OPUS 4 Latest Documents RSS Feed

High functional diversity is related to high nitrogen availability in a deciduous forest - evidence from a functional trait approach

Fri, 24 May 2013 09:59:40 +0200

The current study tested the assumption that floristic and functional diversity patterns are negatively related to soil nitrogen content. We analyzed 20 plots with soil N-contents ranging from 0.63% to 1.06% in a deciduous forest near Munich (Germany). To describe species adaptation strategies to different nitrogen availabilities, we used a plant functional type (PFT) approach. Each identified PFT represents one realized adaptation strategy to the current environment. These were correlated, next to plant species richness and evenness, to soil nitrogen contents. We found that N-efficient species were typical for low soil nitrogen contents, while N-requiring species occur at high N-contents. In contrast to our initial hypotheses, floristic and functional diversity measures (number of PFTs) were positively related to nitrogen content in the soil. Every functional group has its own adaptation to the prevailing environmental conditions; in consequence, these functional groups can co-exist but do not out-compete one another. The increased number of functional groups at high N-contents leads to increased species richness. Hence, for explaining diversity patterns we need to consider species groups representing different adaptations to the current environmental conditions. Such co-existing ecological strategies may even overcome the importance of competition in their effect on biodiversity.

Early otic development depends on autophagy for apoptotic cell clearance and neural differentiation

Thu, 23 May 2013 17:07:25 +0200

Autophagy is a highly regulated program of self-degradation of the cytosolic constituents that has key roles during early development and in adult cell growth and homeostasis. To investigate the role of autophagy in otic neurogenesis, we studied the expression of autophagy genes in early stages of chicken (Gallus gallus) inner ear development and the consequences of inhibiting the autophagic pathway in organotypic cultures of explanted chicken otic vesicles (OVs). Here we show the expression of autophagy-related genes (Atg) Beclin-1 (Atg6), Atg5 and LC3B (Atg8) in the otocyst and the presence of autophagic vesicles by using transmission electron microscopy in the otic neurogenic zone. The inhibition of the transcription of LC3B by using antisense morpholinos and of class III phosphatidylinositol 3-kinase with 3-methyladenine causes an aberrant morphology of the OV with accumulation of apoptotic cells. Moreover, inhibition of autophagy provokes the misregulation of the cell cycle in the otic epithelium, impaired neurogenesis and poor axonal outgrowth. Finally, our results indicate that autophagy provides the energy required for the clearing of neuroepithelial dying cells and suggest that it is required for the migration of otic neuronal precursors. Taken together, our results show for the first time that autophagy is an active and essential process during early inner ear development.

RAF kinase activity regulates neuroepithelial cell proliferation and neuronal progenitor cell differentiation during early inner ear development

Thu, 23 May 2013 15:08:25 +0200

Background: Early inner ear development requires the strict regulation of cell proliferation, survival, migration and differentiation, coordinated by the concerted action of extrinsic and intrinsic factors. Deregulation of these processes is associated with embryonic malformations and deafness. We have shown that insulin-like growth factor I (IGF-I) plays a key role in embryonic and postnatal otic development by triggering the activation of intracellular lipid and protein kinases. RAF kinases are serine/threonine kinases that regulate the highly conserved RAS-RAF-MEK-ERK signaling cascade involved in transducing the signals from extracellular growth factors to the nucleus. However, the regulation of RAF kinase activity by growth factors during development is complex and still not fully understood. Methodology/Principal Findings: By using a combination of qRT-PCR, Western blotting, immunohistochemistry and in situ hybridization, we show that C-RAF and B-RAF are expressed during the early development of the chicken inner ear in specific spatiotemporal patterns. Moreover, later in development B-RAF expression is associated to hair cells in the sensory patches. Experiments in ex vivo cultures of otic vesicle explants demonstrate that the influence of IGF-I on proliferation but not survival depends on RAF kinase activating the MEK-ERK phosphorylation cascade. With the specific RAF inhibitor Sorafenib, we show that blocking RAF activity in organotypic cultures increases apoptosis and diminishes the rate of cell proliferation in the otic epithelia, as well as severely impairing neurogenesis of the acoustic-vestibular ganglion (AVG) and neuron maturation. Conclusions/Significance: We conclude that RAF kinase activity is essential to establish the balance between cell proliferation and death in neuroepithelial otic precursors, and for otic neuron differentiation and axonal growth at the AVG.

AKT signaling mediates IGF-I survival actions on otic neural progenitors

Thu, 23 May 2013 14:55:27 +0200

Background: Otic neurons and sensory cells derive from common progenitors whose transition into mature cells requires the coordination of cell survival, proliferation and differentiation programmes. Neurotrophic support and survival of post-mitotic otic neurons have been intensively studied, but the bases underlying the regulation of programmed cell death in immature proliferative otic neuroblasts remains poorly understood. The protein kinase AKT acts as a node, playing a critical role in controlling cell survival and cell cycle progression. AKT is activated by trophic factors, including insulin-like growth factor I (IGF-I), through the generation of the lipidic second messenger phosphatidylinositol 3-phosphate by phosphatidylinositol 3-kinase (PI3K). Here we have investigated the role of IGF-dependent activation of the PI3K-AKT pathway in maintenance of otic neuroblasts. Methodology/Principal Findings: By using a combination of organotypic cultures of chicken (Gallus gallus) otic vesicles and acoustic-vestibular ganglia, Western blotting, immunohistochemistry and in situ hybridization, we show that IGF-I-activation of AKT protects neural progenitors from programmed cell death. IGF-I maintains otic neuroblasts in an undifferentiated and proliferative state, which is characterised by the upregulation of the forkhead box M1 (FoxM1) transcription factor. By contrast, our results indicate that post-mitotic p27Kip-positive neurons become IGF-I independent as they extend their neuronal processes. Neurons gradually reduce their expression of the Igf1r, while they increase that of the neurotrophin receptor, TrkC. Conclusions/Significance: Proliferative otic neuroblasts are dependent on the activation of the PI3K-AKT pathway by IGF-I for survival during the otic neuronal progenitor phase of early inner ear development.

