Ninjurin 1 contributes to TLR-induced inflammation in endothelial cells

Background: Nerve injury induced protein 1 (Ninjurin 1 (Ninj1)) was first identified in Schwann cells and neurons contributing to cell adhesion and nerve regeneration. Recently, the role of Ninj1 has been linked to inflammatory processes in the central nervous system where functional repression reduced leukocyte infiltration and clinical disease activity during experimental autoimmune encephalomyelitis in mice [1]. But Ninj1 is also expressed outside the nervous system in various organs such as the liver and kidney as well as on leukocytes [2,3]. Therefore, we hypothesized that Ninj1 contributes to inflammation in general; that is, also outside the nervous system, with special interest in the pathogenesis of sepsis. Methods: Ninj1 was repressed by transfecting HMEC-1 cells, a human dermal microvascular endothelial cell line with siRNA targeting Ninj1 (siNinj1) or a negative control (siC). Subsequently, cells were stimulated with 100 ng/ml LPS (TLR4 agonist), 3 μg/ml LTA (TLR2 agonist) or 100 n/ml poly(I:C) (TLR3 agonist) for 3 hours. The inflammatory response was analyzed by real-time PCR. In addition, transmigration of neutrophils across a HMEC-1 monolayer was measured using transwell plates (pore size 3 μm). Results: Repression of Ninj1 by siRNA reduced Ninj1 mRNA expression in HMEC about 90% (Figure 1A). Reduced Ninj1 expression decreased neutrophil migration to 62.5% (Figure 1B) and TLR signaling. In detail, knockdown of Ninj1 significantly reduced TLR-2 and TLR-4 triggered expression of ICAM-1 and IL-6 (Figure 1C,D) while poly(I:C)-induced expression was only slightly reduced. To analyze a more specific TLR-3 target, we measured IP-10 mRNA expression, which was also significantly reduced in siNinj1-transfected cells (Figure 1E). Conclusion: Our in vitro data strongly indicated that Ninj1 is involved in regulation of TLR signaling and therewith contributes to inflammation. In vivo experiments will clarify its impact on systemic inflammation

Longer-Baseline Telescopes Using Quantum Repeaters

We present an approach to building interferometric telescopes using ideas of quantum information. Current optical interferometers have limited baseline lengths, and thus limited resolution, because of noise and loss of signal due to the transmission of photons between the telescopes. The technology of quantum repeaters has the potential to eliminate this limit, allowing in principle interferometers with arbitrarily long baselines.Comment: 10 pages, v2 improved clarit

Perceptions of leadership across cultures: a study of French and German managers and their employees in both their domestic and host environments

The phenomenon of globalisation has contributed greatly to the increasing interest in investigating cross-cultural leadership in recent years (Avolio, Walumbwa and Weber, 2009). To date cross-cultural leadership research has mainly involved comparative studies between countries at manager level (e.g. House et al., 2004) and has focused on potential cultural effects on leadership styles. The aim of this crosscultural study is to address the gap of neglecting employees’ views on leadership by investigating leadership perceptions both from the perspective of managers and employees in their domestic and host environments. This study explores leadership perceptions in the specific context of France and Germany, using a mixed methods approach. Based on implicit leadership theories and connectionist theory (Hanges et al., 2006; Lord et al., 2001), the similarities and differences that exist between the structure and the content of cognitive leadership schemas of French and German managers and employees regarding their perceptions of effective leadership are compared. This study shows that the application of connectionist theory to the field of cross-cultural leadership research provides a meaningful lens to investigate the relationship between culture and leadership. It found that despite the spread of globalisation and an often assumed concomitant convergence of cultures, differences in the approaches to leadership in a French and German business context still persist and should, therefore, be considered by companies planning foreign operations or secondments

Regulation of peroxisome proliferator-activated receptor gamma in macrophages during inflammatory processes

