ABSTRACTS

Editors:Michael BrandPetra SchwilleAndrea A. RobitzkiPeter Seibel

SAXON BIOTECHNOLOGY SYMPOSIUM

2007

Biotechnology Center

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IMPRESSUM

Editors Prof. Dr. Michael Brand Director and Professor of Developmental Genetics,

BIOTEChnology Center Dresden, Technische Universität Dresden

Prof. Dr. Petra Schwille Professorship for Biophysics, BIOTEChnology Center Dresden, Technische Universität Dresden

Prof. Dr. Andrea A. Robitzki Chair of the Managment Board, Center for Biotechnology and Biomedicine, Universität Leipzig Prof. Dr. Peter Seibel Head of Professorship for Molecular Cell Therapy, Center for Biotechnology and Biomedicine, Universität Leipzig Compilation Robert Männel

BIOTEChnology Center Dresden, Technische Universität Dresden

Dr. Svenne Eichler Chief Executive Officer of the Center for Biotechnology and Biomedicine, Universität Leipzig

Layout Antje Ferrier Center for Biotechnology and Biomedicine, Universität Leipzig Robert Männel BIOTEChnology Center Dresden, Technische Universität Dresden Print addprint AG Editorial Deadline November 13, 2007

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CONTENT

FOREWORD 19

PRESENTATIONS

NEW TECHNOLOGICAL DEVELOPMENTS 21

Microfluidics Dr.Andreas Manz 22 Screening with force Spectroscopy Dr. Jens Struckmeyer, nAmbition GmbH 23 ZnO Nanowires Prof. Dr. Marius Grundmann 24 Biomolecular Motors at work Dr. Stefan Diez 25 Feeling for cells with lights Dr. Josef A. Käs 26 Microfluidic components & systems for Biotechnology Dr. Steffen Howitz 27 MEDICAL BIOTECHNOLOGY 29 Tissue Engineering Dr. Simon Hoerstrup 30 Haematology/Oncology Dr. Michael Cross 31 Development of Site-Specific Recombinases for Genome Surgery Dr. Frank Buchholz 32 Tissue Engineering of bone with rat marrow stromal cells Prof. Dr. Michaele Schulz-Siegmund 33 Potential of tissue specific neural stem cells Prof. Dr. Johannes Schwarz 34 DNA Engineering for drug discovery Prof. Dr. Francis Stewart 35 Raft Technology: The new frontier in Pharmaceutical Development Charl van Zyl 36

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POSTERS 39 MOLECULAR MEDICINE ID 1 The role of Wnt/beta-catenin signaling in zebrafish heart regeneration Kristin Schnabel, Katja Pfeifer, Gilbert Weidinger 40 ID 2 Nucleo-cytoplasmic shuttling of the Golgi phosphatidylinositol 4-kinase Pik1 is regulated by 14-3-3 proteins and coordinates Golgi function with cell growth Mike Beck, Lars Demmel, Anne-Lore Schlaitz, Peggy Hsu, Jan Havlis, Andrej Shevchenko, Eberhard Krause, Yannis Kalaidzidis Christiane Walch-Solimena 41 ID 3 Identification of Wnt target genes involved in zebrafish fin regeneration Birgit Kagermeier-Schenk, Herman Spaink, Gilbert Weidinger 42 ID 4 Imitating protein ß-hairpins with more robust ß-peptide folds Carsten Baldauf, Maria Teresa Pisabarro 43 ID 5 Regulator of G-protein signalling 9-, isoform 2 (RGS9-2): modulation of NMDA receptor signalling and neuronal plasticity Kathy Busse, Rainer Strotmann, Torsten Schöneberg, Johannes Schwarz 44

ID 6 Biphasic effect of TGF-beta on Th17-cell development Julia Schumann, Uwe Müller, Jens Knauer, Reinhard Straubinger, Manfred Blessing 45 ID 7 The Transcription Factor Tbx15: Expression and Subcellular Localization During Adipocyte Differentiation David Kosel, Annette Beck-Sickinger 46

ID 8 Distribution of mitofusin 2 (Mfn2) after the formation of megamitochondria Christian Kukat, Ingo Schäfer, Alexandra Kukat, Peter Seibel 47 ID 9 Medicinal plants, nutritional supplements and menopausal health Günter Vollmer, Oliver Zierau, Georg Kretzschmar 48 ID 10 Investigation of the Functionality of Growth Hormone Secretagogue Receptor 1a (GHSR1a) and Motilin Receptor (MTLR) Chimaera Enrico Schild, Annette Beck-Sickinger 49

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ID 11 Evidence for an additional risk factor in the HLA region modifying the risk from known HLA-DRB1 risk alleles in Rheumatoid Arthritis Peter Ahnert, Holger Kirsten, Grit Wolfram, Jörg Reichardt, Dirk Anders, Kathleen Hofmann, Saskia Ruhland 50

ID 12 TGF-β targets in T-cells Samiya Al-Robaiy, Bernhard Hoflack, Manfred Blessing, Cornelia Czupalla, Theresia Pursche 51 ID 13 Reverse transcriptase of HIV as a target for directed evolution Bianca Heyn, Sascha Stumpp, Matthias Exner, Susanne Brakmann 52 ID 14 Chimeric Y-Receptors to understand Receptor Subtype specific Ligand Binding of Neuropeptide Y Jan Stichel, Annette Beck-Sickinger 53 ID 15 Formation of Virus-like particles consisting of the major capsid protein VP1 of avian- and non-human Polyomaviruses in yeast Saccharomyces Cerevisae Anja Zielonka 54 ID 16 Engineering Human Proteases for Therapeutical Use Stefan Kappeler 55 ID 17 Mitochondrial import of peptide conjugated DNA Ingo Schäfer, Christian Kukat, Alexandra Kukat, Peter Seibel 56 ID 18 Characterization of the fibre outgrowth in organotypic dopaminergic slice co-cultures Claudia Heine, Annett Wegner, Jens Grosche, Peter Illes, Heike Franke 57 ID 19 Modification of the Regenerative Chemokine SDF1 α to Allow Fluorescence Imaging Kathrin Bellmann, Lars Baumann, Annette Beck-Sickinger 58 ID 20 Extracellular Nucleotides govern human neural precursor cell Proliferation and dopaminergic Differentiation Javorina Milosevic, Sigrid Schwarz, Patrizia Rubini, Alexander Storch, Peter Illes, Johannes Schwarz 59 ID 21 Long-term multilineage Engraftment of human cells in NOD/SCID Mice Manuela Ackermann, Frank Emmrich, Manja Kamprad 60

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ID 22 Interaction of leucocyte Antigen CD97 and the Activasion associated endothelial Receptor THY-1 (CD90) Elke Wandel, Manja Wobus, Anja Saalbach, Ulf Anderegg, Gabriela Aust 61 ID 23 Profile of GFP Release derived from transformed fetal Hepatocytes in Alginate Microcapsules Aldo Leal-Egana, Jochen Lach, Isabel Rode, Andrea A. Robitzki, Augustinus Bader 62 ID 24 Synthesis and characterization of peptide oligodeoxyribonucleotide Conjugates Lena Schönzart, Jan Michael, René Beutner, Dieter Schwarnweber Hartmut Worch, Bernd Schwenzer 63

ID 25 Ligand Binding at the human Y5-Receptor Diana Lindner, Jan van Diek, Robert Günther, Hans-Jörg Hofmann, Annette Beck-Sickinger 64 ID 26 Functionalised carbon nanotubes for biomedical applications Rüdiger Klingler, Silke Hampel, Anja Wolter, Kamil Lipert, Matthias Lutz, Diana Haase, Yulia Krupskaya, Christopher Mahn, Arthur Taylor, Kai Krämer, Manfred Wirth, Bernd Büchner 65 ID 27 Dendritic cells rather than macrophages produce interleukin (IL)-12 Cytokine family members in response to Salmonella Enteritidis in vitro and in vivo Sabine Siegemund, Nicole Schütze, Marina Freudenberg, Reinhard Straubinger, Gottfried Alber 66 ID 28 Population genetics of the GPR33 pseudogene Iris Böselt, Peter Kovacs, Michael Stumvoll, Holger Römpler Torsten Schöneberg 67 ID 29 Synaptic Localisation of NPY Y1 Receptors in the Rat Cingulate Cortex Kathrin Sichardt, Wolfgang Härtig, Annette Beck-Sickinger, Karen Nieber 68

ID 30 Time-Dependened Desensitisation of the Adnosine A1-Receptor During Hypoxia Cornelia Rufke, Andreas Deussen, Karen Nieber 69

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ID 31 IL-23-deficient mice are restitant against Salmonella Enteritidis infection but are defective in IL-17 production and the delayed-type hypersensitivity (DTH) response Silke Schulz, Alissa Chackerian, Robert Kastelein, Gottfried Alber 70 ID 32 Determination of Cell-Biological Parameters of Non-activated and activated (LPS) Monocytes in response to different synthetic soft Materials under the Influence of shear stress Erik Schilling 71 ID 33 An altered renal sodium-reuptake contributes to osmotic dysregulation in V2 vasopressin receptor-deficient mice Nicole Schliebe, Rainer Strotmann, Torsten Schöneberg 72 ID 34 A unique Infrastructure for High-Content Screening is mediating Technology Transfer from Academia to Industry Eberhard Krauß, Ivan Baines, Marino Zerial 73 ID 35 Small Molecules as Agonists of Hedgehog signaling and their use for wound Healing Katrin Seifert, Thomas Cottin, Anita Büttner, Athanassios Giannis 74 ID 36 Peptide prolyl isomerases can act as a molecular timer to control the Amplitude and duration of Stat3 activity Kay Bauer, Friedemann Horn 75 ID 37 In-situ-biotinylation – An approach to investigate the STAT3 signaling Pathway Conny Blumert, Friedemann Horn 76 ID 38 Expression of human full length adiponectin and its globular variant in E. coli John Heiker, Annette Beck-Sickinger 77

BIOANALYTICS 79 ID 39 Hygienic properties of altenative surfaces Ursula Eschenhagen, Marc Mauermann, Jens-Peter Majschak, Thomas Bley 80

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ID 40 Monitoring gasförmiger Metabolite in Feststoffreaktoren Tommy Opitz 81 ID 41 Myeloperoxidase binds to non-vital spermatozoa on phosphatidylserine epitopes Jacqueline Leßig, Uta Reibetanz, Martin Fischlechner, Jürgen Arnhold, Hans-Jürgen Glander 82 ID 42 Interaction of Cells with Fluorescein-Labeled Polyelectrolyte Multilayer Particles and Capsules Uta Reibetanz, David Haložan, Martin Fischlechner, Milan Brumen, Edwin Donath 83 ID 43 The influence of organic solvents on the enzymatic synthesis of large-ring cyclodextrins produced by an amylomaltase from Thermus aquaticus ATCC 33922 Nicole Weizenmann, Susanne Washeim, Christian Roth, Norbert Straeter, Wolfgang Zimmermann 84 ID 44 The PFKFB3 splice variant UBI2K4 inhibits cell proliferation of U87 glioblastoma cells Kristin Köllner, Renate Kessler, Klaus Eschrich 85 ID 45 Development of a new metabolic labelling approach (Protein-SIP) for proteome based identification of bacteria with specialized functions in mixed cultures Nico Jehmlich, Martin von Bergen, Carsten Vogt, Hans-Hermann Richnow 86 ID 46 High throughput library screening for novel lipase activities Sally Bayer, Meike Ballschmiter, Thomas Greiner-Stöffele 87 ID 47 Searching new enzymes for glucose and lactate biosensors in Metagenomic libraries Michael Hofer, Thomas Griner-Stöffele, Katrin Boensch 88 ID 48 Analysis of microbial processes in a small-scale calorimeter with additional sensors Regina Huettl, Joachim Harmel, Andeas lissner, Gert Wolf, Karl Held, Peter Klare, Winfried Vonau, Sigrun Hermann, Frank Berthold 89

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ID 49 RNase P and MRP: function and architecture in plants Mario Krehan, Sebastian Braun, Janine Dahl, Nikolas Menzel, Christian Heubeck, Astrid Schön 90 ID 50 Immobilization of chitosan from molds onto glass beads for the use in biosorption columns Mirko Trutnau, Thomas Bley, Jelka Ondruschka 91 ID 51 In vitro expression and high throughput screening for novel proteases Antje Eichler, Thomas Greiner-Stöffele, Meike Ballschmiter 92 ID 52 Regeneration of retinal Tissue – clearance of Edema by Triamcinolone Acetonide Ianors Iandiev, Ortrud Uckermann, Antje Wurm, Thomas Pannicke, Peter Wiedemann, Andreas Reichenbach, Andreas Bringmann, Susann Uhlmann 93 ID 53 Optical Tweezers to Study Single Receptor / Ligand Interactions Mathias Salomo, Ulrich Keyser, Marc Struhalla, Friedrich Kremer 94 ID 54 Induction of oxidative stress in the MCF7/NeuT cell line overexpressing the receptor tyrosine kinase ErbB2 Anika Schumann, Matthias Hermes, Matthias Gilbert, Jan G. Hengstler, Christian Wilhelm 95 ID 55 Strategy for altering the substrate specificity of P450-Monooxy-genases Gregor Hoffmann, Katrin Boensch, Thomas Greiner-Stöffele 96 ID 56 Studies of Interaction between a Novel Probe and Raft-borne Sphingolipids Elke Boschke, Richard Wetzel, Steffen Steinert, Sarita Hebbar, Guillaume Tresset, Rachel Kraut 97 ID 57 Structural studies of human cAMP specific phosphodiesterase 4A Roy Eylenstein, Bartholomeus Küttner, Susanne Moschütz Norbert Sträter 98 ID 58 Comparison of quantum dots with an organic fluorescent dye tagged to Cholera Toxin B for bioimaging applications Sylvie Rockel, Rachel Kraut 99

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ID 59 Cloning expressions and purification of a lactone hydrolase from soil Bacteria Christian Roth, Silke Wegener, Michael Schlömann, Norbert Sträter 100 ID 60 Structure and function of ecto-nucleotide diphosphohydrolases in Purinergic signaling Norbert Sträter, Matthias Zebisch 101 ID 61 Combined Ultrasonic and Confocal Laser scanning Microscopy of Biological Objects Evgeny Twerdowski, Albert Kamany, Esam Ahmed Mohamed, Wolfgang Grill, Reinhold Wannemacher 102 ID 62 NMR-micro-probe for mass limited biological samples Thomas Riemer, Stefan Leidich 103 BIOINFORMATICS 105 ID 63 MD analysis of water-mediated interactions in protein interfaces Sergey Samsonov, Joan Teyra, Maria Teresa Pisabarro 106 ID 64 Analysis and classification of the Protein Interactome Joan Teyra, Maria Teresa Pisabarro 107 ID 65 ParaDockS: A Parallel Framework for Molecular Docking Frank Brandt, Carsten Baldauf, Rene Meier, Maria Teresa Pisabarro 108 ID 66 Unfolding pathway recognition of membrane proteins by the use of wavelet techniques William Andreopoulus, Alexander Andreopoulus, Dirk Labudde, Michael Schroeder 109 ID 67 How to manage the massive amounts of biotech data in the future? Christian Panse, Simon Barkow-Oesterreicher, Can Tuerker, Bertran Gerrits, Bernd Roschitzki, Dieter Joho, Ralph Schlapbach 110 ID 68 Understanding of SMFS barriers by means of energy profiles Frank Dressel, Annalisa Marsico, Anne Tuukkanen, Dirk Labudde, Michael Schroeder 111

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ID 69 Development and application of automated structure-based functional protein annotation methods Aurelie Tomczak, Maria Teresa Pisabarro 112 ID 70 Optimization of the biocatalytic synthesis of large-ring cyclodextrins Mohd Noriznan Mokhtar, Melanie Gruner, Wolfgang Zimmermann 113

ID 71 DIAF: A High-Throughput Data Integration and Analysis Framework for Functional Annotation of Genes Maciej Paszkowski-Rogacz, Maria Teresa Pisabarro, Frank Buchholz 114 ID 72 CREx - A Tool for Analyzing Genomic Rearrangement Operations Based on Common Intervals Mathias Bernd, Daniel Merkle, Kai Ramsch, Guido Fritzsch, Marleen Perseke, Detlef Bernhard, Martin Schlegel, Peter F. Stadler, Martin Middendorf 115

ID 73 A Method for Solving the Preserving Reversal Median Problem Matthias Bernt, Daniel Merkle, Martin Middendorf 116 ID 74 An annotation platform for structural bioinformatics Gerd Anders 117 ID 75 An Automatic Analysis of Cartilage Regeneration in a Model of Human/Murine SCID arthritis Nico Scherf, Ulf-Dietrich Baumann, Jens-Peer Kuska, Manja Kamprad, Franziska Lange, Ulrich Sack 118 ID 76 Bioplastic Polyhydroxybutyrate Karl-Michael Meiß 119 ID 77 Evolutionary and functional Analysis of the Redß/RecT Single-Strand annealing Protein Family Axel Erler, Madeleine Teucher, Marcello Maresca, Youming Zhang Bianca Habermann, Francis Stewart, Vineeth Surendranath 120

ID 78 Automated Data Flow and Quality Control Practise in High-Content Screening Karol Kozak 121

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TISSUE ENGINEERING 123 ID 79 Tissue engineering scaffold biomaterial and 3D-culture influence expression of the chondrogenic master transcription factor Sox9 Peter Bernstein, Meng Dong, Sylvi Graupner, Denis Corbeil, Michael Gelinsky, Klaus-Peter Günther, Stefan Fickert 124 ID 80 Interphase- Fluorescence in situ Hybridization- technique for detecting human stem cells in frozen xenogeneic tissue Peter Ahnert, Heidrun Holland 125 ID 81 Cryopreservation of 3D Tissue Engineering Constructs Gabriele Spörl, Günter Lauer, Edith Klingner 126 ID 82 Establishing the Basis of Tissue Engineering: developing in vitro models for bone biomineralization, remodeling, vascularization and nanotoxicity Ralf Rühl, Ulla König, Armin Springer, Anne Bernhardt, Anja Lode, Eckehart Rudolph, Thomas Hanke, Michael Gelinsky 127 ID 83 In vitro cultivation of osteoblast precursor cells on novel scaffolds for hard tissue engineering Anne Bernhardt, Anja Lode, Corina Vater, Beatrice Burmeister, Anja Walther, Thomas Hanke, Michael Gelinsky 128 ID 84 Semi-automated radiosynthesis and in vitro testing of [18F] fluoroetanidazole as a new surrogate marker for PET imaging of stem cell therapy in stroke Udo Großmann, Marianne Patt, Andreas Schildan, Heike Franke, Petter Illes, Frank Emmrich, Osama Sabri, Henryk Barthel 129 ID 85 Investigation of Scaffold Materials for the Tissue Engineering of Cryopreservable Artificial Tissue Constructs Holger Reinsch, Gabriele Spörl 130 ID 86 Isolation and Characterisation of Human Melanocytes for Clinical Use Christina Fieber, Ulf Anderegg, Jan C. Simon, Andreas Emmendörffer 131

ID 87 Development of an automated 3D bioprocess for osteogenic differentiation of primate embryonic stem cells Susanne Trettner, Erika Sasaki, Nicole zur Nieden 132 12

 

ID 88 Comprehensive in vitro model for the engineering of microvasculature - evaluation of morphological and functional data Karsten Werner, Ulf Wehrmeister, Antje Böttner, Jens-Peer Kuska, Bernhard Frerich 133 ID 89 Quantitative evaluation of adipose tissue engineering in SCID mice Bernhard Frerich, Karsten Winter, Konstanze Weinzierl 134 ID 90 rhBMP-2 Delivery From Scaffolds, Microparticles And In Situ-forming Gels Alexander Lochmann, Hagen Nitzsche, Sabrina von Einem, Elisabeth Schwarz, Karsten Mäder 135 ID 91 Xenogeneic infection risk and Restriction of individual Rights: an European Approach (EU, German, Spanish, and English Law) Jorge Guerra 136 ID 92 Correlation between nitriding parameters of medical CoCr alloys and resulting hardness increase and reduction of wear rate Johanna Lutz, Inga-Maria Eichentopf, Antje Lehmann, Stephan Maendl 137 ID 93 Determination of the Cell/Aggregate growth Profile inside Alginate Capsules Aldo Leal-Egana, Felicia Heidebrecht, Isabel Rode, Andrea A. Robitzki Augustinus Bader 138 ID 94 A bead Model for the Realization f 3D Tissue Cytometry Anja Mittag, Attila Tarnok 139 ID 95 rhBMP-2 Delivery from Scaffolds, Microparticles and in Situ-forming Gels Fritzi Siegert, Anja Zlobinsky, Anja Grahnert, Gunther Böttcher, Kathrin Reiß, Andreas Schubert, Sunna Hausschildt, Karen Nieber 140 ID 96 Entwicklung eines High Content Screening Systems zur Untersuchung des Einflusses der extrazellulären Matrix auf Adhäsion und Proliferation und Differenzierung von humanen neuralen Stammzellen Holger Stephan 141 ID 97 Correlation between nitriding parameters of medical CoCr alloys and resulting hardness increase and reduction of wear rate Stefan Schwan, Amanda Hughes, Andrea Staeudte, Frauke Junghans, Uwe Spohn, Andreas Heilmann 142

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ID 98 Characterization of a new Material for human mesenchymal stem cell-based Bone Tissue Engineering Kathleen Schröck, Dirk Schumann, Manja Kamprad 143 ID 99 Stem Cell based Bone Graft Design with a Perfusion and Rotation vs Hydrogal Bioreactor Jan Liese, Susanne Liese, Bernhard Frerich, Augustinus Bader, Alexander Hemprich 144 ID 100 Investigations with scanning electron Microscopy (SEM) and focused Ion Beam (FIB) on Cartilage Tissues Andrea Stäudte, Andreas Cismak, Stefan Schwan, Ronny Schulz Uwe Spohn, Andreas Heilmann 145 CELL ENGINEERING 147 ID 101 Influenza vaccine production with adherent Vero cells Christian Dietzsch, Yvonne Genzel, Thomas Bley 148 ID 102 Characterization of voltage-gated potassium channels in midbrain derived neural precursor cells Grit Schaarschmidt 149 ID 103 New Insights into Redβ-mediated DNA Annealing using Atomic Force Microscopy Axel Erler, Susanne Wegmann, Celine Elie-Caille, Marcello Maresca Ralf Seidel, Tobias Heine, Daniel J. Müller, Francis Stewart 150 ID 104 Flow cytometric and phytochemical investigations with plant cell suspension cultures of sunflower (Helianthus annuus) Christiane Haas, Milen Georgiev, Jost Weber, Jutta Ludwig-Müller Thomas Bley 151 ID 105 Transplantation of human derived neural precursor cells into 6-OHDA-rat-model 11 Johanna Scheibe 152 ID 106 Analyses of primary human fetal cultures and derived neural stem cells in vitro 12 Florian Wegner, Monike Poppe, Javorina Milosevic, Maja Dieterlein, Johanna Scheibe, Andreas Boldt, Sigrid Schwarz, Johannes Schwarz 153

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ID 107 Simulation of embryogenic conditions to differentiate stem cells into functional neurons Sabine Schewtschik, Grit Schaarschmidt 154 ID 108 Flow cytometry investigations of Datura innoxia Jost Weber, Vasil Georgiev, Mladenka Ilieva, Atanas Pavlov, Thomas Bley 155

ID 109 Flow cytometry investigations of Beta vulgaris cv. Egypt and in vitro systems obtained from it Vasil Georgiev, Jost Weber, Atanas Pavlov, Mladenka Ilieva, Thomas Bley 156 ID 110 Cholesterol and apolipoproteins enhance neurite outgrowth and synapse Formation in dorsal root ganglia (DRG) cultures Joanna Kosacka, Martin Gericke, Marcin Nowicki, Jürgen Borlak Katharina Spanel-Borowski 157 ID 111 Fusion and cellular reprogramming Isabelle Hanisch, Alexandra Stolzing 168 ID 112 Development of a screening tool to isolate of E. coli mutant strains that produce native dsulfide-bonded recombinant proteins in the periplasm Brigitte Söhling, Rainer Rudolph 159 ID 113 Membrane tethers as constant force clamps Jonne Helenius, Jens Friedrichs, Yi-Ping Chu, Daniel J. Müller 160 DIAGNOSTICS 163 ID 114 Magnetic Deposition Microscopy for malaria detection in a field , study in Papua New Guinea Stephan Karl, Maciej Zborwowski, Thomas Bley 164 ID 115 Cytogenetic and molecular biological characterization of an adult medulloblastoma Peter Ahnert , Heidrun Holland, Ronald Koschny, Wolfgang Krupp, Jürgen Meixensberger, Manfred Bauer, Holger Kirsten, Ralf Schober 165 ID 116 Micro cavity array (MCA) biosensor for impedance based functional drug screening of 3D tissue models Daniel Kloß, Andrée Rothermehl, Andrea A. Robitzki 166

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ID 117 Alternative instrumentation concepts for bioimpedance spectroscopy for application specific multichannel measurement & diagnosis solutions Sebastian Wegner 167 ID 118 Trial Design of Remyelination Trials in Multiple Sclerosis: Reproducibility of Visual evoked Responses Florian Then Bergh 168 MICROFLUIDICS 171 ID 119 Generation of Microspheres as Drug Using Hydrodynamic Flow Focusing Thomas Schneider, Thomas Bley, Urs Hafeli 172 ID 120 Nanomicroimplants for controlled drug delivery Randy Kurz, Anselm Sickinger, Andrea A. Robitzki 173 ID 121 Controlled dynamic droplet fusion in PDMS based microelectromechanical Systems (MEMS) for GFP in-vitro expression Stefan Harazim 174 BIOTEChnology Center Dresden 177 Center for Biotechnology and Biomedicine 178 INDEX 180

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Foreword

For more than five years the Center for Biotechnology and Biomedicine at the University of Leipzig has staged the Biotechnology Symposium annually. Due to its great success, it was decided to enlarge the symposium and include other regional and extra-regional institutions. This year the Biotechnology Center of the TU Dresden and the Center for Biotechnology and Biomedicine at the University of Leipzig are cooperating to hold the first joint Biotechnology Symposium in Saxony. The main topics of this conference, hosted in Dresden, will be “New Technological Developments” and “Medical Biotechnology”.

Saxony has a long tradition in the industrial field. Yet, biotechnology is a comparatively young and developing albeit booming sector of industry. The region supplies all vital resources for successful advancements in the field of biotechnology: the excellent basic research, the applied research, and finally the producing industries. More than 50 biotechnology companies are presently working and thriving in Saxony. Further 50 companies produce instruments and technological equipment primarily used in the biotechnological and pharmaceutical industries.

Biotechnology is at the brink of emerging as key technology for the future. Inventions and innovations are necessary, indispensable motors for biotechnological progress. We hope this Symposium will stimulate and boost the Saxon biotechnological sector, the universities, research centers and companies.

Biotechnology in Saxony matters!

   

 

 

 

Prof. Dr. Michael Brand     Prof. Dr. Andrea A. Robitzki 

Director        Chair of the Management Board 

Biotechnology Center      Center for Biotechnology and Biomedicine 

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New Technological Developments

 

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Andreas Manz ISAS – Institute for Analytical Sciences Dortmund a.manz@isas.de www.isas.de

Microfluidics for Chemistry and Systems Biology Dr. Andreas Manz, ISAS - Institute for Analytical Sciences, Dortmund

Microfluidic chips have been developed and applied to various fields. Particularly, fast separations and chemical reactions for use in analytical chemistry context have been of interest [1]. Scaling laws predict a higher mass and heat transport for smaller dimensions. However, there is a trade-off between internal volumes and detection limits. Key applications include drug discovery [2], systems biology and regenerative medicine research. In this presentation, I will briefly review scaling laws, show examples of microfluidic chips for electrophoresis, elaborate on work-in-progress with cell differentiation and give an example how mishaps turn out to be quite interesting…. References [1] D.Janasek, J.Franzke, A.Manz, Nature 442, 374-380 (2006); P.S.Dittrich, K.Tachikawa, A.Manz, Anal. Chem. 78, 3887-3908 (2006) [2] P.S.Dittrich, A.Manz, Nature Reviews Drug Discovery, 5, 210-218 (2006)

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Jens Struckmeier nAmbition GmbH Dresden jens.struckmeier@nambition.de www.nambition.com

Screening with Force Spectroscopy

Dr. Jens Struckmeier nAmbition GmbH Dresden

Force spectroscopy is the method of choice, whenever scientists want to understand how individual biomolecules, small compounds, polymers and surfaces interact with each other. The ability to measure binding forces and distances directly on the molecular level and with a label-free technique, makes it a valuable tool for many application areas. Research topics range from material science, structural characterization, folding/unfolding dynamics to molecular design and binding site localization.

In contrast to its undoubted usefulness in many application areas, the technology is not as widespread as to be expected. This is reasoned by the fact that traditional force spectroscopes are complicated to use, need numerous manual adjustments and permanent instrument control by the user. With the aim to improve the situation, we developed a more user-friendly and automated force spectroscope, the ForceRobot®. It overcomes the hurdles of traditional force spectroscopes, automates routine procedures and provides software support for experimental design, data acquisition and evaluation. Ten thousands of force curves are generated in a couple of hours in unattended mode. No-event data are automatically filtered and the useful data are presented for further evaluation. In addition, a dynamic temperature control with integrated drift compensation allows temperature dependency studies and further modules like an automated fluid system for buffer exchange and a newly invented active noise cancellation feature are currently under development. By generating high quality data in short time frames and with its easy accessibility, ForceRobot® will help to establish force spectroscopy as a standard method in molecular analytics at the nano-scale. We will give an outlook how the automation of force spectroscopy opens new fields not only in academic research but also in industrial applications and drug discovery.

 

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Marius Grundmann Universität Leipzig Faculty of Physics and Earth Science Institute of Experimental Physics II grundmann@phyik.uni-leipzig.de www.physik.uni-leipzig.de

Nanowhisker And Their Applications

Prof. Dr. Marius Grundmann Universität Leipzig Institute of Expermental Physics II

 

Various possible applications of nanowhiskers are discussed, including nanophotonics (ultrasmall and efficient light sources), sensors (e.g. for gas or pH-value) and nanomechanics (energy conversion).

Nanowhisker are quasi-onedimensional objects that have a small radius (typically 1-1000 nm), large length (typically 100 nm – 10 μm) and large aspect ratio, typically (10-100). Such nano-objects can be fabricated from several semiconductors such as Si, GaAs, GaN or ZnO.

We have investigated in detail growth, self-assembly and physical properties of ZnO nanowhiskers fabricated by pulsed laser deposition. ZnO is a very interesting material for such nano-objects since it combines multi-functionally superior optical properties, tunable conductivity and piezoelectricity.

 

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Stefan Diez Max Planck Institute for Molecular Cell Biology and Genomics Research group leader buchholz@mpi-cbg.de http://www.mpi-cbg.de/~diez

Motor Proteins at work: Molecular transport in cell biology and nanotechnology Dr. Stefan Diez Groupleader Max Planck Institute for Molecular Cell Biology and Genetics

Inside cells, motor proteins perform a variety of complex tasks such as the transport of vesicles and the separation of chromosomes. We are interested in the novel use of such biological machines as transporters and manipulators for a wide range of nanoobjects in an engineered, cell-free environment. This idea is intriguing because such machines are robust, can work in parallel, their size is in the nanometer range, they work with a high energy efficiency and their application is potentially cheap. However, it is a major challenge to gain precise spatio-temporal control over the activity of the motor proteins when implemented in an artificial (cell-free) environment. One promising strategy to use the force generation of motor proteins in vitro the so called gliding motility assay, where motor proteins (such as kinesin) are immobilized to the substrate and propel cytoskeletal filaments (such as microtubules) over the surface in the presence of ATP. In order to guide microtubules along predefined paths, we work on the generation of nanometer-wide, non-topographical patterns of motor proteins on planar surfaces. Towards the temporal control over motor activity by external signals, we fabricate composite surface layers where functional motor-molecules are adsorbed onto a silicon substrate between surface-grafted stimuli-responsive polymers. In order to observe and optimize the biologically driven transport processes in the nanometer range, it is a second goal of our group to develop optical single molecule techniques. This includes imaging of single fluorescently labelled proteins (such as biomolecular motors) and colloidal semiconductor nanocrystals (quantum dots) by total-internal reflection fluorescence (TIRF) and fluorescence interference contrast (FLIC) microscopy.

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Josef A. Käs Universität Leipzig Faculty of Physics and Earth Science Institute of Experimental Physics jkaes@physik.uni-leipzig.de http://www.uni-leipzig.de/%7Epwm/

Passive and Active Single Cell Biomechanics Prof. Dr. Josef A. Käs Universität Leipzig Institute of Experimental Physics I

The cytoskeleton a compound of highly dynamic polymers and active nano-elements inside biological cells is responsible for a cell’s stability and organization. It mechanically senses a cell’s environment and generates cellular forces sufficiently strong to push rigid AFM-cantilevers out of the way. These forces are generated in the lamellipodium – which can be considered a thin polymeric film – by molecular motor-based nano-muscles, and by polymerization through mechanisms similar to Feynman’s hypothetical thermal ratchet. The active polymer networks as basic element of the lamellipodium are described by a new type of polymer physics since nano-sized molecular motors and active polymerization overcome the inherently slow, often glass-like brownian polymer dynamics. Light has been used to observe cells since Leeuwenhoek’s times; however, we use the forces caused by light described by Maxwell’s surface tensor to feel the cytoskeleton. The optical stretcher exploits the nonlinear, thus amplified response of a cell’s mechanical strength to small changes between different cytoskeletal proteomic compositions as a high precision cell marker that uniquely characterizes different cell types. Consequentially, the optical stretcher detects tumors and their stages with accuracy unparalleled by molecular biology approaches. This precision allows us to isolate adult stem cells for regenerative medicine without contamination through molecular markers.

