Molecular cloning and characterization of serpins as potential immunomodulators

Titelblatt 1 Titelblatt 2 Inhaltsverzeichnis Abkürzungsverzeichnis Zusammenfassung Summary Einleitung Ziele der Arbeit Ergebnisse Diskussion Schlußfolgerung Methoden Material Literatur Vorträge und Poster Anhang Danksagung Lebenslauf SelbständigkeitserklärungFilarien sind permanent einer Vielzahl von Immuneffektormechanismen ihres Wirtes ausgesetzt. Ihr Überleben hängt davon ab, schädlichen Immunantworten auszuweichen oder sie zu modulieren. Eine Strategie, um dies zu erreichen, ist die Freisetzung immunmodulatorischer Komponenten, die die Effektormechanismen blockieren. Zu diesen sekretierten Molekülen gehören Serpine, für die Immunevasionsfunktionen beschrieben worden sind (Zang et al. 1999, 2000, Maizels et al. 2001). In dieser Arbeit wollte ich die immunmodulatorische Kapazität eines Serpins der Nagetierfilarie Acanthocheilonema viteae im Vergleich zu einem homologen Protein des frei lebenden Nematoden Caenorhabditis elegans analysieren. Das Ziel dieser Arbeit war herauszufinden, ob Serpine von A. viteae vom Parasiten speziell genutzt werden, um der Immunantwort des Wirtes auszuweichen oder ob Serpine des frei lebenden Nematoden C. elegans ähnliche immunmodulatorische Aktivitäten besitzen. Ein A. viteae- und ein C. elegans-Serpin wurden kloniert, exprimiert und charakterisiert hinsichtlich ihrer inhibitorischen und immunmodulatorischen Eigenschaften. Das rCe-Serpin zeigte in vitro bei den verwendeten Serinproteasen eine Spezifität für Trypsin und Cathepsin G. Die Proliferation sowohl polyklonal als auch Antigen-spezifisch stimulierter Mausmilzzellen wurde durch das rCe-Serpin inhibiert und die Bildung von IL-10 verstärkt. Die Hemmung des mitogen wirkenden Cathepsin G und die Induktion der IL-10-Produktion sind wichtige Faktoren für die zelluläre Hyporeaktivität. Das rAv-Serpin zeigte weder eine inhibitorische Wirkung auf die eingesetzten Proteasen noch auf die T-Zell-Proliferation. Da das Serpin des frei lebenden C. elegans über immunmodulatorische Fähigkeiten verfügt, sind Serpine vermutlich nicht durch die parasitisch lebenden Nematoden zur Beeinflussung der Wirtsabwehr spezifisch modifiziert worden, sondern stellten für die Nematoden einen Vorteil beim Übergang zur parasitären Lebensform dar. Das ebenfalls im Rahmen dieser Arbeit klonierte und exprimierte Serpin des parasitären Protozoen E. tenella hemmte die Serinproteasen Trypsin, Chymotrypsin, porzine Pankreaselastase und Cathepsin G. Es verminderte die Proliferation polyklonal stimulierter Mausmilzzellen und bewirkte eine tendenziell erhöhte IL-10-Bildung. Antigen-spezifisch stimulierte Mausmilzzellen wurden durch rEt-Serpin nicht in ihrer Proliferation gehemmt.Filarial parasites are constantly exposed to an array of immune effector mechanisms of their hosts. Their survival therefore depends on the evasion or modulation of harmful immune responses. One strategy to cope with the host s immune responses is the release of immunomodulators that block effector mechanisms. Serpins (serine protease inhibitors) are secretory molecules that have been described to have functions in immune evasion (Zang et al. 1999, 2000, Maizels et al. 2001). To determine parasite-specific properties of serpins, the immunomodulatory capacity of a serpin of the rodent filarial nematode Acanthocheilonema viteae was analysed in comparison to a homologous protein of the free-living nematode Caenorhabditis elegans. The aim of the project was to investigate whether serpins of A. viteae are specifically used by the parasite to evade immune responses of the host or whether serpins of the free-living nematode C. elegans have similar immunomodulatory activities. Homologous serpins from A. viteae and C. elegans were cloned, expressed as proteins in E. coli, and their inhibitory and immunomodulatory properties were characterised. The serine proteases from C. elegans (rCe-Serpin) showed trypsin and cathepsin G specificity. In addition, it inhibited the proliferation of polyclonally as well as antigen-stimulated spleen cells of mice, and enhanced the production of IL-10. The inhibition of the mitogenic cathepsin G and the induction of the IL-10 production are important factors in cellular hyporesponsiveness. On the other hand, the serine protease from A. viteae (rAv-Serpin) showed no inhibitory property on the same proteases or T cell proliferation. Since the serpin of the free-living C. elegans posseses immunomodulatory properties, serpins have probably not been specifically modified by parasitic nematodes for influencing the host s immune response. It may therefore be surmised that serpins are a preadaption to a parasitic life style of nematodes. To further verify whether serpins of parasites have specific properties, a serpin of the parasitic protozoon E. tenella (rEt- Serpin) was also cloned and expressed. It inhibited the serine proteases trypsin, chymotrypsin, porcine pancreatic elastase and cathepsin G. The rEt- Serpin inhibited the proliferation of polyclonal stimulated spleen cells of mice and showed a tendency to enhance the IL-10 production. However, proliferation of antigen specific stimulated spleen cells was not inhibited by rEt-Serpin

