FRET compatible long-wavelength labels and their application in immunoassays and hybridization assays

Ziel der Arbeit war die Synthese neuer langwelliger Fluoreszenzfarbstoffe, ihre Kopplung an Biomoleküle und deren Verwendung in Assay

TSLP is a direct trigger for T cell migration in filaggrin-deficient skin equivalents

Mutations in the gene encoding for filaggrin (FLG) are major predisposing factors for atopic dermatitis (AD). Besides genetic predisposition, immunological dysregulations considerably contribute to its pathophysiology. For example, thymic stromal lymphopoietin (TSLP) is highly expressed in lesional atopic skin and significantly contributes to the pathogenesis of AD by activating dendritic cells that then initiate downstream effects on, for example, T cells. However, little is known about the direct interplay between TSLP, filaggrin-deficient skin and other immune cells such as T lymphocytes. In the present study, FLG knockdown skin equivalents, characterised by intrinsically high TSLP levels, were exposed to activated CD4+ T cells. T cell exposure resulted in an inflammatory phenotype of the skin equivalents. Furthermore, a distinct shift from a Th1/Th17 to a Th2/Th22 profile was observed following exposure of T cells to filaggrin-deficient skin equivalents. Interestingly, TSLP directly stimulated T cell migration exclusively in filaggrin-deficient skin equivalents even in the absence of dendritic cells, indicating a hitherto unknown role of TSLP in the pathogenesis of AD

BNip3 regulates mitochondrial function and lipid metabolism in the liver

BNip3 localizes to the outer mitochondrial membrane, where it functions in mitophagy and mitochondrial dynamics. While the BNip3 protein is constitutively expressed in adult liver from fed mice, we have shown that its expression is superinduced by fasting of mice, consistent with a role in responses to nutrient deprivation. Loss of BNip3 resulted in increased lipid synthesis in the liver that was associated with elevated ATP levels, reduced AMP-regulated kinase (AMPK) activity, and increased expression of lipogenic enzymes. Conversely, there was reduced β-oxidation of fatty acids in BNip3 null liver and also defective glucose output under fasting conditions. These metabolic defects in BNip3 null liver were linked to increased mitochondrial mass and increased hepatocellular respiration in the presence of glucose. However, despite elevated mitochondrial mass, an increased proportion of mitochondria exhibited loss of mitochondrial membrane potential, abnormal structure, and reduced oxygen consumption. Elevated reactive oxygen species, inflammation, and features of steatohepatitis were also observed in the livers of BNip3 null mice. These results identify a role for BNip3 in limiting mitochondrial mass and maintaining mitochondrial integrity in the liver that has consequences for lipid metabolism and disease

Modellierung der Motor-Impedanz einer elektrisch erregten Synchronmaschine anhand von Messungen im aktiven und passiven Betrieb

[no abstract available

The BRCT domain of mammalian Pes1 is crucial for nucleolar localization and rRNA processing

The nucleolar protein Pes1 interacts with Bop1 and WDR12 in a stable complex (PeBoW-complex) and its expression is tightly associated with cell proliferation. The yeast homologue Nop7p (Yph1p) functions in both, rRNA processing and cell cycle progression. The presence of a BRCT-domain (BRCA1 C-terminal) within Pes1 is quite unique for an rRNA processing factor, as this domain is normally found in factors involved in DNA-damage or repair pathways. Thus, the function of the BRCT-domain in Pes1 remains elusive. We established a conditional siRNA-based knock-down-knock-in system and analysed a panel of Pes1 truncation mutants for their functionality in ribosome synthesis in the absence of endogenous Pes1. Deletion of the BRCT-domain or single point mutations of highly conserved residues caused diffuse nucleoplasmic distribution and failure to replace endogenous Pes1 in rRNA processing. Further, the BRCT-mutants of Pes1 were less stable and not incorporated into the PeBoW-complex. Hence, the integrity of the BRCT-domain of Pes1 is crucial for nucleolar localization and its function in rRNA processing

Dominant-negative Pes1 mutants inhibit ribosomal RNA processing and cell proliferation via incorporation into the PeBoW-complex

The nucleolar PeBoW-complex, consisting of Pes1, Bop1 and WDR12, is essential for cell proliferation and processing of ribosomal RNA in mammalian cells. Here we have analysed the physical and functional interactions of Pes1 deletion mutants with the PeBoW-complex. Pes1 mutants M1 and M5, with N- and C-terminal truncations, respectively, displayed a dominant-negative phenotype. Both mutants showed nucleolar localization, blocked processing of the 36S/32S precursors to mature 28S rRNA, inhibited cell proliferation, and induced high p53 levels in proliferating, but not in resting cells. Mutant M1 and M5 proteins associated with large pre-ribosomal complexes and co-immunoprecipitated Bop1 and WDR12 proteins indicating their proper incorporation into the PeBoW-complex. We conclude that the dominant-negative effect of the M1 and M5 mutants is mediated by the impaired function of the PeBoW-complex

STAT3 regulated ARF expression suppresses prostate cancer metastasis.