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Steffen Howitz GeSiM GmbH CEO howitz@gesim.de www.gesim.de

Microfluidic Components and Systems for Biotech-Application Steffen Howitz CEO GeSiM GmbH Großerkmannsdorf

 

 

An overview of the currently available microfluidic components from GeSiM will be presented. These microfluidic components concerns piezo-pipettes, hydrogel-valves, PDMS-based valves, flow sensors, heaters, temperature sensors, flowthrough cells and master chips for PDMS-casting technology. GeSiM provides these components as single OEM-products, as small sub-devices for research and development and last but not least as key-components which are integrated into more complex systems for micro-array technology, for microscopy and recently for the nano-technology. The three most essential product lines of GeSiM shall be introduced roughly. The first product line issued the Nano-Plotter™ from GeSiM, a device family to handle biological samples by ink jet printing, pin tool printing, high viscous sample writing and dry powder handling. The basic system features and some applications of our state of the art Nano-Plotter technology will be presented. The second product line covers our flow-through cell technology, summarized under the concept MicCell. MicCell allows the implementation of micro fluidic tasks directly onto the stage of an inverse or upright microscope stage. We have developed different technologies to fabricate customized flow-cell for microscopy and in the presentation examples of glass/glass, glass/silicon and glass/PDMS flow cells will be discussed. Our latest system development concerns the field of μ-contact printing of biomolecules. We have developed a semi-automatically driven μ-contact printer. This device is equipped with our genuine PDMS-stamp. And the key element here is, that we handle precisely a perfectly spanned PDMS-membrane in the device, which contains micron- or sub-micron patterns inked with bio-molecules or nano-particles for transfer to a flat bio-chip surface. In this presentation we will introduce GeSiM as an biotech-instrumentation company with various activities in a wide field of micro-fluidic applications.

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Medical Biotechnology

 

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Simon Hoerstrup Unispital Zürich Simon_philipp.hoerstrup@usz.ch www.herzgefaesschirurgie.usz.ch/german/ LehreUndForschung/default.htm

Tissue Engineering and Cell-Transplantation – Cell Based Therapies for Cardiovascular Applications Prof. Dr. Simon Hoerstrup, Unispital Zürich Department of Surgery Regenerative Medicine Program and Cardiovascular Research

Cell based therapeutic concepts are focused on two mayor principles: a) de novo tissue creation (tissue engineering) for the repair/replacement, and b) cell transplantation for the regeneration of defective organ tissues. With regard to cardiovascular applications an array of new therapeutic options is under development and may revolutionize current treatment modalities. Here, we present research on cell based therapies addressing cardiovascular disorders such as congenital heart disease, as well as degenerative heart valve, vascular and myocardial disease. Tissue engineering technologies are aiming at the creation of living replacements with the capacity of self-repair and growths. Ideally, these replacement are based on the patients own cells preventing the risk of immunological and infectious (e.g. zoonosis) complications. Such autologous tissue engineered constructs can be created by in vitro cell seeding onto biodegradable scaffold matrices. These scaffolds serve as an initial guiding structure for neo-tissue development before degradation. The in vitro tissue maturation can be enhanced by bioreactor technologies mimicking e.g. a physiological environment. For heart valve and blood vessel tissue engineering, pulsatile flow conditions have shown favourable results leading to implantable tissues with adequate functionality in long term animal models. The growth potential of tissue engineered living arteries was demonstrated in preclinical animal tests covering the full biological growth cycle. Several cell sources were investigated ranging from fully differentiated cells to various human stem cells. Bone marrow derived and blood derived cells were used for heart valve tissue engineering. Recently, prenatal stem cells which can be obtained during pregnancy before birth have been successfully utilized for cardiovascular tissue engineering, a technology which may allow the treatment of congenital heart malformations with autologous, growing replacement material directly after birth.

Cell transplantation using bone marrow and blood derived progenitor cells has been recently introduced to clinical use for the treatment of ischemic myocardial disease. Although showing some improvement of cardiac performance, the long-term benefits for patients as well as the underlying cellular mechanisms remain unclear. Mayor limitations as to current single cell transplantation are insufficient cell delivery, integration and function. In order to overcome these limitations, a new micro-scale tissue engineering approach using micro-tissue cell aggregates instead of single cells has been developed and is currently tested in animal models.

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Michael Cross Universitätsklinikum Leipzig Department of Haematology michael.cross@medizin.uni-leipzig.de www.uni-leipzig.de

The metabolic niche hypothesis – implications for stem cell culture Dr. Michael Cross PhD Universität Leipzig Department of Haematology

The establishment in recent years of culture conditions which support the symmetric division and expansion of various stem cell populations provides a basis for promising new strategies in stem cell therapy and tissue engineering. In vivo, however, symmetric amplification appears to be the exception rather than the rule. Indeed, those somatic stem cells which are most productive in vivo appear least prepared to undergo symmetric division in culture, suggesting that amplification of physiologically relevant stem cells is under very strict control.

Based on our studies of haematopoietic stem and progenitor cells, I suggest that specific metabolic parameters of the stem cell niche make a decisive contribution to this control, and that provision of an appropriately limiting metabolic environment will be essential to the successful ex-vivo amplification of HSC. One prediction of this metabolic stem cell niche hypothesis is that the extensive amplification of stem and progenitor populations under nutrient rich, non-physiological conditions may encourage the selection of mutants carrying potentially oncogenic mutations.

 

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Frank Buchholz Max-Planck-Institute for Molecular Cell Biology and Genetics buchholz@mpi-cbg.de http://www.mpi-cbg.de/research/ groups/buchholz/buchholz.html

Development of Site-Specific Recombinases for Genome Surgery Dr. Frank Buchholz Research group leader Max-Planck-Institute for Molecular Cell Biology and Genetics

 

Site-specific recombination describes a process that involves reciprocal exchange between defined DNA sites catalyzed by a specialized recombinase protein that is responsible for recognizing the sites and for breaking and rejoining the DNA. The applied use of site-specific recombinase technology allows for the manipulation of genetic material in order to explore gene function. As such, recombinases have found wide-spread use for genomic manipulations in laboratory animals and plants. To extent the usefulness of site-specific recombinases we apply directed evolution to change the properties of these enzymes. I will present examples of altered recombinases and show that these enzymes can be modified to excise an HIV-provirus from infected human cells. Medical implications of this technology will be discussed.  

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33

Johannes Schwarz Universitätsklinikum Leipzig Department of Neurology johannes@caltech.edu http://neurologie.uniklinikum-leipzig.de/ ParkinsonArbeitsgruppe.htm

The Potential of tissue-specifc human neural stem cells Prof. Dr. Johannes Schwarz NeuroProgen Leipzig/ Universität Leipzig Department of Neurology

Many brain disorders bear a considerable burden for affected patients and are contrasted by the lack of tolerability towards age related handicaps in modern societies. In most cases, conventional therapies can alleviate these diseases only symptomatically; patients remain in a state of permanent treatment. Transplantation of neurons and/or glial cells provides an option but has not been applied with relevant success. This is also related to the lack of appropriate tissue. Advances in regenerative medicine have created an immense hope that such therapies may be available in the near future. Much of such hope is based on the discovery of neural stem or progenitor cells (NPCs) bearing a profound proliferative potential and the potential to differentiate into neurons. Such NPCs can be generated from embryonic stem cells (ESCs), fetal and adult brain or mesenchymal stem cells. While ESCs may have the largest potential long-term, their clinical application is hampered by ethical concerns and their potential to form highly malignant tumors following transplantation. NSCs from the adult brain show limited proliferation and without genetic alteration can only be differentiated into a few cell types. NSC-like cells derived from bone marrow or mesenchymal stem cells may express few neuronal markers but are unlikely to represent a reliable source for mature neurons. Thus, tissue-specific NSCs derived form the fetal brain which can proliferate for many months and retain their potential to differentiate into most cell types of the brain regions that they were derived from are the most promising cell type that will allow for restoration of lost or damaged brain tissue in the near future.We have developed proprietory tissue culture methods that have provided large numbers of human tissue-specific NSCs derived from a huge variety of brain areas including frontal cortex, striatum, cerebellum, thalamus, midbrain and spinal cord. Such cells are currently employed in several animal models of human disorders.

 

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Francis Stewart Technische Universität Dresden BIOTEC Francis.stewart@biotec.tu-dresden.de www.biotec.tu-dresden.de/stewart

Drug discovery by DNA engineering Prof. Dr. Francis Stewart Technische Universität Dresden Group leader BIOTEC

 

 

 

About one third of pharmaceuticals and pesticides are produced from prokaryotic secondary metabolites pathways (polyketide synthase, PKS; non-ribosomal peptide synthase, NRPS). These pathways are typically encoded in DNA as single, large (> 25 kb), operons. The recent explosion in complete prokaryotic genome sequences has revealed a surprising discovery for secondary metabolism; there are many ‘silent’ PKS/NRPS operons whose secondary metabolite product is unknown. However most prokaryotes are difficult to culture and experiments to find conditions whereby the silent pathways would be activated are laborious and empirical guess-work. Because these silent operons are very likely to encode bioactive molecules, they could be avery rewarding sources for new pharmaceuticals and pesticides . Consequently we are developing a generic technology to express the pathway and examine the product.

The technology is based on the application of a DNA engineering methodology we developed, termed recombineering, coupled to the use of heterologous prokaryotic hosts for expression of the pathway. We have established proof-of-principle experiments with several known PKS/NRPS pathways and are now exploring selected silent pathways from recently sequenced myxobacteria.

 

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Charl van Zyl JADO Technologies CEO Charl.vanzyl@jado-tech.de www.jado-tech.com

JADO Technologies GmbH - Raft Technology: The New Frontier in Pharmaceutical Development Charl van Zyl CEO JADO Technologies GmbH

 

 

JADO is a leader in the emerging field of RAFT intervention therapeutics. Representing a paradigm shift in drug development, RAFT therapeutics have the potential to address multiple unmet needs, particularly in allergy, infectious diseases, Alzheimer's disease and cancer.

RAFTS are sub-compartments of cell membranes that play an integral role in key biological pathways. The Company's RAFT Intervention Technology® provides a unique platform, protected by a strong patent position, to drive significant pipeline development opportunities. JADO has leveraged its technology to generate multiple small molecule drug candidates, with its lead program in Phase IIa clinical trials for allergy indications. The Company is supported by a global network of clinical and academic experts, including JADO founders, Prof. Kai Simons, Prof. Marino Zerial, Dr. Teymuras Kurzchalia (Max-Planck Institute of Molecular Cell Biology and Genetics, Dresden) and Prof. Hans-Joachim Knölker (Technical University of Dresden). JADO is headquartered in Dresden, Germany with a subsidiary in Bethlehem, Pennsylvania (USA). 

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Molecular Medicine

 

 

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Kristin Schnabel Technische Universität Dresden

ID 1 BIOTEChnology Center Dresden Research group Weidinger kristin.schnabel@biotec.tu-dresden.de www.biotec.tu-dresden.de/weidinger

The role of Wnt/beta-catenin signaling in zebrafish heart regeneration

Kristin Schnabel, Katja Pfeifer, Gilbert Weidinger

The capacity to regenerate damaged or injured tissue differs widely between higher and lower vertebrates. Whereas damage to the mammalian heart results in permanent scarring without functional recovery, teleost fish, like zebrafish, have the capacity to regenerate the heart after major injury. While very little is known about the molecular mechanisms regulating heart regeneration, a better understanding of these mechanisms holds the promise to reveal why mammals fail to regenerate heart tissue and might offer possibilities for therapeutic interventions in humans. Previous results of our lab demonstrated the importance of Wnt/beta-catenin signaling for initiation, progression and regulation of zebrafish fin regeneration. Wnt/beta-catenin signaling also regulates embryonic heart development and we found that the pathway is activated during heart regeneration. Therefore we are investigating whether wnt/beta-catenin signaling is required for zebrafish heart regeneration.  

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Mike Beck Max-Planck-Institut für

ID 2 molekulare Zellbiologie & Genetik beck@mpi-cbg.de www.mpi-cbg.de/research/groups/ walch-solimena/walch-solimena.html

Nucleo-cytoplasmic shuttling of the Golgi phosphatidylinositol 4-kinase Pik1 is regulated by 14-3-3 proteins and coordinates Golgi function with cell growth

Mike Beck, Lars Demmel, Annelore Schlaitz, Christiane Walch-Solimena, Jan Havlis, Andrej Shevchenko, Eberhard Krause, Yannis Kalaidzidis, Peggy Hsu

ID 10

The yeast phosphatidylinositol 4-kinase Pik1p is essential for growth and normal Golgi morphology and regulates transport of newly synthesized proteins from this compartment. Phosphatidylinositol 4-phosphate, which is generated by Pik1p recruits cytosolic effectors involved in formation of post-Golgi transport vesicles. A second pool of catalytically active Pik1p resides in the nucleus. The physiological significance and regulation of this dual localization of the lipid kinase is not understood. We report here that Pik1p binds to the redundant 14-3-3 proteins Bmh1p and Bmh2p through a phosphorylated consensus motif found in other 14-3-3 targets. We demonstrate that nucleo-cytoplasmic shuttling of Pik1p involves phosphorylation and that 14-3-3 proteins sequester Pik1p in the cytoplasm. A dramatic relocation of Pik1p from the Golgi to the nucleus is observed upon nutrient deprivation, a process rapidly reversed upon restoration of the nutrient supply. Pik1p relocation is accompanied by a transient increase of the Pik1p - 14-3-3 complex. The data presented suggest a role of Pik1p nucleo-cytoplasmic shuttling in coordination of biosynthetic transport from the Golgi with nutrient signaling in a pathway that involves 14-3-3 proteins.

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Birgit Kagermeyer-Schenk Technische Universität Dresden

ID 3 BIOTEChnology Center Dresden Research group Weidinger birgit.kagermeyer-schenk@biotec.tu-dresden.de www.biotec.tu-dresden.de/weidinger

Identification of Wnt target genes involved in zebrafish fin regeneration

Birgit Kagermeyer-Schenk, Herman Spaink, Gilbert Weidinger

Lower vertebrates, including teleost fish, have the intriguing ability to regenerate many organs and structures after damage due to injury or disease. The zebrafish fin provides a convenient model to study the molecular mechanisms regulating regenerative processes. Amputated fins are completely restored in three distinct steps (wound healing, formation of a blastema, a population of pluripotent progenitor cells, and regenerative outgrowth) within 1-2 weeks. Recently, we have shown that Wnt/betacatenin signaling is required for all three steps, since inhibition of this pathway at any time point causes defects in the re-grown structure. Since Wnt/beta-catenin signaling acts via regulation of target gene expression, we attempt to learn more about the molecular mechanisms and cellular functions of Wnt signaling during fin regeneration by identifying Wnt-regulated genes. We use customized AGILENT 44k microarrays to compare the transcriptional profile of wild-type regenerating fins with fins where Wnt/beta-catenin signaling has been repressed or activated at different time points during regeneration. To inducibly inhibit Wnt/beta-catenin signaling, we use hsDkk1GFP and hsAxin1YFP transgenic fish, where a secreted and a cell autonomous inhibitor of the pathway are expressed after heatshock. Overexpression of Wnt8 in hsWnt8GFP fish is used to activate beta-catenin signaling. Initial results of this screen will be presented at the meeting.

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Carsten Baldauf Technische Universität Dresden

ID 4 BIOTEChnology Center Dresden Research group Pisabarro carsten.baldauf@biotec.tu-dresden.de www.biotec.tu-dresden.de/pisabarro

Imitating protein ß-hairpins with more robust ß-peptide folds

Carsten Baldauf, M. Teresa Pisabarro

The ß-hairpin is a structural motif in proteins in which a turn links two ß-strands in a way that the strands can form hydrogen bonds. Unlike helices, ß-hairpins involve interactions between non-local amino acids. Besides their importance in studying folding behavior in a small model system, ß-hairpins seem to be a perfect template to design relatively small molecules that can inhibit, tune or promote protein-protein interactions. But there is a strong limitation for this and one always has to find a compromise between the residues that have the right propensities to form a hairpin and the residues one needs for the desired function[1,2]. It has been shown in the last years that foldamers from non-natural amino acids are promising towards the design of small but still stable-folding peptides. While the most efforts have been spent on the design of helical ß-peptides, we want to focus on the rational design of ß-peptide hairpins [3,4] for the tuning of protein-interactions. Besides Molecular Dynamics simulations in explicit water of a ten residue ß-peptide, where we are able to show folding to a hairpin-like conformation within 10 ns, we focus on a real protein-protein interaction problem. The WW domain recognizes Proline-rich sequences (PRS) and such protein-protein interactions are of importance in many signaling pathways, diseases and viral infections. Thus it is of great interest to have scaffolds that can robustly fold to the desired shape and position the necessary functionalities correctly in 3D. Foldamers, or more precise non-natural peptides, can serve as such scaffolds and are, additionally, stable against proteases. The recognition of PRS by WW domains is based on a hydrophobic patch, formed mainly by Tyrosine and Tryptophan residues. We claim that this binding site can be reduced to a ß-hairpin structure. The resembling of this structural element with a canonical peptide appears to be difficult, maybe due to problems with getting a stable-folding hairpin[5]. We are designing artificial and selective PRS recognition modules based on ß-peptides, there the formation of a hairpin depends on hetero-chiral ß 2,3-amino acids for the strand region and two building blocks forming a ten-membered turn[3]. The great advantage of this scaffold is the high folding tendency towards a hairpin-like structure and the possibility to discriminate two sides: the ‘A-side’, which is supposed to carry the side chains needed for binding, and the ‘B-side’ containing hairpin-stabilizing interactions. The authors thank Dr. Xavier Daura and Dr. David van der Spoel for help with the ß-amino acid topologies for the Gromos force field. The group is funded by Klaus Tschira Stiftung gGmbH. [1] Fisk, J.D., Gellman, S.H. (2001) J. Am. Chem. Soc. 123, 343-344. [2] Gellman, S. H. (1998) Curr. Opin. Chem. Biol. 2, 717-725. [3] Daura, X., Gademann, K., Schafer, H., Jaun, B., Seebach, D., van Gunsteren, W.F. (2001) J. Am. Chem. Soc. 123, 2393-2404. [4] Langenhan, J.M., Gellman, S.H. (2004) Organic Lett. 6, 937-940. [5] Espinosa, J.F., Syud, F.A., Gellman, S.H. (2005) Biopolymers 80, 303-311.

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Kathy Busse Universitätsklinikum Leipzig

ID 5 Department of Neurologie kathy.busse@medizin.uni-leipzig.de neurologie.uniklinikum-leipzig.de

Regulator of G-protein signalling 9-, isoform 2 (RGS9-2): modulation of NMDA receptor signalling and neuronal plasticity

Kathy Busse, Rainer Strotmann, Johannes Schwarz

Regulator of G-protein signaling 9-, isoform 2 (RGS9-2), a member of the RGS family of Gα GTPase acceleration proteins, is specifically expressed in stiatum, a component of the basal ganglia. Despite detailed information on the domain composition and the interaction partners of RGS9-2 is available, its specific function remains poorly understood. However, because of its involvement in dopamine-2-receptor (D2R) signaling, a role of RGS9-2 in dyskinesia and Parkinson’s disease is discussed. To identify functional interaction partners of RGS9-2, we subjected the striata of RGS9-2 knock out and control mice to genome-wide expression profiling using the Affymetrix microarray technology. Expression of selected genes was confirmed by quantitative RT-PCR. The expression data revealed a significant down regulation of genes involved in the long-term depression, long-term potentiation and calcium signaling pathways. These results suggest that RGS9-2 may not only modulate the D2R signal transduction but also influence NMDA receptor signaling and neuronal plasticity.

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Julia Schumann Universität Leipzig

ID 6 Center for Biotechnology and Biomedicine Professorship for Molecular Pathogenesis julia.schumann@bbz.uni-leipzig.de www.uni-leipzig.de/~blessing/

Biphasic effect of TGF-beta on Th17-cell development

Julia Schumann, Uwe Müller, Jens Knauer, Reinhard Konrad Straubinger, Manfred Blessing

Introduction TGF-beta (Transforming Growth Factor-beta), an immune modulating cytokine, plays a central role in controlling the activity of different cells of the immune system. Thereby, the pleiotropic factor elicits both immune suppressive as well as stimulating effects. Th17-cells, a recently described IL-17-producing type of T-helper-cells are potent proinflammatory effectors, combating extracellular bacteria. Likewise Th17-cells are discussed in the context of destructive pathologies. Up to now, TGF-beta is considered essential for the generation of Th17-cells in the mouse. However, our findings suggest that TGF-beta is dispensable or even inhibitory for generation and amplification of Th17-cells. Methods CD4+/IL-17+-cells were detected by intracellular flow cytometry. Numbers of Th17-cells were determined in lymph nodes of lyme-borreliosis infected mice as well as after cultivation of CD4+-lymphocytes isolated from spleen of mice in absence or presence of TGF-beta. In vitro generation of Th17-cells is accomplished by T-cell receptor stimulation in the presence of TGF-beta and IL-6. Used mice strains are FVB/N WT, TgN(CD2Tß)1Mbl (T-cell-specific overexpression of TGF-beta), TgN(CD2δkTsRII)2Mbl (T-cell-specific inhibition of TGF-beta signal transduction). Results 1. Despite the T-cell insensitivity to TGF-beta, TgN(CD2δkTsRII)2Mbl display Th17-cells. 2. TGF-beta action on T-cells is dispensable for Th17 development. 3. Amplification of TH17-cells occurs in TGF-beta insensitive T-cells. Conclusion According hitherto existing literature TGF-beta in coherence with IL-6 is precondition for developing naïve T-helper-cells in proinflammatory Th17-cells. In contrast Th17-cells could be detected in lymph nodes and spleen of the mouse strain TgN(CD2δkTsRII)2Mbl demonstrably insensitive for TGF-beta in T-cells. In addition culturing Th17-cells in absence of TGF-beta was connected with an increased proliferation of this cells. TGF-beta thereby appears to have an inhibitory effect on the amplification of Th17-cells.

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Mike Beck Max-Planck-Institut für Molekular-, Zellbiologie & Genetik Email: beck@mpi-cbg.de www.mpi-cbg.de/research/groups/ walch-solimena/walch-solimena.html

David Kosel Universität Leipzig

ID 7 Faculty of Biosciences, Pharmacy and Psychology Institute of Biochemistry dkosel@uni-leipzig.de www.biochemie.uni-leipzig.de/agbs/default.asp

The Transcription Factor Tbx15: Expression and Subcellular Localization During Adipocyte Differentiation

David Kosel, Annette Beck-Sickinger

Trancription factors regulate a variety of cellular and developmental processes. One family of transcription factors is encoded by T-box (Tbx) genes, known to play a key role in specification and development of various tissues and organs in vertebrates and invertebrates. All members of this family share a conserved DNA-binding motif termed “T-box”. The T-box gene Tbx15 was discovered in 1998. Knock-out studies in mice implicated a crucial role for Tbx15 during skin pigmentation and the skeletal development. However, recent data indicate that Tbx15 also contributes to body fat distribution and obesity. The present study explored the role of Tbx15 during adipogenesis by using the murine fibroblast cell line 3T3-L1 as a model system for adipocyte differentiation. The cells were differentiated into mature adipocytes and Tbx15 expression was examined by Western blotting. Here we could demonstrate for the first time that Tbx15 is expressed in undifferentiated 3T3-L1 cells. Upon differentiation, expression levels of Tbx15 protein decreased nearly 5-fold. However, we observed also a slight increase of Tbx15 in later steps of adipogenesis, implying a stage specific expression. We could also show that Tbx15 contains a putative nuclear localization signal (NLS), consisting of four basic amino acids. To verify the nuclear localization, we fractionated cytoplasmic and nuclear proteins from 3T3-L1 cells. We were able to confirm that Tbx15 is localized exclusively in the nucleus of undifferentiated 3T3-L1 and is nearly absent in mature fat cells. Our results strongly suggest that Tbx15 is expressed early in adipogenesis and is localized in the nucleus and disappears during differentiation. Accordingly, we could also clearly demonstrate that 3T3-L1 is an appropriate model system to study the role of Tbx15 during adipocyte differentiation.

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Christian Kukat Universität Leipzig

ID 8 Center for Biotechnology and Biomedicine Professorship for Molecular Cell Therapy Christian.kukat@bbz.uni-leipzig.de www.uni-leipzig.de/~mct/

Distribution of mitofusin 2 (Mfn2) after the formation of megamitochondria

Christian Kukat, Ingo Schäfer, Alexandra Kukat, Peter Seibel

Extraordinary large mitochondria are known as megamitochondria, the formation of which is characterized not only by simple swelling as in isolated organelles in hypotonic solutions but also by additional fusion events. The accumulation of megamitochondria was detected both in physiological (e.g. mammalian sperm cells) as well as pathological conditions (e.g. diabetes) and can be induced for instance by ethanol, chloramphenicol, hydrazine or – as in our study – by valinomycin. We used valinomycin-induced megamitochondria in human culture cells to examine mitochondrial fusion events, especially by monitoring the distribution of mitofusin 2 (Mfn2). Mitofusins are conserved, large GTPases localised to the mitochondrial outer membrane. In mammals, two closely related but not redundant mitofusin homologs, Mfn1 and Mfn2 can be found. Both the N- and C-terminal regions of these structural similar proteins protrude from the mitochondrial outer membrane into the cytosol. Mitofusins are involved in outer membrane fusion of mitochondria, especially by homo-and heterodimeric interactions via coiled-coil domains. Mutations in the gene for Mfn2 are known to be involved in the hereditary neuropathy Charcot-Marie-Tooth Type 2. As an approach to localise Mfn2 in this study we used a plasmid encoding a fusion protein consisting of EGFP (enhanced green fluorescent protein) and mitofusin 2 (Mfn2). This vector allowed us to observe the distribution of Mfn2 in living cells with valinomycin-induced megamitochondria via confocal microscopy.

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Günter Vollmer Technische Universität Dresden

ID 9 Zoologie - Professur für Molekulare Zellphysiologie und Endokrinologie Guenter.vollmer@ tu-dresden.de www.biologie.tu-dresden.de/zoologie/vollmer/

Medicinal plants, nutritional supplements and menopausal health

Günter Vollmer, Oliver Zierau, Georg Kretzschmar

Cessation of ovarian hormone production can severely compromise female well being and health. During perimenopause women are bothered by symptoms like hot flushes, sleeplessness, vaginal dryness etc. Later in menopause these individuals eventually develop osteoporosis, which is associated with a tremendous increase in the risk for bone fractures. In parallel a significant population develops an obese stature often associated with insulin resistance. Menopausal symptoms are traditionally treated by hormone replacement therapy (HRT) through administration of ovarian estrogens and progestins. However, after two major studies reported an increased risk in the development of breast and endometrial cancer, the majority of affected individuals seek alternatives which can be found as prescribed medicinal plant extracts or nutritional supplements. However, for these so-called alternatives sufficient information about the molecular mode of action, the efficiency and safety of these products is often missing. In addition, due to obvious demands, new products are needed. Our group offers a battery of test systems to a) screen for new substances, b) elucidate the molecular mode of action and c) tackle issues related to safety. The power of these experimental tools will be demonstrated by selected examples. With our screening tools we were the first to describe a mixed estrogenic and antiandrogenic property of the plant derived flavonoid 6-Dimethylallynaringenin. With our cell based assay we were the first to show that hydroxystilbenes contained in the medicinal plant extract of Rheum rhaponticum dominantly stimulates the estrogen receptor-� and exhibits only minimal residual activity on the estrogen receptor-�. In a sophisticated experimental animal model setting we have shown that the soy isoflavone genistein often contained in nutritional supplements presumably is safe regarding proliferative end points. In summary, we offer the research platform to screen for plant derived and nutritional compounds relevant to menopausal health. In cell based in vitro models and in physiological/pharmacological experimental animal models we are able to elucidate the molecular mode of action of these compounds and to test for efficiency and safety.

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Mike Beck Max-Planck-Institut für Molekular-, Zellbiologie & Genetik Email: beck@mpi-cbg.de www.mpi-cbg.de/research/groups/ walch-solimena/walch-solimena.html

Enrico Schild Universität Leipzig

ID 10 Faculty of Biosciences, Pharmacy and Psychology Institute of Biochemistry eschild@uni-leipzig.de www.biochemie.uni-leipzig.de/agbs/default.asp

Investigation of the Functionality of Growth Hormone Secretagogue Receptor 1a (GHSR1a) and Motilin Receptor (MTLR) Chimaera

Enrico Schild, Annette Beck-Sickinger

Motilin is a 22 amino acid peptide, which is expressed throughout the gastrointestinal (GI) tract. It primarily regulates the step three of the intestine peristalsis [1]. The activity is mediated by the motilin receptor (MTLR). This receptor was first isolated from the human stomach. It was predominantly found in the gastric corpus, the antrum pyloricum and the duodenum [2]. Ghrelin is a 28 amino acid peptide, which contains an unusual octanoyl-modification at the side chain of Ser3. It plays an important role in the regulation of energy homeostasis and food intake. The ghrelin receptor (GHSR1a) is mainly expressed in the hypothalamus and pituitary [3] and is considered as a potential target for pharmaceuticals. In contrast to the MTLR the GHSR1a shows constitutive activity. Both receptors, MTLR and GHSR1a, belong to the family of class A rhodopsin like receptors and couple to G�q/13- (MTLR) or G�q/11-proteins (GHSR1a), respectively [3,4,5]. Because of their high sequence-similarity they belong to the same subfamily, which is named ghrelin receptor family. In the presented study we exchanged the N-terminal segment and the extracellular loops of both receptors. Chimeric receptors were investigated in a functional IP3-assay by stimulation with the natural ligands. For the wildtype GHSR1a EC50-value was determined as 5.5 ± 2.8 nM. Chimaera, containing segments of the MTLR, showed a 3-fold (N-terminus), 14-fold (loop 1) or 31-fold (loop 3) increase in EC50-values. In contrast MTLR significantly lost activity when the N-terminus or loop 1 of GHSR1a was introduced, whereas loop 2 and 3 are well tolerated. Accordingly, whereas for GHSR1a the extracellular domains play a minor role, in MTLR this is significantly different. 1. Itoh Z, Aizawa I, Sekiguchi T., The digestive migrating complex and its significance in man. Clin Gastroenterol 1982; 11: 497–521. 2. Bormans V, Peeters T, Vantrappen G., Motilin receptors in rabbit stomach and small intestine. Regul Pept 1986; 15: 143–53. 3. Howard A.D. et al., A receptor in pituitary and hypothalamus that functions in growth hormone release, Science 1996; 273: 974–977. 4. McKee KK, Palyha OC, Feighner SD, Hreniuk DL, Tan CP, Phillips MS, Smith RG, Van der Ploeg LH, Howard AD., Molecular analysis of rat pituitary and hypothalamic growth hormone secretagogue receptors. Mol Endocrinol 1997; 11: 415–423. 5. Huang J., Zhou H., Mahavadi S., Sriwai W., Lyall V.,Murthy K.S., Signaling pathways mediating gastrointestinal smooth muscle contraction and MLC20 phosphorylation by motilin receptors. Am J Physiol Gastrointest Liver Physiol 2005, 288: G23-G31

 

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Peter Ahnert Universität Leipzig

ID 11 Center for Biotechnology and Biomedicine Molecular Diagnostics – Microarray Techniques ahnert@uni-leipzig.de www.uni-leipzig.de/~ahnert/

Evidence for an additional risk factor in the HLA region modifying the risk from known HLA-DRB1 risk alleles in Rheumatoid Arthritis

Peter Ahnert, Holger Kirsten, Grit Wolfram, Jörg Reichardt, Dirk Anders, Kathleen Hofmann, Saskia Ruhland

Rheumatoid Arthritis (RA) is a common complex autoimmune disease of rather unclear etiology with a high social and economic impact. The overall genetic contribution is estimated to be ca. 50 %, whereas roughly one third of the genetic contribution is attributed to certain HLA-DRB1 risk alleles. There is evidence that there are other disease susceptibility loci within the HLA region than DRB1 risk alleles. However, the strong association of the DRB1 risk alleles with Rheumatoid Arthritis and strong linkage disequilibrium within the whole HLA region make the mapping of additional putative disease genes in this region a challenging task. In an association study applying trio design we found significant association for a SNP and a certain haplotype of a gene in the LTA-TNF region in two different Caucasian populations. To clarify independence of the observed association from association of known HLA-DRB1 risk alleles we compared the haplotype distribution between chromosomes from RA patients and controls strictly matched for HLA-DRB1 alleles, studied differences of the linkage disequilibrium structure between RA patients and controls and compared relative risks of alleles and haplotypes. All these analyses gave evidence that the identified associated haplotype together with the HLADRB1* 0401 allele is a marker for at least one unknown, HLA-DRB1 independent risk factor for Rheumatoid Arthritis.

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Samiya Al Robaiy Universität Leipzig

ID 12 Center for Biotechnology and Biomedicine Professorship for Molecular Pathogenesis Samiya.al.robaiy@bbz.uni-leipzig.de www.uni-leipzig.de/~blessing/

TGF-β targets in T-cells

Samiya Al Robaiy, Bernhard Hoflack, Manfred Blessing, Cornelia Czupalla, Theresia Pursche

For the treatment and diagnosis of T-cell mediated autoimmune diseases (i.e. RA, AE etc.) additional agents are needed to be developed. Promising candidates for new prognostic and diagnostic tools are expected from members of transduction pathways involved in inflammation. Major pathways include the Mapk pathways, Smad pathway, and the Jak pathway. A key cytokine activating or modulating these pathways is Transforming growth factor (TGF-β). This cytokine is upregulated in several autoimmune diseases e.g. in the synovial fluid of patients with RA. For the identification of TGF-β targets in T-cells comparative proteomics analysis was performed for TGF-β resistant T-cells from transgenic mice expressing a dominant negative TGF-β type II receptor in T-cells under the control of the CD2 promotor/locus (hCD2 kTGFβRII) and for T-cells overexpressing an activated form of TGF-β (hCD2 kTGFβ). The purified T-cells were lyzed and the extracts were analyzed by two-dimensional gel electrophoresis. Proteins were visualized by coomassie blue and differentially expressed proteins of interest were processed for analysis by MALDI-TOF/TOF mass spectrometry. The comparison between T-cells isolated from transgenic animals and their corresponding controls identified more than 20 different proteins showing statistically significant differences (>1.2-1.4 fold) in expression. The analysis of these proteins revealed that TGF-β is involved in the regulation of cell cycle progression in many ways. The proteins modulated in transgenic mice indicated alteration in the calcium signalling and the results lead to the assumption that the calcium/calcineurin pathway is one of the main affected pathways. Also the serine/threonineprotein phosphatase 2A regulatory SU B which plays a big role in cell activation and proliferation was modulated in the T-cells of these mice. Moreover, many proteins involved in actin-cytoskeleton organization which is necessary for all stages in lymphocyte life and activation as well as different proteins expressed in the proteasome are modulated due to changes in TGF-β signalling. Studies on expression of these proteins are under way, in order to evaluate them as potential prognostic markers.

