyten und Makrophagen vorhanden ist, könnten die Folgen eines Ergothionein-Mangels im Immunsystem dann im Zebrabärbling untersucht werden. Damit kämen dann die weitreichenden experimentellen Vorteile des Zebrabärblings zum Tragen

Polycomb Repressive Complex 2 Regulates Genes Necessary for Intestinal Microfold Cell (M Cell) Development

BACKGROUND & AIMS: Microfold cells (M cells) are immunosurveillance epithelial cells located in the Peyer's patches (PPs) in the intestine and are responsible for monitoring and transcytosis of antigens, microorganisms, and pathogens. Mature M cells use the receptor glycoprotein 2 (GP2) to aid in transcytosis. Recent studies have shown transcription factors, Spi-B and SRY-Box Transcription Factor 8 (Sox8). are necessary for M-cell differentiation, but not sufficient. An exhaustive set of factors sufficient for differentiation and development of a mature GP2+ M cell remains elusive. Our aim was to understand the role of polycomb repressive complex 2 (PRC2) as an epigenetic regulator of M-cell development. Estrogen-related-receptor gamma (Esrrg), identified as a PRC2-regulated gene, was studied in depth, in addition to its relationship with Spi-B and Sox8. METHODS: Comparative chromatin immunoprecipitation and global run-on sequencing analysis of mouse intestinal organoids were performed in stem condition, enterocyte conditions, and receptor activator of nuclear factor kappa B ligand-induced M-cell condition. Esrrg, which was identified as one of the PRC2-regulated transcription factors, was studied in wild-type mice and knocked out in intestinal organoids using guide RNA's. Sox8 null mice were used to study Esrrg and its relation to Sox8. RESULTS: chromatin immunoprecipitation and global run-on sequencing analysis showed 12 novel PRC2 regulated transcription factors, PRC2-regulated Esrrg is a novel M-cell-specific transcription factor acting on a receptor activator of nuclear factor kappa B ligand-receptor activator of nuclear factor kappa B-induced nuclear factor-kappa B pathway, upstream of Sox8, and necessary but not sufficient for a mature M-cell marker of Gp2 expression. CONCLUSIONS: PRC2 regulates a significant set of genes in M cells including Esrrg, which is critical for M-cell development and differentiation. Loss of Esrrg led to an immature M-cell phenotype lacking in Sox8 and Gp2 expression. Transcript profiling: the data have been deposited in the NCBI Gene Expression Omnibus database (GSE157629).Peer reviewe

Financial analysis of company DENSO Manufacturing Czech, s.r.o.

The main aim of the Bachelor's thesis "Financial Analysis DENSO Manufacturing Czech, s.r.o." is to conduct a comprehensive financial analysis company Denso Manucfacturing Czech s.r.o. and to assess its financial health in the years 2009 - 2014 and the supplement development of company in the year 2015. The thesis consists of theoretical and practical parts. The theoretical part provides an overview of financial indicators. Among the methods of the financial analysis include horizontal and vertical analysis, ratio analysis , Du pont ROE, bankruptcy and credibility models, analysis of differential indicators, economic value added, intercompany comparison. In the practical part is description of the company and the application of different methods

Functional validation of microRNA-126-3p as a platelet reactivity regulator using human haematopoietic stem cells

BACKGROUND:  Platelets are an abundant source of micro-ribonucleic acids (miRNAs) that may play a role in the regulation of platelet function. Some miRNAs, such as miR-126-3p, have been noted as potential biomarkers of platelet reactivity and the recurrence of cardiovascular events. However, the biological relevance of these associations remains uncertain, and the functional validation of candidate miRNAs on human-derived cells is lacking. OBJECTIVE:  This article functionally validates miR-126-3p as a regulator of platelet reactivity in platelet-like structures (PLS) derived from human haematopoietic stem cells. MATERIALS AND METHODS:  CD34+-derived megakaryocytes were transfected with miR-126-3p and differentiated in PLS. PLS reactivity was assessed using perfusion in a fibrinogen-coated flow chamber. miR-126-3p's selected gene targets were validated using quantitative polymerase chain reaction, protein quantification and a reporter gene assay. RESULTS:  CD34+-derived megakaryocytes transfected with miR-126-3p generated PLS exhibiting 156% more reactivity than the control. These functional data were in line with those obtained analysing CD62P expression. Moreover, miR-126-3p transfection was associated with the down-regulation of a disintegrin and metalloproteinase-9 (ADAM9) messenger RNA (mRNA), a validated target of miR-126-3p, and of Plexin B2 (PLXNB2) mRNA and protein, an actin dynamics regulator. Silencing PLXNB2 led to similar functional results to miR-126-3p transfection. Finally, using a reporter gene assay, we validated PLXNB2 as a direct target of miR-126-3p. CONCLUSION:  We functionally validated miR-126-3p as a regulator of platelet reactivity in PLS derived from human haematopoietic stem cells. Moreover, PLXNB2 was validated as a new gene target of miR-126-3p in human cells, suggesting that miR-126-3p mediates its effect on platelets, at least in part, through actin dynamics regulation

Kappe neurons, a novel population of olfactory sensory neurons

Perception of olfactory stimuli is mediated by distinct populations of olfactory sensory neurons, each with a characteristic set of morphological as well as functional parameters. Beyond two large populations of ciliated and microvillous neurons, a third population, crypt neurons, has been identified in teleost and cartilaginous fishes. We report here a novel, fourth olfactory sensory neuron population in zebrafish, which we named kappe neurons for their characteristic shape. Kappe neurons are identified by their G(o)-like immunoreactivity, and show a distinct spatial distribution within the olfactory epithelium, similar to, but significantly different from that of crypt neurons. Furthermore, kappe neurons project to a single identified target glomerulus within the olfactory bulb, mdg5 of the mediodorsal cluster, whereas crypt neurons are known to project exclusively to the mdg2 glomerulus. Kappe neurons are negative for established markers of ciliated, microvillous and crypt neurons, but appear to have microvilli. Kappe neurons constitute the fourth type of olfactory sensory neurons reported in teleost fishes and their existence suggests that encoding of olfactory stimuli may require a higher complexity than hitherto assumed already in the peripheral olfactory system

MicroRNA-126 is a regulator of platelet-supported thrombin generation

Circulating microRNA (miRNA) expression profiles correlate with platelet reactivity. MiR-126 is a promising candidates in this regard. We generated a transgenic zebrafish line with thrombocyte-specific overexpression of miR-126. Laser injury of the posterior cardinal vein of 5 day-old larvae was performed with or without antithrombotic pre-treatment. Platelet-like structures (PLS) derived from human megakaryocytes transfected with miR-126 were also evaluated for procoagulant activity. Finally, we studied the correlation between miR-126 level and thrombin generation markers in a cohort of stable cardiovascular patients. Control zebrafish developed small thrombocyte-rich thrombi at the site of vessel injury, without vessel occlusion. The miR-126 transgenic line developed an occluding thrombus in 75% (95% CI: 51–91%) of larvae. Pre-treatment with the direct thrombin inhibitor argatroban, but not aspirin, prevented vessel occlusion in the transgenic line (0% occlusion, 95%CI: 0–18%). Upon activation, human PLS showed an increased procoagulant profile after miR-126 transfection compared to control. Finally, the plasma levels of miR-126, but not a control platelet-derived miRNA, correlated with markers of in vivo thrombin generation in a cohort of 185 cardiovascular patients. Our results from three complementary approaches support a key role for miR-126 in platelet-supported thrombin generation and open new avenues in the tailoring of antithrombotic treatment