Prostate cancer (PCa) is the most prevalent cancer in men. Hyperactive STAT3 is thought to be oncogenic in PCa. However, targeting of the IL-6/STAT3 axis in PCa patients has failed to provide therapeutic benefit. Here we show that genetic inactivation of Stat3 or IL-6 signalling in a Pten-deficient PCa mouse model accelerates cancer progression leading to metastasis. Mechanistically, we identify p19(ARF) as a direct Stat3 target. Loss of Stat3 signalling disrupts the ARF-Mdm2-p53 tumour suppressor axis bypassing senescence. Strikingly, we also identify STAT3 and CDKN2A mutations in primary human PCa. STAT3 and CDKN2A deletions co-occurred with high frequency in PCa metastases. In accordance, loss of STAT3 and p14(ARF) expression in patient tumours correlates with increased risk of disease recurrence and metastatic PCa. Thus, STAT3 and ARF may be prognostic markers to stratify high from low risk PCa patients. Our findings challenge the current discussion on therapeutic benefit or risk of IL-6/STAT3 inhibition.Lukas Kenner and Jan Pencik are supported by FWF, P26011 and the Genome Research-Austria project “Inflammobiota” grants. Helmut Dolznig is supported by the Herzfelder Family Foundation and the Niederösterr. Forschungs-und Bildungsges.m.b.H (nfb). Richard Moriggl is supported by grant SFB-F2807 and SFB-F4707 from the Austrian Science Fund (FWF), Ali Moazzami is supported by Infrastructure for biosciences-Strategic fund, SciLifeLab and Formas, Zoran Culig is supported by FWF, P24428, Athena Chalaris and Stefan Rose-John are supported by the Deutsche Forschungsgemeinschaft (Grant SFB 877, Project A1and the Cluster of Excellence --“Inflammation at Interfaces”). Work of the Aberger lab was supported by the Austrian Science Fund FWF (Projects P25629 and W1213), the European FP7 Marie-Curie Initial Training Network HEALING and the priority program Biosciences and Health of the Paris-Lodron University of Salzburg. Valeria Poli is supported by the Italian Association for Cancer Research (AIRC, No IG13009). Richard Kennedy and Steven Walker are supported by the McClay Foundation and the Movember Centre of Excellence (PC-UK and Movember). Gerda Egger is supported by FWF, P27616. Tim Malcolm and Suzanne Turner are supported by Leukaemia and Lymphoma Research.This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/ncomms873

Motor-Impedanzmessungen im aktiven Betriebszustand anhand einer permanentmagneterregten Synchronmaschine

Aufgrund ihres Schaltverhaltens können Wechselrichter (WR) hohe elektromagnetische Emissionen erzeugen. Zur Sicherstellung der elektromagnetischen Verträglichkeit (EMV) werden deshalb u.a. Emissionsmessungen durchgeführt. Bei Emissionsmessungen von Kfz-Wechselrichtern nach CISPR 25 [1] wird der zugehörige Motor als Peripherie betrachtet. Aus diesem Grund kann stattdessen eine möglichst realistische Ersatzlast verwendet werden. Häufig wird hierzu eine passive Impedanznachbildung eingesetzt, welche zwar eine gute Reproduzierbarkeit gewährleistet, allerdings nicht den realen Lastbedingungen entspricht. Für möglichst reale Lastbedingungen bieten einige wenige EMV-Labore Emissionsmessungen mit einer externen Last am Motor (hydraulisch oder mithilfe einer zusätzlichen Lastmaschine) an. Nachteilig sind hierbei u.a. die erhöhten Sicherheitsanforderungen aufgrund rotierender Teile sowie hohe Anschaffungs- und Prüfkosten. Vor diesem Hintergrund entstand die Idee, eine aktive elektrische Ersatzlast zu verwenden. Die aktive Impedanznachbildung soll sowohl das niederfrequente Funktionsverhalten als auch die hochfrequenten (HF) Koppelpfade einer belasteten elektrischen Maschine emulieren können. Für funktionale Prüfungen von Wechselrichtern werden bereits Systeme angeboten, welche das funktionale Verhalten abbilden und als elektrische Maschinenemulatoren bezeichnet werden (u.a. [2]). Bild 1 zeigt den zu prüfenden Wechselrichter (DUT, engl. device under test) sowie den über ein Koppelnetzwerk angeschlossenen Emulator. Um die Maschinenemulation auch bei EMV-Prüfungen verwenden zu können, muss diese die HF-Impedanz der belasteten Maschine ausreichend genau nachbilden, bei vernachlässigbar geringen Eigenemissionen. Eine genauere Ausführung zu diesem Ansatz und den Anforderungen sowie erste Messergebnisse werden in [3] präsentiert. Ziel dieses Papers ist die Untersuchung der Impedanzen für Gleichtakt (CM) und Gegentakt (DM) von elektrischen Maschinen im aktiven Betrieb. Die Ergebnisse sollen anschließend zur Nachbildung des HF-Verhaltens bei der elektrischen Maschinenemulation verwendet werden. Zunächst wird die verwendete Messmethode vorgestellt und validiert. Dann werden beispielhaft die CM- und DM-Impedanzen einer permanentmagneterregten Synchronmaschine (PMSM) bei verschiedenen Betriebsmodi analysiert. Abschließend werden Verhaltensmodelle zur Nachbildung der Motorimpedanz erstellt und die Werte der Streuparameter aus den Messkurven extrahiert