Autophagy during vertebrate development

Maria Rodriguez Aburto; Juan M. Hurlé; Isabel Varela-Nieto; Marta Magariños — Thu, 23 May 2013 13:03:42 +0200

Autophagy is an evolutionarily conserved catabolic process by which cells degrade their own components through the lysosomal machinery. In physiological conditions, the mechanism is tightly regulated and contributes to maintain a balance between synthesis and degradation in cells undergoing intense metabolic activities. Autophagy is associated with major tissue remodeling processes occurring through the embryonic, fetal and early postnatal periods of vertebrates. Here we survey current information implicating autophagy in cellular death, proliferation or differentiation in developing vertebrates. In developing systems, activation of the autophagic machinery could promote different outcomes depending on the cellular context. Autophagy is thus an extraordinary tool for the developing organs and tissues.

Do non-genomically encoded fusion transcripts cause recurrent chromosomal translocations?

Eric Kowarz; Theodor Dingermann; Rolf Marschalek — Thu, 23 May 2013 12:21:54 +0200

We among others have recently demonstrated that normal cells produce “fusion mRNAs”. These fusion mRNAs do not derive from rearranged genomic loci, but rather they are derived from “early-terminated transcripts” (ETTs). Premature transcriptional termination takes place in intronic sequences that belong to “breakpoint cluster regions”. One important property of ETTs is that they exhibit an unsaturated splice donor site. This results in: (1) splicing to “cryptic exons” present in the final intron; (2) Splicing to another transcript of the same gene (intragenic trans-splicing), resulting in “exon repetitions”; (3) splicing to a transcript of another gene (intergenic trans-splicing), leading to “non-genomically encoded fusion transcripts” (NGEFTs). These NGEFTs bear the potential risk to influence DNA repair processes, since they share identical nucleotides with their DNA of origin, and thus, could be used as “guidance RNA” for DNA repair processes. Here, we present experimental data about four other genes. Three of them are associated with hemato-malignancies (ETV6, NUP98 and RUNX1), while one is associated with solid tumors (EWSR1). Our results demonstrate that all genes investigated so far (MLL, AF4, AF9, ENL, ELL, ETV6, NUP98, RUNX1 and EWSR1) display ETTs and produce transpliced mRNA species, indicating that this is a genuine property of translocating genes.

Venomous Secretions from Marine Snails of the Terebridae Family Target Acetylcholine Receptors

Thu, 23 May 2013 11:19:28 +0200

Venoms from cone snails (Conidae) have been extensively studied during the last decades, but those from other members of the suborder Toxoglossa, such as of Terebridae and Turridae superfamilies attracted less interest so far. Here, we report the effects of venom and gland extracts from three species of the superfamily Terebridae. By 2-electrode voltage-clamp technique the gland extracts were tested on Xenopus oocytes expressing nicotinic acetylcholine receptors (nAChRs) of rat neuronal (α3β2, α3β4, α4β2, α4β4, α7) and muscle subtypes (α1β1γδ), and expressing potassium (Kv1.2 and Kv1.3) and sodium channels (Nav1.2, 1.3, 1.4, 1.6). The extracts were shown to exhibit remarkably high inhibitory activities on almost all nAChRs tested, in particular on the α7 subtype suggesting the presence of peptides of the A-superfamily from the venom of Conus species. In contrast, no effects on the potassium and sodium channels tested were observed. The venoms of terebrid snails may offer an additional source of novel biologically active peptides.

Biological activities of ethanolic extracts from deep-sea antarctic marine sponges

Thu, 23 May 2013 11:05:02 +0200

We report on the screening of ethanolic extracts from 33 deep-sea Antarctic marine sponges for different biological activities. We monitored hemolysis, inhibition of acetylcholinesterase, cytotoxicity towards normal and transformed cells and growth inhibition of laboratory, commensal and clinically and ecologically relevant bacteria. The most prominent activities were associated with the extracts from sponges belonging to the genus Latrunculia, which show all of these activities. While most of these activities are associated to already known secondary metabolites, the extremely strong acetylcholinesterase inhibitory potential appears to be related to a compound unknown to date. Extracts from Tetilla leptoderma, Bathydorus cf. spinosus, Xestospongia sp., Rossella sp., Rossella cf. racovitzae and Halichondria osculum were hemolytic, with the last two also showing moderate cytotoxic potential. The antibacterial tests showed significantly greater activities of the extracts of these Antarctic sponges towards ecologically relevant bacteria from sea water and from Arctic ice. This indicates their ecological relevance for inhibition of bacterial microfouling.

Unmanned Aerial Vehicle (UAV) for Monitoring Soil Erosion in Morocco

Thu, 23 May 2013 10:45:29 +0200

This article presents an environmental remote sensing application using a UAV that is specifically aimed at reducing the data gap between field scale and satellite scale in soil erosion monitoring in Morocco. A fixed-wing aircraft type Sirius I (MAVinci, Germany) equipped with a digital system camera (Panasonic) is employed. UAV surveys are conducted over different study sites with varying extents and flying heights in order to provide both very high resolution site-specific data and lower-resolution overviews, thus fully exploiting the large potential of the chosen UAV for multi-scale mapping purposes. Depending on the scale and area coverage, two different approaches for georeferencing are used, based on high-precision GCPs or the UAV’s log file with exterior orientation values respectively. The photogrammetric image processing enables the creation of Digital Terrain Models (DTMs) and ortho-image mosaics with very high resolution on a sub-decimetre level. The created data products were used for quantifying gully and badland erosion in 2D and 3D as well as for the analysis of the surrounding areas and landscape development for larger extents.

Molecular Mechanisms of Adaptation of the Moderately Halophilic Bacterium Halobacillis halophilus to Its Environment

Inga Hänelt; Volker Müller — Thu, 23 May 2013 10:28:33 +0200

The capability of osmoadaptation is a prerequisite of organisms that live in an environment with changing salinities. Halobacillus halophilus is a moderately halophilic bacterium that grows between 0.4 and 3 M NaCl by accumulating both chloride and compatible solutes as osmolytes. Chloride is absolutely essential for growth and, moreover, was shown to modulate gene expression and activity of enzymes involved in osmoadaptation. The synthesis of different compatible solutes is strictly salinity- and growth phase-dependent. This unique hybrid strategy of H. halophilus will be reviewed here taking into account the recently published genome sequence. Based on identified genes we will speculate about possible scenarios of the synthesis of compatible solutes and the uptake of potassium ion which would complete our knowledge of the fine-tuned osmoregulation and intracellular osmolyte balance in H. halophilus.