Bonus oder Bias? Mechanismen geschlechtsspezifischer Entlohnung

To what extent is the gender wage gap of fulltime employees due to differences in productiv-ity- related characteristics, to discrimination of female employees, and how is the wage gap affected by firm specific features? To analyze these problems we use a linked employer-employee database for East and West Germany in 2000, made available by the Institute for Labour Market Research. In particular, we address the effect on wage levels of the sex com-position of occupations within firms (job cells). Our estimations rely on the decomposition of the wage differential proposed by Oaxaca and Blinder. We find that only one tenth of the gender gap in wages is explained by human capital differences between men and women. Re-garding the gender wage gap in Germany we conclude that discrimination in the allocation of men and women to different jobs is an important mechanism: With increasing proportions of women within job cells we observe decreasing wage levels for men and women but with higher rates of decline for women than for men. While the presence of worker's councils are shown to have a positive impact on wage levels, women are found to profit most from their presence within firms

Bonus oder Bias? Mechanismen geschlechtsspezifischer Entlohnung

"To what extent is the gender wage gap of fulltime employees due to differences in productiv-ity- related characteristics, to discrimination of female employees, and how is the wage gap affected by firm specific features? To analyze these problems we use a linked employer-employee database for East and West Germany in 2000, made available by the Institute for Labour Market Research. In particular, we address the effect on wage levels of the sex com-position of occupations within firms (job cells). Our estimations rely on the decomposition of the wage differential proposed by Oaxaca and Blinder. We find that only one tenth of the gender gap in wages is explained by human capital differences between men and women. Re-garding the gender wage gap in Germany we conclude that discrimination in the allocation of men and women to different jobs is an important mechanism: With increasing proportions of women within job cells we observe decreasing wage levels for men and women but with higher rates of decline for women than for men. While the presence of worker's councils are shown to have a positive impact on wage levels, women are found to profit most from their presence within firms." (Author's abstract, IAB-Doku) ((en))Lohnunterschied - Determinanten, erwerbstätige Frauen, erwerbstätige Männer, Arbeitsproduktivität, Qualifikationsniveau, Betriebsrat, regionaler Vergleich, IAB-Linked-Employer-Employee-Datensatz, Ostdeutschland, Westdeutschland, Bundesrepublik Deutschland

Effect of substrate properties and phosphorus supply on facilitating the uptake of rare earth elements (REE) in mixed culture cropping systems of Hordeum vulgare, Lupinus albus and Lupinus angustifolius