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Mike Beck Max-Planck-Institut für Molekular-, Zellbiologie & Genetik Email: beck@mpi-cbg.de www.mpi-cbg.de/research/groups/ walch-solimena/walch-solimena.html

Bianca Heyn Universität Dortmund

ID 13 Chemical Biology Bianca.heyn@uni-dortmund.de www.chemie.uni-dortmund.de/ groups/niemeyer/index.html

Reverse transcriptase of HIV as a target for directed evolution

Bianca Heyn, Sascha Nico Strumpp, Susanne Brakmann, Matthias Exner

The reverse transcriptase of HIV is an interesting enzyme for directed evolution. It is responsible for the transcription of the RNA genome into DNA that can integrate into the human genome. This function is a main target in antiretroviral strategies. Since the fidelity of HIV reverse transcriptase is low [1] the viral offspring is highly diverse and only a small portion is infectious. Recently [2] a mutant library of HIV reverse transcriptase was created by error-prone PCR. E. coli JS200 (recA718 polA12 trpE65) [3] was transformed with the mutant library. The selection for active variants of HIV reverse transcriptase could be performed because of functional complementation of the temperature sensitive DNA polymerase I at higher temperatures. The screening of active variants for a reduced fidelity was based on the reversion of a stop codon in the trpE gene which enabled the cells to grow on minimal media without the presence of tryptophan. With this directed evolution approach a variant of HIV reverse transcriptase was found which showed the same activity and an even lower fidelity as compared to the wildtype enzyme. In this variant 5 mutations on the amino acid level were found. In further studies, we are investigating the influence of these mutations on activity and error rate. The position E138 is one of the mutations found in the active variant. It seems to be important for protein folding and is therefore a target for a saturation mutagenesis. Here we present first results of our biochemical analyses that contribute to an understanding of structure-function relationships of HIV-1 reverse transcriptase.

References

[1] Preston, B.D. et al., Science, 1998, 242(4884): 1168-71 [2] Stumpp, S.N., Dissertation, 2007 [3] Witkin, E.M. et al., Mol Gen Genet, 1982, 185(1): 43-50

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Jan Stichel Universität Leipzig

ID 14 Faculty of Biosciences, Pharmacy and Psychology Institute of Biochemistry stichelj@rz.uni-leipzig.de www.biochemie.uni-leipzig.de/agbs/default.asp

Chimeric Y-Receptors to Understand Receptor Subtype Specific Ligand Binding of Neuropeptide Y

Jan Stichel, Annette Beck-Sickinger

Neuropeptide Y (NPY) is one of the most abundant neuropeptides in the mammalian nervous system. It belongs to the so-called PP-family and exerts its diverse physiological effects by acting at 4 different receptor subtypes in human (Y1, Y2, Y4, Y5). Earlier investigations showed that acidic residues in the extracellular loops of the Y1-receptor play a crucial role in NPY-binding [1, 2]. In order to identify the molecular basis of ligand-receptor interaction and the influence of the extracellular loops on receptor subtype specificity, we systematically created Y1/Y2-receptor chimera with single or multiple extracellular loops being exchanged. Furthermore, Y1/Y4 and Y2/Y5-receptor loop and segment chimera were constructed. The chimeric receptors were obtained by site-directed mutagenesis and the ligand binding properties were characterized by radioligand binding assays on transiently transfected COS-7 cells. To probe the signal transduction ability of the receptor chimera, COS-7 cells were co-transfected with a chimeric Gq-protein that links Y-receptors to the Gq-signalling pathway and allowed the monitoring of receptor activation by intracellular accumulation of radiolabelled inositol phosphates. No binding of [3H]-NPY could be observed for most of the chimeric receptors. Multiple loop exchanges could not restore binding. However, we identified segments of the first and third extracellular loop of the Y1-receptor to be very important for [3H]-NPY binding and downstream signalling. Especially the third extracellular loop of the Y1-receptor plays an important role in receptor activation, as its replacement by the corresponding Y2-receptor loop completely inhibits the production of inositol phosphates. Additionally, the first loop may play a role in subtype specificity, as partial mutation of this loop of the Y2 receptor by the corresponding Y1 receptor sequence led to Y1-like binding behaviour. Exchanging the third extracellular loop of the Y4-receptor with the corresponding sequence of Y1 led to an improved binding of NPY, whereas the affinity for pancreatic polypeptide PP is unaltered. A similar behaviour can be observed for a chimeric receptor of Y1 and Y4, containing the N-Terminus, TM1, ICL1 and TM2 from Y1 and ECL1 to C-Terminus from Y4. Based on these results, we suggest a complex involvement of more than one extracellular loop and of the transmembrane helices for the ligand binding site of Y-receptors. Moreover, intramolecular interactions between different extracellular loops and the 3-dimensional loop structure seem to be responsible for subtype specificity. Additionally, the arrangement of the transmembrane helices may play an important role. [1] M. Sautel, K. Rudolf, H. Wittneben, H.Herzog, R. Martinez, M. Munoz, W. Eberlein, W. Engel, P. Walker, A. G. Beck-Sickinger. Mol. Pharmacol. 50, 282-292. 1996 [2] Merten N, Lindner D, Rabe N, Römpler H, Mörl K, Schöneberg T, Beck- Sickinger AG. J. Biol. Chem. 282, 10, 7543-7551, 2007

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Anja Zielonka Universität Leipzig

ID 15 Faculty of Verterinary Medicine Institute of Virology

anja.zielonka@gmx.de www.vmf.uni-leipzig.de/ik/wvirologie/index.html

Formation of Virus-like particles consisting of the major capsid Protein VP1 of avian- and non-human Polyomaviruses in yeast Saccharomyces Cerevisae

Anja Zielonka

Polyomaviruses are small non-enveloped icosahedral viruses with a diameter of approximately 45 nm. The capsid contains a circular double-stranded DNA genome with about 5000 bp in size. The mammalian polyomaviruses mainly cause persistent subclinical infections in their natural hosts. In contrast, the polyomaviruses of birds, like avian polyomavirus (APV) and goose hemorrhagic polyomavirus (GHPV), cause acute and chronic diseases with high mortality rates in young birds. Recently the genome of two novel polyomaviruses of birds, the finch polyomavirus (FPyV) and crow polyomavirus (CPyV), was detected in samples of diseased birds. It has been shown for several mammalian and avian polyomaviruses that recombinant expression of the major structural protein VP1 in eukaryotic systems leads to the formation of virus-like particles (VLPs). These VLPs represent a promising carrier for encapsidation of foreign nucleic acids for gene therapy and are known as excellent antigens. In order to provide suitable antigens for diagnostic tests and vaccines, the recombinant expression of the major structural protein VP1 of the avian polyomaviruses FPyV, CPyV, and of the chimpanzee polyomavirus (ChPyV) and its ability to form VLPs was investigated in a galactose-inducible Saccharomyces cerevisiae yeast expression system. The expression of VP1 and its ability to build up VLPs was confirmed by cesium chloride ultracentrifugation, SDS-PAGE and electron microscopy. With all three viruses VLPs were formed. Yeast-derived VLPs are generally free of toxins, host cell DNA and proteins. These VLPs are currently tested for their application as gene delivery systems, new diagnostical tools and antiviral vaccines. The established protocols will be modified for the efficient generation of APV-VLPs, which are very urgently needed as a potential vaccine. Supported by Deutsche Forschungsgemeinschaft.

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Stefan Kappler c-LEcta GmbH

ID 16 stefan.kappeler@c-lecta.de www.c-lecta.de

Engineering Human Proteases for Therapeutical Use

Stefan Kappeler

Thanks to recent advances in biomedical research, the molecular mechanisms behind some major diseases have been elucidated. In many cases, an excess of key proteins or peptides is involved in disease progression. Several new drugs have been designed on the basis of antibodies or inhibitors for down-regulation of such targets. A promising alternative to these drug substances will be the use of proteases for the inactivation of abundant target proteins. The advantage over antibodies and inhibitors is the catalytic mechanism behind this drug concept. The active ingredient may act repeatedly on the target molecules, as it is known for several mechanisms, in which proteases are involved, e.g. the complement system or the hormonal regulation of blood pressure. In this way, much lower dosage forms will lead to improved tolerance of the active ingredient and thereby to fewer side-effects. In a proof-of-concept study, human caspase-3 is currently being modified to act on a validated cancer target. Some side-chain modifications in the active site cleft have been introduced, which changed the protease selectivity towards a peptide bond within the target molecule. The proprietary c-LEcta cluster screening technology was applied to screen caspase-3 libraries for variants with high specificity in the cleavage of the cancer target. The libraries were focused on the exchange of a few positions within the substrate binding pockets to alter the cleavage preference towards the target sequence. Primary screens were done in complexities of up to 500 variants per cluster. Variants with proteolytic activity towards the novel substrate were found and singled out. Amongst several caspase-3 variants exhibiting the desired target activity, one was found with zero wild-type activity. Residual activities of the variants were between 0.10% and 0.01% of the original wild-type activity. Application of this screening approach led to a reduction of the number of experimental samples required to screen the full library from 1.1 million to less than 3,000. The drug candidate will be further optimized and validated towards the cancer target.

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Mike Beck Max-Planck-Institut für Molekular-, Zellbiologie & Genetik Email: beck@mpi-cbg.de www.mpi-cbg.de/research/groups/ walch-solimena/walch-solimena.html

Ingo Schäfer Universität Leipzig

ID 17 Center for Biotechnology and Biomedicine Professorship for Molecular Cell Therapy ingo.schaefer@bbz.uni-leipzig.de www.uni-leipzig.de/~mct/

Mitochondrial import of peptide conjugated DNA

Ingo Schäfer, Christian Kukat, Alexandra Kukat, Peter Seibel

Mitochondria are the compartment where the energy of eukaryotic cells is generated by the oxidative phosphorylation system. The compounds of this system are encoded either in the nuclear or the mitochondrial genome. In the last decade, a variety of devastating neuromuscular diseases have been associated with mutations of the mitochondrial genome. These variations can range from single point replacements in tRNA and rRNA genes to deletions of DNA fragments encoding several genes. Import of DNA into the mitochondrial matrix is an obligatory step for a site directed mutagenesis or gene therapy approach on mitochondrial DNA diseases. Up to now, no endogenous system mediating this transfer is known in mammalian cells. To close this gap we developed peptide conjugated DNA vectors that are capable of delivering nucleic acids to distinct cellular organelles after microinjection. The vector is made up of the mitochondrial signal peptide of the ornithine transcarbamylase (a nuclear encoded protein finally located in mitochondria) which is chemically cross linked to a nucleic acid component. Due to the unique physical structure of the attached DNA, self-replication is initiated upon reaching the mitochondrial matrix.

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Mike Beck Max-Planck-Institut für Molekular-, Zellbiologie & Genetik Email: beck@mpi-cbg.de www.mpi-cbg.de/research/groups/ walch-solimena/walch-solimena.html

Claudia Heine Universität Leipzig

ID 18 Translational Center for Regenerative Medicine REMOD cheine@trm.uni-leipzig.de www.trm.uni-leipzig.de/remod.html

Characterization of the fibre Outgrowth in organotypic dopaminergic slice co-cultures

Claudia Heine, Annett Wegner, Jens Grosche, Peter Illes, Heike Franke

The mesolimbic dopaminergic system of the rat was reconstructed in vitro using an organotypic co-culture model, whereby tissue slices including the ventral tegmental area/substantia nigra-complex (VTA/SN-complex) and the prefrontal cortex (PFC) were cultured for 10 days. Multiple fluorescence labelling with antibodies against microtubule associated protein-2 (MAP2) and glial fibrillary acidic protein (GFAP) combined with laser scanning microscopy indicated the presence of neurons and astrocytes as well as a neuronal fibre outgrowth interconnecting the VTA/SN with the PFC. To specify the outgrowing fibres, antibodies raised against tyrosine hydroxylase (TH), Gamma-aminobutyric acid (GABA) and the vesicular glutamate transporters (vGLUT1-3) were used. Qualitative analysis indicated TH- and GABA-positive fibres as well as vGLUT1-marked projections in the border region. In addition, the expression of different dopaminergic (D1, D2) and purinergic (P2X1,2,3,4,6,7, P2Y1,2,4,6,12) receptors was investigated and revealed a localization of D1, D2 and P2X1, P2Y1 on the outgrowing projections. To support the hypothesis of the involvement of purinergic signalling in growth and development, different P2 receptor agonists were used to modulate the fibre outgrowth in the border region. The incubation with the ATP analogues 2-methylthio-ATP (P2X/Y receptor agonist) and α,β-methylene-ATP (P2X1/3 receptor agonist) induced an increase in axonal outgrowth and fibre density, which could be inhibited by pretreatment with the P2X/Y receptor antagonist pyridoxalphosphate-6-azophenyl-2´,4´-disulphonic acid (PPADS). In conclusion, the organotypic co-culture system provides a valuable model for studying neurocircuitries of the brain. Moreover, our results suggest a trophic role of ATP and the involvement of purinergic receptors in development and growth.

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Kathrin Bellmann Universität Leipzig

ID 19 Faculty of Biosciences, Pharmacy and Psychology Institute of Biochemistry bellmann@uni-leipzig.de www.biochemie.uni-leipzig.de/agbs/default.asp

Modification of the Regenerative Chemokine SDF1α to Allow Fluorescence Imaging

Kathrin Bellmann, Lars Baumann, Annette Beck-Sickinger

SDF-1 is a chemokine that plays a major role in trafficking of hematopoietic stem cells (HSC). Thus it enables the formation of bone marrow during embryogenesis and later in adult life it supports retention and homing of these cells in the bone marrow. Furthermore it is involved in organogenesis and regeneration, respectively [1]. Due to these promising features the subform SDF-1α could serve as a therapeutic target. For these purposes the small protein needs to be modified chemically in order to improve stability against proteolysis, localization and over all bioavailability. In order to reach these goals, the N-terminus SDF-1α(1-49) has been cloned and expressed recombinantly in E. coli ER 2566, while the C-terminus SDF-1α(50-68) has been synthesized via solid phase peptide synthesis. Modifications are thereby introduced at the C-terminus at Lys56. Up to now carboxyfluorescein has been coupled to the ε-amino group of the lysine residue. The two fragments then have been ligated via Expressed Protein Ligation (EPL), a subform of the Native Chemical Ligation (NCL) [2]. Ligation procedure was tested at different reaction temperatures and time points. Formation of a reaction product with the corresponding molecular weight could be observed after 96 hours at 22 °C. References [1] Kucia, M. et al., Journal of Molecular Histology, 2004, 35, 233-245. [2] David, R. et al., European Journal of Biochemistry, 2004, 271, 663-677.

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Javorina Milosevic Universität Leipzig

ID 20 Translational Center for Regenerative Medicine REMOD jmilosevic@trm.uni-leipzig.de www.trm.uni-leipzig.de/remod.html

Extracellular Nucleotides govern human neural precursor cell Proliferation and dopaminergic Differentiation

Javorina Milosevic, Sigrid Schwarz, Patrizia Rubini, Alexander Storch, Peter Illes, Johannes Schwarz

In human midbrain derived neural precursor cells (hmNPCs) we detected functional P2 receptors via calcium imaging and identified them as P2Y1, P2Y4 and P2X4 receptors via RNA analyses. We have developed a novel procedure for increasing both the yield and dopaminergic differentiation of NPCs isolated from human fetal midbrain. Extracellular UTP, a P2Y2/4 agonist, stimulated proliferation (1 μM) in the presence of the mitogens EGF and FGF-2, which was abrogated by the P2Y receptor blocker PPADS (100 μM). In addition, UTP and UDP, a potential extracellular hydrolysis product of UTP, increased dopaminergic differentiation when added to the growth factor-free differentiation medium. Treatment with UTP (100 μM) during differentiation increased the number of TH-positive cells and TH protein by 267 % and 319 %, respectively. During differentiation, treatment with UTP stimulated the extracellular signal-regulated kinase (ERK) pathway. Both, ERK1/2 phosphorylation and dopaminergic differentiation were reverted by U0126, a selective ERK kinase (MEK) inhibitor, as well as by suramin. When other P2 receptor agonists (ATP, ADP and ADPßS, all 100 μM) were applied, both proliferation and dopaminergic differentiation of hNPCs were compromised. We conclude that uracil nucleotides exert specific P2Y receptor-mediated effects on midbrain-derived hNPC, enhancing both proliferation and dopaminergic differentiation.

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Mike Beck Max-Planck-Institut für Molekular-, Zellbiologie & Genetik Email: beck@mpi-cbg.de www.mpi-cbg.de/research/groups/ walch-solimena/walch-solimena.html

Manuela Ackermann Universität Leipzig

ID 21 Translational Center for Regenerative Medicine IMONIT fzientek@trm.uni-leipzig.de www.trm.uni-leipzig.de/imonit.html

Long-term Multilineage Engraftment of human cells in NOD/SCID Mice Manuela Ackermann, Frank Emmrich, Manja Kamprad

The immunodeficient characteristics of NOD/SCID mice are defined in a complete lack of lymphocytes, a reduced number of NK-cells, defects in differentiation and function of antigen-presenting cells and absence of circulating complement. Consequently, the NOD/SCID mouse is an excellent model to prove the potential of human haematopoietic stem cells in regard to stable long-term engraftment and in-vivo functionality. By using xenotransplantation, cryopreserved mononuclear cells from human cord blood (MNC CB), including CD34+ haematopoietic stem cells, were injected intravenously. Mice were preconditioned with a total body irradiation (350cGy) before application. Relative numbers of human leucocytes in peripheral blood and lymphatic organs will be meassured at particular time points (biweekly and monthly). Analysis of human engraftment occurs by flow cytometry with human lineage-specific antibodies. We determine human lymphoid, myeloid and stem cell populations, including subpopulations and regulatory cell populations. MNC CB support a stable long-term engraftment in all transplanted mice with an individually increase and multilineage development with the main focus on human B-cells but also monocytes, granulocytes, dendritic cells and stem cells. By using an optimised protocol it is possible to facilitate the development of stable T-cells populations and other haematopoietic regulatory cell populations. We will evaluate humanised NOD/SCID mice representing the main functions of a human adaptive immune system. Functionality of reconstructed human immune system in mice is one of the main criterions on the further use as a research and preclinical animal model.

60

Mike Beck Max-Planck-Institut für Molekular-, Zellbiologie & Genetik Email: beck@mpi-cbg.de www.mpi-cbg.de/research/groups/ walch-solimena/walch-solimena.html

Elke Wandel Universität Leipzig

ID 22 Translational Center for Regenerative Medicine CELLT ewandel@trm.uni-leipzig.de www.trm.uni-leipzig.de/cellt.html

Interaction of Leucocyte Antigen CD97 and the Activation associated Endothelial Receptor THY-1 (CD90)

Elke Wandel, Manja Wobus, Anja Saalbach, Ulf Anderegg, Gabriela Aust

Objective: Leukocyte recruitment in response to inflammatory signals is governed by interactions between endothelial cell (EC) receptors and leukocyte adhesion molecules. CD97 is expressed at the surface of peripheral activated lymphocytes and myeloid cells. Here, we identified Thy-1, an activation-associated cell adhesion molecule on human dermal microvascular endothelial cells, as a new ligand of CD97. Methods and Results: In a cell-cell adhesion assay CD97 overexpressing cells were shown to specifically adhere to transfectants expressing human Thy-1 in comparison to vector transfected cells used as control. This effect was shown to be independent of the cell types and expression systems used. Furthermore, granulocyte adhesion to purified Thy-1 could be inhibited by 50 % with antibodies against the stalk region of CD97 and thus was shown to be in part dependent on the CD97-Thy-1 interaction. Antibodies against the first EGF-domain of CD97 did not block the adhesion, suggesting involvement of the stalk region of CD97 in ligand binding. Conclusion: This new ligand pair in leukocyte-endothelium interaction may play a role in the regulation of leukocyte recruitment to sites of inflammation. Further experiments will focus on the influence of (soluble) CD97 on phenotype and activation state of endothelial cells and its functional relevance for angiogenesis.

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Aldo Leal-Egana Universität Leipzig

ID 23 Translational Center for Regenerative Medicine TEMAT aleal@trm.uni-leipzig.de www.trm.uni-leipzig.de/temat.html

Profile of GFP Release derived from transformed foetal Hepatocytes in Alginate Microcapsules.

Aldo Leal-Egana, Jochen Lach, Isabel Rode, Andrea A. Robitzki, Augustinus Bader

Ca2+-Alginate is widely used as an artificial support in cell and gene therapy where its well known physicomechanic properties allow its use in most biomedical applications. Nevertheless, the state of cross-linking causes variations in elasticity and in the superficial area of the alginate (Leonard et al, 2004). Therefore it could prevent proper cell growth and proliferation, leading thus to cellular apoptosis. In order to demonstrate the dependence of the process of cellular growth and death on the biopolymer concentration of the capsules, we designed an experiment where the release of an intracellular target protein was used to indicate apoptosis. Operating under this premise, we characterized the in vitro release of a target protein in a system with cells immobilized in alginate beads, where the cellular proliferation and apoptosis could be controlled by regulating the mechanical properties of this biomaterial. The experiment was designed employing the immortalized foetal hepatocytes line, transformed with a plasmids which is able to produce constitutive intracellular GFP in high amounts. The experiments were carried out using 500 μm capsules made from two different alginate concentrations. Different cells concentrations were analyzed too. The experiment took place within a period of 7 days, characterizing the release of GFP (27 kD) into the media, as well as the cellular vitality and the cell proliferation. So far our results show that the release of GFP is strongly dependent on the alginate concentration, with better vitality and proliferation in the cells entrapped in lowest alginate concentration. Therefore the mechanical properties of the biomaterial have to be considered prior to designing a bioreactor as the release of specific proteins during the cellular death can lead to an undesired immuno-response caused by the use of immobilized cells in artificial organs. References. Leonard M., Rastello De Boisseson M., Hubert P., Dalencon F., Dellacherie E., (2004), Hydrophobically modified alginate hydrogels as protein carriers with specific controlled release properties. Journal of Controlled Release, 98: 395-405

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Mike Beck Max-Planck-Institut für Molekular-, Zellbiologie & Genetik Email: beck@mpi-cbg.de www.mpi-cbg.de/research/groups/ walch-solimena/walch-solimena.html

Lena Schönzart Technische Universität Dresden

ID 24 Faculty of Science Professorship for common Biochemistry Lena.schoenzart@chemie.tu-dresden.de www.chm.tu-dresden.de/bc1/ 

Synthesis and characterization of peptide oligodeoxyribonucleotide conjugates

Lena Schönzart, Jan Michael, Rene Beutner, Dieter Scharnweber, Hartmut Worch, Bernd Schwenzer

A modular system for bio-surface engineering of titanium implant materials based on partial anodic entrapment of oligodeoxyribonucleotides (ODN) in a titanium oxide layer and subsequent hybridization with different conjugated ODN was subject to further characterization. We focussed on various peptide conjugates of ODN whose linker molecules were different with respect to their hydrophobicity. A 31-mer ODN and the hexapeptide GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro), containing the recognition sequence RGD for osteoblast integrins, were coupled. Although hydrophobic properties were varied reversed-phase HPLC was not suitable for a successful separation of the conjugates. The Sakaguchi reaction for arginine was adapted to our system and proved successful conjugate syntheses. The test is based on the reaction of arginine’s guanidine group with 1-naphthol in the presence of hypobromite and the product shows a characteristic red colour. Whilst the Sakaguchi product spectra of arginine, GRGDSP, and mixtures of ODN and GRGDSP show a broad maximum between 450 and 570 nm, the arginine-containing conjugates show a maximum that is shifted to shorter wavelengths. Thus, the formation of ODN conjugates of GRGDSP can be proven via the characteristic wavelength shift of the Sakaguchi products, even if the product is not yet purified. All conjugates showed biological activity and could bind to integrins on osteoblasts.

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Mike Beck Max-Planck-Institut für Molekular-, Zellbiologie & Genetik Email: beck@mpi-cbg.de www.mpi-cbg.de/research/groups/ walch-solimena/walch-solimena.html

Diana Lindner Universität Leipzig

ID 25 Faculty of Bosciences, Pharmacy and Psychology Institute of Biochemistry dlindner@ uni-leipzig.de www.biochemie.uni-leipzig.de/agbs/

Ligand Binding at the Human Y5-Receptor

Diana Lindner, Jan van Diek, Robert Günther, Hans-Jörg Hofmann, Annette Beck-Sickinger

Y-receptors belong to the large superfamily of heptahelical G-protein couple receptors. Four Y-receptor subtypes have been cloned from human tissue (Y1, Y2, Y4 and Y5). These receptors are activated by a family of neuroendocrine hormones, the so-called NPY hormone family. Neuropeptide Y (NPY), pancreatic polypeptide (PP) and peptide YY (PYY) consist of 36 amino acids and are C-terminally amidated. We already know that NPY and PYY show a higher affinity at the hY5-receptor compared to PP. In contrast to the hY5-receptor, the hY4-receptor preferentially binds PP and has a lower affinity to NPY and PYY. One contact point between the ligands and the hY4- and hY5-receptors has already been identified. Arg35 of PP interacts with the highly conserved aspartate residue Asp6.59 in the third extracellular loop of the hY4-receptor, whereas Asp6.59 interacts with Arg33 of NPY in the hY5-receptor. Accordingly, this is an excellent example for a multi-receptor/multi-ligand system, in which we can study the binding mode at the molecular level. We were able to characterize the mode of interaction between the ligands and the hY5-receptor in a deeper manner. Here, we present a rational explanation for the different affinities of the various ligands to the hY5-receptor. We can demonstrate that the ligand binding mode is determined by the sequence and structure of the receptor subtype. To obtain further insight into the ligand binding, a computer model of the human Y5-receptor has been established and docking studies have been performed.

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Mike Beck Max-Planck-Institut für Molekular-, Zellbiologie & Genetik Email: beck@mpi-cbg.de www.mpi-cbg.de/research/groups/ walch-solimena/walch-solimena.html

Rüdiger Klingeler Leibniz Institute for Solid State and

ID 26 Materials Research Dresden r.klingeler@ifw-dresden.de www.ifw-dresden.de/ 

Functionalised carbon nanotubes for biomedical applications

Rüdiger Klingeler, Silke Hampel, Anja Wolter, Kamil Lipert, Matthias Lutz, Diana Haase, Yulia Krupskaya, Christopher Mahn, Arthur Taylor, Kai Krämer, Manfred Wirth, Bernd Büchner

There is a fastly increasing interest in applying nanoscaled materials in biomedicine aiming at the manipulation and sensoring of biological systems. A promising approach concerns the synthesis of functional elements by filling carbon nanotubes (CNT) with tailored materials. In this case, CNT act as chemically and mechanically stable nanocontainers which protect the filling materials and the biological systems against each other. The carbon shells provide wear resistance and oxidation protection, can stabilize novel magnetic molecules and enhance the possibilities for exohedral (e.g. bio-) functionalisation of the nanoparticles. In the EU Network CARBIO (Multifunctional Carbon Nanotubes for Biomedical Applications) we systematically exploit the potential of filled CNT to act as magnetic nano-heaters, drug carrier systems and sensors for a diagnostic and therapeutic usage at the cellular level. We have studied the magnetic properties of individual, iron-filled multi-walled CNT which imply their potential for magnetic nano-heaters. We successfully inserted biofunctionalised Fe-CNT into cancer cells and have proved their applicability for local in-situ-heating (hyperthermia) by applying AC magnetic fields. Filled CNT can also be used for a diagnostic purpose since the nanocontainers can be filled with appropriate sensor materials. One example is their filling with CuI or AgCl, which exhibit a temperature dependent NMR signal so that nanoscaled contactless temperature sensors are realised. The potential for drug-delivery is demonstrated by inserting conventional cytostatics into CNT and their release after transferring the filled CNT into a cancer cell culture.  

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Mike Beck Max-Planck-Institut für Molekular-, Zellbiologie & Genetik Email: beck@mpi-cbg.de www.mpi-cbg.de/research/groups/ walch-solimena/walch-solimena.html

Sabine Siegemund Universität Leipzig

ID 27 Faculty of Veterinary Medicine Institute of Immunology siegemund@vetmed.uni-leipzig.de www.vetmed.uni-leipzig.de/ 

Dendritic cells rather than macrophages produce interleukin (IL)-12 cytokine family members in response to Salmonella Enteritidis in vitro and in vivo

Sabine Siegemund, Nicole Schütze, Marina Freudenberg, Reinhard Straubinger, Gottfried Alber

Production of IL-12 is essential for protection against Salmonella infection. Previously macrophages (MΦ) were suggested to be the main producers of IL-12 in response to Salmonella. However, more recent data point to dendritic cells (DC) as important target cells for Salmonella and as source for IL-12. We compared the release of the IL-12 family members IL-12, IL-23, IL-27p28 and p40 by DC and MΦ in response to Salmonella Enteritidis (SE) in vitro and in vivo. Bone marrow-derived conventional DC (BMDC) and MΦ (BMMΦ) were generated by incubating bone marrow cells with GM-CSF and M-CSF, respectively. Comparison of the release of the IL-12 family members by BMDC and BMMΦ after in vitro stimulation with SE revealed BMDC as more potent producers of all IL-12 family members than BMMΦ. To investigate whether these in vitro results are valid in vivo, we infected mice intraperitoneally with SE and analyzed the secretion of IL-12, IL-23, and p40 at the site of infection and at the site of immune response induction by studying the cytokine release from isolated peritoneal exudate cells (PEC) and from lymph node cells draining the peritoneal cavity (DLN), respectively. Both, PEC und DLN released ex vivo p40 in response to SE. To identify the p40-producing cell type at the site of infection we depleted either DC or MΦ from isolated PEC and analyzed the release of p40. No p40 was produced by PEC depleted of DC, whereas p40 production was unchanged in PEC depleted of MΦ. These data confirm our in vitro results and point to DC rather than MΦ as major producers of the IL-12 cytokine family members in response to SE.  

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Iris Böselt Universität Leipzig

ID 28 Faculty of Biosciences, Pharmacy and Psychology Institute of Biochemistry iris.boeselt@medizin.uni-leipzig.de www.biochemie.uni-leipzig.de/

Population genetics of the GPR33 pseudogene

Iris Böselt, Peter Kovacs, Michael Stumvoll, Holger Römpler, Torsten Schönberg

The orphan chemoattractant receptor GPR33 was originally identified as a pseudogene in humans and as an intact gene in mouse. The human GPR33 pseudogene carries a premature stop codon but no other obvious structural defects, suggesting a recent inactivation of the receptor. Cloning and analysis of far more than hundred GPR33 orthologs disclosed inactivation of this GPCR not only in humans, but also in several great ape and rodent species. Deep genomic analysis revealed an independent, simultaneous inactivation of this chemoattractant GPCR in species from distantly related mammalian orders. This points to a recent selective pressure, for instance by interaction of a common pathogen on this receptor. Surprisingly, in all ape and some rodent species where inactivation were found, samples harbored both pseudogene and intact gene variants. Analysis of a human DNA panel representing all major linguistic groups revealed that the intact allele of GPR33 is still present in the human population with an allele frequency of 2 %. The coexistence of intact allele and pseudogene over such a long period of time could indicate a balanced selection. Haplotype analyses of certain geographically determined human populations displayed significant differences in allele frequencies. There is a tenfold decreased frequency if the intact wild-type allele in Catholic Sorbs as well as in the whole German population, compared to the overall frequency, furthermore no intact allele was detected in about 1000 Pima Indians. In contrast, Asian populations show an increased frequency of the intact GPR33 allele compared to the global population. Significant differences in allele frequencies may suggest an evolutionary advantage for humans carrying either the intact or the non-functional GPR33 depending on certain evolutionary forces in time and locality. Henceforth we plan to investigate the geographically associated allele distribution in more detail and we will also focus on a possible disease association. Additionally, access to human remains of the Sorbs population dating back several centuries ago will allow for studies on temporal aspects of allele frequencies at a defined locality.

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Mike Beck Max-Planck-Institut für Molekular-, Zellbiologie & Genetik Email: beck@mpi-cbg.de www.mpi-cbg.de/research/groups/ walch-solimena/walch-solimena.html

Kathrin Sichardt Universität Leipzig

ID 29 Faculty of Biosciences, Pharmacy and Psychology Institute of Pharmacy

sichardt@uni-leipzig.de www.uni-leipzig.de/~pharm/ 

Synaptic Localisation of NPY Y1 Receptors in the Rat Cingulate Cortex Kathrin Sichardt, Wolfgang Härtig, Annette Beck-Sickinger, Karen Nieber

Neuropeptide Y (NPY) is one of the most abundant neuropeptides in the CNS and exerts various functions on at least six receptor subtypes (Y1R-Y5R,y6R). Activation of Y1Rs results in an inhibition of excitatory synaptic transmission postulating a presynaptic influence on cortical neurons. Previous studies of our group confirmed the inhibitory effect of NPY via Y1Rs on pyramidal cells of the rat cingulate cortex but did not differentiate between a pre- and/or postsynaptic site of action. Therefore the aim of the present study was to elucidate on which site the inhibition occurs. Intracellular recordings were performed in rat brain slice preparations containing pyramidal cells of the cingulate cortex using glass microelectrodes placed in layer V. Superfusion of NPY and the selective Y1R agonist [F7, P34]pNPY caused inhibition of postsynaptic potentials (PSPs) but did not significantly influence the membrane potential and input resistance. In the presence of CNQX, a non-NMDA receptor antagonist, the inhibitory effect of [F7, P34]pNPY on PSPs was present. Inhibition of PSPs failed when preincubation with APV, a NMDA receptor antagonist, occurred. Furthermore, the influence of [F7, P34]pNPY on glutamate induced depolarisation of the cell membrane and attenuation of input resistance was investigated. [F7, P34]pNPY decreased depolarisation as well as changes of input resistance. The data indicate an interaction with NMDA receptors and reveal that Y1Rs are not exclusively located presynaptically.