Predator avoidance in extremophile fish

Thu, 23 May 2013 10:05:45 +0200

Extreme habitats are often characterized by reduced predation pressures, thus representing refuges for the inhabiting species. The present study was designed to investigate predator avoidance of extremophile populations of Poecilia mexicana and P. sulphuraria that either live in hydrogen sulfide-rich (sulfidic) springs or cave habitats, both of which are known to have impoverished piscine predator regimes. Focal fishes that inhabited sulfidic springs showed slightly weaker avoidance reactions when presented with several naturally occurring predatory cichlids, but strongest differences to populations from non-sulfidic habitats were found in a decreased shoaling tendency with non-predatory swordtail (Xiphophorus hellerii) females. When comparing avoidance reactions between P. mexicana from a sulfidic cave (Cueva del Azufre) and the adjacent sulfidic surface creek (El Azufre), we found only slight differences in predator avoidance, but surface fish reacted much more strongly to the non-predatory cichlid Vieja bifasciata. Our third experiment was designed to disentangle learned from innate effects of predator recognition. We compared laboratory-reared (i.e., predator-naïve) and wild-caught (i.e., predator-experienced) individuals of P. mexicana from a non-sulfidic river and found no differences in their reaction towards the presented predators. Overall, our results indicate (1) that predator avoidance is still functional in extremophile Poecilia spp. and (2) that predator recognition and avoidance reactions have a strong genetic basis.

Impact of climate change on renewable groundwater resources: assessing the benefits of avoided greenhouse gas emissions using selected CMIP5 climate projections

Felix Theodor Portmann; Petra Döll; Stephanie Eisner; Martina Flörke — Thu, 23 May 2013 09:52:28 +0200

Reduction of greenhouse gas (GHG) emissions to minimize climate change requires very significant societal effort. To motivate this effort, it is important to clarify the benefits of avoided emissions. To this end, we analysed the impact of four emissions scenarios on future renewable groundwater resources, which range from 1600 GtCO2 during the 21st century (RCP2.6) to 7300 GtCO2 (RCP8.5). Climate modelling uncertainty was taken into account by applying the bias-corrected output of a small ensemble of five CMIP5 global climate models (GCM) as provided by the ISI-MIP effort to the global hydrological model WaterGAP. Despite significant climate model uncertainty, the benefits of avoided emissions with respect to renewable groundwater resources (i.e. groundwater recharge (GWR)) are obvious. The percentage of projected global population (SSP2 population scenario) suffering from a significant decrease of GWR of more than 10% by the 2080s as compared to 1971–2000 decreases from 38% (GCM range 27–50%) for RCP8.5 to 24% (11–39%) for RCP2.6. The population fraction that is spared from any significant GWR change would increase from 29% to 47% if emissions were restricted to RCP2.6. Increases of GWR are more likely to occur in areas with below average population density, while GWR decreases of more than 30% affect especially (semi)arid regions, across all GCMs. Considering change of renewable groundwater resources as a function of mean global temperature (GMT) rise, the land area that is affected by GWR decreases of more than 30% and 70% increases linearly with global warming from 0 to 3 ° C. For each degree of GMT rise, an additional 4% of the global land area (except Greenland and Antarctica) is affected by a GWR decrease of more than 30%, and an additional 1% is affected by a decrease of more than 70%.

Implementing transparency in an Eastern European office market: preparing Warsaw for global investments

Nadine Bitterer; Susanne Heeg — Wed, 22 May 2013 15:57:03 +0200

In the aftermath of an increasing integration of property and financial markets, the real estate industry is subject to soaring internationalization processes. Since international institutional investors appeared, transnational real estate investments have increased tremendously. In recent years, Central and Eastern European countries have been becoming more attractive to institutional investors and are therefore being integrated into international market structures. Within these countries, Warsaw emerged as the most dynamic and important real estate market. But what are the mechanisms and practices through which the real estate market of Warsaw becomes international? Which networks, intermediaries and frames are necessary to constitute a mature real estate market? The article argues that international real estate consultants are playing a crucial role in the underlying internationalization process. They are acting at the interface between investors, developers, construction companies and tenants and are therefore becoming a crucial hinge between real estate actors. With the example of the Warsaw real estate market we argue that international real estate consultancies are key drivers of the transformation process from a local to a global market. They transfer global knowledge, competence and practices and implement transparent and professional structures in the emerging Warsaw real estate market.

Jahresbericht / Johann Wolfgang Goethe-Universität Frankfurt am Main, Fachbereich 09: Sprach- und Kulturwissenschaften, Japanologie. April 2010 bis März 2011

Wed, 22 May 2013 13:37:59 +0200

Bericht 2011 / Goethe-Universität Frankfurt am Main

Wed, 22 May 2013 13:20:33 +0200

Geoprax : Newsletter ; Nr. 25 - 3/2013

Wed, 22 May 2013 10:58:36 +0200

Libraries around the world / compiled by Andreas J. Werner

Andreas Werner — Wed, 22 May 2013 09:42:04 +0200

"Library Buildings around the World" is a survey based on researches of several years. The objective was to gather library buildings on an international level starting with 1990.