The peroxisome proliferator activated receptor gamma (PPARgamma) plays an eminent role during alternative activation of macrophages and resolution of inflammation. As an antiinflammatory signaling molecule, it seems likely that it is tightly regulated dependent on the state of the immune response. There is growing evidence that PPARgamma expression is reduced during inflammation, whereas molecular mechanisms are illdefined. Even though, its role in immunosuppression is getting more definite. Apoptotic cells (AC) provoke an active repression of pro-inflammatory responses inter alia by the inhibition of pro-inflammatory cytokine expression or attenuated generation of reactive oxygen species (ROS). The reduced formation of ROS was attributed to PPARgamma activation, while mechanisms behind the reduced cytokine expression remained unclear. Therefore, my Ph.D. thesis addressed the role of PPARgamma during inhibited cytokine synthesis in response to AC and the regulation of PPARgamma expression during an inflammatory response, which was initiated by lipopolysaccharide (LPS) exposure. In the first part of the thesis, I investigated the role of PPARgamma in coordinating the attenuation of pro-inflammatory cytokine expression in response to AC. Exposing murine RAW264.7 macrophages to AC prior to LPS-stimulation, reduced NFKB transactivation and lowered target gene expression of e.g. TNFalpha and IL-6 compared to controls. In macrophages over-expressing a dominant negative (d/n) mutant of PPARgamma, NFKB transactivation in response to LPS was restored, while using macrophages from myeloid lineage-specific conditional PPARgamma knock-out mice proved that PPARgamma transmitted the anti-inflammatory response delivered by AC. Domain analysis revealed that amino acids 32-250 are essential for inhibition of NFKB. Mutation of a SUMOylation (SUMO: small-ubiquitin related modifier) site in this region (K77R) and interfering SUMOylation by silencing the SUMO E3 ligase PIAS1 (protein inhibitor of activated Stat1) eliminated AC-provoked NFKB inhibition and concomitant TNFalpha expression. Chromatin-immunoprecipitation assays demonstrated that AC prevented the LPS-induced removal of nuclear receptor co-repressor (NCoR) from the KB response element within the TNFalpha promoter. I concluded that AC induce PPARgamma SUMOylation to attenuate the removal of NCoR, thereby blocking transactivation of NFKB. This contributes to an anti-inflammatory phenotype shift in macrophages in response to AC, by lowering pro-inflammatory cytokine production. The second part addressed molecular mechanisms responsible for reduced PPARgamma expression upon LPS exposure. PPARgamma gained considerable interest as a therapeutic target during chronic inflammatory diseases. Remarkably, the pathogenesis of diseases such as multiple sclerosis or Alzheimer’s disease is associated with impaired PPARgamma expression. Initiation of an inflammatory response by exposing primary human macrophages to LPS revealed a rapid decline of PPARgamma1 expression. PPARgamma1 mRNA decrease was prevented by inhibition of NFKB and also after pre-treatment with the PPARgamma agonist rosiglitazone, suggesting a NFKB-dependent pathway, because activated PPARgamma is known to inhibit NFKB transactivation. Since promoter activities were not affected by LPS, I focused on mRNA stability and noticed a decreased PPARgamma1 mRNA half-life. RNA stability is often regulated via 3’ untranslated regions (UTRs). Therefore, I analyzed the impact of the PPARgamma-3’UTR by luciferase assays. LPS significantly reduced luciferase activity of pGL3-PPARgamma-3’UTR, suggesting that PPARgamma1 mRNA is destabilized. Deletion of a potential miR-27a/b binding site within the 3’UTR completely restored luciferase activity. Moreover, inhibition of miR-27b, which was induced upon LPS-exposure, partially reversed PPARgamma1 mRNA decay, whereas the mature miR-27 mimicked the effect of LPS. MiR-27b was at least partially induced by NFKB, thus correlating with NFKB-dependent PPARgamma1 mRNA decrease. Since deletion of the miR-27 site also containing an AU-rich element (ARE) completely abrogated LPS-induced reduction but inhibition of miR-27b only partially restored PPARgamma1 mRNA expression, I suggested an additional implication of an ARE-binding protein. I provide evidence that LPS induces miR-27b, which in turn destabilizes PPARgamma1 mRNA. Understanding the molecular mechanism of PPARgamma mRNA destabilization, might help to rationalize inflammatory diseases associated with impaired PPARgamma expression. Even though, further experiments are needed to clarify the potential involvement of ARE-binding proteins.Chronische Entzündungskrankheiten entstehen häufig in Folge einer unkontrollierten