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Mike Beck Max-Planck-Institut für Molekular-, Zellbiologie & Genetik Email: beck@mpi-cbg.de www.mpi-cbg.de/research/groups/ walch-solimena/walch-solimena.html

Cornelia Rufke Universität Leipzig

ID 30 Faculty of Biosciences, Pharmacy and Psychology Institute of Pharmacy rufke@uni-leipzig.de www.uni-leipzig.de/~pharm/ 

Time-Dependened Desensitisation of the Adenosine A1-Receptor during Hypoxia

Cornelia Rufke, Andreas Deussen, Karen Nieber

The release of adenosine, as the degradation product of ATP, is increased since of a higher energy consumption under hypoxic conditions. It can modulate the neuronal function and is known to act neuroprotectively mainly due to activation of adenosine A1 receptor (A1R), one of its four G-Protein-coupled-receptor subtypes. Endogenously released adenosine as well as selective A1R agonists inhibit the postsynaptic potential in cortical neurones. A desensitisation of this receptor subtype yields to the loss of neuroprotection. Therefore the extracellular level of adenosine during hypoxia as well as the time-dependent desensitisation of the A1R were investigated on CHO cells stably transfected with A1R by the measurement of intracellular markers using calcium imaging and ALPHA-Screen-technology. The activation of A1R by N6-Cyclopentyladenosine, a selective receptor agonist, leeds to an increase of the intracellular calcium concentration, which decreases time dependently during persistent agonist exposure. The extracellular level of adenosine is increased under hypoxic conditions. Moreover, the endogenous released adenosine desensitizes the A1R induced inhibition of the adenylate cyclase. This effect occurs after 12 and 24 hours of hypoxia exposure. Our results contribute to the understanding of the cellular mechanisms which underlie the time dependent A1R desensitisation.

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Mike Beck Max-Planck-Institut für Molekular-, Zellbiologie & Genetik Email: beck@mpi-cbg.de www.mpi-cbg.de/research/groups/ walch-solimena/walch-solimena.html

Silke Schulz Universität Leipzig

ID 31 Faculty of Veterinary Medicine Istitute of Immunology sischulz@vetmed.uni-leipzig.de http://www.vmf.uni-leipzig.de/  

IL-23-deficient mice are resistant against Salmonella Enteritidis infection but are defective in IL-17 production and the delayed-type hypersensitivity (DTH) response

Silke Schulz, Alissa Chackerian, Robert Kastelein, Gottfried Alber

IL-12 and IL-23 are both members of the IL-12 cytokine family. They share the p40 subunit, but have distinct IL-12p35 and IL 23p19 subunits. IL-12 has been shown to play a major role for protective T-cell-mediated immunity against intracellular pathogens such as Salmonella Enteritidis (SE). To characterize the effects of IL-23 in immunity to SE, wild-type (wt) and IL 23p19-/- mice on a C57BL/6 background were infected with varying doses of SE and monitored for at least 80 days. IL-23 deficient mice survived high inocula (up to 2,5 x 106 SE) exactly like wild-type mice. Antigenspecific re-stimulation of splenocytes revealed similar levels of IFN-� compared to wt mice, but a defective IL-17 production in the absence of IL-23. Furthermore, IL- 23p19-/- mice did not differ from wt mice in bacterial burden of spleen and liver on days 20 and 80 post infectionem. Delayed-type hypersensitivity (DTH) reactivity though was compromised in IL-23p19-/- mice. These data indicate that although IL-23 is required for T-cell dependent effector and IL-17 responses, IL-23 is dispensable for protection against SE when IL-12 is present. To analyze a potential protective role of IL-23 in the absence of IL-12, IL-12p35-/- mice (lacking IL-12 only) were compared with p35/p40-/- mice (lacking IL-12 and IL-23 as well as p40 monomers and homodimers) using low infective doses of SE. Following SE infection with only 2,500 cfu per mouse, we found long-term survival of IL-12p35-/- mice, whereas all p35/p40-/- mice died within 3-6 weeks and exhibited significantly higher bacterial burden in spleen and liver 20 days post infectionem. In contrast to p35/p40-/- mice, IL-12p35-/- mice developed a DTH reaction indicating that IL-23 (and/or p40) is responsible for DTH responses against SE. Splenocytes from infected IL-12p35-/- mice but not from p35/p40-/- mice produced IFN-� and IL-17 in response to stimulation with SE antigen. This indicates that p40-dependent cytokines are essential for protection in the absence of IL-12 and points to a role of IFN-�and/or IL-17. To investigate whether IL-23 and/or p40 monomers and homodimers are involved in protection, experiments with p35/19 gene-disrupted mice (lacking IL-12 and IL-23 but not p40) are in progress.

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Erik Schilling Universität Leipzig

ID 32 Faculty of Biosciences, Pharmacy and Psychology Institute of Biology II eschilling@trm.uni-leipzig.de www.uni-leipzig.de/~biowiss/biologie_ii/index.html

Determination of Cell-Biological Parameters of Non-activated and activated (LPS) Monocytes in response to different synthetic soft Materials under the Influence of shear Stress Erik Schilling

Monocytes from healthy donors are incubated on culture plates coated with different synthetic soft materials. Activation of the cells is achieved by the addition of lipopolysaccharide (LPS). The response of monocytes to fluid shear stress is investigated in a cone-plate system. After having shown that the viability of monocytes varies between resting monocytes and monocytes exposed to shear stress, the tests are repeated with stimulated monocytes. Besides viability tests other immunological parameters (cytokine production, oxidative burst, and phagocytosis) and electrophysiological parameters (patch-clamp studies) and Ca2+-imaging are measured. The results will be discussed with respect to the effect of shear stress and synthetic soft materials on the viability and functions of human monocytes.

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Mike Beck Max-Planck-Institut für Molekular-, Zellbiologie & Genetik Email: beck@mpi-cbg.de www.mpi-cbg.de/research/groups/ walch-solimena/walch-solimena.html

Nicole Schliebe Universität Leipzig

ID 33 Faculty of Biosciences, Pharmacy and Psychology Institute of Biochemistry nicole.schliebe@medizin.uni-leipzig.de www.biochemie.uni-leipzig.de/ 

An altered renal sodium-reuptake contributes to osmotic dysregulation in V2 vasopressin receptor-deficient mice

Nicole Schliebe, Rainer Strotmann, Torsten Schöneberg

Nephrogenic diabetes insipidus (NDI) is an inherited disease that is characterized by the kidneys insensitivity towards the action of the vasopressin, resulting in severe disorder of the water and electrolyte homeostasis. Typical symptoms of NDI are polyuria, hypovolemia and hypernatremia. Hypernatremia is commonly explained as resulting symptom caused by the net loss of water due to insufficient insertion of aquaporin 2 into the apical membrane of the collecting duct cells. However, our genome-wide expression analysis of the kidneys from V2 vasopressin receptor (AVPR2)-deficient mice suggests an additional mechanism where up-regulation of components involved in sodium reuptake significantly contribute to hypernatremia. The increased expression of several key components of the sodium reuptake, such as Na+/K+-ATPase and carboanhydrase, was accompanied by significantly increased activity of these enzymes. In comparison with previous studies on rats with lithium-induced NDI, AVPR2-deficiency causes specific changes in renal gene expression. Further, we found significant changes in components of the eicosanoid and thyroid hormone pathway, including cyclooxygenases and deiodinases. Our data highlights the involvement of renin-angiotensin-aldosterone and thyroid hormone systems in pathogenesis of NDI symptoms and provides further clues to explain the effectiveness of thiazide diuretics and indomethacin in treatment of NDI.

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Mike Beck Max-Planck-Institut für Molekular-, Zellbiologie & Genetik Email: beck@mpi-cbg.de www.mpi-cbg.de/research/groups/ walch-solimena/walch-solimena.html

Eberhard Krauß Max-Planck-Institute of Molecular

ID 34 Cell Biology and Genetics Technology Development Studio (TDS) krausz@mpi-cbg.de http://tds.mpi-cbg.de 

A unique Infrastructure for High-Content Screening is Mediating Technology Transfer from Academia to Industry

Eberhard Krauß, Ivan Baines, Marino Zerial

With combined forces of our institute, the Max-Planck Society and the BMBF, a European-wide unique academic high-content screening centre for Functional and Chemical Genomics has been established. Due to successful accomplishment of a number of scientifically as well as technically outstanding assay development and RNA interference screening projects, the long-term funding of the operations has been secured.

The infrastructure will be presented along with a number of innovative and disease-relevant cell-based screening assays and correlating image analysis solutions that have promising potential for application in drug discovery programmes in cancer, metabolic diseases and infection.

 

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Mike Beck Max-Planck-Institut für Molekular-, Zellbiologie & Genetik Email: beck@mpi-cbg.de www.mpi-cbg.de/research/groups/ walch-solimena/walch-solimena.html

Katrin Seifert Universität Leipzig

ID 35 Translational Center for Regenerative Medicine REMOD kseifert@trm.uni-leipzig.de www.trm.uni-leipzig.de/remod 

Small Molecules as Agonists of Hedgehog Signaling and their use for wound Healing

Katrin Seifert, Thomas Cottin, Anita Büttner, Athanassios Giannis

The Hedgehog (Hh) pathway controls the formation of limb, lung, gut, hair follicles, heart, bone, eye, neurons and pancreas. Activating Hedgehog pathway leads to increased angiogenesis. Angiogenesis is a critical component of wound healing. After wounding, the formation of a rich vascular bed is essential for optimal repair. Pre-clinical studies document that angiogenic growth factors, such as VEGF, FGF-1, FGF-2, TGF-ß, hepatocyte growth factor (HGF), angiopoietin-related growth factor (AGF) and placenta growth factor (PIGF) can promote vessel growth in vivo and microvascular endothelial cell morphogenesis in vitro. However, the use of these growth factors for wound healing of the skin is limited due to the rapid degradation of these proteins by several proteinases in the injured tissues. The aim of this project is the development of small molecule Hh agonists and their use for sustained stimulation of mesenchymal stem cells to express VEGF and angiopoietins-1 and -2 in order to stimulate angiogenesis. Thereby, a recently published Hh-agonist (SAG) will be used as a lead structure. Until now, it was possible to synthesize a library of about 20 SAG derivatives, which exhibit interesting activities in Shh-LIGHT2 cell line.

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Mike Beck Max-Planck-Institut für Molekular-, Zellbiologie & Genetik Email: beck@mpi-cbg.de www.mpi-cbg.de/research/groups/ walch-solimena/walch-solimena.html

Kay Bauer Universität Leipzig

ID 36 Medical Faculty Institute of Clinical Immunology Kay.bauer@medizin.uni-leipzig.de http://ikit.uniklinikum-leipzig.de 

Peptidyl prolyl isomerases can act as a molecular timer to control the amplitude and duration of Stat3 activity

Kay Bauer, Friedemann Horn

Signal transducer and activator of transcription 3 (Stat3) is the major mediator of interleukin-6 (IL-6) family cytokines and involved in the pathophysiology of many malignancies. Recently, we were able to identify an interaction of Stat3 with cyclophilin B (CypB), a cis-trans peptidyl-prolyl isomerase described to be involved in autoimmune diseases and in different types of cancer. Interestingly the highly related cyclophilin A (CypA) does not interact with Stat3, but coimmunoprecipitation studies also showed an association of CypA with Jak1 and Jak2. Furthermore our studies show that both cyclophilins can modulate the transcription of Stat3 target genes. But only CypB could be found on Stat3-specific promotors in the presence as well as absence of IL-6. CypA knock-down inhibited tyrosine phosphorylation of Stat3, while CypB knock-down does not. Interestingly, we found CypA and -B also to be involved in the survival of myeloma cell lines. Knock-down of CypA or -B induced apoptosis in these cells. Taken together, CypA and CypB both play pivotal roles, yet at different signaling levels, for Stat3 activation and function.

 

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Mike Beck Max-Planck-Institut für Molekular-, Zellbiologie & Genetik Email: beck@mpi-cbg.de www.mpi-cbg.de/research/groups/ walch-solimena/walch-solimena.html

Conny Blumert Universität Leipzig

ID 37 Medical Faculty Institute of Clinical Immunology Conny.blumert@medizin.uni-leipzig.de http://ikit.uniklinikum-leipzig.de 

In-situ-biotinylation – An approach to investigate the STAT3 signaling Pathway

Conny Blumert, Friedemann Horn

For in-situ-biotinylation we have chosen an approach using a small peptide tag of 23 amino acids, that is fused to the protein of interest. For biotinylation of this tag the enzyme BirA is co-expressed in the same cells with the protein containing the biotin acceptor domain. Once the protein is biotinylated, the moiety remains attached through the whole lifetime of the molecule.

The association between biotin and streptavidin is one of the strongest known non-covalent interactions. This fact can be used to create very stringent conditions for binding and washing procedures within different applications like western blotting, co-immunoprecipitation, immunofluoreszenz or chromatin-immunoprecipitation studies.

To determine expression, biotinylation and function of biotinylated signal transducer and activator of transcription (STAT)3, western blot analysis as well as reporter gene analysis were carried out. The results show that biotinylated STAT3 becomes expressed, phosphorylated at tyrosine residue 705 and thereby activated. We further demonstrated that biotinylated STAT3 is able to activate STAT3-dependent promotors with almost the same amplitude as wildtype STAT3.

 

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Mike Beck Max-Planck-Institut für Molekular-, Zellbiologie & Genetik Email: beck@mpi-cbg.de www.mpi-cbg.de/research/groups/ walch-solimena/walch-solimena.html

John Heiker Universität Leipzig

ID 38 Institute of Biochemistry AG Beck-Sickinger jheiker@uni-leipzig.de www.biochemie.uni-leipzig.de/agbs/default.asp

Expression of human full length adiponectin and its globular variant in E. coli

John Heiker, Annette Beck-Sickinger

Adiponectin is an adipokine with anti-atherogenic, anti-diabetic and insulin sensitizing properties predominantly expressed in and secreted from adipocytes mainly in the white adipose tissue. Its effects on energy homeostasis and glucose and lipid metabolism are mediated by two ubiquitously expressed seven-transmembrane receptors, AdipoR1 and AdipoR2, yet signaling pathways downstream of these receptors remain poorly defined. Here, we have recombinantly expressed human adiponectin (fAd) and its globular domain (gAd) as His-tag fusion proteins in E. coli BL21(DE3) to obtain adiponectin in large amounts for biochemical and functional studies. Inducible prokaryotic expression vectors fAd_pET15b and gAd_pET15b (with 6xHis-tag) were constructed with pET15b and the cDNA of adiponectin gene cloned in our lab. His-Ad was almost exclusively present as inclusion bodies and urea-solubilised His-Ad was purified by metal ion affinity chromatography with on-column refolding. The purified recombinant adiponectin was characterized by SDS-PAGE, Western Blot and MALDI-TOF analysis. Both variants, His-tag full length and globular adiponectin, have shown biological activity as evidenced by induction of the phosphorylation of ACC in differentiated C2C12 myotubes.

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BIOANALYTICS

 

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Ursula Eschenhagen Technische Universität Dresden

ID 39 Inst. of Food Techn. and Bioprocess Engineering Ursula.eschenhagen@tu-dresden.de www.tu-dresden.de/die_tu_dresden/fakultaeten/ fakultaet_maschinenwesen/ilb

Hygienic properties of altenative surfaces

Ursula Eschenhagen, Marc Mauermann, Jens-Peter Majschak, Thomas Bley

Microbial contaminations in plants of food or pharmaceutical processing plants are a great risk for the industry and the consumers. To avoid such contaminations, the companies have to make great efforts in optimizing the cleaning procedure, what is time- and cost-intensive. To reduce the cleaning periods and plants downtime alternative material and surfaces with lower affinity for contamination are discussed. There are several so called “easy to clean surfaces” available on the market. To make it easier for the customers to choose the appropriate material for his application, several materials are compared according to their hygienic and technical characteristics. To characterize the materials according to their anti-adhesive properties a corresponding method has been developed. We divided the method into several steps: • Defined contamination of the surfaces with microorganisms or non-microbial matrices • Detection of cell adhesion or non-microbial contamination • Detection of non-microbial contamination • Defined cleaning • Verification of the cleaning result The investigations were made with different samples from the following surface coating groups: Polymer coatings, Sol-Gel coatings, Carbon coatings, Ceramic coatings and further coatings deposited by physical or chemical vapour deposition. As contamination matrix whey protein, potato starch and the microorganisms Pseudomonas fluorescence and Saccheromyces cerevisiae were used. The non-microbial contamination was determined gravimetrically at first. To detect marginal amounts, fluorescence microscopic methods were used. The microbial contamination was detected by help of epifluorescence technique and electronic image processing. As material properties roughness and surface tension were measured and compared with the contamination and cleaning characteristics of the materials. Furthermore different investigations to test the material stability will be made.

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Tommy Opitz Technische Universität Dresden

ID 40 Inst. of Food Techn. and Bioprocess Engineering Tommy.opitz@tu-dresden.de www.tu-dresden.de/die_tu_dresden/fakultaeten/ fakultaet_maschinenwesen/ilb

Monitoring gasförmiger Metabolite in Feststoffreaktoren

Tommy Opitz,

Als Solid State Fermentationen (SSF) werden Kultivierungen von Mikroorganismen auf festen feuchten Substraten bezeichnet. Biotechnologisch und wirtschaftlich interessante Produkte aus Pilzen wie Enzyme, Sporen oder sekundäre Metabolite werden für Applikationen auf festen Substraten hergestellt. Im Gasraum über einer Feststoffkultur kommt es im Gegensatz zum klassischen Rührkessel-Fermenter zu erheblichen Gradienten von Temperatur, Substratkonzentration, Wasser und Sauerstoff, die ein homogenes Zellwachstum behindern. Eine Möglichkeit zur Beurteilung der biologischen Aktivität dieser Kulturen besteht in der Messung typischer Stoffwechselprodukte im Headspace. Im Posterbeitrag wird eine Möglichkeit zum Monitoring biotechnologischer Fermentationsprozesse als Online-Messung der Konzentration von gasförmigen Kohlenwasserstoffen und Ammoniak mittels hochempfindlicher Gassensoren vorgestellt. Die Ergebnisse zeigen, dass die Zusammensetzung der Atmosphäre im Headspace über einer Feststoffkultur stark vom Substratangebot und den Kultivierungsbedingungen abhängt. Die geringe Konzentration an Zielmolekülen in Gegenwart eines hohen Anteils an Wasserdampf und CO2 stellt für viele klassische spektroskopische Methoden ein Problem dar. Die Analytik der Gasräume mittels GC-MS ist ohne selektive Anreicherung auf Adsorbensmaterialien nicht möglich. Es konnten neben der Hauptkomponente Ethanol verschiedene Furan-Derivate, Terpene, aromatische Kohlenwasserstoffe und Alkansäuren nachgewiesen werden. Der eingesetzte potentiometrische Gassensor auf Basis fester Elektrolyte liefert hingegen online und ohne Unterbrechungen ein Summensignal aller emittierten Kohlenwasserstoffe. Eine Kopplung mehrerer empfindlicher Sensoren wird über eine Mustererkennung Rückschlüsse auf spezifische Gaskomponenten zulassen.

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Jacqueline Leßig Universität Leipzig

ID 41 Faculty of Medicine Institute of Medical Physics and Biophysics Jacqueline.leßig@medizin.uni-leipzig.de www.uni-leipzig.de/~biophys/

Myeloperoxidase binds to non-vital spermatozoa on phosphatidylserine epitopes

Jacqueline Leßig, Uta Reibetanz, Martin Fischlechner, Hans-Jürgen Glandner, Jürgen Arnhold

The heme protein myeloperoxidase is released from stimulated polymorphonuclear leukocytes, a cell species found in increasing amounts in the male and female genital tract of patients with genital tract inflammations. Myeloperoxidase binds only to a fraction of freshly prepared human spermatozoa. The number of spermatozoa able to bind myeloperoxidase raised considerably in samples containing pre-damaged cells or in acrosome-reacted samples. In addition, myeloperoxidase released from zymosanstimulated polymorphonuclear leukocytes was also able to bind to pre-damaged spermatozoa. The ability of spermatozoa to bind myeloperoxidase coincided with the binding of annexin V to externalised phosphatidylserine epitopes indicating the loss of plasma membrane integrity and with the incorporation of ethidium homodimer I. Myeloperoxidase did not interact with intact spermatozoa. Annexin V and myeloperoxidase bind to the same binding sites as verified by double fluorescence techniques, flowcytometry analyses as well as competition experiments. We demonstrated also that myeloperoxidase is eluted together with pure phosphatidylserine liposomes or liposomes composed of phosphatidylserine and phosphatidylcholine in gel filtration, but not with pure phosphatidylcholine liposomes. In conclusion, myeloperoxidase interacts with apoptotic spermatozoa via binding to externalised phosphatidylserine indicating a yet unknown role of this protein in recognition and removal of apoptotic cells during inflammation.

82

Uta Reibetanz Universität Leipzig

ID 42 Faculty of Medicine Institute of Medical Physics and Biophysics Uta.reibetanz@medizin.uni-leipzig.de www.uni-leipzig.de/~biophys/

Interaction of Cells with Fluorescein-Labeled Polyelectrolyte Multilayer Particles and Capsules

Uta Reibetanz, David Haložan, Martin Fischlechner, Milan Brumen, Edwin Donath

The Layer-by-Layer (LbL) coating of particles and capsules with biocompatible polycations, polyanions and active agents opens an interesting field of application in cell biology and drug delivery: Bioagents like DNA, RNA or enzymes as well as sensor molecules like fluorescence probes can be either integrated within the polymer multilayer or encapsulated in the interior [1,2,3]. Especially the investigation of uptake and processing of particles and capsules by cells is important with regard to transport and release of the bioagents into the cytoplasm: Only a small expression rate was found after uptake of particles coated with plasmid encoding for the green fluorescent protein (GFP) as reporter molecule [1]. One hypothesis for the small rate is, that most of the particles remain located in endolysosomes and undergo the lysosomal degradation process, which leads to the decomposition of the polymer shell and the inactivation of the plasmid. The aim of this work is to develop a tool that would permit to localize the particles or capsules in their intracellular environment under in vivo conditions and to quantify the number of them in the cytoplasm in relation to those in endolysosomes. So we engineered particles and capsules, 5μm in diameter, with an integrated sensor function which is based on a pH sensitive fluorescent probe. The intracellular pH can be used as a marker to localize the particles: the pH varies from pH 4.5-5 within lysosomes to pH 7.5 in the cytoplasm [4]. The fluorescent probe, amino fluorescein-labeled poly(acrylic acid) (PAAAF), was integrated into the device to protect it from the undesirable interaction with cellular components. These particles and capsules were employed to explore their endocytotic uptake into HEK 293T cells by flow cytometry. The percentage of capsules residing in the endolysosomal compartment was estimated from the fluorescence intensity decrease caused by acidification. Capsules attached to the extracellular surface of the plasma membrane were identified by trypan blue quenching [5]. [1] Reibetanz, U. et al. Macromol. Biosci. 6 (2006) 153-160 [2] Javier, A. et al. Small 2 (2006) 394-400 [3] Cortez, C. et al. Adv. Mater. 18 (2006) 1998-2003 [4] Mellman, I. et al. Annu. Rev. Biochem. 55 (1986) 663-700 [5] Reibetanz, U. et al. Biomacromolecules 8 (2007) 1927-1933

83

Nicole Weizenmann Universität Leipzig

ID 43 Faculty of Bioscience, Pharmacy and Psychology Institute of Biochemistry weizenmann@uni-leipzig.de www.biochemie.uni-leipzig.de/agz/

The influence of organic solvents on the enzymatic synthesis of large-ring cyclodextrins produced by an amylomaltase from Thermus aquaticus ATCC 33922

Nicole Weizenmann, Susanne Washeim, Christian Roth, Norbert Straeter, Wolfgang Zimmermann

Amylomaltases are intracellular 4-α-glucanotransferases and belong to the family 13 (α-anylase family) of the glycosyl-hydrolases. They catalyse the transfer of maltooligo-saccharides from one α-1,4-glucan to another α-1,4-glucan or to glucose. The amylomaltase (EC 2.4.1.25) from Thermus aquaticus ATCC 33922 acts on amylose in different ways. It catalyses an intermolecular glucan transfer reaction (disproportionation reaction) and shows a significant hydrolytic activity. The enzyme catalyses furthermore a cyclization reaction (intramolecular transglycosylation reaction) to form large-ring cyclodextrins with a degree of polymerisation of 22 up to several hundred. Cyclodextrins have the ability to form inclusion complexes with a wide range of guest molecules thereby modifying their physicochemical properties. The corresponding gene was expressed in E. coli K12 UT5600 and the protein was purified. The influence of organic solvents (ethanol, methanol and isopropanol) on the enzymatic synthesis of large-ring cyclodextrins with a degree of polymerization (DP) of 22 to 40 was investigated. With 45 % of ethanol no cyclic products were obtained while in the presence of 5 % to 25 % of ethanol, an increased yield of large-ring cyclodextrins with a DP of 23 to 36 and a higher remaining activity of the amylomaltase after 4 h of incubation at 70 °C was found. With 45 % of methanol only a small amount of cyclic products were obtained while in the presence of 5 % to 35 % of methanol, a slightly increased yield of large-ring cyclodextrins with a DP of 25 to 37 was found. With 35 % and 45 % of isopropanol very small amounts of cyclic products were obtained while in the presence of 5 % to 15 % of isopropanol, an increased yield of large-ring cyclodextrins with a DP of 23 to 36 was detected. Ethanol may facilitate the binding of the amylose to amylomaltase. Another possibility is that the addition of ethanol reduces the amount of water or acceptor molecules at the active centre thereby protecting the large-ring cyclodextrins against their degradation

84

Kristin Köllner Universität Leipzig

ID 44 Faculty of Bioscience, Pharmacy and Psychology Institute of Biochemistry kristin.koellner@medizin.uni-leipzig.de www.uni-leipzig.de/~biochem/abch_cms/index.php

The PFKFB3 splice variant UBI2K4 inhibits cell proliferation of U87 glioblastoma cells

Kristin Köllner, Renate Kessler, Klaus Eschrich

Tumor cells maintain a high rate of glycolysis even in the presence of oxygen (Warburg effect). Fructose-2,6-bisphosphate (F2,6P2) is the most potent activator of 6-phosphofructo-1-kinase, a rate-controlling enzyme of glycolysis. The synthesis and degradation of F2,6P2 is catalyzed by the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2) which is expressed in four tissue-specific isoforms (PFKFB1-4). PFKFB3 protein is expressed strongly in several human tumors and tumor cell lines. In human, six alternatively spliced isoforms of PFKFB3 are known (UBI2K1-6). RT-PCR analysis revealed the expression of three splice variants in U87 glioblastoma cells: UBI2K4, UBI2K5 and UBI2K6. We analyzed the influence of these splice variants on metabolic profile and proliferation of U87 cells. Overexpression of UBI2K5 or UBI2K6 activates the glycolytic flux via F2,6P2 synthesis leading to enhanced lactate production. In contrast, cells with UBI2K4 overexpression showed no increase of lactate production compared with control cells resulting from a lower cell number caused by inhibition of proliferation. Additionally, U87 cell lines stably expressing the splice variants UBI2K4 or UBI2K5 were generated. The doubling time of cells stably expressing UBI2K4 is decelerated compared to cells stably expressing UBI2K5 confirming the inhibitory effect of UBI2K4 expression on cell proliferation. For elucidating the role of PFKFB3 splice variants in more detail protein-protein interactions were analyzed using an improved ‘tandem affinity purification’ (TAP) method. The FLAG-tag was used for the secondary chromatography step instead of the calmodulin binding peptide resulting in higher recovery during purification. Until now, only heat shock proteins associated with the recombinantly expressed UBI2K5-TAP protein as well as with the TAP-tag protein alone were identified as interacting proteins.

85

Nico Jehmlich Helmholtz Center for Environmental Research -

ID 45 UFZ Leipzig Department Proteomics nico.jehmlich@ufz.de www.ufz.de/index.php?de=6693

Development of a new metabolic labeling approach (Protein-SIP) for proteome based identification of bacteria with specialized functions in mixed cultures

Nico Jehmlich, Martin von Bergen, Hans-Herman Richnow, Carsten Vogt

For biotechnological and environmental microbiology the identification of bacteria with specialized function in mixed cultures is a question of utmost importance. In cases, the function of interest is linked with the metabolism a suitable approach is to use substrate labelled with stable isotopes. These are metabolized and incorporated into the biomass of the cell, in particular proteins. By applying state of the art 2D gel electrophoresis and MALDI-MS/MS it is possible we were able to analyse incorporation of stable isotopes into proteins with sensitivity down to 2 % incorporation. This technique can be applied in several fields ranging from screening schemes for micro-organisms with defined functions (e.g. proteolytic activities) to degradation of environmental harmful xenobiotics. We conducted a model experiment for identifying the species responsible for anaerobic toluene degradation in an artificial mixed culture fed with gluconate and isotope labelled [7-13C]-toluene under denitrifying conditions. Labelled proteins were subsequently analysed by 2D gel electrophoresis and mass spectrometry to characterize their identity, as well as their 13C content as an indicator for function and activity of the degrading species organism in a bacterial community. The toluene degrading bacteria Aromatoleum aromaticum strain EbN1 was grown under anoxic conditions with [7-12C]- and [7-13C]-toluene as carbon source and nitrate as electron acceptor. Selections of six proteins from 2D gels were identified. The levels of 13C incorporation in peptides were determined as about 94 %. Subsequently, we cultivated strain EbN1 together with the enrichment culture UFZ-1, which is not able to degrade toluene. The both substrates [7-13C]-toluene and [12-12C]-gluconate were added as carbon sources and nitrate as an electron acceptor. Incorporation of 13C was exclusively found in proteins from EbN1 at contents of about 85 % and not in proteins from the UFZ1 culture. The results enabled us to identify the active degrading species and to unravel partially the food chain in a bacterial consortium.

86

Sally Bayer Universität Leipzig

ID 46 Center for Biotechnology and Biomedicine Junior Research Group “White Biotechnology” sbayer@rz.uni-leipzig.de

www.uni-leipzig.de/~tgs 

High throughput library screening for novel lipase activities

Sally Bayer, Meike Ballschmiter, Thomas Greiner-Stöffele

Lipases (triacylglycerol hydrolases EC.3.1.1.3.) are biotechnologically relevant enzymes. Especially bacterial lipases are important biocatalysts. Being the most used enzymes in organic and fat chemistry they have been successfully applied in triglyceride synthesis, production of mono- and diacylglyceroles and the alcoholation of fats and oils. Lipases are also applied in pharmaceutical, detergent and food industries. Culturable microorganisms as the source of lipases to date represent only a small fraction of the organisms known to be present in the environment. Metagenome libraries, containing directly isolated DNA of all organisms of a particular ecosystem, open an attractive alternative to cultured microorganisms for finding desired biocatalysts. After establishing different high throughput lipase activity assays the untapped resources of the metagenome and the high throughput screening approach were combined to successfully screen environmental libraries. Several hydrolases acting on ester bonds and their corresponding genes could be identified.

87

Michael Hofer Universität Leipzig

ID 47 Center for Biotechnology and Biomedicine Junior Research Group “White Biotechnology” hofer@rz.uni-leipzig.de

www.uni-leipzig.de/~tgs 

Searching new enzymes for glucose and lactate biosensors in metagenomic libraries

Michael Hofer, Katrin Boensch, Thomas Greiner-Stöffele

The enzyme “Pyrroloquinoline quinone dependent glucose dehydrogenase-B” (PQQ GDH-B) [1] is frequently used as an enzymatic biosensor for measuring blood glucose levels. In this system Glucose is reduced by PQQ GDH-B to glucono-1,5-lactone, which is followed by an electron transfer first from Glucose to the Cofactor PQQ and finally to an electron acceptor in the biosensor. As this electron flow is proportional to the glucose concentration, it can be used to visualize the glucose concentration by the biosensor unit. PQQ GDH-B has a high turnover number and it is not influenced by oxygen, which makes it faster and more reliable than other glucose biosensor enzymes like glucose oxidase (GOD). Beside these beneficial properties, there are still some further features which could be improved, like substrate specificity or heat stability. Our group is interested in finding new PQQ GDH-B like enzymes with such improved properties. For this purpose we developed a sensitive assay for the screening of PQQ GDH-B like active enzymes and their isolation out of metagenomic libraries. Furthermore we are working on the isolation of enzymes with similar enzymatic activities like lactate oxidase (LOD)[2], which is widely used in lactate biosensors. The monitoring of lactate levels nowadays is very important for diagnostic purpose and for fitness control in professional sports. We therefore developed a screening assay to find LOD like enzymes in metagenomic libraries with better features in terms of stability and activity compared to commercial available lactate oxidases. References: 1.) A. M. Cleton Jahnsen, Mol. Gen. Genetic, 217, 430-436, 1989. 2.) J.D. Duncan, Bioch. and Bioph. Research Comm., 164, 919-926, 1989

88

Regina Huettl TU Bergakademie Freiberg

ID 48 Institute of Physical Chemistry Regina.huettl@chemie.tu-freiberg.de www.chemie.tu-freiberg.de/phy/new/ index.php?menue=1&lang=1

Analysis of microbial processes in a small-scale calorimeter with additional sensors

Regina Huettl, Andreas Lissner, Joachim Harmel, Gert Wolf, Karl Held, Peter Klare, Winfried Vonau, Sigrun Herrmann, Frank Berthold

The application of calorimetric methods for the investigation of the metabolism of cell cultures using different devices is well known. In earlier presentations /1, 2/ we show that for the interpretation of calorimetrically detected metabolic processes the simultaneous registration of additional culture parameters is necessary. However the possibilities to integrate additional sensors in conventional calorimetric systems, especially using small reactor volumes, are limited. This contribution presents a miniaturized chemostatic measuring system based on a small-scale calorimeter (20 ml) equipped with two additional sensors. In our system the universal thermal power, characterizing the physiological state of the microorganisms is combined with two special electrochemical sensors. The two miniaturized sensors can be chosen from a pool of potentiometric and amperometric sensors, measuring pH-value, redox potential, nitrate activity, dissolved carbon dioxide and glucose content, according to the fermentation process or to the analytical problem. The miniaturized sensors were developed by the Kurt-Schwabe-Institute (Meinsberg) and the circuit design of the chemostatic measuring system carried out by the other project partner IMM (Mittweida). This system is applicable for batch and continuous cultivations. The calorimetrically monitored continuous cultivation of P. denitrificans over a period of more than 14 days and the influence of nutrient insertion is demonstrated. The work is supported by the European Funds for Regional Development (EFRE) 2000 – 2006 and the Free State of Saxony (Department of State for economics and work). [1] Ullrich, F.; Winkelmann, M.; Hüttl, R.; Wolf, G: Analytical and Bioanalytical Chemistry 383 (2005) 747-7518 [2] Ullrich, F.; Winkelmann, M.; Hüttl, R.; Wolf, G: Presentation on the XIIIth ISBC conference 2003 in Würzburg

89

Mario Krehan Universität Leipzig

ID 49 Center for Biotechnology and Biomedicine Professorship for Molecular Cell Therapy Mario.krehan@bbz.uni-leipzig.de www.uni-leipzig.de/~mct/

RNase P and MRP: function and architecture in plants

Mario Krehan, Sebastian Braun, Janine Dahl, Nikolas Menzel, Christian Heubeck, Astrid Schön

RNase P is the single enzyme responsible for the 5’ maturation of all precursor tRNAs (pre tRNAs) and thus essential for cellular protein biosynthesis. This ribonucleoprotein enzyme is composed of one RNA and one to several proteins in all organisms. Whereas bacterial RNase P consists of one protein and one RNA that possesses strong ribozyme activity in vitro, the RNA component of eukaryotic nuclear RNase P has also recently been proven as ribozyme, but of weak activity. However, there is unexpected variability in composition and function of RNase P, even between eukaryotic cells. The structurally related RNase P and RNase MRP share up to 8 protein subunits, but have different RNA components and substrates. In plants no RNase P RNA but two RNase MRP RNAs could be identified. In mitochondria and chloroplasts of multi¬cellular organisms, no RNase P RNA has been found to date. This raises the question of enzyme evolution in these organelles and in plants. In animal and human cells, it is currently unclear whether the resident nuclear RNase P and MRP enzymes are also present in mitochondria. In this case, there must be a mechanism for the sorting of both RNA and protein components to the nucleus as well as to the mitochondria. Our aim is to understand composition, function and transport of RNase P and MRP in plants. From the plant model organism A. thaliana we have therefore identified and cloned a number of protein subunits which share similarities to those from human and yeast as well as the two slightly different RNase MRP RNA variants. In present, we are experimentally verifying the relationship of the RNA and protein subunits to the RNase P and MRP holoenzymes, respectively.