Resonant pickups for non-destructive single-particle detection in heavy-ion storage rings and first experimental results

Mohammad Shahab Sanjari — Tue, 21 May 2013 09:51:40 +0200

Soziale Interaktion auf Finanzmärkten

Frederik König — Tue, 21 May 2013 09:22:43 +0200

Ziel meiner Dissertation ist die empirische Analyse von Auswirkungen der sozialen Interaktion zwischen Akteuren auf Finanzmärkten. In meinem ersten Aufsatz stelle ich ein Marktpreismodell vor, welches dem Einfluss durch soziale Interaktion Rechnung trägt. Mit Hilfe dieses Modells gehe ich der Fragestellung nach, ob soziale Interaktion zwischen Marktteilnehmern eine stabilisierende oder eine destabilisierende Wirkung auf Finanzmärkte hat. Mit meinem zweiten Aufsatz untersuche ich das Verhalten von Aktienanalysten, die als wesentlicher Impulsgeber für Finanzmärkte gelten. Konkret stelle ich heraus, ob Analysten stärker von anderen Analysten beeinflusst werden, wenn diese im gleichen Land bzw. in der gleichen Stadt arbeiten oder wenn sogar ein regelmäßiger Meinungsaustausch erfolgt. Beides setzte ich ins Verhältnis zum vorherrschenden Marktumfeld. In meinem dritten Aufsatz beschäftige ich mich mit der sozialen Interaktion zwischen Fondsmanagern. Diese verwalten in etwa ein Drittel des frei handelbaren Aktienvermögens und haben folglich einen nennenswerten Einfluss auf Finanzmärkte. Mit Hilfe einer neuartigen Schätzmethode bestimme ich die Größe des sozialen Einflusses und untersuche auch hier temporale Variationen im Verhältnis zum zu Grunde liegenden Marktumfeld. Des Weiteren zerlege ich die Gesamtgröße des sozialen Einflusses in zwei Komponenten, die zum einen den Einfluss im Rahmen der reinen Beobachtung und zum anderen den Einfluss durch Kommunikation reflektieren.

CD69 Is a TGF-β/1α,25-dihydroxyvitamin D3 Target Gene in Monocytes

Thea K. Wöbke; Andreas von Knethen; Dieter Steinhilber; Bernd L. Sorg — Fri, 17 May 2013 13:28:34 +0200

CD69 is a transmembrane lectin that can be expressed on most hematopoietic cells. In monocytes, it has been functionally linked to the 5-lipoxygenase pathway in which the leukotrienes, a class of highly potent inflammatory mediators, are produced. However, regarding CD69 gene expression and its regulatory mechanisms in monocytes, only scarce data are available. Here, we report that CD69 mRNA expression, analogous to that of 5-lipoxygenase, is induced by the physiologic stimuli transforming growth factor-β (TGF-β) and 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) in monocytic cells. Comparison with T- and B-cell lines showed that the effect was specific for monocytes. CD69 expression levels were increased in a concentration-dependent manner, and kinetic analysis revealed a rapid onset of mRNA expression, indicating that CD69 is a primary TGF-β/1α,25(OH)2D3 target gene. PCR analysis of different regions of the CD69 mRNA revealed that de novo transcription was initiated and proximal and distal parts were induced concomitantly. In common with 5-lipoxygenase, no activation of 0.7 kb or ~2.3 kb promoter fragments by TGF-β and 1α,25(OH)2D3 could be observed in transient reporter assays for CD69. Analysis of mRNA stability using a transcription inhibitor and a 3′UTR reporter construct showed that TGF-β and 1α,25(OH)2D3 do not influence CD69 mRNA stability. Functional knockdown of Smad3 clearly demonstrated that upregulation of CD69 mRNA, in contrast to 5-LO, depends on Smad3. Comparative studies with different inhibitors for mitogen activated protein kinases (MAPKs) revealed that MAPK signalling is involved in CD69 gene regulation, whereas 5-lipoxygenase gene expression was only partly affected. Mechanistically, we found evidence that CD69 gene upregulation depends on TAK1-mediated p38 activation. In summary, our data indicate that CD69 gene expression, conforming with 5-lipoxygenase, is regulated monocyte-specifically by the physiologic stimuli TGF-β and 1α,25(OH)2D3 on mRNA level, although different mechanisms account for the upregulation of each gene.

On the geometry, topology and approximation of amoebas

Timo de Wolff — Fri, 17 May 2013 12:31:07 +0200

We investigate multivariate Laurent polynomials f \in \C[\mathbf{z}^{\pm 1}] = \C[z_1^{\pm 1},\ldots,z_n^{\pm 1}] with varieties \mathcal{V}(f) restricted to the algebraic torus (\C^*)^n = (\C \setminus \{0\})^n. For such Laurent polynomials f one defines the amoeba \mathcal{A}(f) of f as the image of the variety \mathcal{V}(f) under the \Log-map \Log : (\C^*)^n \to \R^n, (z_1,\ldots,z_n) \mapsto (\log|z_1|, \ldots, \log|z_n|). I.e., the amoeba \mathcal{A}(f) is the projection of the variety \mathcal{V}(f) on its (componentwise logarithmized) absolute values. Amoebas were first defined in 1994 by Gelfand, Kapranov and Zelevinksy. Amoeba theory has been strongly developed since the beginning of the new century. It is related to various mathematical subjects, e.g., complex analysis or real algebraic curves. In particular, amoeba theory can be understood as a natural connection between algebraic and tropical geometry. In this thesis we investigate the geometry, topology and methods for the approximation of amoebas. Let \C^A denote the space of all Laurent polynomials with a given, finite support set A \subset \Z^n and coefficients in \C^*. It is well known that, in general, the existence of specific complement components of the amoebas \mathcal{A}(f) for f \in \C^A depends on the choice of coefficients of f. One prominent key problem is to provide bounds on the coefficients in order to guarantee the existence of certain complement components. A second key problem is the question whether the set U_\alpha^A \subseteq \C^A of all polynomials whose amoeba has a complement component of order \alpha \in \conv(A) \cap \Z^n is always connected. We prove such (upper and lower) bounds for multivariate Laurent polynomials supported on a circuit. If the support set A \subset \Z^n satisfies some additional barycentric condition, we can even give an exact description of the particular sets U_\alpha^A and, especially, prove that they are path-connected. For the univariate case of polynomials supported on a circuit, i.e., trinomials f = z^{s+t} + p z^t + q (with p,q \in \C^*), we show that a couple of classical questions from the late 19th / early 20th century regarding the connection between the coefficients and the roots of trinomials can be traced back to questions in amoeba theory. This yields nice geometrical and topological counterparts for classical algebraic results. We show for example that a trinomial has a root of a certain, given modulus if and only if the coefficient p is located on a particular hypotrochoid curve. Furthermore, there exist two roots with the same modulus if and only if the coefficient p is located on a particular 1-fan. This local description of the configuration space \C^A yields in particular that all sets U_\alpha^A for \alpha \in \{0,1,\ldots,s+t\} \setminus \{t\} are connected but not simply connected. We show that for a given lattice polytope P the set of all configuration spaces \C^A of amoebas with \conv(A) = P is a boolean lattice with respect to some order relation \sqsubseteq induced by the set theoretic order relation \subseteq. This boolean lattice turns out to have some nice structural properties and gives in particular an independent motivation for Passare's and Rullgard's conjecture about solidness of amoebas of maximally sparse polynomials. We prove this conjecture for special instances of support sets. A further key problem in the theory of amoebas is the description of their boundaries. Obviously, every boundary point \mathbf{w} \in \partial \mathcal{A}(f) is the image of a critical point under the \Log-map (where \mathcal{V}(f) is supposed to be non-singular here). Mikhalkin showed that this is equivalent to the fact that there exists a point in the intersection of the variety \mathcal{V}(f) and the fiber \F_{\mathbf{w}} of \mathbf{w} (w.r.t. the \Log-map), which has a (projective) real image under the logarithmic Gauss map. We strengthen this result by showing that a point \mathbf{w} may only be contained in the boundary of \mathcal{A}(f), if every point in the intersection of \mathcal{V}(f) and \F_{\mathbf{w}} has a (projective) real image under the logarithmic Gauss map. With respect to the approximation of amoebas one is in particular interested in deciding membership, i.e., whether a given point \mathbf{w} \in \R^n is contained in a given amoeba \mathcal{A}(f). We show that this problem can be traced back to a semidefinite optimization problem (SDP), basically via usage of the Real Nullstellensatz. This SDP can be implemented and solved with standard software (we use SOSTools and SeDuMi here). As main theoretic result we show that, from the complexity point of view, our approach is at least as good as Purbhoo's approximation process (which is state of the art).

