Studienauftrag «Potenzial von Gebäuden für Biodiversität und Landschaftsqualität in Agglomerationen» : Projekt A2.2 Aktionsplan Strategie Biodiversität Schweiz

Die zunehmende bauliche Verdichtung setzt das Grün und damit die Lebensbedingungen für Flora, Fauna und Menschen unter Druck. Es braucht neue Wege für qualitativ hochwertige Frei- und Grünräume innerhalb einer nachhaltigen Stadt- und Siedlungsentwicklung. Hierbei kommt der Begrünung von Gebäuden (Fassade und Dächer) und Massnahmen, welche die Ansiedlung von Tieren ermöglichen (Animal-Aided Design), eine bedeutende Rolle zu. Im Auftrag des BAFU wurden Handlungsanleitungen erarbeitet, die es sowohl Kantons- und Gemeindeverwaltungen als auch den Akteuren des privaten Planungs- und Bausektors erleichtern, das Potenzial von Gebäuden zur Förderung von Biodiversität und Landschaftsqualität besser auszuschöpfen

A Study of the PDGF Signaling Pathway with PRISM

In this paper, we apply the probabilistic model checker PRISM to the analysis of a biological system -- the Platelet-Derived Growth Factor (PDGF) signaling pathway, demonstrating in detail how this pathway can be analyzed in PRISM. We show that quantitative verification can yield a better understanding of the PDGF signaling pathway.Comment: In Proceedings CompMod 2011, arXiv:1109.104

Entwicklung und Charakterisierung eines IL-6-Inhibitors basierend auf Funktionsanalysen des murinen IL-6R alpha

Among other humoral factors, cytokines get secreted by a variety of cell types in response to different external stimuli and induce a multitude of biological effects. IL-6-type cytokine family members activate the Jak/STAT signalling pathway and hence modulate processes such as apoptosis, proliferation, angiogenesis and migration of cells. The cytokine IL-6 possesses great clinical relevance because dysregulation of IL-6 expression can be observed in many chronic inflammatory diseases as well as in cancer. A particular concept for IL-6 inhibition was achieved by preventing receptor activation through sequestration of the cytokine. In a previous study, an IL-6 inhibitor was constructed by a fusion of the ligand binding domains of the human IL-6 receptor components, namely domains D1 to D3 of gp130 and D2-D3 of IL-6R alpha. The present study aimed at the generation of an IL-6-inhibitor based on the murine receptor proteins for the future validation of the fusion protein concept in aminal studies. Although murine and human IL-6R alpha and gp130 proteins show a very high degree of homology, no IL-6 binding could be achieved by an analogous fusion of murine receptor domains. This unexpected result illustrates functional differences in respect of ligand binding between human and murine IL-6R alpha. Based on a model structure of murine IL-6R alpha derived from the solved crystal structure of the human IL-6 receptor complex, a set of amino acids was identified which could potentially influence IL-6 binding and account for the observed differences in the human and murine systems. By generation of specific deletion- and point-mutations within the immunoglobulin-like domain of the full-length murine IL-6R alpha protein the functional relevance of particular amino acid residues within a 12 amino acid stretch (preD1-region) was investigated. This region at the amino-terminus of the protein is connected to domain D2 via a disulfide bond linking the cysteine residues C6 and C171. The disulfide bond is important for the positioning of several hydrophobic N-terminal amino acids (LVL-motif) which interact with amino acid residues located in the beta-strands C and D of domain D2, a region which is in the immediate vicinity of the so-called site I-interface between IL-6R alpha and IL-6. Using life cell imaging and flow cytometry, it was shown that the deletion or substitution of Cysteine C6 within the preD1-region of murine IL-6R alpha shows a dominant negative effect both on localization (surface expression) and protein function (IL-6 binding). By the mutation of this particular cysteine a mutation outside the „classical“ CBM was generated which impairs IL-6 binding. Another motif of functional relevance in murine IL-6R alpha consists of three hydrophobic N-terminal amino acid residues (L1V2L3-motif). By the generation of a triple mutant where the hydrophobic amino acids were replaced by amino acids with small polar side chains, the cooperative effect of these residues in regard to IL-6 binding was characterized. Substitution of this motif resulted in a significantly diminished activation of the JAK/STAT-signal transduction pathway despite a comparable surface expression of the receptor constructs. At a later stage, it could also be shown that this mutation showed reduced IL-6 binding in the context of a mIL-6R alpha/gp130 fusion protein (inhibitor concept). Based on these findings, the protein domains which are crucial for ligand binding in the murine receptor system were defined and a functional IL-6 inhibitor was generated. For this, the domains D1 to D3 of murine gp130 were connected to domains D1 to D3 of murine IL-6R alpha. This mIL-6-R fusion protein (mIL-6-RFP) was capable of neutralizing IL-6 of different species (human, mouse, rat) with comparable efficiency, whereas no inhibition of structurally related cytokines of the IL-6-type-cytokine-family such as IL-11 and OSM was detectable. In vitro, mIL-6-RFP showed a 30-times stronger inhibition of IL-6-dependent proliferation of cells than an antagonistic IL-6R alpha antibody. Moreover, it was demonstrated that mIL-6-RFP efficiently inhibited the so-called trans-signalling which is elicited by IL-6 in combination with a soluble form of IL-6 receptor. In the course of this study a strategy for optimization of protein secretion was established, resulting in a considerable increase of protein amount in mammalian expression systems. The generated mIL-6-RFP also displayed a satisfactory stability in plasma samples of animals if expressed in-vivo in rats. With the generation of mIL-6-RFP a highly potent and specific IL-6 inhibitor is available for the use in animal studies (mouse/ rat). This will permit a further characterization of the role of IL-6 in various disease models and could ultimately lead to the use of this macromolecular inhibitor in anti-IL-6 therapy