90

Mirko Trutnau Technische Universität Dresden

ID 50 Ins. of Food Techn. and Bioprocess Engineering Mirko.trutnau@tu-dresden.de

tu-dresden.de/die_tu_dresden/fakultaeten/ fakultaet_maschinenwesen/ilb 

Immobilization of chitosan from molds onto glass beads for the use in biosorption columns

Mirko Trutnau, Jelka Ondruschka, Thomas Bley

 

In the kingdom of molds Mucor rouxii belongs to the Phycomycota to the class of Zygomycetes. In their ubiquitous appearance as food spoilage agents Mucorales are highly saccharolytic and proteolytic. Therefor M. rouxii can be readily cultured in simple nutrients like for example in waste from food industries such as distiller’wash or molasses. Among chitin chitosan is also component of the cell wall of M. rouxii with a fraction up to one third of the cell wall dry weight, in contrast to other molds. Chitosan finds numerous applications especially in water purification, the agriculture, medical applications and as dietary in the food industries. It is also derivable by deacetylation of chitin with sodium hydroxide from crustacea and other fungi. For the first step of chitosan extraction from hyphal wall cells were deproteinized in a solution of sodium hydroxide followed by demineralization in hydrochlorid acid. For the separation from other cell wall components chitosan was solved in acetic acid. By neutralization the supernatant with sodium hydroxide to neutral chitosan was precipitated and washed successively with destilled water ethanol and aceton. Finally the resulted chitosan was freeze dried. Notably the demineralization step is of great importance because its influence to the ash content. By using distiller’s wash as substrate ash contents up to 6 % are typical without demineralization because of calcium adsorption to the amino group of chitosan. After demineralization ash contents decrease to 1.5 % and are comparable with commercially available chitosan from shrimp and crabshell. The degree of deacetylation was determined to 87 %. The degree of polymerization from mold Chitosan is comparatively low with a dynamic viscosity of 2.6 mPa at 20°C. For using a high active surface of chitosan and to avoid a high leakage of pressure across the filter bed the polymer was immobilized onto a carrier material (glass beads with an average diameter to 150 to 212 μm). The immobilization was achieved in four steps according to Liu (2002): 1.) activation of the glass beads surface with NaOH 2.) amination with 3-aminopropyltriethoxysilane (APTES) 3.) introduction of a glutaraldehyd spacer 4.) immobilization of chitosans through a Schiff’s reaction. The quantification of bounded chitosan was done by a modified Svennerholm method (Liu, 2002) for determination of the amount of glucosamines.

91

Antje Eichler Universität Leipzig

ID 51 Center for Biotechnology and Biomedicine Junior Research Group „White Biotechnology“ eichler@uni-leipzig.de www.uni-leipzig.de/~tgs

In vitro expression and high throughput screening for novel proteases

Antje Eichler, Thomas Greiner-Stöffele, Meike Ballschmiter

Proteases are enzymes which catalyse the hydrolytical cleavage of peptide bonds. Because of their specificity and stability they have a wide range of applications in industrial processes and clinical research. Since proteases are enzymes which are potentially harmful to bacterial expression hosts in vitro expression is the method of choice for finding new proteases in natural or artificial libraries. The use of such a system decreases the occurrence of insoluble inclusion bodies, the reduced expression of proteins or the need for a functional secretion system, which is often encountered in heterologous in vivo expression. We have established a screening method for proteases using a model protease from Bacillus clausii for the optimization of the linked transcription-translation system we use. We are able to detect proteases in a genomic library of Bacillus subtilis by high throughput screening. In the same way artificial libraries can be screened for proteases with novel properties, thereby avoiding the disadvantages of the commonly applied E. coli expression system.

92

Ianors Iandiev Universität Leipzig

ID 52 Translation Center for Regerative Medicine REMOD iiandiev@trm.uni-leipzig.de www.trm.uni-leipzig.de/remod.html

Regeneration of Retinal Tissue – Clearance of Edema by Triamcinolone Acetonide

Ianors Iandiev, Ortrud Uckermann, Antje Wurm, Thomas Pannicke, Peter Wiedemann, Andreas Reichenbach, Andreas Bringmann, Susann Uhlmann

 

Objective: Edema develops in retinas after transient ischemia, retinal detachment, ocular inflammation and diabetes. The accumulation of fluid in the retinal tissue causes a compression of retinal cells and blood vessels. For retinal regeneration a sufficient clearance of edema is necessary. The fluid clearance is normally carried out by the pigment epithelium and Müller glial cells. Material and methods: Alterations of retinal Müller cells under pathological conditions were investigated by electrophysiologic methods, immunohistochemistry and recording their cell size under osmotic stress. The effect of triamcionolone acetonide on the osmotic swelling of Müller glial cells was determined in acutely isolated retinal slices. Results: Müller cells under pathological conditions decreased their expression of potassium channels. This was associated with a decrease in the potassium currents and a decrease in the water transport through the cells, leading to swelling of Müller cells. Triamcinolone inhibited the swelling of Müller cells. The effect of triamcionolone was mediated by stimulation of transporter-mediated release of endogenous adenosine and subsequent A1 receptor activation, resulting in an elevation of the intracellular cAMP level and activation of the protein kinase A, and finally, in an opening of extrusion pathways for K+ and Cl- ions. The ion efflux is associated with a water efflux which prevents cellular swelling under hypoosmotic conditions. Conclusion: The data suggest that a disturbed fluid transport through Müller cells is a pathogenic factor contributing to the development of retinal edema. The inhibitory effect on the cytotoxic swelling of glial cells may contribute to the fast edema-resolving effects of vitreal triamcinolone observed in human patients.

93

Mathias Salomo Universität Leipzig

ID 53 Faculty of Biosciences, Pharmacy and Psychology Institute of Biochemistry msalomo@rz.uni-leipzig.de

www.biochemie.uni-leipzig.de 

Optical Tweezers to Study Single Receptor / Ligand Interactions

Mathias Salomo, Ulrich F. Kaiser, Marc Struhalla, Friedrich Kremer

 

Optical tweezers (OT) are ideally suited to study the interaction of single receptorligand bonds. Here a newly developed assay is introduced using optical tweezers to investigate the interactions between Protein A from Staphylococcus aureus and Immunoglobulin G from rabbit serum (RIgG). It is demonstrated that the rupture forces depend on the loading rate and sodium chloride concentration. The measured loading rate effect is well known in the literature and the obtained data were found to be in good agreement with an already published theoretical model. The dependence of the rupture forces on the salt concentration demonstrates the influence of hydrophobic interactions on the bond strength. Our experimental setup can probe the interaction between a single receptor and its specific ligand under changing conditions and hence is applicable to biotechnological purposes.

94

Anika Schumann Universität Leipzig

ID 54 Faculty of Bioscience, Pharmacy and Psychology Institute of Biology I anikaschumann@gmx.de www.uni-leipzig.de/ ~pflaphys/

Induction of oxidative stress in the MCF7/NeuT cell line overexpressing the receptor tyrosine kinase ErbB2

Annika Schumann, Matthias Hermes, Matthias Gilbert, Jan G. Hengstler, Christian Wilhelm

Oxidative stress plays a major role in the induction and development of disease in human, animal and plant pathology as well. In biomedical research the evaluation of the cellular oxidative stress level is important for diagnosis, treatment and testing the effectiveness of new promising drugs. Normally, different oxidative stress markers are analysed by time-consuming biochemical extraction followed by analytical methods. Instead, thermoluminescence (TL) measurements can use complex samples without further time-consuming preparation. Different peroxidic compounds formed by oxidative stress decompose in the heating step of TL measurements at temperatures between 100 to 180°C under chemiluminescent light emission. This thermoinduced light emission can be quantified and correlates well with conventional biochemical estimations of lipid or protein peroxidation products. In this poster we present data for the induction of oxidative stress in doxycyclin treated MCF7/Neu T cells overexpressing the receptor tyrosine kinase ErbB2. ErbB2 is thought to be a key player in governing the cell cycle, potentially leading to cancer or premature senescence. After 14 days exposure to doxycyclin the oxidative stress markers malondialdehyde, malondialdehyde + 4-hydroxynonenal and thiobarbituric acid reactive substances increased significantly. Likewise, a significant increase of the TL signal could be observed in nitrogen atmosphere for doxycyclin treated MCF7/NeuT cells. Finally, we could show that TL measurements can monitor the oxidative stress status of MCF7/NeuT cells overexpressing the receptor tyrosine kinase ErbB2.

95

Gregor Hoffmann Universität Leipzig

ID 55 Center for Biotechnology and Biomedicine Junior Research Group “White Biotechnology” gregor.hoffman@bbz.uni-leipzig.de http://www.uni-leipzig.de/~tgs/

Strategy for altering the substrate specificity of P450-Monooxygenases

Gregor Hoffmann, Katrin Boensch, Thomas Greiner-Stöffele

 

 

 

 

Cytochrom P450-Monooxygenases are versatile catalysts. The property to hydroxylate a wide variety of organic compounds making them interesting for a lot of chemical applications. In contrast to established methods they work under mild conditions and are regio- and stereoselective. As a model system for the alteration of substrate specificity we use the D-camphor Monooxygenase (P450cam) from Pseudomonas putida. The system of P450cam together with NADH-Ferredoxin-Reduktase (pdR) and Ferredoxin (pdX) catalyse the hydroxylation of D-camphor at the 5-exo carbon atom. P450cam is one of the best characterized P450-Monooxygenase and leads to the first crystallographic structure of P450. That was the fundament for numerous site directed mutagenesis studies on P450cam, successfully showing the feasibility to change the substrate range. Against the laborious try-and-error of pure rational design our approach is the preparation of semi rational libraries based on the available structural information, varying the substrate contacting residues in the active site and screening on catalytic activity with different non natural substrates. Here we present the creation of a library, the cloning and expression of the necessary factors pdR and pdX and the development of an assay for high throughput screening.

96

Elke Boschke Technische Universität Dresden

ID 56 Ins. of Food Techn. and Bioprocess Engineering Elke.boschke@tu-dresden.de http://tu-dresden.de/die_tu_dresden/ fakultaeten/fakultaet_maschinenwesen/ilb/

Studies of Interaction between a Novel Probe and Raft-borne Sphingolipids

Elke Boschke, Richard Wetzel, Steffen Steinert, Sarita Hebbar, Giullaume Tresset, Rachel Kraut

Lipid rafts (microdomains) at the plasma membrane of cells are nanoscale clusters enriched in cholesterol and sphingolipids. These domains and the lipids within them are linked to neuro degenerative diseases such as Alzheimer’s and the sphingolipid storage diseases. Sphingolipids and cholesterol traffic through intracellular endolysosomal pathways that also play a role in the disease pathology. Currently, very few tools are available for live imaging of the uptake and trafficking of lipid raft domains and raft-borne sphingolipids. In an attempt to fill this gap, we are developing novel probes to study these processes. The goal of the current study was to characterize the interaction between sphingolipids and a novel sphingolipid binding probe, the SBD (Sphingolipid Binding Domain; see Fantini et al, 2002). Binding studies were carried out to assess SBD’s binding characteristics to different glycolipid-containing artificial membranes, using surface plasmon resonance (SPR). The SBD consists of a 25aa (DAEFRHDSGYEVHHQELVFFAEDVG) fragment derived from the sphingolipid binding domain of Aβ (Mahfoud et al, 2002 and Fantini et al, 2002), conjugated via a neutral linker with an organic fluorophore. Fantini et al showed the interaction of similar SBD domains within Prp and HIVgp120 with glycolipids, and postulated a mechanism whereby aromatic amino acids in the V3 loop domain formed by the SBD would interact with sugar containing glycolipid headgroups. Biacore’s surface plasmon resonance (SPR) instrument was used to assay SBD’s affinity for various sphingolipid-containing raft-like mixtures in artificial membranes in real time, and in the absence of detection labels. The detection principle of SPR instead relies on an electron charge density wave phenomenon that arises at the surface of a metallic film when light is reflected at the film under specific conditions. The resonance is a result of energy and momentum being transformed from incident photons into surface plasmons, and is sensitive to the refractive index of the medium on the opposite side of the film from the reflected light. In our experiments, interactions of unlabelled SBD with a lipid bilayer surface (liposomes immobilized on a gold chip) were studied by measuring changes in the solute concentration at this surface as a result of the interactions with SBD ligands. In agreement with our SPR studies, and with the model of Fantini et al, SBD interaction with certain glycosphingolipids and sphingomyelin could be shown by lipid blotting and interaction with detergent resistant membranes of neurons (Hebbar et al, submitted). Future studies will focus on characterizing the interaction of SBD and other glycosphingolipid-binding probes with artificial membranes. In this way we hope to elucidate the binding mechanisms between raft-borne sphingolipids and SBD and other toxins that are taken up into cells by sphingolipid-rich raft-mediated trafficking pathways. Singaporean Institute of Bioengineering and Nanotechnology, Biomedical Research Council and A-Star provided funding. J Fantini, N Garmy, R Mahfoud, and N Yahi. Lipid rafts: structure, function and role in HIV, Alzheimer’s and prion diseases. expert reviews in molecular medicine

(http://www.expertreviews.org/), 20 December 2002. R Mahfoud, N Garmy, M Maresca, N Yahi, Ax Puigserver, and J Fantini. Identification of a Common Sphingolipid-binding Domain in Alzheimer, Prion, and HIV-1 Proteins. The Journal of Biological Chemistry, 277(13):11292–11296, 2002.

97

Roy Eylenstein Universität Leipzig

ID 57 Center for Biotechnology and Biomedicine Professorship for Structural Analysis of Biopolymers roy.eylenstein@bbz.uni-leipzig.de www.uni-leipzig.de/~straeter/

Structural studies of human cAMP specific phosphodiesterase 4A

Roy Eylenstein, Bartholomeus Küttner, Susanne Moschütz, Norbert Sträter

 

Phosphodiesterases catalyze the hydrolysis of the intracellular second messenger 3’, 5’-cyclic AMP (cAMP) to AMP and / or cGMP to GMP. Phosphodiesterase 4 (PDE4), the major cAMP-specific PDE in inflammatory and immune cells, is an attractive target for the treatment of asthma and chronic obstructive pulmonary disease. X-ray structures of the PDEs allow for a rational design of specific inhibitors as therapeutics. The aim of our project is to study the catalytic mechanism of PDEs and the binding mode of synthetic inhibitors to PDE4A. Expression of the catalytic domain of PDE4A in E. coli BL21 (DE3) yields inclusion bodies that were refolded by fast dilution and purified by ion exchange chromatography and size exclusion chromatography. Crystallization conditions were found by sparse matrix screens and were further refined to obtain crystals suitable for X-ray diffraction experiments. Orthorhombic crystals diffract synchrotron light up to 1.9 Å resolution. Furthermore, in collaboration with Curacyte AG in Leipzig and with Elbion GmbH in Dresden-Radebeul we studied the binding mode of several synthetic inhibitors. A complex with a substrate analog was determined, which binds predominantly via hydrophobic interactions in the active site. A coupled two-step enzymatic assay followed by fluorescence analysis is in progress to assess the kinetic parameters of the enzymatic activity. These results will be complemented by isothermal titration calorimetry.

98

Sylvie Rockel Technische Universität Dresden

ID 58 Ins. of Food Techn. and Bioprocess Engineering Sylvie.rockel@gmx.de http://tu-dresden.de/die_tu_dresden/ fakultaeten/fakultaet_maschinenwesen/ilb/

Comparison of quantum dots with an organic fluorescent dye tagged to Cholera Toxin B for bioimaging applications

Sylvie Rockel, Rachel Kraut

Fluorescently tagged toxins that recognize specific glycosphingolipid receptors in the plasma membrane (PM) of cells have emerged as a powerful tool to study the spatial and temporal heterogeneity of microdomains in the lipid bilayer, as well as their trafficking routes following endocytosis. Intracellular tracking of these labelled molecules to compartments in living cells can reveal alterations in normal subcellular trafficking pathways, and reflect the effects of certain pathological conditions and/or drug treatments. Labelling of toxin-derived probes with conventional organic dyes presents practical limitations in live imaging due to photobleaching and other fluorescence properties, such as their narrow excitation spectra. Bright and photostable quantum dots (QDs) with tuneable emission and broad absorption spectra have surfaced as an alternative for biological imaging applications. However, molecular imaging with QDs inside living cells is still faced with challenges such as cell-penetration, valency of the QDs, ligand ratio, and correct targeting to specific organelles. For the validation of QDs to be used in intracellular tracking applications, it remains to be shown that internalization and trafficking in living cells follows the same routes/rules as probes labelled with small organic fluorophores. The uptake and trafficking of non-toxic B subunit of Cholera Toxin (CTxB), which binds specifically to GM1 ganglioside in the PM has been described extensively in the literature. As an example of a well-characterized toxin that recognizes glycosphingolipids and enters a known trafficking route, the present study aims to compare the membrane dynamics and trafficking behaviour of QD-tagged CTxB in live cells with fluorophore labelled protein. Trafficking behaviour of QD-labelled CTxB inside the cell was by live cell imaging after uptake, and colocalization with fluorescent reference markers for various intracellular compartments. The degree of colocalization over time of the CtxB in combination with a number of trafficking markers gives a description of the intracellular pathway taken by the probe. Colocalizations were quantitatively determined using the Mander’s coefficient for each channel (tM1 and tM2) with a calculated threshold according to the algorithm of Costes, in the “colocalization threshold” macro of ImageJ (written by W. Rasband and T. Collins). This work will describe the molecular dynamics of a QD- vs. a dye-labelled probe and discuss the implications for applications of QDs in studying live cell trafficking.

99

Christian Roth Universität Leipzig

ID 59 Center for Biotechnology and Biomedicine Professorship for Structural Analysis of Biopolymers christian.roth@bbz.uni-leipzig.de www.uni-leipzig.de/~straeter/

Cloning expression and purification of a lactone hydrolase from soil bacteria

Christian Roth, Silke Wegener, Michael Schlömann, Norbert Sträter

Some soil bacteria of the beta-proteobacteria family are able to hydrolyse fluororoaromatic compounds commonly present as soil and water pollutants. The presence of fluoroaromatic compounds induce the production of enzymes involved in the degradation of aromatic compounds including a lactone hydrolase. This hydrolase is able to cleave the lactone intermediate of this pathway. The enzyme is able to degrade unmodified and fluorinated variants of this compound. The native protein was cloned and successfully overexpressed in Escherichia coli. The purification involves heat enrichment followed by hydrophobic interaction chromatography and high resolution ion exchange chromatography. The final gelfitration shows one peak corresponding to the monomeric enzyme. Initial crystallisation trials show few crystallisation conditions however the crystals are not suitable for structure determination. Currently we are designing variants of the protein with different Tags, as well as truncations and mutations to obtain a construct suitable for crystallisation and subsequent structure determination.

100

Norbert Sträter Universität Leipzig

ID 60 Center for Biotechnology and Biomedicine Professorship for Structural Analysis of Biopolymers strater@bbz.uni-leipzig.de www.uni-leipzig.de/~straeter/

Structure and function of ecto-nucleotide diphosphohydrolases in purinergic signaling

Norbert Sträter, Matthias Zebisch

 

 

 

ATP and other nucleotides and nucleosides act as extracellular messengers thereby mediating shortterm signaling functions in neurotransmission, mechanosensory transduction, secretion and vasodilatation, and long-term signaling function in cell proliferation, differentiation and death (1). The nucleotides act on the P2X ionotropic ligand-gated ion channel receptors and P2Y metabotropic G protein-coupled receptors. The signaling function is terminated by the action of extracellular membrane-bound phosphatases. Since purinergic signaling is involved in cardiovascular diseases, migraine, pain perception, inflammation, respiratory diseases etc., the receptors as well as the nucleotidases are interesting pharmacological targets. Ecto-Nucleoside triphosphate diphosphohydrolases or NTPDases catalyze the sequential removal of γ- and β- phosphate from ATP, ADP and other nucleotides (2). NTPDase 1, 2, 3 and 8 are localized on the cell surface, anchored to the membrane by two transmembrane helices. For biochemical and structural characterization we established an E. coli expression system for insoluble production of the extracellular domains of rat NTPDases 1, 2 and 3 and optimized their in vitro refolding. After refolding the proteins could be purified to near homogeneity and have been characterized in respect to substrate specificity, KM values for ATP and ADP, pH dependance and metal cofactor activation (3). For NTPDase 2 suitable crystallization conditions could be found. Needles and rodlike crystals grew from 2 % PEG6000, 100 mM NaHEPES pH 7.3 with a protein concentration of 2 mg/ml. The crystals belong to space group P212121, contain one molecule in the asymmetric unit and diffract up to 1.8 Å using synchrotron radiation. References 1. Burnstock, G. (2006) Pharmacol. Rev. 58, 58-86. 2. Zimmermann, H. (2000) Naunyn Schmiedebergs Arch Pharmacol 362, 299-309. 3. Zebisch, M. and Sträter, N. (2007) Biochemistry 46, 11945-11956

Evgeny Twerdowski Universität Leipzig

ID 61 Translational Center for Regenerative Medicine IMONIT etwerdowski@trm.uni-leipzig.de www.trm.uni-leipzig.de/imonit

Combined Ultrasonic and Confocal Laser scanning Microscopy of Biological Objects

Evgeny Twerdowski, Albert Kamany, Esam Ahmed Mohamed, Wolfgang Grill, Reinhold Wannemacher

Combinatory and synchronous imaging by confocal laser scanning and phase-sensitive acoustic microscopy (CLSM and PSAM, respectively) has been developed on the basis of a commercial confocal laser scanning microscope ZEISS LSM 510 and a custom-designed confocal phase-sensitive acoustic microscope operating at an ultrasonic frequency of 1.2GHz. Both instruments are used in the reflection mode and the acoustic and optical lenses are mounted on opposite sides of the sample. The different types of contrast available for CLSM including reflection, transmission, and fluorescence, have been combined with the contrast modes available for PSAM including topographical, extinction and phase contrast. The detection schemes are presented together with applications of the described multi-contrast imaging scheme to living and fixated biological cells, for example red blood cells and fibroblasts.

102

Thomas Riemer Universität Leipzig

ID 62 Medical Faculty IZKF riemer@ uni-leipzig.de www.uni-leipzig.de/~izkf

NMR-micro-probe for mass limited biological samples

Thomas Riemer, Stefan Leidich

 

 

 

NMR-spectroscopy is a powerful spectroscopic tool. It allows for physical and chemical characterisation of biological systems. In the field of metabolomics, which is concerned with the metabolic charaterisation of biological systems, NMR-spectrscopy is a powerfull alternative to gas chromatography combined with mass spectroscopy (GC-MS). Mass limited samples, such as adult hematopoetic stem cells and their extracts, are just coming into the range of a NMR-spectroscopic characterisation due to the development of very sensitive NMR-micro-resonators. We will illustrate our newly developed, very sensitive NMR-micro-resontor, which is the result of an ongoing two years collaboration between the IZKF-Leipzig, University of Leipzig and the Center for Microtechnologies of Chemnitz University of Technology.

103

 

104

BIOINFORMATICS

 

 

105

Sergey Samsonov Technische Universität Dresden

ID 63 BIOTEChnology Center Dresden Research group Pisabarro Sergeys@biotec.tu-dresden.de www.biotec.tu-dresden.de/pisabarro

MD analysis of water-mediated interactions in protein interfaces

Sergey Samsonov, Joan Teyra, M. Teresa Pisabarro

Water plays an extremely important role in all biological processes regarding its unique physical and chemical properties. Being the universal solvent for biopolymers, water does not only form passive environment but also actively participates in such processes as folding, catalysis, molecular interactions, and influences dynamical properties of macromolecules. However, in some fields of fundamental research the importance of water is often ignored. Particularly, it is widely assumed to define protein interactions disregarding water molecules present in the interface. Recent studies carried out in our group suggest that residues interacting through water (wet spots) may play a crucial role in protein binding affinity and specificity, and their consideration in the protein interface description might improve the performance of empirical methods used for docking, potential energy functions for protein folding and ligand design. We are currently studying protein interfacial solvent, in particular, the behavior of wet spots and water molecules in small protein domains (i.e. SH3) interfaces using molecular dynamics approach. SH3 domains and their binding partners were extensively studied during the last couple of decades. Several complexes structures are available at high resolution. We have been describing some conservative interfacial water sites as well as characteristics like fluctuations and free energy decomposed components of the interacting residues, time and energetic parameters for the water sites. These results point out the high heterogenity in chemical, mechanic, thermodynamic and kinetic properties of the residues participating in the water mediated protein interactions. These residues, however, make up a distinct class of the interface participants that should not be disregarded in, both qualitative and quantitative, description of the protein-protein interactions.

106

Mike Beck Max-Planck-Institut für Molekular-, Zellbiologie & Genetik Email: beck@mpi-cbg.de www.mpi-cbg.de/research/groups/ walch-solimena/walch-solimena.html

Joan Teyra Technische Universität Dresden

ID 64 BIOTEChnology Center Research group Pisabarro Joan.teyra@biotec.tu-dresden.de www.biotec.tu-dresden.de/pisabarro

Analysis and classification of the Protein Interactome

Joan Teyra, M. Teresa Pisabarro

Experimentally determined structures of protein complexes at atomic detail are deposited in the Brookhaven Protein Data Bank (PDB). Many efforts have been recently made towards the development of tools that allow mining of such enormous amount of three-dimensional data. However, there is still a need for accurate description and clustering of protein interfaces to be used for comparative analysis in large-scale. We have developed the SCOWLP database for a complete characterization and visualization of protein interfaces. It allows us to extract three-dimensional information of interfacial residues and solvent from all protein-protein and protein-peptide complexes of the PDB at atom, residue and domain level. We use the classification scheme of SCOP for fold and protein family definitions. The inclusion of water enriches the definition of protein interfaces by considering residues interacting exclusively by water, defined as wet spots. Mapping the interacting residues and wet spots into the protein sequences allows us to cluster the domains of a protein family based on Pairwise structural alignments and to catalog SCOP family complexes at binding site (BS) and interface (IF) levels. The SCOWLP web-server (www.scowlp.org) allows the user to search and display all interfacial information contained in the PDB in an automatic and user-friendly fashion. The user can navigate and query our SCOP-extended hierarchy to search and/or select a particular protein family or PDB Id. The analysis and comparison of the detailed interaction information is performed by using sorting tables and an interactive 3D viewer.

107

Frank Brandt Technische Universität Dresden

ID 65 BIOTEChnology Center Dresden Research group Pisabarro Frank.brandt@biotec.tu-dresden.de www.biotec.tu-dresden.de/pisabarro

ParaDockS: A Parallel Framework for Molecular Docking

Frank Brandt, Carsten Baldauf, M. Teresa Pisabarro

The aim of molecular docking is to find the optimal binding orientation of two molecules in space and to estimate the stability of the formed complex. Due to the nature and number of the degrees of freedom the resulting fitness landscape is highly irregular. To tackle this continuous and multi-dimensional search space, at present mostly Genetic Algorithms (GA), Lamarckian-GA (LGA), etc. are being employed. Recent developments have shown that other bio-inspired algorithms can perform better. With the wide availability of parallel architectures, methods making use of these advantages are desired. A docking program should be designed for such architectures like multi-core workstation and small workstation clusters, but also for massive parallel systems as found at the Centre for Information Service and High Performance Computing (ZIH) of the TU Dresden. We have developed a flexible, modular, parallel docking program and incorporated, as a reference for future implementations, a known scoring function and a Particle Swarm Optimizer (PSO). Our framework is suited for productive docking tasks but also to test newly developed scoring functions and optimization algorithms for accuracy and parallel performance.

108

Bill Andreopoulus Technische Universität Dresden

ID 66 BIOTEChnology Center Research group Schroeder William.andreopoulus@biotec.tu-dresden.de www.biotec.tu-dresden.de/schroeder

Unfolding pathway recognition of membrane proteins by the use of wavelet techniques

Bill Andreopoulus, Alexander Andreoploulus, Michael Schroeder, Dirk Labudde

We use a new approach to detect possible unfolding pathways of membrane proteins that are investigated with single molecular force spectroscopy (SMFS). The approach is based on smoothing the SMFS curves via wavelets and then looking for peaks and subpeaks, independent of their size. We adapted the well-known apriori mining method for finding trajectories to work with derivatives of the SMFS curves. As a result of our approach, we could detect modular units of unfolding events, as well as larger unfolding pathways, with a very fast runtime.

109

Christian Panse Eidgenössische Technische Hochschule Zürich

ID 67 Functional Genomics Center Zürich Bioinformatics Scientist Proteomics cp@fgcz.ehtz.ch www.fgcz.ethz.ch

How to manage the massive amounts of biotech data in the future?

Christian Panse, Simon Barkow-Oesterreicher, Can Tuerker, Bertran Gerrits, Dieter Joho, Bernd Roschitzki, Ralph Schlapbach

Our experiences in proteomics during the last years show that every new generation of instruments and analysis software produce larger amounts of data in the range of one order of magnitude. To avoid that most of these complex semi structured data end up undefined and unannotated, one needs to organize this information in a sophisticated way. The current situation in many biological labs is that the complexity of necessary information technology is often underestimated. Collaboration between life scientists and systems and software engineers is rare. Besides guaranteeing availability and consistency of the produced data, more demanding requirements for a data storage and management system are coming up that will allow advanced and larger scale analysis such as automatic procedures for high throughput processing and analysis, comparison of experiments across different projects, or providing a platform for plugging in various analysis tools into existing workflows. Our current data infrastructure already provides solutions for some of these requirements dealing with data from a number of different mass spectrometers (MS). Approaches taken by systems biologists working at our facility already benefit from a structured data pipeline that deals with, e. g., large-scale analysis of whole proteomes. Here we will present our interdisciplinary multilayered system architecture and are going to address the specific problems that arise with such large-scale environment. Our infrastructure is proven successful by managing over 300 different diverse research projects within the last three years. We believe that the whole systems biology effort in particular and life science in general depend on the successful implementation of an elaborate system for processing, management, validation, interpretation, and integration of biological data.

110

Mike Beck Max-Planck-Institut für Molekular-, Zellbiologie & Genetik Email: beck@mpi-cbg.de www.mpi-cbg.de/research/groups/ walch-solimena/walch-solimena.html

Frank Dressel Technische Universität Dresden

ID 68 BIOTEChnology Center Research group Schroeder Frank.dressel@biotec.tu-dresden.de www.biotec.tu-dresden.de/schroeder

Understanding of SMFS barriers by means of energy profiles

Frank Dressel, Annalisa Marsico, Dirk Labudde, Anne Tuukkanen, Michael Schroeder

In the last years, Single Molecule Force Spectroscopy was more and more used to gain insight into the fundamental principles behind protein structure and stability. Nevertheless, the interpretation of the experimental findings is not so easy and additional computational approaches are needed to interpret them. Here, we proposed an approach based on interaction patterns between amino acids to explain the emergence of SMFS unfolding barriers in the experiment. With our approach, we can predict around 64 % of the experimentally detectable barriers.

111

Aurelie Tomczak Technische Universität Dresden

ID 69 BIOTEChnology Center Research group Pisabarro Aurelie.tomczak@biotec.tu-dresden.de www.biotec.tu-dresden.de/pisabarro

Development and application of automated structure-based functional protein annotation methods

Aurelie Tomczak, M. Teresa Pisabarro

If the function of a protein is unknown the classic bioinformatics approaches based on sequence similarity are often useful to find homologous proteins. In case sequence-based annotation methods fail, the application of structure-based methods (i.e. Fold recognition) can provide functional clues or confirm tentative functional assignments. These methods have already proven to be successful for single proteins but, due to the large amount of data obtained by sequencing and other high throughput approaches automation of structure-based functional annotation of proteins is needed. The aim of this project is to develop a framework for automatic structure-based annotation of novel proteins. Therefore we are going to integrate public databases containing experimental structural and sequence data (i.e. PDB, SwissProt), structure-based computational methods (i.e. Fold recognition), functional information (i.e. RNAi) and 3D descriptors derived from common protein family motifs. This framework will be applied to discover novel chemokines with remote sequence identity by screening public databases for not annotated protein sequences having cysteine residues. Those can possibly be part of the conserved motif with intramolecular disulfide bond formation used as 3D descriptors for chemokines. Furthermore it will be used for structure-based characterisation of novel genes obtained by large-scale RNAi screenings in human cells.