This study presents how phosphate (P) availability and intercropping may influence the migration of rare earth elements (REEs) in legume–grass associations. In a replacement model, Hordeum vulgare was intercropped with 11% Lupinus albus and 11% Lupinus angustifolius. They were cultivated on two substrates, A (pH = 7.8) and B (pH = 6.6), and treated with 1.5 g P m−2 or 3 g P m−2. Simultaneously, a greenhouse experiment was conducted to quantify carboxylate release. There, one group of L. albus and L. angustifolius was supplied with either 200 µmol L-1 P or 20 µmol L-1 P. L. albus released higher amounts of carboxylates at low P supply than L. angustifolius, while L. angustifolius showed the opposite response. Plants cultivated on substrate B accumulated substantially higher amounts of nutrients and REE, compared to substrate A. Higher P supply did not influence the leaf and stem P concentrations of H. vulgare. Addition of P decreased REE accumulation in barley monocultures on alkaline soil A. However, when H. vulgare was cultivated in mixed culture with L. angustifolius on alkaline substrate A with high P supply, the accumulation of REE in H. vulgare significantly increased. Conversely, on acidic substrate B, intercropping with L. albus decreased REE accumulation in H. vulgare. Our findings suggest a predominant effect of soil properties on the soil–plant transfer of REEs. However, in plant communities and within a certain soil environment, interspecific root interactions determined by species-specific strategies related to P acquisition in concert with the plant’s nutrient supply impact REE fluxes between neighbouring plants

Bonus oder Bias? Mechanismen geschlechtsspezifischer Entlohnung

"In welchem Ausmaß ist das Lohngefälle zwischen vollzeitbeschäftigten Frauen und Männern auf Produktivitätsunterschiede zurückzuführen? Welchen Anteil hat die Diskriminierung von Arbeitnehmerinnen und wie wirken sich betriebliche Merkmale auf die Lohnunterschiede aus? Diese Fragen untersuchen wir mit einem verbundenen Personen- und Betriebsdatensatz des Instituts für Arbeitsmarkt- und Berufsforschung für das Jahr 2000 in Ost- und Westdeutschland. Im Mittelpunkt steht die Frage, wie sich die Geschlechterzusammensetzung von Berufsgruppen in Betrieben (Jobzellen) auf die Lohnhöhe auswirkt. Um zwischen Ausstattungs- und Entlohnungseffekten trennen zu können, wird eine von Oaxaca und Blinder vorgeschlagene Zerlegung des Lohndifferentials vorgenommen. Wie sich zeigt, kann nur etwa ein Zehntel der Lohnlücke zwischen Frauen und Männern durch die Unterschiede im gemessenen Humankapitalbestand erklärt werden. Mit steigendem Frauenanteil in den Jobzellen sinken die Löhne, wobei die Lohnabschläge von Frauen höher ausfallen als die von Männern. Diskriminierung, so die Folgerung, greift insbesondere bei der geschlechtsbasierten Zuweisung von Arbeitsplätzen. Mit dem Vorhandensein von Betriebsräten steigen die Löhne, was vorrangig den beschäftigten Frauen zu Gute kommt." (Autorenreferat)"To what extent is the gender wage gap of fulltime employees due to differences in productivity-related characteristics, to discrimination of female employees, and how is the wage gap affected by firm specific features? To analyze these problems we use a linked employer-employee database for East and West Germany in 2000, made available by the Institute for Labour Market Research. In particular, we address the effect on wage levels of the sex com-position of occupations within firms (job cells). Our estimations rely on the decomposition of the wage differential proposed by Oaxaca and Blinder. We find that only one tenth of the gender gap in wages is explained by human capital differences between men and women. Regarding the gender wage gap in Germany we conclude that discrimination in the allocation of men and women to different jobs is an important mechanism: With increasing proportions of women within job cells we observe decreasing wage levels for men and women but with higher rates of decline for women than for men. While the presence of worker's councils are shown to have a positive impact on wage levels, women are found to profit most from their presence within firms." (author's abstract

АСР температуры и расхода гелеобразного топлива в системе топливоподачи испытательного стенда