Generation and analysis of mouse lines with a defect in platelet secretion and application in mouse models of atherosclerosis and tumor metastasis

Dagmar Schumacher — Fri, 17 May 2013 11:53:25 +0200

Introduction: The involvement of platelets in various diseases has been increasingly recognized in the recent decades. This contribution is believed to involve platelet secretion and formation of reactive microparticles. Platelets contain two functionally important forms of vesicles, alpha and dense granules, which are secreted upon activation of platelets. Alpha granules incorporate larger molecules such as adhesive proteins, e.g. P-selectin, vWF and fibrinogen; chemokines like PF4 and RANTES and growth hormones like VEGF and PDGF are among the most important proteins attributed to the involvement of platelets in pathological conditions. In contrast, dense granules contain small molecules like ADP, ATP, serotonin and histamine, and they are more rapidly and completely secreted than alpha granules. Like in all secreting cells, regulated exocytosis in platelets is mediated by “zippering” of three different classes of SNARE proteins. The subtypes of these proteins found to be involved in platelet secretion are SNAP-23, syntaxin-2 and -4 and VAMP-3 and -8. Apart from SNARE proteins, other conserved proteins influencing exocytosis by e.g. acting on SNARE proteins have been described, one of the most important ones being Munc13. Platelets contribute to the progression of atherosclerosis by local deposition of inflammatory mediators like PF4, RANTES and CD40L, which leads to enhanced leukocyte recruitment and plaque formation. In 1865, Armand Trousseau first described the correlation between cancer and thrombotic events. Since the 1960s, an increasing number of studies have found an involvement of platelets also in the progression of cancer, especially in the formation of metastases. Platelets bind to circulating tumor cells and may shield them from NK cell attacks and shear stress. Platelets may also facilitate the interaction of tumor cells with other cell types and the vessel wall. Lastly, they may secrete molecules that influence the tumor cell phenotype and invasiveness. Aims of this study: We sought to generate and describe genetically modified mouse lines with defective platelet secretion and to employ these mouse lines in murine models of atherosclerosis and tumor progression to study the role of platelet secretion under pathological in vivo conditions. Results: Clostridial toxins cleave members of the SNARE protein family and can thus completely block exocytosis of neuronal and other cells. We generated three transgenic mouse lines expressing tetanus, botulinum-E or -C light chains and two transgenic mouse lines with dominant-negative mutations of SNAP-23 under the control of the platelet-specific PF4 promotor. None of these constructs was able to interfere with platelet secretion despite expression of the transgene. A functional null mutant of the only Munc13 isoform expressed in platelets, Munc13-4, showed complete lack of dense granule secretion, measured by ATP release, while alpha granule release as determined by PF4 and vWF secretion, was unaltered. Morphology, composition and adhesion of these platelets were also normal. Aggregation in response to U46619 and collagen and formation of large aggregates in flow chamber assays was attenuated. Munc13-4-deficient mice showed a severe defect in bleeding time and no formation of stable aggregates in FeCl3 thrombosis model. In response to B16 melanoma and LLC1 carcinoma cells, Munc13-4 KO platelets also showed complete abrogation of dense granule secretion, whereas alpha granule secretion and binding of platelets to tumor cells was unchanged. Interestingly, wild-type platelets, but not Munc13-4 KO platelets, enhanced transmigration of B16 and LLC1 cells through an endothelial cell layer. Exogenous ATP was able to mimic the effect of wild-type platelets and the ATP-degrading enzyme apyrase blocked platelet-mediated tumor cell transmigration. Platelets incubated with tumor cells secreted large amounts of ATP. Murine endothelial cells showed perturbed adherens junctions identified by irregular VE-cadherin staining and gap formation when incubated with supernatants from tumor cell-activated platelets as well as increased permeability under the same conditions. Addition of apyrase preserved normal endothelial morphology and function. In vivo, primary tumor growth and weight was comparable in wild-type and Munc13-4 KO mice upon B16 or LLC1 flank injection but formation of lung metastases was strongly reduced. Number, but not size of metastases was also reduced upon i.v. injection of B16 and LLC1 cells. We found P2Y2 and P2X4 receptors to be the most abundantly expressed endothelial metabotropic and ionotropic ATP receptors, respectively. Neither knock-down nor inhibition of P2X4 in endothelial cells influenced platelet-mediated transendothelial migration of B16 cells, but knock-down of P2Y2, for which no specific antagonist is available, strongly reduced plateletdependent tumor cell transmigration. When B16 melanoma cells were injected i.v. shortly after FITC-dextran (70 kDa) into wild-type mice, prominent leakage of FITC-dextran was observed three hours post-injection at extraluminal sites in the lung. In contrast, leakage into the lung parenchyma was at basal levels in Munc13-4 KO and P2Y2 KO mice after B16 cell injection. Marginal vascular leakage in Munc13-4 KO mice lacking platelet ATP secretion and in P2Y2 KO mice lacking the main endothelial ATP receptor correlated with strongly reduced extravasation of CFSE-labeled B16 melanoma cells 6 hours post-injection in these mice. Consistently, P2Y2 KO mice showed strongly reduced formation of metastases in the lung after i.v. injection of B16 or LLC1 tumor cells. Bone marrow-transplanted LDLR KO mice reconstituted with Munc13-4-deficient or wildtype bone marrow and subjected to 16 weeks of high fat diet showed no significant difference in atherosclerotic plaque formation in the aorta. Discussion: We hereby provide a thorough analysis of a mouse line with an exclusive defect in platelet dense granule secretion, thus representing a unique genetic tool to study the role of dense granule secretion in various contexts without interfering with other platelet functions. We also provide evidence how extravasation of circulating tumor cells is facilitated by tumor cell-induced ATP release from platelets. This ATP release destabilizes endothelial barriers and facilitates tumor cell extravasation and formation of metastases in the target organ. Since metastasis is the leading cause of cancer death, pharmacological interference with endothelial P2Y2 receptor function may represent a promising therapeutic strategy.