112

Mohd Noriznan Mokhtar Universität Leipig

ID 70 Faculty of Biosciences, Pharmacy and Psychology Institute of Biochemistry noriznan@uni-leipzig.de www.biochemie.uni-leipzig.de/agz/

Optimization of the biocatalytic synthesis of large-ring cyclodextrins

Mohd Norizan Mokhtar, Melanie Gruner, Wolfgang Zimmermann

Cyclodextrin glucanotransferase (EC 2.4.1.19, CGTase) from Bacillus macerans synthesizes cyclodextrins (CD) composed of 6 to more than 21 glucose units from high amylose starches. Larger CD composed of nine to 21 glucose units have different size and structural features distinct from the commercially available CD6 to CD8, and could find applications as novel host compounds in molecular recognition processes in nanobiotechnology [1]. The amount and the size distribution of CD produced by CGTase will be strongly influenced by the combined effects of the three transglycosylation reactions such as cyclization, coupling and disproportionation, as well as the hydrolytic activity of the enzyme. The biocatalytic process needs to be controlled very well due to the conversion of larger CD into smaller CD during extended incubation times. The optimum yield of larger CD could be predicted by optimizing incubation time, CGTase activity and pea starch concentration using response surface methodology. Yield improvements could also be obtained by adding polar organic solvents. The results show that the yield of larger CD could be increased by suppressing the secondary degradation reactions of the CGTase resulting in their conversion to smaller CD [2]. [1] Endo, T., Zheng, M.Y. and Zimmermann, W.: Enzymatic synthesis and analysis of large ring cyclodextrins. Aust. J. Chem. 55 (2002) 39 – 48 [2] Qi, Q., Mokhtar, M.N. and Zimmermann, W.: Effect of ethanol on the synthesis of large-ring cyclodextrins by cyclodextrin glucanotransferases. J. Incl. Phenom. Macrocycl. Chem. 57 (2007) 95 - 99

113

Maciej Paszkowski-Rogacz Max Planck Institute of

ID 71 Molecular Cell Biology and Genetics paszkows@ mpi-cbg.de www.mpi-cbg.de/research/groups/buchholz/ buchholz.html

DIAF: A High-Throughput Data Integration and Analysis Framework for Functional Annotation of Genes

Maceij Paszkowski-Rogacz, M. Teresa Pisabarro, Frank Buchholz

Biological research is currently changing from a more genes based focus to an analysis of gene networks and hence, a systematic understanding of biological processes is now at reach. In this process large amounts of data are accumulated that need to be interpreted. We are developing tools and methods to store, browse and analyze data from high-throughput experiments. In particular, we perform metaanalysis of phenotypic data generated by RNAi screens and data from gene expression profiling. The aim of our studies is to functionally characterize genes by theoretical and experimental verification of hypothesis based on data analysis results. We have developed a Java 2 Enterprise Edition web application that implements functionalities such as browsing, searching, filtering and cross-comparing data sets gathered in a relational database. The database schema allows storing any kind of biological data in a gene-centered fashion. Selected gene sets of interest can be exported to different tools such as annotation enrichment analysis. Currently we are mining cell viability phenotypes acquired from genome-wide esiRNA screens performed in different cancer cell lines. First results show that most essential genes are the same in the different cell lines. However, there are small groups of genes that might be essential for cell viability in specific cell lines only. These genes may be explored as potential novel candidates in cancer therapy.

114

Mike Beck Max-Planck-Institut für Molekular-, Zellbiologie & Genetik Email: beck@mpi-cbg.de www.mpi-cbg.de/research/groups/ walch-solimena/walch-solimena.html

Matthias Bernt Universität Leipzig

ID 72 Faculty of Mathematics and Computer Science Institute of Computer Science bernt@informatik.uni-leipzig.de http://pacosy.informatik.uni-leipzig.de/

CREx - A Tool for Analyzing Genomic Rearrangement Operations Based on Common Intervals

Matthias Bernt, Daniel Merkle, Kai Ramsch, Guido Fritzsch, Marleen Perseke, Detlef Bernhard, Martin Schlegel, Peter F. Stadler, Martin Middendorf

Genomic rearrangement operations present a powerful approach to determining the phylogenetic relationship of species. In this context, unichromosomal genomes are usually encoded as signed permutations (usually just called sequences), in which each element represents a gene and the sign determines the orientation of the gene. Inversions (often referred to as “reversals” in the bioinformatics literature), are by far the best-studied rearrangement operations. Phylogenetic reconstruction methods based on more general sets of rearrangement operations are rare, presumably because for some of the operations of interest even computing the distance between two sequences is computationally hard. On the other hand, there is ample evidence in the biological literature that in particular mitochondrial genome rearrangements are the consequence of multiple mechanisms. We have developed the web-based program CREx for heuristically determining pairwise rearrangements events in unichromosomal genomes. CREx considers transpositions, reverse transpositions, reversals, and tandem-duplication-random-loss (TDRL) events. It supports the user in finding parsimonious rearrangement scenarios given a phylogenetic hypothesis. CREx is based on common intervals, which reflect genes that appear consecutively in several of the input gene orders.

115

Matthias Bernt Universität Leipzig

ID 73 Faculty of Mathematics and Computer Science Institute of Computer Science bernt@informatik.uni-leipzig.de http://pacosy.informatik.uni-leipzig.de/

A Method for Solving the Preserving Reversal Median Problem

Matthias Bernt, Martin Middendorf, Daniel Merkle

Genomic rearrangement operations are a powerful approach to infer the phylogenetic relationship of gene orders representing species. One basic problem in this field of research is to find for the gene orders of three taxa potential ancestral gene orders such that the corresponding rearrangement scenario has a minimal number of reversal operations and where each of the reversals has to preserve the common intervals of the given input gene orders. Common intervals identify sets of genes that occur consecutively in all input gene orders. The problem of finding such an ancestral gene order is called the preserving reversal median problem (pRMP). We have developed a new method called TCIP for solving the perfect reversal median problem (pRMP). TCIP is based on a tree based data structure for the representation of the common intervals of all input gene orders. We have shown empirically on data sets of mitochondrial gene orders and for randomly generated data sets that TCIP is faster than the fastest exact algorithm for the reversal median problem (RMP), i.e., the corresponding problem in which common intervals are not considered.

116

Gerd Anders Technische Universität Dresden

ID 74 BIOTEChnology Center Research group Pisabarro Gerd.anders@biotec.tu-dresden.de www.biotec.tu-dresden.de/pisabarro

An annotation platform for structural bioinformatics

Gerd Anders

Annotating proteins structurally and functionally is one of the most important tasks in structural bioinformatics. Many current annotation approaches are knowledge -based, e. g., threading (fold recognition) strongly relies on a collection of template proteins with known fold to predict the structure of a given protein sequence without sequence similarity to know ones. Increasing the number of template proteins may improve chances of matching a query protein sequence to a particular protein structure and furthermore a function. Commonly used protein classification schemes like SCOP or CATH independently annotate protein structures from the PDB. Unfortunately, both are non - frequently updated and currently lack about one third of all known and available PDB structures (about 46,000, October 2007). To use the full potential of fold recognition methods and all available data of protein structures, we set up a relational database - driven platform, including PDB, SCOP and other data sources. This provides fast data access as well as easy data linking among the different data sources. Furthermore, we established an automatic fold annotation approach based on the ASTRAL95 dataset and the classification scheme of the SCOP database. Using BLAST and ASTRAL95 for sequence alignments as well as MAMMOTH for structural alignments and representatives from the SCOP V. 1.71 database on family level, we fully automatically annotated about 90 percent of all the PDB structures missing in SCOP.

117

Nico Scherf Universität Leipzig

ID 75 Translational Center for Regerative Medicine IMONIT nscherf@trm.uni-leipzig.de www.trm.uni-leipzig.de/imonit.html

An Automatic Analysis of Cartilage Regeneration in a Model of Human/Murine SCID arthritis

Nico Scherf, Ulf Braumann, Jens Kuska, Manja Kamprad, Franziska Lange, Ulrich Sack

Objective The purpose of this work is to automatically analyse the differences in cartilage regeneration of different therapies (mesenchymal stem cells from bone marrow versus Wharton`s jelly) in an model of human/murine SCID arthritis, on the basis of digital images of stained histological sections. Usually, the experimenter manually measures the thickness of the cartilage layer at a single position in the images. Manual analysis of the whole spatial dimensions of the cartilage layer is not feasible, although such an complete analysis would provide significant measures for the subsequent statistical comparison of the observed therapeutic effects. Therefore, we decided to automate the process. Method The cartilage layer has to be identified in the digitised image at first. The segmentation of such colour images is difficult and for the most part remains an open problem in current research on image processing. Since the differences in the structures to be discerned in such images are rather subtle, fully automatic segmentation methods usually cannot be applied. Therefore, we decided to use a semi-automatic approach here. The experimenter can roughly mark the regions of cartilage in the image, and the final segmentation of the structures is computed afterwards. The result is further processed by morphological algorithms to obtain a spatially coherent solution and several statistical features of the cartilage layer are computed, e.g. mean thickness, standard deviation, thickness profiles etc. Conclusions An automatic calculation of several statistical measures of the thickness of the cartilage layer will improve the quantitative assessment of the experiments by providing objective and reproducible measures.

118

Karl-Michael Meiß Arnold-Sommerfeld-Gesellschaft e.V.

ID 76 Leipzig Dr.meiss@usa.net www.dr-meiss.de/ASG/

Bioplastic Polyhydroxybutyrate

Karl-Michael Meiß

 

 

 

A new way in the production of polyhydroxybutyrate (PHB) is the solvent free processing. We make it possible to use biomass for bioplastic. The process is neutral in CO2 - balance and thermo energy.

 

119

Axel Erler Technische Universität Dresden

ID 77 BIOTEChnology Center Professorship Stewart - Genomics erler@biotec.tu-dresden.de www.biotec.tu-dresden.de/stewart

Evolutionary and Functional Analysis of the Redβ/RecT Single-Strand Annealing Protein Family

Axel Erler, Madeleine Teucher, Marcello Maresca, Vineeth Surendranath, Bianca Habermann, Francis Stewart, Youming Zhang

 

DNA single-strand annealing proteins (SSAPs) are primarily of bacteriophage origin and function in RecA-dependent and RecA-independent DNA recombination pathways. Recent computational sequence analysis identified three evolutionarily distinct superfamilies, namely RecT/Redβ, ERF, and RAD52. SSAPs are usually encoded in predicted operons and neighboring genes therefore represent potential functional partners. Among these especially Redβ and RecT gained prominence through the development of the DNA engineering technology known as ‘recombineering’ or ‘Red/ET’. Both are coliphage proteins found in operons (red and recET) with a co-operating 5’ to 3’ exonuclease (Redα and RecE, respectively). Additionally, the red-operon consists of Redγ, a DNA mimetic and RecBCD inhibitor. Based on the recent increase of fully sequenced bacterial genomes we re-examined the RecT/Redβ SSAP superfamily and identified 106 individual sequences in 78 host strains, which cluster into four separate subfamilies. To gain better insight into their evolutionary relationships and to identify potential common functional neighbors, we also investigated their genomic environment by comparative analysis of the operon structures. Complete red-like operon structures could be identified in 15 sequences in 11 host strains of which 5 sequences were lacking the redγ gene. Furthermore, 8 sequences in 5 host strains showed an recET-like operon structure. These candidate operons provide an important source for systematic functional analysis towards the identification of novel recombinases.

120

Karol Kozak Max-Planck-Institute of Molecular Cell

ID 78 Biology and Genetics Screening Facility kozak@mpi-cbg.de www.MPI-CBG.de

Automated Data Flow and Quality Control Practice in High-Content Screening

Karol Kozak

 

High-Content Screening (HCS), applying automated microscopy, requires informatics solution for quality control (QC) and correction of systematic errors coming in HCS data sets. Informatics platform should automate data flow from any data generating instrument and image processing software. Informatics platform should turn raw data files into fully reduced data in a single process. Such system detects plate local effects as well as trends within a sequence of microtiter plates without user interaction. The recognized patterns should be automatically compensated. A unified data publishing system should alerts scientists immediately when results are available. Here we present as example automated data flow for QC and data validation for assay development and screening process.

 

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122

TISSUE ENGINEERING

 

 

123

Peter Bernstein Universitätsklinikum Dresden

ID 79 Orthopedic Surgery peter.bernstein@uniklinikum-dresden.de http://ortho.uniklinikum-dresden.de/

 

Tissue engineering scaffold biomaterial and 3D-culture influence expression of the chondrogenic master transcription factor Sox9

Peter Bernstein, Meng Dong, Sylvi Graupner, Denis Corbeil, Michael Gelinsky, Klaus-Peter Günther, Stefan Fickert

Sox9 is known to be an important chondrogenic master transcription factor. In our cartilage tissue engineering experiments we used quantitative RT-PCR to study Sox9 expression levels during different modes of 3D culturing. summary : We demonstrated a 3D-biomaterial- and time-dependend manner of Sox9 expression. The time dependend effect supports the hypothesis of Sox9 being a marker for differentiation dynamics. The biomaterial-factor proves the observation of chondrocytes‘ environment-dependend differentiation behaviour. We could demonstrate favourable materials (alginate or alginatechitosan) and favourable tissue engineering methods (condensation). Bringing both together will probably result in synergistic effects and should be investigated further.  

124

Peter Ahnert Universität Leipzig

ID 80 Center for Biotechnology and Biomedicine Molecular Diagnostics – Microarray Techniques ahnert@uni-leipzig.de http://www.uni-leipzig.de/~ahnert/

Interphase-Fluorescence in situ Hybridization-technique for detecting human stem cells in frozen xenogeneic tissue

Peter Ahnert, Heidrun Holland,

Fluorescence in situ hybridization (FISH) is a nonisotopic labelling and detection method that provides a direct way to determine human cells in frozen xenogeneic tissue. With recent advancements, this technique has found increased application in a number of clinical and research areas, including prenatal/postnatal cytogenetics, cancer research, nuclear organization, gene loss/amplification, and gene mapping. FISH on interphase cells allows the simultaneous evaluation of various DNA-regions independent of the cell cycle, without the need of cell culture and chromosome preparation and - if applied to tissue sections - within the natural tissue context. Genetic analysis using FISH methods applied to intact tissue sections is well known to be relatively difficult. The technical problems include unsuccessful hybridization as a result of poor probe penetration, excessive background, auto-fluorescence, and overlapping or incomplete interphase cells. For accurate identification of human cells in xenogeneic tissue we optimized the interphase- fluorescence in situ hybridization (I-FISH) technique for this application. Therefore, we used β-satellite-DNA probes for all human or mouse chromosomes (spectrum orange, Q BIOgene) and performed the hybridization directly on the xenogeneic tissue. We demonstrated that this I-FISH-technique could be employed for the identification and localization of individual human stem cells in xenogeneic tissues.  

125

Gabriele Spörl Institute for Air Conditioning and

ID 81 Refrigeration Dresden Cryophysics, Material Development gabriele.spoerl@ilkdresden.de www.ilkdresden.de/mambo/home.php

Cryopreservation of 3D Tissue Engineering Constructs

Gabriele Spörl, Günter Lauer, Edith Klingner

Cryopreservation should be the first choice for long time storage of tissues. Cryopreservation of single cell suspensions is well established, but protocols and procedures to cryopreserve complete tissues are not yet available. That’s why we started the investigation of the behaviour of tissue engineered (TE) constructs during freezing. We used two kinds of constructs. First, after primary culture fibroblasts and gingival keratinocytes were seeded in a collagen sponge (collagen resorb) to tissue engineer the human mucosa transplants. Second, after primary culture osteoblasts were seeded in a biomaterial for bone regeneration (Cerasorb M®). The development of a low temperature fluorescence assay allowed the permanent microscopic observation of cells and TE constructs related to time and temperature during freezing and thawing. For both TE constructs cryoprotocols were developed down to -80 °C including special equipment for the handling during culturing, freezing and thawing. The transfer of such a protocol can take place to a tissue bank as a standard routine within next two years.  

126

Ralf Rühl Technische Universität Dresden

ID 82 Max Bergmann Center for Biomaterials MBZ Tissue Engineering and Biomineralisation ruehl@tmfs.mpgfk.tu-dresden.de www.mpgfk.tu-dresden.de/

Establishing the Basis of Tissue Engineering: developing in vitro models for bone biomineralization, remodeling, vascularization and nanotoxicity

Ralf Rühl, Ulla König, Anne Bernhardt, Armin Springer, Anja Lode, Eckehart Rudolph, Thomas Hanke, Michael Gelinsky

In the group “Tissue Engineering and Biomineralisation” at the Max Bergmann Center of Biomaterials (TU Dresden) research is focused on development of novel scaffolds for hard and soft tissue engineering, biochemical functionalisation thereof and cell biological testing. The present poster describes the establishment of in vitro models for the complex biological processes bone biomineralisation, remodeling and vascularisation. In addition, investigation of potential toxicity of nanoparticles using cell culture models will be explained. summary : Synthesis, mineralisation and remodeling of extracellular matrix of bone are complex and multicellular processes which can hardly be investigated in vivo. We are therefore trying to establish in vitro models, using either cell free systems (as for biomineralisation) or co-cultures of osteoblasts (bone forming cells) and osteoclasts (resorbing cells) on artificial extracellular matrices (mineralised collagen). A crucial point for successful use of cell-seeded scaffolds for healing of critical size defects is a fast vascularisation of the constructs. Several approaches to analysis of vascularisation processes in vitro and to an enhancement of blood vessel ingrowth by tuning the porosity of the scaffold and specific functionalisations are discussed.  

127

Anne Bernhardt Technische Universität Dresden

ID 83 Max Bergmann Center for Biomaterials MBZ Tissue Engineering and Biomineralisation bernhardt@tmfs.mpgfk.tu-dresden.de www.mpgfk.tu-dresden.de/

In vitro cultivation of osteoblast precursor cells on novel scaffolds for hard Tissue engineering

Anne Bernhardt, Anja Lode, Corina Vater, Beatrice Burmeister, Anja Walther, Thomas Hanke, Michael Gelinsky

In the group “Tissue Engineering and Biomineralisation” at the Max Bergmann Center of Biomaterials (TU Dresden) research is focused on development of novel scaffolds for hard and soft tissue engineering, biochemical functionalisation thereof and cell biological testing. In addition, in vitro models for complex processes like bone biomineralisation, remodelling and vascularisation are under investigation. Here we present in vitro investigations with osteoblast precursor cells on different novel scaffolds, prepared in our group. summary : Human mesenchymal stromal cells (hMSC) were seeded on 3D scaffolds from mineralized collagen, calcium alginate gels with an anisotropic pore structure, highly porous, but mechanically stable devices, prepared by electrostatic flocking and fast resorbable calcium phosphate bone cements and were stimulated towards osteogenic differentiation. Adhesion, proliferation and osteogenic differentiation as well as penetration of the cells into the porous 3D scaffold were studied using biochemical and molecular biological assays and microscopic methods.  

128

Udo Großmann Universität Leipzig

ID 84 Translation Center for Regenerative Medicine IMONIT ugrossmann@trm.uni-leipzig.de www.trm.uni-leipzig.de/imonit.html

Semi-automated radiosynthesis and in vitro testing of [18F]fluoroetanidazole as a new surrogate marker for PET imaging of stem cell therapy in stroke

Udo Großmann, Marianne Patt, Andreas Schildan, Heike Franke, Peter Illes, Osama Sabri, Frank Emmrich, Henryk Barthel

In stroke, new stem cell treatment approaches target the ischemic penumbra, a brain area which can so far not directly be visualized and quantified with the standard brain imaging techniques in use. [18F]fluoroetanidazole ([18F]FETA) has recently been shown to be a useful marker for hypoxia positron emission tomography (PET) imaging in tumours (Barthel et al., Br J Canc 2004). This project aimed to establish a semiautomated radiosynthesis of [18F]FETA and to evaluate this promising radiotracer with respect to its potential to detect hypoxic tissue in the brain. [18F]FETA was synthesized according to the protocol described by Tewson et al. (Nucl Med Biol 1997) with minor modifications. This was accomplished by a 5-step approach for which four steps were realised in an automated synthesis module (GE Medical Systems) and one step was performed manually in a shielded hood. The oxygendependency of the [18F]FETA uptake was assessed ex vivo in primary corticoencephalic cells obtained from Wistar rats. The cells were incubated with nitrogen (5 % CO2 + 95 % N2) or air (20 % O2). The oxygen concentration in the cell suspension was measured using a Licox probe (Integra NeuroSciences). The cells were incubated with 5 MBq [18F]FETA for 5, 15, 30, 60 and 120 min, respectively, harvested and the cell-bound radioactivity was measured. [18F]FETA was produced with an overall radiochemical yield of 20 % in a total synthesis time of 70 min. In the in vitro experiments, the oxygen concentration in the cell suspension was < 1 mm Hg and ~70 mm Hg under nitrogen and air, respectively. The [18F]FETA uptake under normoxia was low and constant over time (0.3 ± 0.08 % ID.mio cells-1). In contrast, there was a timedependent linear increase of the [18F]FETA uptake by the cells incubated with nitrogen, which was 1.5 and 2.8-fold as compared to the normoxia values at 60 min and 120 min (p &lt; 0.05), respectively. In conclusion, a semi-automated radiosynthesis of [18F]FETA was successfully established for the first time. The in vitro results point to a hypoxia-selective radiotracer uptake in corticoencephalic rat cells. Therefore, a further in vivo evaluation of [18F]FETA in animal stroke and stem cell treatment models is warranted. Supported by the Translation Centre for Regenerative Medicine of the University of Leipzig, Germany (project no. 1095MN)  

129

Holger Reinsch Institute for Air Conditioning and

ID 85 Refrigeration Dresden Tissue Engineering holger.reinsch@ilkdresden.de www.ilkdresden.de/mambo/home.php

Investigation of Scaffold Materials for the Tissue Engineering of Cryopreservable Artificial Tissue Constructs

Holger Reinsch, Gabriele Spörl

Tissue engineering is a new and spreading technology which may bring substantial benefit to the field of regenerative medicine. Displacing autogenic and allogenic tissue grafts by in vitro generated artificial tissues can solve a lot of consisting problems such as an increased morbidity in the donor area as well as the risks of inflammatory reactions or virus transmission. However, in most cases reconstructive surgery is hard to schedule because it strictly depends on the patient’s condition. That represents a major difficulty for a transplantation of artificial tissue constructs which can only be bred in due time. A possibility to solve that problem is the cryopreservation of the in vitro generated tissue constructs. Cryopreservation allows long term storage and assures disposability according to the surgeon’s requirements if temporal flexibility is needed. For this reason the ILK Dresden gGmbH is highly engaged in the field of evaluation and qualification of commercially available biomaterials. Within an extensive screening process different biomaterials (collagen sponges, fleeces, and membranes as well as mineral materials of synthetic and biologic origin) from several manufacturers were tested regarding to their usability as cryopreservable scaffold materials in tissue engineering. First structural analyses were made to acquire basic material parameters such as surface condition, porosity, pore size, and interconnectivity. If geometrical properties referred to the suitability for use as scaffold material, material behaviour under cell culture conditions was tested. Particularly we checked media uptake, hydrolytic degradation as well as the mechanical and dimensional long term stability under in vitro conditions. Referring to the cryopreservation, low temperature properties were checked. The thermal stability down to 133 K was measured in a differential scanning calorimeter. Furthermore the behaviour of dry and media soaked materials during the cooling process was observed microscopically. Some materials were found suitable for tissue engineering as well as stable under the conditions of cryopreservation. These materials were seeded with several cell types and were cultivated for several weeks. The resulting artificial tissue constructs were successfully cryopreserved under various conditions.  

130

Christina Fieber Universität Leipzig

ID 86 Translation Center for Regenerative Medicine TEMAT cfieber@trm.uni-leipzig.de www.trm.uni-leipzig.de/temat.html

Isolation and Characterisation of Human Melanocytes for Clinical Use

Christina Fieber, Ulf Anderegg, Jan C. Simon, Andreas Emmendörffer

Despite significant progresses in tissue engineering and cell biology there is still an unmet need for treating patients suffering from vitiligo. The disease is characterized by depletion of melanocytes in more or less extended areas of the skin. In correlation to the extent and localisation of the depigmented areas patients ask for medical treatment. Today the treatment regimen includes dermabrasio followed by transplantation of a mixture of keratinocytes and melanocytes taken from biopsies of non-affected areas. The transplantation is supported by treatment with an excimer laser. However, this modern treatment is restricted to few centres, less than five at least in Germany. The hair follicle bulge area is an abundant easily accessible source of actively growing pluripotent adult stem cells. These cells can be differentiated into various cell lineages, e. g. keratinocytes and melanocytes amongst others. Euroderm GmbH uses ORS (outer root sheath) technology and possesses the technological know-how for generating autologous keratinocytes. The strategic concept of this translational project is to isolate and propagate melanocytes and keratinocytes, preferentially from ORS cells, or alternatively from skin biopsies, in order to achieve sufficient quantities. A mixture of these cells will then be administered to the affected skin of a patient. The technical challenge lies in the definition of optimal culture conditions to foster the growth of melanocyte precursors. Additionally the culture conditions have to meet GMP requirements. The results of these preclinical studies will then be the prerequisite for starting clinical trial (Phase I).

131

Susanne Trettner Fraunhofer Institute for Cell Therapy and

ID 87 Immunology Leipzig Group Applied Stem Cell Technologies susanne.trettner@izi.fraunhofer.de

http://www.izi.fraunhofer.de/ 

Development of an automated 3D bioprocess for osteogenic differentiation of primate embryonic stem cells

Susanne Trettner, Erika Sasaki, Nicole zur Nieden

Owing to their pluripotency, embryonic stem cells (ESCs) have the developmental potential to differentiate into nearly all cell types of the adult organism. Earlier studies have shown the possibility to differentiate ESCs in vitro into mature cell types of all three germ layers, including haematopoietic and neural precursors, cardiomyocytes, hepatocytes, osteoblasts and various other cell types. Because of this developmental capacity, ESCs have been suggested to find applications not only in cellular therapies of otherwise incurable diseases, but also as unlimited cell sources of human origin to evaluate toxic properties of compounds. The differentiation of murine ESCs to osteoblasts in static culture is well described [1]. The long-term goal of this study is to develop an optimal automated bioprocess for a large-scale culture system for the osteogenic differentiation of ESCs derived from mouse and the common marmoset monkey, Callithrix jacchus. As a first step, we describe here the in vitro differentiation of Callithrix ESCs towards osteoblasts using the active form of vitamin D3 in static culture. Due to the similarity between Callithrix and human ESCs with regard to their colony morphology and expression of extracellular pluripotency markers, we expect this study to directly translate into the identification of up-scaling conditions for human ESCs. This bioprocess will enable an osteogenic differentiation of ESCs with limited human influence. This controlled and well-defined system could then build a basis to be used when differentiating cells especially for cell-based therapies and embryotoxicity assays. [1] zur Nieden et al., Differentiation 71, 2003

132

Karsten Winter Universität Leipzig

ID 88 Translation Center for Regenerative Medicine IMONIT kwinter@trm.uni-leipzig.de www.trm.uni-leipzig.de/imonit.html

Comprehensive in vitro model for the engineering of microvasculature - evaluation of morphological and functional data

Karsten Winter, Ulf Wehrmeister, Antje Böttner, Jens-Peer Kuska, Bernhard Frerich

Objectives: The engineering of functional microvasculature has special impact for voluminous and metabolically active tissues. In order to quantify formation of coherent vascular networks, appropriate three-dimensional imaging and image processing is needed. We present a model for in vitro engineering, imaging and comprehensive analysis of microvessel-like structures. Material and methods: Small-diameter vessel equivalents were fabricated from collagenous scaffolds, adipose tissue stromal cells and endothelial cells. Specimens were compared after 16 days of rotation or pulsatile perfusion with regard to “capillary” density, recruitment of α-actin-positive cells and branching from the central lumen. Algorithms were developed for 3D measurement of various characteristic quantities of capillary-like structures as well as for pericytal recruitment from CLSM (confocal laser scanning microscopy) data. Results: The aggregates were imaged and measured three-dimensionally by use of CLSM and image analysis. Capillary density and the maturation of capillary-like structures ascertained by the recruitment of α-actin-positive cells reached the highest degree in the luminal portion of the perfused specimens. Manually acquired histomorphometrical data were compared with computed CLSM derived data and used for the validation of the algorithms. Already interactively measured datasets were measured again automatically and results from histomorphometry agree with results from automated 3D measurement. Conclusion: Application of hydrodynamic forces (flow and pressure) is essential for microvascular engineering in vitro and has impact for the engineering of “feeder donor vessel” with the ability to supply surrounding tissues for the purposes of plastic and reconstructive surgery.

133

Bernhard Frerich Universität Leipzig

ID 89 Department of Oral & Maxillofacial Surgery bernhard.frerich@medizin.uni-leipzig.de http://mkg.uniklinikum-leipzig.de/

Quantitative evaluation of adipose tissue engineering in SCID mice

Bernhard Frerich, Karsten Winter, Konstanze Weinzierl

Introduction: Adipose tissue has strong significance in plastic and reconstructive surgery (body-contour, mobility of tissue layers). The aim of our present study was to evaluate the feasibility of adipose tissue engineering in a SCID-mouse model. Methods: Adipose tissue stromal cells (ATSC) were harvested from human adipose tissue obtained during elective surgery. Differentiation was induced with two cycles of IBMX administration. Adipose tissue equivalents were generated in vitro and transplanted into NOD-SCID-mice (n=90) with and without co-transplantation of endothelial cells (HUVEC). Histomorphometric evaluation of adipose tissue formation was performed on whole sections imaged by a ZEISS MIRAX SCAN slide scanner. The resulting images were segmented into regions of different tissue types and evaluated quantitatively by means of image processing. Results: After explantation of SCID mice 10 days, 4 weeks, 6 months postoperatively, mature adipose tissue could be observed. Transplanted cells were S-100 positive (adipocyte-typic). Thickness of the implants diminished from 10 days to 4 weeks, but increased after 6 months with completion of adipocytic differentiation. There was no significant advantage for endothelial co-transplantation and small sized implants showed significant better differentiation than larger sized implants (ca. 10mm). Conclusion: Differentiation of three-dimensional adipose tissue constructs is feasible and reproducible in vitro and in vivo, however reliable solutions for microvascular network formation still have to be found.

134

Alexander Lochmann Universität Leipzig

ID 90 Translation Center for Regenerative Medicine REMOD alochmann@trm.uni-leipzig.de www.trm.uni-leipzig.de/remod.html

rhBMP-2 Delivery From Scaffolds, Microparticles And in Situ-forming Gels

Alexander Lochmann, Hagen Nitzsche, Sabrina von Einem, Elisabeth Schwarz, Karsten Mäder

Objectives In previous approaches the action of recombinant human Bone Morphogenetic Protein 2 (rhBMP-2) administered in bone defect regeneration has proven effective, but its use is, especially in higher doses, often accompanied by an increase of inflammation processes. It is vital to minimize these disease prolonging effects by finding the right release pattern in an appropriate dosage form. Two approaches towards optimization have been made, (I) the fixation of BMP-2 on a scaffold and (II) the incorporation of BMP-2 into in situ-forming implants. Materials and Methods (I) The composite material for scaffolds made of collagene and hydroxyapatite was produced by a continuous precipitation method. It was further processed by suspending with chitosan and subsequently freeze-dried to form a porous scaffold. rhBMP-2 was derived from E.coli inclusion bodies and purified via affinity chromatography after a renaturation step. The growth factor was applied in different ways in order to achieve a variation in release kinetics. (II) Furthermore, in situ-forming implants with different compositions have been prepared. rhBMP-2 was incorporated during the manufacturing process to get a homogenous distribution. Results rhBMP-2 has been produced and purified successfully. A technique to incorporate BMP into in situ-forming gels could be developed. The growth factor could be formulated with polymer films to be put on highly porous, biodegradable scaffolds. Conclusion rhBMP-2 can be formulated into various delivery systems. Effects on the integrity of the growth factor after fixation/ incorporation are still to be examined.

135

Jorge Guerra Universität Leipzig

ID 91 Translation Center for Regenerative Medicine CELLT jguerra@trm.uni-leipzig.de www.trm.uni-leipzig.de/cellt.html

Xenogeneic Infection Risk and Restriction of Individual Rights: An European Approach (EU, German, Spanish and English Law)

Jorge Guerra

Objectives: One day clinical organ xenotransplantation (XTx) will be a reality; this will mean assuming an uncertain infection risk for patients and society in general. Legislation will have to consider and cope with this xenogeneic infection risk (XIR) as well as reduce it as much as possible. It has to be determined, when, if, how and which legal instruments may be used to prevent XIR. The chief focus here will concern individual rights restrictions. The utility of this analysis is double: to have a legal basis ready when XTxs are deemed prepared for the first clinical experiments; and to offer new arguments to the discussion of if XTx should be authorized—on the basis of what would be currently legally possible, or if the law would have to be changed instead. Method: The view of current European law (especially EU, Spanish, German and English Law) facing these—to date—hypothetical problems will be considered. The well-established legal institutions of proportionality and reasonableness will provide the foundations for the legal analysis. They will answer the questions of whether and how law should or must act as well as which individual rights can be constrained in a concrete situation, and consider time or rather risk (from the XTx until the appearance of a XZn or beyond) as an independent variable. Results: Individual risk reduction would imply that at least XTx recipients would have to observe certain measures in order to prevent xenogeneic transmissions. Either these patients may oblige themselves with a private law instrument to do so; or they can be obliged to with a public law instrument, as it is always allowed to constrain individual rights if higher public interests are threatened. According to the legal systems analysed, the XIR must be a real or direct menace if individual rights are to be significantly constrained. Hence any use of the precautionary principle cannot be a firm legal basis for such individual rights restrictions. Conclusion: Factual and theoretical legal aspects limit the present and traditional law as an instrument to prevent XIR. Due to the essential value of fundamental rights, law can only react when a real XIR directly threatens society. And even in this case it is doubtful if legal repression is the best way to face infectious diseases with regard to human beings.