Объектом автоматизации является система топливоподачи испытательного стенда. Цель работы – проектирование автоматической системы регулирования температуры и расхода гелеобразного топлива в системе топливоподачи испытательного стенда. В процессе выполнения работы проводились экспериментальные исследования, анализ объекта автоматизации, составление структурной схемы автоматической системы регулирования температуры и расхода гелеобразного топлива в системе топливоподачи испытательного стенда, проектирование функциональной схемы, монтажной и принципиальной электрической схем, а также разработка чертежа общего вида щита автоматизации, выбор технических средств и приборов автоматизации с последующим составлением заказной спецификации, разработка мнемосхемы проекта.The object of automation is the fuel supply system of the test stand. The purpose of the work is to design an automatic system for controlling the temperature and the flow rate of gel-like fuel in the fuel dispensing system of the test bench. In the course of the work carried out, experimental studies, analysis of the automation facility, drafting structural diagram of the automatic temperature and flow gel-like fuel management system in the test bench system were carried out, Design of the functional circuit, mounting diagram and principle electrical circuits, as well as drawing of the general kind of automation shield, selection of technical means and automation devices with subsequent elaboration of the custom specification, development of the design mnemonic diagram

The nuclear receptor PPARγ selectively inhibits Th17 differentiation in a T cell–intrinsic fashion and suppresses CNS autoimmunity

T helper cells secreting interleukin (IL)-17 (Th17 cells) play a crucial role in autoimmune diseases like multiple sclerosis (MS). Th17 differentiation, which is induced by a combination of transforming growth factor (TGF)-β/IL-6 or IL-21, requires expression of the transcription factor retinoic acid receptor–related orphan receptor γt (RORγt). We identify the nuclear receptor peroxisome proliferator–activated receptor γ (PPARγ) as a key negative regulator of human and mouse Th17 differentiation. PPARγ activation in CD4+ T cells selectively suppressed Th17 differentiation, but not differentiation into Th1, Th2, or regulatory T cells. Control of Th17 differentiation by PPARγ involved inhibition of TGF-β/IL-6–induced expression of RORγt in T cells. Pharmacologic activation of PPARγ prevented removal of the silencing mediator for retinoid and thyroid hormone receptors corepressor from the RORγt promoter in T cells, thus interfering with RORγt transcription. Both T cell–specific PPARγ knockout and endogenous ligand activation revealed the physiological role of PPARγ for continuous T cell–intrinsic control of Th17 differentiation and development of autoimmunity. Importantly, human CD4+ T cells from healthy controls and MS patients were strongly susceptible to PPARγ-mediated suppression of Th17 differentiation. In summary, we report a PPARγ-mediated T cell–intrinsic molecular mechanism that selectively controls Th17 differentiation in mice and in humans and that is amenable to pharmacologic modulation. We therefore propose that PPARγ represents a promising molecular target for specific immunointervention in Th17-mediated autoimmune diseases such as MS

A new face of Borjeson–Forssman–Lehmann syndrome? De novo mutations in PHF6 in seven females with a distinct phenotype

Background: Borjeson–Forssman–Lehmann syndrome (BFLS) is an X-linked recessive intellectual disability (ID) disorder caused by mutations in the PHF6 gene and characterised by variable cognitive impairment, a distinct facial gestalt, obesity, and hypogonadism. Female carriers are usually not affected or only mildly affected, and so far only two females with de novo mutations or deletions in PHF6 have been reported. Methods and results: We performed PHF6 mutational analysis and screening for intragenic deletions and duplications by quantitative real-time PCR and multiplex ligation dependent probe amplification (MLPA) in female patients with variable ID and a distinct appearance of sparse hair, remarkable facial features, hypoplastic nails, and teeth anomalies. We detected two truncating mutations and two duplications of exons 4 and 5. Furthermore, two female patients with PHF6 deletions and a similar phenotype were identified by routine molecular karyotyping. Recently, two patients with a clinical diagnosis of Coffin-Siris syndrome in early infancy had been found to harbour mutations in PHF6, and their phenotype in advanced ages is now described. Further studies revealed skewed X-inactivation in blood lymphocytes, while it was normal in fibroblasts, thus indicating functional mosaicism. Conclusions: Our findings indicate that de novo defects in PHF6 in females result in a recognisable phenotype which might have been under-recognised so far and which comprises variable ID, a characteristic facial gestalt, hypoplastic nails, brachydactyly, clinodactyly mainly of fingers IV and V, dental anomalies, and linear skin hyperpigmentation. It shows overlap with BFLS but also additional distinct features, thus adding a new facet to this disorder