A Probabilistic Boolean Network Approach for the Analysis of Cancer-Specific Signalling: A Case Study of Deregulated PDGF Signalling in GIST.

BACKGROUND: Signal transduction networks are increasingly studied with mathematical modelling approaches while each of them is suited for a particular problem. For the contextualisation and analysis of signalling networks with steady-state protein data, we identified probabilistic Boolean network (PBN) as a promising framework which could capture quantitative changes of molecular changes at steady-state with a minimal parameterisation. RESULTS AND CONCLUSION: In our case study, we successfully applied the PBN approach to model and analyse the deregulated Platelet-Derived Growth Factor (PDGF) signalling pathway in Gastrointestinal Stromal Tumour (GIST). We experimentally determined a rich and accurate dataset of steady-state profiles of selected downstream kinases of PDGF-receptor-alpha mutants in combination with inhibitor treatments. Applying the tool optPBN, we fitted a literature-derived candidate network model to the training dataset consisting of single perturbation conditions. Model analysis suggested several important crosstalk interactions. The validity of these predictions was further investigated experimentally pointing to relevant ongoing crosstalk from PI3K to MAPK signalling in tumour cells. The refined model was evaluated with a validation dataset comprising multiple perturbation conditions. The model thereby showed excellent performance allowing to quantitatively predict the combinatorial responses from the individual treatment results in this cancer setting. The established optPBN pipeline is also widely applicable to gain a better understanding of other signalling networks at steady-state in a context-specific fashion

Characterization of the Interleukin (IL)-6 Inhibitor IL-6-RFP: fused receptor domains act as high affinity cytokine-binding proteins.

Although fusion proteins of the extracellular parts of receptor subunits termed cytokine traps turned out to be promising cytokine inhibitors for anti-cytokine therapies, their mode of action has not been analyzed. We developed a fusion protein consisting of the ligand binding domains of the IL-6 receptor subunits IL-6Ralpha and gp130 that acts as a highly potent IL-6 inhibitor. Gp130 is a shared cytokine receptor also used by the IL-6-related cytokines oncostatin M and leukemia inhibitory factor. In this study, we have shown that the IL-6 receptor fusion protein (IL-6-RFP) is a specific IL-6 inhibitor that does not block oncostatin M or leukemia inhibitory factor. We characterized the complex of IL-6-RFP and fluorescently labeled IL-6 (YFPIL-6) by blue native PAGE and gel filtration. A 2-fold molar excess of IL-6-RFP over IL-6 was sufficient to entirely bind IL-6 in a complex with IL-6-RFP. As shown by treatment with urea and binding competition experiments, the complex of IL-6 and IL-6-RFP is more stable than the complex of IL-6, soluble IL-6Ralpha, and soluble gp130. By live cell imaging, we have demonstrated that YFP-IL-6 bound to the surface of cells expressing gp130-CFP is removed from the plasma membrane upon the addition of IL-6-RFP. The apparent molecular mass of the IL-6.IL-6-RFP complex determined by blue native PAGE and gel filtration suggests that IL-6 is trapped in a structure analogous to the native hexameric IL-6 receptor complex. Thus, fusion of the ligand binding domains of heteromeric receptors leads to highly specific cytokine inhibitors with superior activity compared with the separate soluble receptors

Approximation of the steady-state distribution in the <i>optPBN</i> framework.

<p>The dynamic behaviour of the PBN model was represented as a Markov chain. A small perturbation parameter ‘p’ was introduced to ensure the ergodicity of the respective Markov chain which is rendered <i>irreducible</i> (all states can be reached by any other states) and <i>aperiodic</i> (all states can be revisited in a non-periodic manner) thus possessing a unique steady-state distribution independent of the initial conditions. The two-state Markov chain approach was subsequently applied to determine the number of necessary time steps to reach the steady-state (burn-in period) and to collect sufficiently large number of samples in order to approximate the marginalised steady-state distribution of the output states for a defined (adjustable) accuracy. Note that multiple evaluations of the burn-in period and collected samples might be needed. The approximated steady-state distribution corresponds to the probability of the output node(s) being ON, i.e. being 1, as determined from the collected samples.</p