Untersuchungen zur Bedeutung von Superoxid-Dismutasen für die Alterung von Podospora anserina

Sandra Zintel — Fri, 17 May 2013 11:16:13 +0200

Im Rahmen dieser vorliegenden Doktorarbeit sollte die Bedeutung von Superoxid-Dismutasen für das Resistenzverhalten und den Alterungsprozess bei P. anserina untersucht werden. Folgende Befunde aus den Analysen konnten erhalten werden: 1. Lokalisationsstudien der drei PaSods Aus den biochemischen und fluoreszenzmikroskopischen Untersuchungen der drei verschiedenen PaSODs geht hervor, dass PaSOD1, eine Cu/ZnSOD, überwiegend im Cytosol und zu einem geringen Anteil im mitochondrialen Intermembranraum lokalisiert ist. Eine der beiden MnSODs, PaSOD2, wird vermutlich zur Abwehr von exogenem Superoxid sekretiert. Bei PaSOD3 handelt es sich um eine mitochondriale MnSOD. 2. Generierung von verschiedenen PaSod-Mutanten Im Rahmen dieser Arbeit wurden von jeder PaSod mindestens drei unabhängige Überexpressionsstämme, ein GFP-Stamm- und ein Deletionsstamm hergestellt. Weiterhin wurden alle möglichen Doppel-Deletionsstämme und die Dreifach-Deletionsmutante erzeugt. Alle Stämme wurden auf DNA-Ebene verifiziert, zusätzlich wurde die Proteinmenge bzw. –Aktivität überprüft. 3. Einfluss der PaSODs auf die ROS-Toleranz Die Analysen der ROS-Resistenzen haben gezeigt, dass PaSODs eine wichtige Rolle in der Entgiftung von Superoxiden spielt. So ließ sich bei den Deletionsstämmen der PaSods eine gesteigerte Sensitivität gegenüber Paraquat feststellen. Eine Aufsummierung der Sensitivität gegenüber Paraquat ist bei der PaSod-Tripelmutante (ΔPaSod1/2/3) zu erkennen. Überraschenderweise kann durch die gesteigerten Mengen an aktiver PaSOD in den Überexpressionsstämmen (PaSod1-3_OEx) keine verbesserte Resistenz gegenüber Paraquat erzielt werden. Darüber hinaus führt die Überexpression des Gens für die mitochondriale SOD, PaSOD3, zu massiven negativen Effekten. 4. Einfluss auf die Lebensspanne Durch eine fehlende Entgiftung von Superoxid in den PaSod-Deletionsmutanten ist eine Verminderung der Lebensspanne nicht festzustellen. Bei PaSod-Mutantenstämme, die eine erhöhte PaSOD-Aktivität und damit eine gesteigerte Abbaurate des Superoxids aufweisen, kann bei den PaSod1- und PaSod2-Überexpressionsstämmen keine verbesserte Lebensspanne unter den gewählten Standardbedingungen erzielt werden. Vielmehr noch ist die Lebensspanne der PaSod3-Überexpressionsstämme stark reduziert. 5. Einfluss der PaSod-Modulation auf andere Komponenten des ROS-Abbausystems Die PaSOD-Aktivitäten scheinen miteinander co-reguliert zu werden. Des Weiteren scheint es ein Zusammenhang zwischen den beiden sekretierten Enzymen PaSOD2 und PaCATB zu geben. Deutlich wird auch, dass die Modulation der Superoxid-Dismutasen eine weitreichende Auswirkung auf andere Schutzsysteme hat. Beispielweise konnte gezeigt werden, dass Komponenten des mitochondrialen ROS-Schutzsystems und der Protein-Qualitätskontrolle in den PaSod3-Überexpressionsstämmen verändert sind. Zusammenfassend lassen die Analysen der PaSod-modulierten Stämme den Schluss zu, dass die Superoxid-Dismutase in P. anserina ein wichtiges Enzym zum Abbau des schädlichen Superoxids darstellt, welches aber nur eine untergeordnete Rolle bei der Kontrolle der Lebensspanne unter den gewählten Wachstumsbedingungen im Labor ausübt. Des Weiteren haben die Analysen gezeigt, dass es durch die Modulation der PaSod-Gene zu weitreichenden Änderungen, die das ROS-Schutzsystem (PaSOD, PaCATB und PaPRX1) sowie die Protein-Qualitätskontrolle (PaHSP60, PaLON und PaCLPP) betreffen, kommt. Welche Auswirkung dabei diese Veränderungen in Bezug auf die Lebensspanne hat, kann nur schwer abgeschätzt werden und muss mit weiteren Untersuchungen geklärt werden.