Entzündungsreaktion und damit verbundenen irreversiblen Schäden des umliegenden Gewebes. Die Ausbildung eines anti-inflammatorischen Makrophagen-Phänotyps ist ein wichtiger Bestandteil zur Beendigung von Entzündungen. Charakteristisch für diesen Phänotyp ist eine verminderte Synthese pro-inflammatorischer Zytokine, welche teilweise auf die Aktivierung des Transkriptionsfaktors PPARgamma (‚peroxisome proliferator activated receptor gamma‘) zurückzuführen ist. Daher ist die Regulation der Aktivierung als auch der Expression von PPARgamma entscheidend für die Immunantwort von Makrophagen. Es konnte bereits gezeigt werden, dass durch die Phagozytose apoptotischer Zellen (AZ) zum einen PPARgamma aktiviert und zum anderen die Zytokinexpression durch eine Hemmung von NFKB (‚nuclear factor KB‘) vermindert wird. Daher untersuchte ich im ersten Teil meiner Arbeit die Rolle von PPARgamma bei der Inhibition von NFKB nach Interaktion mit AZ. Die Stimulation von RAW264.7-Makrophagen mit AZ führte zu einer Hemmung der NFKB-Aktivität, welche durch Überexpression einer dominantnegativen Mutante von PPARgamma reduziert war. Weiterhin konnte in primären PPARgamma-knock-out Makrophagen keine Hemmung der TNFalpha-Expression, als klassisches NFKB-Zielgen, festgestellt werden. Analysen der PPARgamma-Protein Domänen zeigten, dass die Aminosäuren 32-250 essentiell für die NFKB-Inhibition sind. Mutation der in diesem Bereich liegenden SUMOylierungsstelle K77 (SUMO: „small-ubiquitin related modifier“) als auch das Ausschalten der essentiellen SUMO-E3-Ligase PIAS1 („protein inhibitor of activated Stat1“) verhinderte die Hemmung von NFKB und bestätigte die SUMOylierung von PPARgamma als zugrunde liegenden Mechanismus. Als verantwortlichen Repressor identifizierte ich NCoR („nuclear receptor co-repressor“), welcher im Ruhezustand konstitutiv an NFKB-Bindestellen verschiedener pro-inflammatorischer Promotoren gebunden ist. Nach TLR4-Aktivierung dissoziiert dieser von der Promotorregion und wird abgebaut. Durch Chromatin-Immunpräzipitationen konnte ich zeigen, dass vermutlich SUMOyliertes PPARgamma nach Interaktion mit AZ die Dissoziation von NCoR und damit die Zielgen-Expression verhindert. Die Aufklärung dieses Mechanismus trägt damit zum weiteren Verständnis bei, wie AZ einen anti-inflammatorischen Makrophagen-Phänotyp hervorrufen und damit zur Eindämmung einer Entzündungsreaktion beitragen. Bei verschiedenen Entzündungskrankheiten wie Alzheimer oder auch Multipler Sklerose konnte eine Verringerung der PPARgamma-Expression nachgewiesen werden. Da der Mechanismus dieser Reduktion jedoch weitgehend unbekannt ist, beschäftigte ich mich im zweiten Teil meiner Arbeit mit der Expressionsregulation von PPARgamma in Makrophagen. Die Stimulation von primären humanen Makrophagen mit LPS verringerte den PPARgamma1 mRNA-Gehalt. Diese mRNA-Reduktion konnte durch Hemmung von NFKB als auch durch Vorstimulation mit dem PPARgamma-Agonisten Rosiglitazone verhindert werden, was auf einen NFKB-abhängigen Mechanismus hinwies. Durch Promotor-Reporteranalysen konnte eine Reduktion der PPARgamma1 mRNA auf transkriptioneller Ebene ausgeschlossen werden. LPS führte vielmehr zu einer 3‘-UTR (‚untranslated region‘)-abhängigen Destabilisierung der PPARgamma1 mRNA. Aufgrund einer potentiellen Bindestelle für microRNA-27a/b (miR-27a/b), untersuchte ich deren Expression. LPS führte - zum Teil NFKB abhängig - zur Induktion von miR-27a und b. Eine Depletion der miR-27 Bindestelle innerhalb der PPARgamma-3’UTR verhinderte vollständig den destabilisierenden Effekt von LPS. Weiterhin führte die Inhibition von miR-27b, nicht aber von miR-27a, zur teilweisen Aufhebung der LPS-induzierten Reduktion. Die Destabilisierung von PPARgamma konnte außerdem durch Transfektion mit miR-27b simuliert werden, wobei die additive Zugabe von LPS den Effekt nur wenig verstärkte. Meine Daten beweisen, dass LPS-induzierte miR-27b zur Destabilisierung der PPARgamma1 mRNA führt. Die Aufklärung des vorliegenden molekularen Mechanismus könnte dazu beitragen, das Verständnis und damit verbundene Behandlungsmethoden von Entzündungskrankheiten, welche eine reduzierte PPARgamma-Expression zeigen, zu erweitern

Entangling ability of a beam splitter in the presence of temporal which-path information

We calculate the amount of polarization-entanglement induced by two-photon interference at a lossless beam splitter. Entanglement and its witness are quantified respectively by concurrence and the Bell-CHSH parameter. In the presence of a Mandel dip, the interplay of two kinds of which-path information -- temporal and polarization -- gives rise to the existence of entangled polarization-states that cannot violate the Bell-CHSH inequality.Comment: 8 pages including 2 figure