136

Johanna Lutz Universität Leipzig

ID 92 Translation Center for Regenerative Medicine TEMAT jlutz@trm.uni-leipzig.de www.trm.uni-leipzig.de/temat.html

Correlation between nitriding parameters of medical CoCr alloys and resulting hardness increase and reduction of wear rate

Johanna Lutz, Inga-Maria Eichentopf, Antje Lehmann, Stephan Maendl

CoCr-alloys are commonly used for medical implants in prosthetic replacements and cardiovascular applications due to their good mechanical properties and biocompatibility. However, a low hardness and wear resistance still results in a rather high rate of release of toxic metal particles and ions into the surrounding tissue. Nitrogen insertion into the surface of CoCr alloys by plasma immersion ion implantation allows the formation of hard and wear resistant surface layers, which can additionally act as a diffusion barrier against the release of toxic ions. In this presentation the correlation between nitriding parameters and resulting mechanical properties, e.g. hardness and wear of three different CoCr alloys, covering the main application areas, is investigated. Nitrogen ion implantation was performed in a temperature range between 300 – 600 °C, at ion energies of 10 – 25 kV, and different process times up to 3 hours. The resulting layer thickness was determined with secondary ion mass spectroscopy, while the hardness and the wear rate were analysed using a Berkovich indenter and an oscillating ball-on-disc configuration, respectively. A fast thermally activated interstitial diffusion is observed for nitrogen in CoCr alloys. Thus, with increasing implantation temperature and time, an increased layer thickness up to 5 μm is found. At the same time, an increase of a factor of 3 – 5 in the surface hardness to values of 20 – 25 GPa is measured, which is directly correlated with an improved wear resistance by a factor of up to 10 for an optimized set of parameters.

137

Aldo Leal-Egana Universität Leipzig

ID 93 Translation Center for Regenerative Medicine TEMAT aleal@trm.uni-leipzig.de www.trm.uni-leipzig.de/temat.html

Determination of the Cell/Aggregate growth Profile inside Alginate Capsules

Aldo Leal-Egana, Felicia Heidebrecht, Isabel Rode, Andrea Robitzki, Augustinus Bader

 

 

In Tissue Engineering, Biomaterials play a very important role, being used as in the generation of artificial constructs, directly in cell therapies, as well improving cell differentiation process (Leal et al, 2006). One of these biomaterials, the alginate, remains as one of the most used hydrogels to immobilize and/or encapsulate cells, due to the absence of immune response, easy handling and low cost. In the current study we focused on the influence of alginate on the cell proliferation profile. For our experiments we employed two types of cells cultured independently: An uncontrolled growing cell line, and cells with normal contact inhibition. The cells were entrapped in 500 μm diameter capsules in different alginate concentrations, and cultivated for 12 days. Our results show that there is a high dependence between the duplication characteristics (controlled or uncontrolled), cell surviving profile and the alginate concentration, suggesting that probably this biomaterial would be not suitable for the use of all types of cells, especially those that increase their number rapidly.

References.

1.- Leal-Egana A, Heinrich JM, Smith MD, Nowicki M, Bader A., (2006), A simple non-destructive method for the fixation and immunostaining of cultured cells encapsulated in alginate, Biotechnology and Applied Biochemistry, Jun;44(Pt 3):143-50

 

138

Anja Mittag Universität Leipzig

ID 94 Translational Center for Regenerative Medicine IMONIT amittag@trm.uni-leipzig.de www.trm.uni-leipzig.de/imonit.html

A Bead Model for the Realization of 3D Tissue Cytometry

Anja Mittag, Attila Tarnok

Stoichiometric and quantitative analysis of tissues is of eminent importance in the understanding of all interactions between cells in their natural environment, e.g. in regeneration processes. For such demands (Tissomics) multiplex analyses are required. Slide Based Cytometry (SBC) analysis yields quantitative cell related data on various cell constituents. To be cytometric SBC measurement needs high focal depth in order to acquire the fluorescence of the whole cell. For tissue section analysis it means a section thickness of at least one whole cell (i.e. >30 μm). On the other hand cell fragments are unavoidable due to sectioning. Another problem for quantitative cytometric analysis of tissue is the fact that not all cells are in the same focus. Based on the triggering signal, in most cases the DNA fluorescence information of the cells, and the properties of the objective used cells outside the focus will be included into the analysis. We will demonstrate here the problems of threedimensional cytometric analyses using a simple model of fluorescent beads. The fluorescence of these beads is detectable up to 100 μm of both sides off the focus. Whereas the fluorescence integral is comparatively stable over a broad range the MaxPixel Signal shows a peak in the focus. For measurements of tissues it means that fluorescence signals of cells over or under the cells of interest (focus level) can easily be interpreted as part of the cell in the focus. To be sure to avoid such signals multiple scans at different focus levels are necessary. By combining the measurements the “right” focus for the cells of interest can be found due to the values of the MaxPixel Signal and area. We hope that our model could be a step forward to correct interpretation of cytometric data of tissue.

139

Fritzi Siegert Universität Leipzig

ID 95 Translation Center for Regenerative Medicine TEMAT fsiegert@trm.uni-leipzig.de www.trm.uni-leipzig.de/temat.html

rhBMP-2 Delivery From Scaffolds, Microparticles And In Situ-forming Gels

Fritzi Siegert, Anja Zlobinsky, Anja Grahnert, Gunther Böttcher, Kathrin Reiß, Andreas Schubert, Sunna Hauschildt, Karen Nieber

The longevity and the effective function of an implant is determined by events occurring at the tissue-material interface and by the surrounding environment. The acute or chronic inflammatory reaction of the host as his biological response is characterized particularly by the formation of endothelial cells and monocytes/macrophages. Due to the high potential of these cells to influence inflammatory processes the present study was initiated to develop a co-culture system consisting of HUVECs and macrophages in order to test the biocompatibility of modified silicones. The growth and morphology of the cells was visually observed and the electrophysiological parameters were recorded using the whole-cell configuration of the patch clamp technique 2, 24 and 48 hours after colonisation. When using glass as tissue culture surface the growth and morphology of the macrophages in the coculture did not differ from macrophages cultured alone. Differences were found in the electrophysiological parameters as well as in the ATP-induced inward current. On tissue culture plate treated with ELASTOSIL® P7613 A/B (KETW 7613), a networked silicon rubber designed for flexible implants, neither the morphology nor the electrophysiological parameters of macrophages co-cultured with HUVECs changed when compared to macrophages co-cultured with HUVECs on glass cover slips. Thus, this material seem to be suited for implants. Structure or surface modifications of the silicon rubber (KETW 7684, KETW 7686) resulted in altered morphology and damage of the cells indicating that these materials do not fulfil the requirement for a biocompatible synthetic material. Key words: macrophages - silicone - patch clamp technique

140

Holger Stephan Universität Leipzig

ID 96 Head of Research and Development NeuroProgen GmbH hstephan@neuroprogen.de www.neuroprogen.de

Entwicklung eines High Content Screening Systems zur Untersuchung des Einflusses der extrazellulären Matrix auf Adhäsion und Proliferation und Differenzierung von humanen neuralen Stammzellen

Holger Stephan

Hintergrund: Grundsätzlich ist es möglich humane neurale Stammzellen (NSZ) in-vitro zu kultivieren. Dabei weitestgehend unberücksichtigt ist jedoch der Einfluss der extrazellulären Matrix (ECM) auf das Adhäsions- und Proliferations- und Differenzierungsverhalten der NSZ. Um eine große Anzahl an ECM-Proteinen darauf hin zu untersuchen, ist es notwendig ein High Content Screening System (HCS) zu entwickeln. Methoden: Multiple Substrat Arrays (MSA™), bestehend aus 64 ECM Dots (jeder Spot = 300 μm Durchmesser), wurden auf Objektträger gespottet (10 Microarrays pro Slide = 640 Spots). Auf jedem Spot sind danach humane fötale NSC für 24 h bis zu einer Woche kultiviert worden. Die Zellen wurden anschließend immun-histochemisch mit anti-Ki67-Alexa 594 (neuraler Proliferationsmarker), anti-Nestin-Alexa 488 (neuraler Stammzellmarker) und DAPI (Zellkern) gefärbt. Ein automatisiertes Fluoreszenzmikroskop in Kombination mit einer erweiterten Bildanalysesoftware ist zur Detektion und Auswertung der Spots benutzt worden. Schlussfolgerung: Unter Berücksichtigung der unterschiedlichen Einflüsse der ECM Proteine auf Adhäsion, heterogene Clusterbildung, Proliferation und Differenzierung ist es notwendig das Zellkultursystem in Abhängigkeit von den zu kultivierenden Zelltypen und hinsichtlich Adhäsion, Proliferation oder Differenzierung zu optimieren. Dabei spielen die Eigenschaften der ECM auch in Kombination eine herausragende Rolle.

141

Stefan Schwan Universität Leipzig

ID 97 Translation Center for Regenerative Medicine TEMAT sschwan@trm.uni-leipzig.de www.trm.uni-leipzig.de/temat.html

Correlation between nitriding parameters of medical CoCr alloys and resulting hardness increase and reduction of wear rate

Stefan Schwan, Amanda Hughes, Andrea Staeudte, Frauke Junghans, Uwe Spohn, Andreas Heilmann

For cartilage implants, three-dimensional biodegradable scaffolds with high porosity and define fatigue strength are in focus. Scaffold materials made from bio-resorbable proteins are an alternative to conventional polymeric materials. Based on earlier investigations dealing with the deposition of protein films [1], silk foams were produced using the protocol described. Silk proteins were dissolved in aqueous systems or hexafluoropropanol (HFIP). Beside to foam production in an solid-liquid system [2], we established our own solid-liquid-vapours system to achieve higher porosity and interconnects between the single pores. Using different ratios between protein solution, sodium carbonate and carbon dioxide, different foam parameters like porosity and pore size were obtained. Furthermore, the foams were tested by standard mechanical pressure tests and cyclical mechanical stress tests to analyse the relaxation parameters and shrinking. The mechanically optimized foams were used to form scaffolds with condrocytes. [1] Junghans, F.; Morawietz, M.; Conrad, U.; Scheibel, T.; Heilmann, A. &amp; Spohn, U. Preparation and mechanical properties of layers made of recombinant spider silk proteins and silk from silk worm, Applied Physics A: Materials Science and Processing, 2006, 82, 253-260 [2] Kim, U.; Park, J.; Joo Kim, H.; Wada, M. &amp; Kaplan, D., Three-dimensional aqueousderived biomaterial scaffolds from silk fibroin, Biomaterials, 2005, 26, 2775-2785

142

Kathleen Schröck Universität Leipzig

ID 98 Translation Center for Regenerative Medicine TEMAT kkintzel@trm.uni-leipzig.de www.trm.uni-leipzig.de/temat.html

Characterization of a new Material for human mesenchymal Stem Cell-based Bone Tissue Engineering

Kathleen Schröck, Dirk Schumann, Manja Kamprad

 

In the field of regenerative and translational medicine, it is a main goal to find and characterize new materials for the use in scaffold based tissue engineering to restore and improve physiologic functions. Here, we investigate a new innovative material, its characterization and utilization for three-dimensional (3-D) in vitro cell culture systems and its future use as a tissue engineered bone graft. Based on human mesenchymal stem cells (MSC) from different donors, the scaffold based 3-D culture system needs a broad spectrum of analysis and testing. The used scaffold, an open-porous material, was proofed for biocompatibility and viability using phagocytosis assay for monocytes and viability and adhesion assays for MSC under short and long-term conditions. Since it is not possible to ensure an adequate oxygen supply in 3-D culture systems, reaching certain dimensions, it is of great interest to determine proliferation and differentiation of MSC under hypoxic conditions. Therefore, hypoxia (1 % O2) was applied on cell cultures through a hypoxic incubator. After developing a protocol for optimal cell seeding, proliferation of MSC on the material was observed under normal and hypoxic conditions. The differentiation of MSC was determined on polystyrene multi-well plates and will be proofed on the 3-D scaffold based system. Despite intensive research and the already used inorganic metals and ceramics or natural and synthetic polymeric structures, the final and optimal material for bone grafts has not yet been found.

143

Jan Liese Universität Leipzig

ID 99 Translation Center for Regenerative Medicine TEMAT jliese@trm.uni-leipzig.de www.trm.uni-leipzig.de/temat.html

Stem Cell based Bone Graft Design with a Perfusion and Rotation vs Hydrogel Bioreactor

Jan Liese, Susanne Liese, Bernhard Frerich, Augustinus Bader, Alexander Hemprich

 

Reconstructive procedures in maxillofacial surgery often require transplantation of autologous bone. Reduction of morbidity and costs concerning bone graft harvesting can be realized by using MSC based grafts. Parallel seeding, expansion and differentiation of MSCs on a highly porous mineral scaffold using a rotation and perfusion based bioreactor vs hydrogel seeding is evaluated. Porcine bone marrow derived MSCs were isolated and expanded under autologous conditions. Multilineage potential capacity in osteogenic, adipogenic and chondrogenic differentiation was performed and cells were characterised by flow cytometry postive for CD105, CD166 and CD90 and negative for CD31. Seeding was performed by placing the scaffold in between silicone funnels and sealing of the remaining surfaces with MSC loaded gelcoat creating a perfusion chamber. A highly concentrated MSC suspension was injected through the scaffold. Following initial rotation, intermitted perfusion and addition of osteogenic supplements was continued for 10 days. A fibrin hydrogel was used for seeding of the scaffold and cell distribution was compared. Porcine bone marrow derived MSCs were isolated and expanded under autologous conditions. Multilineage potential capacity in osteogenic, adipogenic and chondrogenic differentiation was performed and cells were characterised by flow cytometry. Osteogenic differentiation was evaluated histochemically by staining of alkaline phosphatase-activity (AP-activity) and deposition of calcium by von Kossa staining. Seeding of MSCs on a highly porous b-TCP scaffold was carried out using an autologous plasma derived fibrinhydrogel and a perfusion and rotation bioreactor system. The bioreactor was set up placing the scaffold in between silicone funnels and sealing of the remaining surfaces with MSC loaded fibrinhydrogel creating a perfusion chamber. The chamber was filled with a highly concentrated MSC suspension. Following initial rotation, intermitted perfusion continued for 10 to 14 days. Distribution of cells was analysed by Calcein AM and Ethidiumbromid stains. PorMSCs satisfy requirements of multilineage potential capacity. The cells showed surface antigens for CD105, CD166 and CD90 and were negative for CD31. Osteogenic differentiation could be induced by cultivation with autologous serum as well as fetal calf serum. It could be observed after 10 days by rise in AP-activity. Von Kossa stains showed Ca-deposition on the third week of cultivation. Seeding of cells on the highly porous mineral scaffold could be achieved reproducible only using fibrinhydrogel method, which inherits low density of cells in the central areas of the scaffold. The rotation/perfusion bioreactor system was not reproducibly capable of maintaining optimal conditions within the centre of the scaffold. Osteogenic differentiation of porMSCs under autologous culture conditions must be further evaluated. RT-PCR of osteogenic proteins is performed at present. The bioreactor setup must be modified to a hydrogel based seeding strategy with a more stable hydrogel to withstand perfusional stress. From our experience hydrogel seeding strategies of scaffolds larger than two centimeter require a central perfusion system. The suitability of our graft will be further evaluated with onlay grafting in a large animal model.

144

Andrea Stäudte Universität Leipzig

ID 100 Translation Center for Regenerative Medicine IMONIT astaeudte@trm.uni-leipzig.de www.trm.uni-leipzig.de/imonit.html

Investigations with scanning electron Microscopy (SEM) and focused ion Beam (FIB) on Cartilage Tissues

Andrea Stäudte, Andreas Cismak, Stefan Schwan, Ronny Schulz, Uwe Spohn, Andreas Heilmann

 

Various scanning electron microscopic techniques and the corresponding sample preparation with Focused Ion beam technology were used to achieve more detailed and deeper insights into the morphology and microstructure of cartilage samples and in vitro cultivated cartilage tissues including osteochondral constructs. The investigations were directed to composites consisting of chondrocytes/chondroblasts, and selected scaffold materials. The study is aimed also to contribute to an improvement of the mechanical properties of artificial cartilage tissues. Isolated articular cartilage explants from porcine femoral heads and chondrocyte constructs were investigated. Using environmental scanning electron microscopy techniques (ESEM) without further sample preparation as well as SEM with chemical fixation, cartilage tissue within cartilage explants and chondrocyte constructs were studied. From cross-sections of this materials produced by FIB in slice to slice technique, results about chondrocyte allocation and the volume of the extracellular matrix were achieved.

145

 

146

CELL ENGINEERING

 

 

147

Christian Dietzsch Technische Universität Dresden

ID 101 Ins. Of Food Tech. And Bioprocess Engineering Christian.dietzsch@gmx.de http://tu-dresden.de/die_tu_dresden/fakultaeten/ fakultaet_maschinenwesen/ilb

Influenza vaccine production with adherent Vero cells

Christian Dietzsch, Yvonne Genzel, Thomas Bley

Production of animal cell culture derived influenza virus seems to be suitable for industrial scale production of human vaccines to prevent pandemics. The bioprocess engineering group at the MPI Magdeburg works on a process with MDCK cells focusing on different aspects like virus replication, cell physiology, proteomics a. o.. For possible comparative studies the establishment of the alternative production process with Vero cells, as described in literature, was the aim of this work. Experiments regarding cell growth and virus replication were carried out in static and microcarrier systems. The influence of multiplicity of infection (moi) and trypsin concentration on infection in several media (serum containing/serum free) were experimentally investigated. It was seen, that the moi had an influence on the course of infection but not on the virus yield. The trypsin concentration for infection had to be adapted to each medium and cultivation system for optimization of the virus yield. A twofold higher concentration of trypsin was necessary in serum free media. By adapting the virus to the Vero cells, virus yield as well as infection course could be improved. The comparison of two bioreactors (Wave Bioreactor/5 L stirred tank reactor) showed a higher virus yield for cultivation in Wave Bioreactor. The obtained data on cell growth, metabolism and virus production for the process with Vero cells were compared to the data on MDCK cells and can be used to set-up further experiments for comparative studies. Presentation of this work at the Bioperspectives 2007 was honored with the first prize of the Poster award (Cologne BioInnovation Award).  

148

Grit Schaarschmidt Universität Leipzig

ID 102 University Hospital Clinic and Polyclinic for Neurology

Grit.schaarschmidt@medizin.uni-leipzig.de http://neurologie.uniklinikum-leipzig.de

Characterization of voltage-gated potassium channels in midbrain derived neural precursor cells

Grit Schaarschmidt

Based on midbrain derived neural precursor cells (hNPCs) a cell therapy for the treatment of Parkinson´s disease shall be established. Kv channel types were analyzed with RT-PCR. Patch Clamp recordings were performed in whole-cell voltage-clamp mode to measure potassium outward currents. Kv channels were blocked by different concentrations of the antagonists 4-Aminopyridine (4-AP) and Quinidine (Q) and EC50 values were determined. MTT assay as a standard colorimetric assay for measuring cellular proliferation was carried out after 3 days of potassium blockade. Several Kv1 and Kv4 channel subtypes could be identified by RT-PCR. Cell proliferation was significantly blocked by 4-AP whereas Q had no impact on cell viability. These results suggest fast inactivating Kv4 channels which are blocked with 4-AP may protect proliferating hNPCs from sustained depolarisation. In contrast the inhibition of slowly inactivating Kv1 channels with Q had no detectable impact on proliferation of hNPCs. Summary : Undifferentiated precursor cells have less functional properties. They generate characteristic potassium currents but neither sodium currents nor expression of ligand-gated channels can be observed. We investigated early voltage-gated potassium channels (Kv) to find out more about their importance for electrophysiological behavior. Therefore, we analysed the types of Kv channels that are expressed in proliferating hNPCs and how they can be blocked. The application of potassium channel antagonists during proliferation gave information about whether these voltage-gated channels have an essential influence on cell growth and developement or not.

149

Axel Erler Technische Universität Dresden

ID 103 BIOTEC Genomics – Prof. Stewart Axel.erler@biotec.tu-dresden.de www.biotec.tu-dresden.de/stewart

New Insights into Redβ-mediated DNA Annealing using Atomic Force Microscopy

Axel Erler, Susanne Wegmann, Celine Elie-Caille, Marcello Maresca, Ralf Seidel, Tobias Heine, Daniel Müller, Francis Stewart

Redβ anneals DNA to initiate homologous recombination and has gained recent prominence through the development of the DNA engineering technology known as ‘recombineering’ or ‘Red/ET’. It originates from the red-operon of λ phage where it is co-expressed in the early life cycle stage with Redα, a processive 5’-3’ exonuclease and Redγ, a DNA mimetic and RecBCD inhibitor. Unlike RecA/RAD51, Redβ is not an ATPase and it’s mechanism for initiating homologous recombination is poorly understood. To examine the structure and dynamics of Redβ complexes at sub-molecular resolution we performed tapping mode atomic force microscopy (AFM) of Redβ protein alone and in complex with DNA. Without DNA, Redβ forms a ‘split lock washer’ structure with a shallow right-handed helicity. Sequentially adding complementary ssDNA generates a stable left-handed helical filament. Importantly, the contour length of the helical filament equated linearly to the lengths of complementary ssDNA, giving the number of nucleotides per Redβ monomer. Additionally, the monomer width along the filament was quantified. These new quantities as well as the observed helical transition reveals new insights into the mechanism of DNA annealing mediated by Redβ and led us to suggest new mechanistic models.

150

Christiane Haas Technische Universität Dresden

ID 104 Ins. Of Food Tech. And Bioproces Engineering christianehaas@web.de http://tu-dresden.de/die_tu_dresden/fakultaeten/ fakultaet_maschinenwesen/ilb 

Flow cytometric and phytochemical investigations with plant cell suspension cultures of sunflower (Helianthus annuus)

Christiane Haas, Milen Georgiev, Jost Weber, Jutta Ludwig-Müller, Thomas Bley

Plants are an unbounded source of valuable secondary metabolites used from centuries as pharmaceuticals, food additives, fragrances, dues and agrochemicals. Production of plant derived metabolites through classical technologies lead to several difficulties, resulting mainly from seasonal, geographical and soil features. Furthermore the isolation of such metabolites (usually in small amounts) from huge plant mass is labour- and time-consuming and makes the process more expensive. Plant cell, tissues and organ cultures offer an alternative opportunity for production of biological active substances. A critical step in the creation of industrial process is a development of on-/off-line methods for determination of cell’s growth and the physiological behaviour of the cells during their growth in different cultivation systems. Recently flow cytometry became a popular method for ploidy screening, detection of mixoploidy and aneuploidy, cell cycle analysis, estimation of absolute DNA amount or genome size etc. Cell suspension cultures of Helianthus annuus possess stable growth and morphological characteristics and were found to produce biologically-active substances (immunologically-active polysaccharides and Vitamin E) and therefore could be used as a good model system for determination of kinetics of growth and metabolite(s) production. The results from flow cytometry measurements (cell cycle analysis) and Vitamin E production obtained during the cultivation of Helianthus annuus cell suspension culture in shake-flasks and 5-L stirred tank reactor are presented and discussed.

151

Johanna Scheibe Universitätsklinikum Leipzig

ID 105 Clinic and Polyclinic for Neurology Johanna.scheibe@medizin.uni-leipzig.de http://neurologie.uniklinikum-leipzig.de/ ParkinsonArbeitsgruppe.htm

Transplantation of human derived neural precursor cells into 6-OHDA-rat-model

Johanna Scheibe

In this study we use in vitro cultivated hNPCs for transplantation into 6-OHDA unilateral lesioned rats, which is a useful model for Parkinson’s Disease (PD). We like to show that the cells have the ability to replace the lost dopaminergic neurons in these animals and therefore playing a great role as a potental stem cell therapeutic in PD patients. Summary : Pre-differentiated hNPCs have the potential to partial compensate the loss of dopaminergic neurons of the substantia nigra. This leads in some 6-OHDA lesioned animals to an improvement of their motor activity.

152

Florian Wegner Universität Leipzig

ID 106 Translation Center for Regenerative Medicine CELLT fwegner@trm.uni-leipzig.de www.trm.uni-leipzig.de/cellt.html

Analyses of primary human fetal cultures and derived neural stem cells in vitro

Florian Wegner, Monike Poppe, Javorina Milosevic, Maja Dieterlen, Johanna Scheibe, Andreas Boldt, Sigrid Schwarz, Johannes Schwarz

BACKGROUND: Neural precursor cells (NPCs) derived from human fetal primary cultures can be expanded and differentiated in vitro. Prior to transplantation studies in patients with Parkinson’s disease detailed analyses of primary cultures and NPCs are necessary. METHODS: RT-PCR, immunocytochemistry, expansion and differentiation of NPCs, DAT-, and PI-FACS, dopamine-ELISA, electrophysiology, striatal transplantations of GFP-labelled NPCs, laserscanning microscopy. RESULTS: Mesencephalic tissue samples contained significantly higher transcription rates of the dopaminergic marker genes TH, DAT, and EN1 than samples from nonmesencephalic tissues. FACS analysis of NPCs revealed a typical cell cycle distribution with well defined G1 and G2 peaks (background cell death 1.3 – 5.0 % over 3 months expansion, 25 – 30 % healthy NPCs in S-phase). More than 70 % of proliferating NPCs expanded for three months expressed nestin, among them 25 % were Ki67-positive. Dopamine release by NPCs significantly elevated after 2 weeks differentiation to 644 pg/ml. During the process of NPC-differentiation maximal GABA- and glutamate-induced current amplitudes increased significantly. After 4 weeks of in vitro differentiation, 47 % of investigated NPCs showed large sodium currents and were able to fire single action potentials in response to depolarizing current pulses. Currentvoltage plots of NPCs showed a significant increase in the maximal amplitudes of sodium and potassium currents during development. 3 weeks after transplantations of GFP-labelled NPCs into rat striatum, confocal laserscans and whole cell recordings indicated survival and differentiation of grafted cells. CONCLUSIONS: These data suggest that primary human fetal mesencephalic tissue can be identified by combined expression of markers TH, DAT, EN1. After differentiation for 3-4 weeks derived neural precursor cells bear essential properties of functional neurons providing a cell source for transplantation in Parkinson‘s disease.

153

Sabine Schewtschik Universität Leipzig ID 107 sschewtschik@freenet.de

 

Simulation of embryogenic conditions to differentiate stem cells into functional neurons

Sabine Schewtschik, Grit Schaarschmidt

 

Parkinson is a neurodegenerative disease characterized by loss of midbrain dopaminergic neurons in the Substantia nigra. Cell replacement therapies are a promising strategy to compensate this cell loss. Culture conditions that promote differentiation of functional dopaminergic neurons are required. The aim of our group is to differentiate human fetal neural mesencephalic progenitor cells (hmNPCs) into dopaminergic neurons. Cells cultivated in standard differentiation media (DM1) provide neurons of limited functional differentiation. Thus, the aim of the present study was to mimic physiological neuronal differentiation. During brain development there is a well described shift from depolarization to hyperpolarization mainly depending on the neurotransmitter GABA. This environmental change results in neuronal maturation and development of essential neuronal connections. We transferred this principle into the hNPCs differentiation protocol. By realising the two phases via changes of ion concentrations we were able to increase the rate of dopaminergic neurons and advance maturation. Besides, neurite growth was accelerated and expression of ion channels increased. Thus, modulating the resting membrane potential may enhance the potential of dopaminergic neurons derived from stem cells. summary : We are eager to establish a cell replacement therapy for patients with Parkinson’s disease based on hmNPCs. Improvement of differentiation protocols is necessary. Using a modified protocol derived from the in vivo situation of a developing neuron during embryogenesis we are able to produce more mature neurons with improved functional activity.

154

Jost Weber Technische Universität Dresden

ID 108 Ins. of Food Techn. and Bioprocess Engineering Jost.weber@tu-dresden.de http://tu-dresden.de/die_tu_dresden/fakultaeten/ fakultaet_maschinenwesen/ilb

Flow cytometry investigations of Datura innoxia.

Jost Weber, Vasil Georgiev, Mladenka Ilieva, Atanas Pavlov, Thomas Bley

The nuclei genomes of the higher plant Datura innoxia both callus and hairy roots cultures obtained from the former, were investigated using the method of flow cytometry. It was established that in vivo plants and the in vitro cultures obtained from them possess the same genome size. However we found differences in ploidity of the investigated plant systems: The hairy roots cultures undergo one endoreduplication cycle and therefore consisted mainly of tetraploid cells, while the callus undergo two endoreduplication cycles. The obtained data showed the same endoreduplication pattern for all 10 investigated transformed root lines, which is in contradiction to the wide spread thesis, that transformed root cultures have identical genome as the plants from which they were obtained. Acknowledgments This work has been supported by DAAD (D/05/01303) and Bulgarian National Science Fund (D-01-84/2006).

155

Vasil Georgiev The Stephan Angeloff

ID 109 Institute for Microbiology Sofia vasgeorgiev@gmail.com www.microbio.bas.bg/

Flow cytometry investigations of Beta vulgaris cv. Egypt and in vitro systems obtained from it.

Vasil Georgiev, Jost Weber, Atanas Pavlov, Mladenka Ilieva, Thomas Bley

The object of investigation was red beet Beta vulgaris cv. Egypt as well as callus and hairy roots, obtained from it. Flow cytometry investigations showed the presence of haploid nuclei in higher plant (32.59 %) and in transformed root cultures (16.76 %). No haploid cells were found in nuclei, isolated from cells of callus culture. The presence of additional levels of ploidy in the investigated in vitro systems was observed and discussed. Acknowledgments This work has been supported by DAAD (D/05/01303) and Bulgarian National Science Fund (D-01-84/2006).

156

Joanna Kosacka Universität Leipzig

ID 110 Medical Faculty Institute of Anatomy joanna.kosacka@medizin.uni-leipzig.de www.uni-leipzig.de/~anatomie/ 

Cholesterol and apolipoproteins enhance neurite outgrowth and synapse formation in dorsal root ganglia (DRG) cultures

Joanna Kosacka, Martin Gericke, Marcin Nowicki, Jürgen Borlak, Katharina Spanel-Borowski

 

The adipose tissue is purported to act like an endocrine organ. Our recent study on cocultures of 3T3-L1 adipocytes with DRG – neurons (isolated from 2-day-old rats) also indicated the neuroprotective function of adipocytes i.e. an enhanced neuritogenesis and synaptogenesis. The gene expression analysis indicated that adipocyte-secreted cholesterol transporter proteins ApoD and ApoE were up-regulated and hence, might be involved in these processes. Our present aim was to investigate the neuritogenesis and synaptogenesis in DRG cultures under the influence of exogenous cholesterol, ApoD and ApoE. After 6 days of treatment, DRG cultures displayed increased neurite outgrowth as confirmed by immunostaining of neurofilament 200 (NF-200) and by Western blot analysis of neurofilament 68 (NF-68) and the growth associated protein-43 (GAP-43). Moreover, the Western blotting demonstrated that the treatment of DRG cultures with cholesterol, ApoD and ApoE respectively, up-regulated the presynaptic synaptophysin, synaptotagmin and the postsynaptic density protein 95. The neuritotrophic effect of apolipoproteins and cholesterol was accompanied by the increased expression of both, trkA and CXCR4 receptors. Summary: These results demonstrate a supportive function of cholesterol and apolipoproteins by neuritogenesis possibly through modulation of chemokine and neurotrophin signalling.

157

Isabelle Hanisch Fraunhofer Institute for Immunology

ID 111 and Cell Therapy Leipzig Group Stem Cell Biology isabelle.hanisch@izi.fraunhofer.de www.izi.fraunhofer.de/ 

Fusion and cellular reprogramming

Isabelle Hanisch, Alexandra Stolzing

 

 

Cell-cell fusion between tissue cells and stem cells has been found after stem cell transplantation. Cellular fusion of adult progenitor cells and somatic cells can contribute a way to rejuvenate senescent cells, reprogram gene expression, viability and proliferation capability and enhance tissue replenishment. In vivo stem cells fuse directly with somatic cells, or activate silenced local tissue progenitor cells by fusion. Fusion incidences increase when tissue is damaged, stressed, or diseased. Therefore we isolate bone-marrow derived adult stem cells and aged astrocytes from mouse and fuse them using different protocols including cold or heat-shock, PEG- or H2O2-treatment. The parental MSC, astrocytes, and hybrids are analyzed regarding to fusion-associated changes in gene-expression, like multipotent markers including sox-2, klf-4 and nanog and changes in proliferation capacity (CFU-F assay) before and after fusion. Furthermore we evaluate reprogramming using growth curves, antioxidative enzyme activity assays, carbonyl-content, senescence associated β-galactosidase staining, telomerase activity and telomere length, and ROS production. A better understanding of the reprogramming and rejuvenation of adult stem cells raise the prospect of using fusion as a tool in medical research and therapy. The goal of (F)using MSC is to maintain and replenish various tissues, via assembly of a stable heterokaryon between stem cell and somatic tissue cell.

158

Brigitte Söhling Martin-Luther-Universität Halle-Wittenberg

ID 112 Faculty of Mathematics, Natural Science and Technology Institute of Biotechnology Brigitte.soehling@biochemtech.uni-halle.de www.biochemtech.uni-halle.de/ 

Development of a screening tool to isolate of E. coli mutant strains that produce native disulfide-bonded recombinant proteins in the periplasm

Brigitte Söhling, Rainer Rudolph

 

 

Most proteins applied in therapy and diagnosis (e.g. hormones, growth factors, or recombinant antibodies) are secreted proteins, and most contain essential disulfide bonds that are required for structure formation in the extracellular oxidizing environment. High-level cytoplasmic expression of these proteins in E. coli is frequently accompanied by the generation of inclusion bodies. This can be a benefit for the matter of easy purification in some cases, but in other situations refolding of these proteins and production of a correct disulfide pattern can be quite difficult. Secretion of such proteins into the E. coli periplasm gives a better chance of proper folding due to the more oxidizing conditions in this extracellular compartment. However, the yield of secreted recombinant proteins is comparably low. The amount of product is limited by the secretion and folding capacity, leading to aggregation in the cytoplasm or periplasm. E. coli mutant strains with better secretion and folding properties would be a benefit to overcome these limitations.