Grundlagen zum potentiellen therapeutischen Einsatz humaner natürlicher Killer-Zellen und spezifischer T-Zellen zur Bekämpfung invasiver Mykosen bei Patienten nach allogener Stammzelltransplantation

Stanislaw Schmidt — Fri, 17 May 2013 10:31:02 +0200

Nach allogenen Stammzelltransplantation und der damit einhergehenden lang andauernden Immuninkompetenz bzw. Immunsuppression sind die betroffenen Patienten stark für lebensgefährliche invasive Pilzinfektionen empfänglich. Trotz der derzeit verfügbaren antimykotischen Medikamente liegt die mit diesen Infektionen assoziierte Mortalität je nach Literatur bei 90 %. Aus diesem Grund rücken zelltherapeutische Behandlungen, die die Immunität der Patienten mit Pilzinfektionen verbessern und so die Sterblichkeit verringern könnten, immer mehr in den Fokus der Forschung auf diesem Gebiet. Leider ist die Interaktion von humanen Natürlichen Killer Zellen mit den wichtigen Verursachern invasiver Mykosen A. fumigatus und R. oryzae bisher nicht untersucht. Die Wichtigkeit von TH-Zellen in der Pilzabwehr wird zunehmend erkannt. Daher wurde bereits versucht die Prognose von Patienten mit invasiven Aspergillosen nach SZT durch den Transfer von Spender-Aspergillus-spezifischen T-Zellen zu verbessern. Die Generierung anti-R. oryzae T-Zellen sowie die Charakterisierung dieser Zellen ist bisher nicht durchgeführt worden. Deshalb sollte im ersten Teil dieser Arbeit sollte die antifungale Aktivität humaner NK-Zellen gegenüber A. fumigatus sowie R. oryzae untersucht und charakterisiert werden. Im zweiten Teil dieser Arbeit sollte die Generierung von spezifischen T-Zellen gegen R. oryzae aus Zellen des peripheren Blutes gesunder Individuen durchgeführt werden und die generierten Zellen im Hinblick auf ihre Funktionalität, Sicherheit und Wirkspektrum in vitro charakterisiert werden. In der vorliegenden Arbeit konnte die direkte antimykotische Aktivität humaner NK-Zellen gegenüber Hyphen von A. fumigatus und R. oryzae gezeigt werden und ein Mechanismus dieser Aktivität identifiziert werden. So induzierte der Kontakt von IL-2 stimulierten NK-Zellen mit Pilzhyphen die Degranulierung der NK-Zellen und die Ausschüttung von Perforin, welches zumindest partiell, die antimykotische Aktivität humaner NK-Zellen gegenüber A. fumigatus und R. oryzae Hyphen vermittelte. Die Ergebnisse dieser Arbeit zeigen allerdings auch, dass der therapeutische Nutzen adoptiv transferierter NK-Zellen durch einige Faktoren begrenzt werden könnte. Einerseits hatten NK-Zellen keinen zytotoxischen Effekt auf Konidien beider getesteten Pilze, andererseits deutet die beobachtete Immunsuppression darauf hin, dass ein stimulierender Effekt durch die transferierten NK-Zellen auf andere Zellen des Immunsystems durch die Hyphen der beiden Pilze inhibiert werden könnte. Die Ergebnisse der vorliegenden Arbeit zeigen, dass sich T-Zellen gegen R. oryzae in allen getesteten gesunden Individuen nachweisen lassen und diese isoliert und kultiviert werden können. Das deutet darauf hin, dass hinsichtlich anti-R. oryzae T-Zellen keine Restriktionen bei der Spenderauswahl zu erwarten sind. Die hergestellten Zellen konnten anhand der Expression ihrer Oberflächenantigene sowie des Profils der ausgeschütteten Zytokine dem TH1-Typ zugeordnet werden, welcher mit der protektive Immunantwort bei Pilzinfektionen assoziiert wird. Nach spezifischer Stimulation produzierten die generierten anti-R. oryzae T-Zellen die bei der Bekämpfung von Pilzinfektionen eine wichtige Rolle spielenden pro-inflammatorischen Zytokine IFN-γ und TNF-α und erhöhten die Aktivität der für die Abwehr von Pilzen wichtigen Granulozyten und Monozyten in vitro. Zwar zeigten die generierten T-Zellen bei Kontakt mit allogenen APZ eine geringe Proliferationsantwort, diese war jedoch mit der nach Stimulation mit autologen, unbeladenen APZ gesehenen Proliferation vergleichbar. Diese Ergebnisse zeigen, dass die generierten anti-R. oryzae T-Zellen zumindest in vitro ein deutlich geringeres alloreaktives Potential als unselektionierte T-Zellen haben. Entsprechende Resultate zeigten sich auch bei Betrachtung der IFN-γ Sekretion der generierten anti-R.oryzae T-Zellen. Die Stimulation mit Fremdspender-APZ beeinflusste die Sekretion von IFN-γ durch die anti-R. oryzae T-Zellen im Vergleich zu der IFN-γ-Sekretion durch anti-R. oryzae T-Zellen, mit autologen, unbeladenen APZ stimulierten wurden, kaum. Des Weiteren wiesen die hergestellten anti-R. oryzae T-Zellen zahlreiche Kreuzreaktivitäten gegenüber anderen Pilzspezies, auch Genera übergreifend, auf. So zeigten sie Kreuzreaktivität gegenüber klinisch bedeutenden Pilzen wie Rhizopus microsporus, Mucor circinelloides, Rhizomucor pusillus, Aspergillus fumigatus, Aspergillus niger, Candida albicans und Candida glabrata, die alle als Erreger invasiver Mykosen bei immunsupprimierten Patienten bekannt sind. Die Stimulation mit Antigenen dieser Pilze mittels Antigenpräsentierenden Zellen führte zur einen deutlichen IFN-γ-Produktion durch die generierten Zellen.

Validierung einer neuen Messmethode zur direkten Bestimmung der Heparin-Konzentration im Blut

Stephanie Haselbach — Thu, 16 May 2013 13:58:18 +0200

In dieser Arbeit wurde ein neues Verfahren zur Heparin-Bestimmung (LiSA-H, light scattering assay of heparin) evaluiert. Dieses wurde an der Universität Frankfurt a. M. am Institut für Biophysik entwickelt und ermittelt erstmals die direkte Heparin-Konzentration im Blutplasma. Durch die Analyse der Lichtstreuung einer Plasmaprobe wird die Bildung von Nanopartikeln aus Heparin und Protamin verfolgt. Die Lichtstreuintensität ist dabei proportional zu der in der Probe enthaltenen Heparin-Plasmakonzentration (Heparin-PK). Das Antikoagulans Heparin wird bei Herz-OPs mit Einsatz der Herz-Lungen-Maschine (HLM) verwendet und soll perioperativ eine lutgerinnselbildung in der HLM, sowie Thromboembolien im Patienten verhindern. Am OP-Ende wird die Wirkung durch Protamin antagonisiert, um eine suffiziente Gerinnung wieder herzustellen. Das derzeitige Gerinnungsmanagement basiert auf einem indirekten Messverfahren, der ACT (activated clotting time), welches starken Störeinflüssen, wie z.B. der Hypothermie, Hämodilution und bestimmten Medikamenten unterliegt. Durch die mögliche Falschdosierung der beiden Medikamente, steigt die Gefahr einer Blutung und Thrombose. LiSA-H soll in Zukunft eine zuverlässigere und kostengünstige „point of care― Analyse der Gerinnung und hierdurch eine gezielte Dosierung von Heparin und Protamin ermöglichen, die die Komplikationsrate verringert. In der vorliegenden Studie handelt es sich um eine offene, nicht kontrollierte, prospektive Multicenter-Studie, die mit 50 Patienten am Universitätsklinikum Frankfurt a.M. und 30 Patienten am Kinderherzzentrum Gießen durchgeführt wurde. Es wurde gezeigt, dass die durchschnittliche perioperative Heparin-PK bei Erwachsenen und Kindern bei ca. 5,4 I.E./ml liegt. Es wurde nachgewiesen, dass die Heparin-Clearance bei Kindern (~113 Min.) um das zweifache im Vergleich zu Erwachsenen (~254 Min.), erhöht ist. Besonders hervorzuheben ist die hohe Fehlerquote der ACT-Messung, die bei Erwachsenen in bis zu 1,8 % und bei Kindern in bis zu 20 % der Messungen keinen aussagekräftigen Wert lieferte. Das bedeutet, dass bei Kindern während einer OP bis zu zwei und bei Erwachsenen bis zu drei Stunden keine Information über den aktuellen Gerinnungszustand vorlag. Um eine Validierung der Messergebnisse vorzunehmen, wurden Rückstellproben mit dem Standardlaborverfahren PiCT (Prothrombinase induced clotting time) gemessen. Die Daten aus dem PiCT korrelieren mit den Ergebnissen aus der LiSA-H-Messung wesentlich besser (r² = 0,80), als mit der herkömmlichen ACT-Messmethode (r² = 0,57). Die ermittelten Heparin-PK und die ACT-Werte während einer OP wurden in Chronogrammen dargestellt. Es wurde gezeigt, dass in 30 % der OPs bei Erwachsenen und in 60 % bei Kindern die Messdaten aus der ACT und LiSA-H nur unzureichend synchron bei Nachdosierung mit Heparin anstiegen oder entsprechend der Heparin-Clearance im OP-Verlauf abfielen. Dies zeigte sich besonders kritisch während langandauernder, komplikationsreicher OPs, die einen erhöhten Blutverlust oder sogar Rethorakotomien nach sich zogen, in denen der ACT-Wert eine suffiziente Gerinnung nahe legte, die LiSA-HMessung aber eine noch hohe Heparin-PK nachwies. Erfahrungen aus den klinischen Studien zeigten, dass die Kombination aus der Messung der Heparin-PK und einer Gerinnungsanalyse bei einem ATIII-Mangel von Vorteil ist. Erst die Kombination aus einerseits mehrfach niedrig gemessener ACT-Werte, trotz ggf. Nachdosierungen von Heparin und andererseits ausreichend gemessener Heparin-PK im LiSA-H, kann einen ATIII-Mangelzustand aufdecken. Dadurch können Nach- bzw. Überdosierungen vermieden und damit die Wahrscheinlichkeit für postoperative Komplikationen verringert werden. Der wichtigste Einflussfaktor auf die LiSA-H-Messung ist die Hämodilution, die durch Einbeziehung des Patienten-Blutvolumens (z.B. mit der Nadler-Formel) durch mathematische Korrektur berücksichtigt werden kann. Patientenindividuelle Reaktionen auf gleiche Heparin- und Protamin-Dosierungen sowie eine patientenspezifische Heparin-Clearance zeigten in diesen Studien auf, dass das derzeitige Antikoagulationsmanagement mit den Dosierungsempfehlungen (Körpergewichtsbezogene Dosierung, 1:1 Dosierung von Protamin zur initialen Heparin-Dosis oder der summierten Heparin-Dosis, „pauschale― Nachdosierungen von 5.000 oder 10.000 I.E. Heparin bei ACT < 480) für eine optimale Dosierung der Medikamente unzureichend ist. In Outcome-Studien soll mit der LiSA-H-Messung Dosierungsempfehlungen von Heparin und Protamin ausgearbeitet werden. Außerhalb der Herz-Thorax-Chirurgie eröffnen sich weitere Möglichkeiten, wie z.B. in Dialysezentren und in der Neurochirurgie, für die bereits Studien geplant sind.