This work focuses on the development of a screening tool to identify E. coli clones that secrete high amounts of a native recombinant protein. In this study, a single chain antibody directed against fluorescein was chosen as a model protein. Secretion was monitored by a two-membrane sandwich technique directly on agar plates (1). E. coli colonies were first grown on a hydrophilic PVDF membrane, and were then transferred onto a second, hydrophobic membrane that is coated with a ligand. Upon incubation, the single-chain antibody is secreted and binds to its ligand fluorescein immobilized on the second membrane. The antibody fragment can subsequently be detected by immunological means. E. coli host cells were then mutagenized by UV treatment are now screened with this method for enhanced secretion and folding properties.

1. Schlehuber, S., G. Beste, and A. Skerra. (2000) J. Mol. Biol. 297:1105-1120.

 

159

Jonne Helenius Technische Universität Dresden

ID 113 BIOTEC Research group Müller jonneh@biotec.tu-dresden.de www.biotec.tu-dresden.de/mueller

Membrane tethers as constant force clamps

Jonne Helenius, Jens Friedrichs, Yi-Ping Chu, Daniel J. Müller

 

 

Cells adhesion is critical for tissue function. Because tissues are frequently under constant strain it is not only important to study the rupture force of single adhesive interact but also their life-time under constant loads. Membrane tethers are very thin tubes that can be pulled out of liposomes and cells. The force required to maintain or extend a tethers is chiefly dependent on membrane properties and only weakly on extension speed. Thus, membrane tethers extracted from cells during single cell force spectroscopy are good constant force actuators. We propose that this allows tether life-time measurements to be used to study the life-times of single receptor-ligand interaction at the tips of tethers. Here, we attempt to validate this hypothesis using a well defined cell-substrate system. The data should augment available force-ramp measurements.

160

 

161

 

162

Diagnostics

 

 

163

Stephan Karl Technische Universität Dresden

ID 114 Ins. Of Food Tech. And Bioprocess Engineering stephanunkarl@googlemail.com http://tu-dresden.de/die_tu_dresden/fakultaeten/ fakultaet_maschinenwesen/ilb

Magnetic Deposition Microscopy for malaria detection in a field study in Papua New Guinea

Stephan Karl, Maciej Zborwowski, Thomas Bley

susceptibility of parasitized cells to magnetic capture. We have exploited this property to demonstrate that trophozoites, schizonts and gametocytes, and all four human malaria species are enriched on microscope slides exposed to a magnetic field. We conducted a trial to test the utility of this diagnostic method in a malariaendemic region of Papua New Guinea (PNG). Individuals observed with malaria symptoms were asked if blood samples provided for malaria blood smear diagnosis could be evaluated by the magnetic deposition microscopy (MDM) method. Blood samples (20 μL) were mixed with media (1 mL) and applied to transparent mylar slides exposed to a high gradient magnetic field (Bmax=1.8 Tesla) under constant flow (8.3 μL/minute). Light microscopic examination was performed following standard methanol fixation and Giemsa staining. We observed that P. falciparum (Pf) parasitemia, measured by conventional blood smear (CBS), was significantly amplified by MDM. Of 55 infected individuals MDM detected gametocytes in 47.1 % of study participants compared to 7.8 % by CBS. Comparing MDM vs. CBS, we observed consistent fold-increased capture of gametocyte (&gt;419,7), schizont (45.6 - 431.8), trophozoite (2.2 - 6.4) and ring (0.8 - 2.0)erythrocyte developmental stages, and a 1.3 – 2.5 fold increase in overall parasitemia. MDM increased detection sensitivity of Pf-infected, hemozoin-containing erythrocytes from infected humans. At approximately 50 %, gametocyte prevalence in the human population is significantly higher in malaria-endemic sites in PNG than estimates based on detection of gametocytes by CBS.

164

Peter Ahnert Universität Leipzig

ID 115 Center for Biotechnology and Biomedicine Molecular Diagnostics – Microarray Techniques ahnert@ uni-leipzig.de www.uni-leipzig.de/~ahnert/

Cytogenetic and molecular biological characterization of an adult medulloblastoma

Peter Ahnert, Heidrun Holland, Ronald Koschny, Wolfgang Krupp, Holger Kirsten, Jürgen Meixensberger, Manfred Bauer, R. Schober

Medulloblastoma (WHO grade IV) is a malignant, invasive embryonal tumor which mainly occurs in children and represents less than 1 % of all adult brain tumors. Therefore comprehensive cytogenetic and molecular biological data on adult medulloblastomas are very limited. Since conventional therapies provide disappointing long-term disease control new therapeutic options are being tested. We performed comprehensive cytogenetic analyses of an adult medulloblastoma, WHO grade IV, using trypsin-Giemsa staining (GTG-banding), multicolor fluorescence in situ hybridization (M-FISH), and locus-specific FISH complemented by molecular karyotyping using high density SNParrays. GTG-banding of 25 metaphases revealed 31 structural chromosomal aberrations, predominantly located on 4q, 9q, 10q, 11p, and 20q, which were confirmed by M-FISH analysis. Interestingly, we found two novel, so far not described translocations, t(4;11)(q25;p15) and t(9;20)(p23;p12). Using GTG-banding, locus-specific FISH, and M-FISH we detected frequent numerical changes of chromosomes 8, 14, 18, 19, 20, 21, and 22. Molecular karyotyping by SNP-array confirmed the chromosomal changes +4q, +9q, -10q, and –11p, and revealed, for medulloblastoma, de novo partial uniparental disomy (UPD) 1q and 9q. Primary medulloblastoma cells were resistant to TRAIL, a novel anti-cancer cytokine, but could be efficiently sensitized by co-treatment with the proteasome inhibitor Bortezomib. Applying complementary methods for cytogenetic analysis we detected novel chromosomal aberrations in medulloblastoma. Bortezomib/TRAIL co-treatment may serve as a powerful therapeutic option for medulloblastoma patients.

165

Daniel Kloß Universität Leipzig

ID 116 Center for Biotechnology and Biomedicine Prof. for Molecular Biological-Biochemical Process Technology Daniel.kloss@bbz.uni-leipzig.de www.uni-leipzig.de/~dmpt/

Micro cavity array (MCA) biosensor for impedance based functional drug screening of 3D tissue models

Daniel Kloß, André Rothermehl, Andrea A. Robitzki

 

Impedance spectroscopy is widely used for real-time analysis of cellular events and morphological changes of living cells and tissues on planar sensor systems. For non-destructive and labelling-free measurements of 3D tissue-models (spheroids), a silicon-based biosensor with implemented microelectrodes was developed and tested. An increased rate of apoptosis (induced by Camptothecin) in melanoma cells was detected and confirmed via caspase-3 assay. Analysed tumour spheroids formed adequate cell-cell- and cell-extracellular matrix contacts and hence mimicked a native three-dimensional cellular environment. In vivo-like conditions are favourable for screenings of pharmacological substances and cytotoxicity tests. Spheroids can be positioned between two pairs of electrodes in 15 squared individual microcavities (200, 300 and 400 μm, depth: 100 μm). The innovative microarray was designed and fabricated especially for both cell- and tissue-based impedance spectroscopy with 3D cultures and provides a basis for drug screening without costly and time-consuming biological assays.

166

Sebastian Wegner HTWK Leipzig

ID 117 Department of Electrical Engineering and Information Technology swegner@fbeit.htwk-leipzig.de http://portal.fbeit.htwk-leipzig.de/

Alternative instrumentation concepts for bioimpedance spectroscopy for application specific multichannel measurement & diagnosis solutions

Sebastian Wegner,

Abstract – Today´s conventional setups for the instrumentation of bioimpedance spectroscopy mostly include generic impedance analyzers aimed at laboratory needs. Hardly any hardware specially designed for electrical bioimpedance spectroscopy is available. This - and the fact that laboratory impedance analyzers are price intensive, lack the ability of simultaneous measurements in multichannel setups and are not suited for application specific and compact solutions - raises the question of a scalable, to different applications adjustable and compact alternative to conventional instrumentation concepts. The standard methods for impedance analysis are in their original form not fit for that task and therefore a new approach based on the vector rectifier-concept and the lock-in effect was developed. For this measurement method two different implementation approaches, the analog lock-in method and the direct-sampling method, were conceived. The resulting designs were modelled both numerically and algebraically to enable derivation of calculation specifications including application specific nonlinearities and minimizing errors and noise influences. Further, a prototypic example application aimed at multi-electrode-array based cell-impedance spectroscopy was designed to allow for later evaluation of the generated concepts.

 

167

Florian Then Bergh Universitätsklinikum Leipzig

ID 118 Clinic and Polyclinic of Neurology thenberf@medizin.uni-leipzig.de http://neurologie.uniklinikum-leipzig.de/

Trial Design of Remyelination Trials in Multiple Sclerosis: Reproducibility of visual evoked Responses

Florian Then Bergh,

 

Background: Remyelination is an important repair strategy in multiple sclerosis (MS). Latencies of visual evoked responses are a suitable surrogate for remyelination of the optic nerve. However, the test-retest variability has been incompletely evaluated, especially in the case of pathologically delayed potentials.

Methods: Visual evoked responses were recorded on two occasions, one to three days apart, in 30 patients with definite MS or evaluated for possible MS. Steroid treatment between the two recordings was an exclusion criterion. For N70 and P100 latencies and N70/P100 amplitude, mean and difference of the two recordings were calculated before and after verification of cursor positioning by a physician blinded for the sequence of recordings.

Results: Before verification, the difference between first and second VEP was 0.97 +/- 8.3 msec for N70 latency, 0.8 +/- 9.4 msec for P100 latency (n=58 eyes) and 0.49 +/- 2.6 μV for N70/P100 amplitude (mean +/- SD). Independent verification did not change the mean values of these variables (p>0.8 for all variables); two eyes were judged as unsuitable for analysis. The differences between first and second recording could be reduced to 0.07 +/- 4.4 msec (N70), 0.8 +/- 4.24 msec (P100) and 0.38 +/- 2.5 μV (amplitude). The 95 % confidence interval of P100 latency differences after verification was 0.49 - 1.98 msec.

Conclusions: Similar to magnetic resonance imaging, use of evoked potentials in MS remyelination trials will require a central evaluating facility, verification of longitudinally consistent recording and interpretation. During the screening period, reproducibility should be verified individually, and our data provide the basis for defining a maximum of test-retest variability in inclusion criteria.

 

168

 

169

 

170

Microfluidics

 

 

171

Thomas Schneider Technische Universität Dresden

ID 119 Ins. Of Food Tech. And Bioprocess Engineering Schneit2@ccf.org http://tu-dresden.de/die_tu_dresden/fakultaeten/ fakultaet_maschinenwesen/ilb

Generation of Microspheres as Drug Carriers Using Hydrodynamic Flow Focusing

Thomas Schneider, Thomas Bley, Urs Hafeli

 

Conventional chemotherapy for the treatment of various cancer types employs standard dose administration of drugs that act on rapidly dividing cells in order to kill them. Due to the necessity of high drug concentrations, patients often experience severe side effects. The search for new anti-cancer agents is therefore focused on low dose administration (concerning the whole body) and high specificity for cancerogenic cells. One way to achieve this goal is localized drug targeting, where the anti-cancer drug is enclosed in a matrix (e.g., a core shell, a solid particle, a polymeric film) and either transplanted or captured/accumulated on site (e.g., by magnetic targeting). The drug is then released in a time-dependent manner based on properties of the carrier (e.g., volume and size distribution of the particles, release profile) and acts directly and locally in a high concentration on the cell population or tumor to be treated. A seminal and easy to use technology that enables the generation of uniform and monodisperse microspheres in a single step, at low cost, and high efficiency, is the so called hydrodynamic flow focusing. First preparations of microspheres encapsulating fluorochrome dyes with the hydrodynamic flow focusing technique were successful. For medical applications, however, the use of biodegradable polymers is preferable to allow for controlled (slow) drug release, increase biocompatibility, and completely eliminate the need for removal of the implants. The goal of this thesis is to employ and study the process of hydrodynamic flow focusing for the generation of uniform biodegradable polymer microspheres. To achieve this goal, a flow focusing apparatus was designed, constructed, and subsequently tested for the influence of various process parameters. Furthermore, protocols for the successful encapsulation of anti-cancer agents were tested and established. The generated drug-loaded and biodegradable microspheres were classified statistically for their size distribution and evaluated for their surface morphology and drug release profile using standardized methods (ISO-DIN), UV spectroscopy, as well as scanning electron microscopy. summary : In conclusion, a comprehensive parameter study established the effect of chemical and physical parameters on the final microsphere size distribution. A dispersity index with a coefficient of variation of as low as 6 % was achieved for microspheres composed of the biodegradable polymer poly(D,L-lactide-co-glycolic acid). Furthermore, the anti-cancer drug camptothecin was encapsulated in biodegradable poly(D,L-lactide-co-glycolic acid) and poly(lactic acid) microspheres. The drug-loaded microspheres showed both, a polymer- and dose dependent drug release profile in vitro over a period of 14 days and a drug-induced cytotoxicity.

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Randy Kurz Universität Leipzig

ID 120 Translational Center for Regenerative Medicine IMONIT kurz@uni-leipzig.de www.trm.uni-leipzig.de/imonit.html

Nanomicroimplants for controlled drug delivery

Randy Kurz,

 

The main goal of this project will be the development of a first prototype of an electrically controlled drug delivery microimplant for a physiologically mediated in vivo release of active substances in the case of critical space. The micro-implant will be designed for controlled release of an active substance from a reservoire comprising a thin carrier substrate made of a material that is impermeable with regard to the active substance but that has adjustable pores for substance release. Electrodes are arranged in the vicinity of these pores and can be controlled by means of an electronic system which is integrated into the carrier substrate (synthetic material). The pores consist of micro-gaps or micro-channels with electrodes on both ends and are covered with a layer of electro-porous material on the inner side of the carrier substrate. The project includes the designing, developing and testing of this electronic micro-device, of the drug immobilisation techniques, and of the establishment of in vitro biological test stations for proof-of-principle of controlled drug release. Finally the micro-system is to be used for a controlled release of active substances in applications where space availability in vivo is critical (e.g. spinal cord, CNS etc.).

 

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Stefan Harazim Technische Universität Dresden

ID 121 BIOTEChnology Center Professorship Schwille - Biophysics Stefan.harazim@biotec.tu-dresden.de www.biotec.tu-dresden.de/schwille

Controlled dynamic droplet fusion in PDMS based microelectromechanical systems (MEMS) for GFP in-vitro expression

Stefan Harazim

 

A multifunctional and high-efficiency microelectromechanical microfluidic system for droplet formation and forced fusion is presented. It is able to generate pairs of droplets in oil with a constant diameter down to 5 micrometer and different content (here an in-vitro GFP kit and the DNA pQBi-63 are used). These droplet pairs can be fused and stored immobile. The structure of this MEMS is based on PDMS and an ITO-electrode-structure for electro fusion. Shear forces caused by the oil flow allow the droplet formation in the two T-junctions. The fusion takes place between the droplet generators and the storage area where an alternating high voltage field is applied. To avoid spontaneous droplet coalescence the surfactant Span 80 (4 %, v/v) is added to the oil. When a droplet containing the DNA and one with the GFP-kit (include T7 polymerase) merged together the translation/transcription starts and GFP will be produced. After droplet immobilisation this biochemical reaction can be detected by fluorescence measurement.

 

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BIOTEC

The Biotechnology Center (BIOTEC) is a unique interdisciplinary center focusing on research and teaching in molecular bioengineering, a combination of modern cell biology and genetics with traditional material and engineering sciences. The center hosts 13 outstanding international research groups and employs 200 scientists and support staff from 30 nations who are dedicated to advancing fields that include genomics, proteomics, biophysics, cellular machinery, tissue engineering, and bioinformatics. All research is facilitated by the BIOTEC, which supplies essential central technology platforms and supports startup companies. A unique feature of the BIOTEC is that, in addition to supplying basic biotechnology research requirements, it makes the latest devices and cutting edge technology available to both research groups and companies. Scientists and entrepreneurs have access to facilities for microscopy and imaging, genomic engineering, mass spectrometry and zebrafish, and they take advantage of the media kitchen service.

The BIOTEC is housed in the BioInnovationsCenter (BIOZ), a research entity that sits at the heart of the young biotechnology cluster BIOPOLIS Dresden and was created with the mission of Economy and Science consolidated. The BIOTEC serves as the central scientific unit of the Technische Universität Dresden and is also home to many companies working in fields that include medical technology, computer software, agent characterization, gene therapy, biomaterial development, organic light emitting diodes, and cell membrane research, e.g. for treating influenza. This pairing of scientific and economic competence in the BIOTEC sets the stage for the transfer of basic research results to startup companies who can ultimately generate marketable products. Thus far, uniting the latest basic research with successful companies has generated three spin-offs led by BIOTEC professors– Genebridges by Francis Stewart, nAmbition by Daniel J. Müller and Transinsight by Michael Schroeder.

Biotechnology in Dresden is a young and dynamic economy sector that has already received international kudos. One particularly outstanding feature of Dresden Biotechnology is the strong network of high-level research institutes, such as the Max-Planck-Institutes, Leibnitz and Fraunhofer Institutes, and the Technische Universität Dresden, that cover a large scientific spectrum and achieve groundbreaking results through efficient interdisciplinary cooperation.

The following scientists head research groups in the BIOTEC:

- Dr. Konstantinos Anastassiadis (Stem Cell Engineering) - Dr. Andreas Beyer (Systems Biology and Cellular Networks) - Prof. Dr. Michael Brand (Director BIOTEC, Developmental Genetics) - Dr. Denis Corbeil (Tissue Engineering) - Prof. Dr. Bernhard Hoflack (Proteomics) - Prof. Dr. Daniel J. Müller (Cellular Machines) - Dr. Maria Teresa Pisabarro (Structural Bioinformatics) - Dr. Erik Schäffer (Nanomechanics) - Prof. Dr. Michael Schroeder (Bioinformatics) - Prof. Dr. Petra Schwille (Biophysics) - Dr. Ralf Seidel (DNA Motors) - Prof. Dr. Francis Stewart (Genomics) - Dr. Gilbert Weidinger (Wnt signaling in Development and Regeneration)

Prof. Dr. Michael Brand Technische Universität Dresden BIOTEChnology Center Email: katrin.grosser@biotec.tu-dresden.de www.biotec.tu-dresden.de

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The Center for Biotechnology and Biomedicine (BBZ)

The BBZ is an internationally competitive center which promotes research and development as well as teaching and advanced training in the areas Protein Engineering & Bioanalytics, Molecular Medicine & Therapeutics as well as Biomedical and Cell Engineering. Platform technologies and methods in biotechnology, biomedicine and nanotechnology are offered as services for academic institutions and industry. Here, the BBZ strengthens and supports collaborations between institutional and university research teams but also forms a unique interface between academia and industry in Europe. With expertise and competence, innovations are transferred to industrial utilization. With a focus on red biotechnology and following the motto "From molecule to patient" the BBZ develops and offers chemical compounds, proteins and other biomolecules, cells as well as tissues as instruments and products for a wide variety of biotechnological and biomedical applications. To this end, novel analytical methods and preparative procedures are developed. The center collaborates closely with the University Hospital and the Interdisciplinary Center for Bioinformatics (IZBI) at the University of Leipzig. A central office for management and acquisition supports the networking between science and business. In the BIO CITY LEIPZIG six academic research groups from the chemical, biological and medical sciences work under one roof with innovative biotechnological companies including many startups. In addition, 32 life science research groups in the Leipzig area are currently members of the BBZ. The BBZ harbors the following members: From the Faculty of Biology, Pharmacy and Psychology: - Prof. Annette Beck-Sickinger, Biochemistry / Bioorganic Chemistry - Prof. Sunna Hauschildt, Immunbiology - Prof. Karen Nieber, Pharmacology for Natural Scientists - Prof. Andrea Robitzki, Molecular Biological-Biochemical Process Technology (Professorship

of BBZ) - Prof. Martin Schlegel, Molecular Evolution and Animal Systematics with focus on Molecular

Phylogeny - Prof. Christian Wilhelm, Plant Physiology - Dr. Thomas Greiner-Stöffele, White Biotechnology From the Faculty of Chemistry and Mineralogy: - Prof. Stefan Berger, Analytical Chemistry - Prof. Athanassios Giannis, Organic Chemistry / Natural Products Chemistry - Prof. Evamarie Hey-Hawkins, Inorganic Chemistry - Prof. Ralf Hoffmann, Bioanalytics (Professorship of BBZ) - Prof. Helmut Papp, Technical Chemistry / Heterogeneous Catalysis - Prof. Norbert Sträter, Structural Analysis of Biopolymers (Professorship of BBZ) From the Faculty of Mathematics and Computer Science: - Prof. Martin Middendorf, Parallel Computing and Complex Systems - Prof. Erhard Rahm, Informatics - Prof. Gerik Scheuermann, Image and Signal Processing - Prof. Peter Stadler, Bioinformatics From the Faculty of Medicine: - Prof. Klaus Arnold, Medical Physics and Biophysics - Prof. Augustinus Bader, Cell Techniques and Applied Stem Cell Biology (Professorship of

BBZ) - Prof. Frank Emmrich, Clinical Immunology - Prof. Kurt Engeland, Molecular Oncology - Prof. Markus Löffler, Medical Informatics, Statistics and Epidemiology - Dr. Astrid Schön, Molecular Cell Therapy - Prof. Peter Seibel, Molecular Cell Therapy (Professorship of BBZ) - Prof. Jan C. Simon, Dermatology and Allergology - Dr. Peter Ahnert, Molecular Diagnostics - Microarray Techniques

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From the Faculty of Veterinary Medicine: - Prof. Gottfried Alber, Immunology - Prof. Manfred Blessing, Molecular Pathogenesis (Professorship of BBZ) - Prof. Hermann Müller, Veterinary Virology including Diagnostics - Dr. Reinhard Straubinger, Molecular Medicine of Contagious Diseases From the Faculty of Physics and Earth Science: - Prof. Josef Alfons Käs, Experimental Physics / Soft Matter Physics with focus on

Cellbiophysics - Prof. Friedrich Kremer, Experimental Physics, Molecular Physics, Polymer Physics, Material

Science Prof. Dr. Andrea Robitzki (Chair) Dr. Svenne Eichler (Chief Executive Officer) Universität Leipzig Center for Biotechnology and Biomedicine (BBZ) E-Mail: kontakt@bbz.uni-leipzig.de www.bbz.uni-leipzig.de

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A ACKERMANN, Manuela – 60 AHNERT, Peter – 50, 125, 165 ALBER, Gottfried – 66, 70 AL-ROBAIY, Samiya – 51 ANDEREGG, Ulf – 61, 131 ANDERS, Dirk – 50 ANDERS, Gerd – 117 ANDREOPOULOS, Alexander – 109 ANDREOPOULOS, Bill – 109 ARNHOLD, Jürgen – 82 AUST, Gabriela – 61

B BADER, Augustinus – 62, 138, 144 BAINES, Ivan – 73 BALDAUF, Carsten – 43, 108 BALLSCHMITER, Meike – 87, 92 BARKOW-OESTERREICHER, Simon – 110 BARTHEL, Henryk – 129 BAUER, Manfred – 165 BAUMANN, Lars – 58 BAUMANN, Ulf - Dietrich - 64 BAYER, Sally – 87 BECK, Mike – 41 BECK-SICKINGER, Annette – 46, 49, 53, 58, 64, 68, 77 BELLMANN, Kathrin – 58 BERNHARD, Detlef – 115 BERNHARDT, Anne – 127, 128 BERNSTEIN, Peter – 124 BERNT, Matthias – 116, BERTHOLD, Frank – 89 BEUTNER, René – 63 BLESSING, Manfred – 45, 51 BLEY, Thomas – 80, 91, 148, 151, 155, 156, 164, 170 BOENSCH, Katrin – 88, 96 BOLDT, Andreas – 153 BORLAK, Jürgen – 157 BOSCHKE, Elke – 97 BÖSELT, Iris – 67 BÖTTCHER, Gunther – 140 BöTTNER, Antje – 133 BRAKMANN, Susanne – 52 BRANDT, Frank – 108 BRAUN, Sebastian – 90 BRINGMANN, Andreas – 93 BRUMEN, Milan – 83 BUCHHOLZ, Frank – 114 BÜCHNER, Bernd – 65 BÜTTNER, Anita – 74 BURMEISTER, Beatrice – 128   GRAUPNER, Sylvi – 124 GREINER-STÖFFELE, Thomas – 87, 92, 96

BUSSE, Kathy – 44

C CHACKERIAN, Alissa – 70 CORBEIL, Denis – 124 COTTIN, Thomas – 74 CZUPALLA, Cornelia – 51

D DAHL, Janine – 90 DEMMEL, Lars – 41 DEUSSEN, Andreas – 69 DIETERLEN, Maja – 153 DIETZSCH, Christian – 148 DONATH, Edwin – 83 DONG, Meng – 124 DRESSEL, Frank – 111

E EICHENTOPF, Inga-Maria – 137 EICHLER, Antje – 92 ELIE-CAILLE, Celine – 150 EMMENDÖRFFER, Andreas – 131 EMMRICH, Frank – 60, 129 ERLER, Axel – 120, 150 ESCHENHAGEN, Ursula – 80 ESCHRICH, Klaus – 85 EXNER, Matthias – 52 EYLENSTEIN, Roy – 98

F FICKERT, Stefan – 124 FIEBER, Christina – 131 FISCHLECHNER, Martin – 82, 83 FRANKE, Heike – 57, 129 FRERICH, Bernhard – 133, 134, 144 FREUDENBERG, Marina A. – 66 FRITZSCH, Guido – 115

G GELINSKY, Michael – 124, 127, 128 GENZEL, Yvonne – 148 GEORGIEV, Milen – 151 GEORGIEV, Vasil – 155, 156 GERICKE, Martin – 157 GERRITS, Bertran – 110 GIANNIS, Athanassios – 74 GILBERT, Matthias – 95 GLANDER, Hans-Jürgen – 82 GRAHNERT, Anja – 140   GROSCHE, Jens – 57 GROßMANN, Udo – 129

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GRUNER, Melanie – 113 GUERRA, Jorge – 136 GÜNTHER, Klaus-Peter – 124 GÜNTHER, Robert – 64

H HAAS, Christiane – 151 HAASE, Diana – 65 HABERMANN, Bianca – 120 HÄRTIG, Wolfgang – 68 HAFELI, Urs – 170 HALOžAN, David – 83 HAMPEL, Silke – 65 HANKE, Thomas – 127, 128 HANISCH, Isabelle – 168 HARAZIM, Stefan - 172 HARMEL, Joachim – 89 HAUSSCHILDT, Sunna – 140 HAVLIS, Jan – 41 HEBBAR, Sarita – 97 HEIDEBRECHT, Felicia – 138 HEILMANN, Andreas – 142 HEINE, Claudia – 57 HEINE, Tobias – 150 HELD, Karl – 89 HEMPRICH, Alexander – 144 HENGSTLER, Jan G. – 95 HERMES, Matthias – 95 HERMANN, Sigrun – 89 HEUBECK, Christian – 90 HEYN, Bianca – 52 HOFER, Michael – 88 HOFLACK, Bernard - 51 HOFFMAN, Gregor – 96 HOFMANN, Hans-Jörg – 64 HOFMANN, Kathleen – 50 HOLLAND, Heidrun – 125, 165 HSU, Peggy – 41 HUGHES, Amanda – 142 HUETTL, Regina – 89 I IANDIEV, Ianors – 93 ILIEVA, Mladenka – 155, 165 ILLES, Peter – 57, 59, 129

J JEHMLICH, Nico – 86 JOHO, Dieter – 110 JUNGHANS, Frauke – 142

K KAGERMEIER-SCHENK, Birgit – 42 KALAIDZIDIS, Yannis – 41 KAMPRAD, Manja – 60, 118, 143 KAPPELER, Stefan – 55 KARL, Stephan – 164 KASTELEIN, Robert A. – 70 KESSLER, Renate – 85 KEYSER, Ulrich F. – 94 KIRSTEN, Holger - 50, 165 KLARE, Peter – 89 KLINGLER, Rüdiger – 65 KLINGNER, Edith – 126 KLOß, Daniel – 166 KNAUER, Jens – 45 KöLLNER, Kristin – 85 KOSACKA, Johanna – 157 KOSCHNY, Ronald – 165 KOSEL, David – 46 KOVACS, Peter – 67 KRÄMER, Kai – 65 KRAUß, Eberhard – 73 KRAUT, Rachel – 97, 99 KREHAN, Mario – 90 KREMER, Friedrich – 94 KRETZSCHMAR, Georg – 48 KRUPP, Wolfgang – 165 KRUPSKAYA, Yulia – 65 KÜTTNER, Bartholomeus – 98 KUKAT, Alexandra – 47, 56 KUKAT, Christian – 47, 56 KURZ, Randy – 171 KUSKA, Jens - Peer – 118, 133 KöNIG, Ulla – 127

L LABUDDE, Dirk – 109, 111 LACH, Jochen – 62 LANGE, Franziska – 118 LAUER, Günter – 126 LEAL-EGANA, Aldo – 62, 138 LEHMANN, Antje – 137 LEIDICH, Stefan – 103 LEßIG, Jacqueline – 82 LIESE, Jan – 144 LIESE, Susanne – 144 LINDNER, Diana – 64 LIPERT, Kamil – 65 LISSNER, Andreas – 89      LOCHMANN, Alexander – 135 LODE, Anja – 127, 128

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LUDWIG-MüLLER, Jutta – 151 LUTZ, Johanna – 137 LUTZ, Matthias – 65

M MÄDER, Karsten – 135 MAENDL, Stephan – 137 MAHN, Christopher – 65 MAJSCHAK, Jens-Peter – 80 MARESCA, Marcello – 120, 150 MARSICO, Annalisa – 111 MAUERMANN, Marc – 80 MEIER, Rene – 108 MEIß, Karl-Michael – 119 MEIXENSBERGER, Jürgen – 165   MENZEL, Nikolas – 90 MERKLE, Daniel – 115, 116 MICHAEL, Jan – 63 MIDDENDORF, Martin – 115, 116 MILOSEVIC, Javorina – 59, 153 MITTAG, Anja – 139 MOKHTAR, Mohd Noriznan – 113 MOSCHÜTZ, Susanne - 98 MüLLER, Daniel J. – 150, 160 MüLLER, Uwe – 45

N NIEBER, Karen – 68, 69, 140    NITZSCHE, Hagen – 135 NOWICKI, Marcin – 157

O ONDRUSCHKA, Jelka – 91 OPITZ, Tommy – 81

P PANNICKE, Thomas – 93 PANSE, Christian – 110 PASZKOWSKI-ROGACZ, Maciej – 114 PATT, Marianne – 129 PAVLOV, Atanas – 155, 156 PERSEKE, Marleen – 115 PFEIFER, Katja – 40 PISABARRO, M. Teresa – 43, 106, 107, 108, 112, 114 POPPE, Monike – 153 PURSCHE, Theresia – 51

R RAMSCH, Kai – 115 REIBETANZ, Uta – 82, 83 REICHARDT, Jörg – 50 REICHENBACH, Andreas – 93 REINSCH, Holger – 130 RICHNOW, Hans-Hermann – 86 RIEMER, Thomas – 103 RIEß, Kathrin – 140 RÖMPLER, Holger – 67 ROBITZKI, Andrea A. – 62, 138, 166, 171 ROCKEL, Sylvie – 99 RODE, Isabel – 62, 138 ROSCHITZKI, Bernd – 110 ROTH, Christian – 84, 100 ROTHERMEL, Andrée – 166 RUBINI, Patrizia – 59 RUDOLPH, Eckehart – 127 RUDOLPH, Rainer – 159 RUFKE, Cornelia – 69 RUHLAND, Saskia – 50 RüHL, Ralf – 127

S SAALBACH, Anja – 61 SABRI, Osama – 129 SACK, Ulrich – 118 SALOMO, Mathias – 94 SAMSONOV, Sergey – 106 SASAKI, Erika – 132 SCHAARSCHMIDT, Grit – 149, 154 SCHÄFER, Ingo – 47, 56 SCHARNWEBER, Dieter – 63 SCHEIBE, Johanna – 152, 153 SCHERF, Nico – 118 SCHEWTSCHIK, Sabine – 154 SCHILD, Enrico – 49 SCHILDAN, Andreas – 129 SCHILLING, Erik – 71 SCHLAITZ, Anne-Lore - 41 SCHLAPBACH, Ralph – 110 SCHLEGEL, Martin – 115 SCHLIEBE, Nicole – 72 SCHLÖMANN, Michael – 100 SCHNABEL, Kristin - 40 SCHNEIDER, Thomas – 170 SCHOBER, Ralf – 165 SCHÖN, Astrid – 90 SCHÖNEBERG, Torsten – 44, 67, 72 SCHÖNZART, Lena – 63 SCHROECK, Kathleen – 143

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SCHROEDER, Michael – 109, 111 SCHUBERT, Andreas – 140 SCHÜTZE, Nicole – 66 SCHULZ, Silke – 70 SCHUMANN, Anika – 95 SCHUMANN, Dirk – 143 SCHUMANN, Julia – 45 SCHWAN, Stefan – 142 SCHWARZ, Elisabeth – 135 SCHWARZ, Johannes – 44, 59, 153, SCHWARZ, Sigrid – 59, 153 SCHWENZER, Bernd – 63 SEIBEL, Peter – 47, 56 SEIDEL, Ralf – 150 SEIFERT, Katrin – 74 SHEVCHENKO, Andrej – 41 SICHARDT, Kathrin – 68 SICKINGER, Anselm – 171 SIEGEMUND, Sabine – 66 SIEGERT, Fritzi – 140 SIMON, Jan C. – 131 SÖHLING, Brigitte – 159 SPAINK, Herman – 42 SPANEL-BOROWSKI, Katharina – 157 SPRINGER, Armin – 127 SPöRL, Gabriele – 126, 130 SPOHN, Uwe – 142 STADLER, Peter F. – 115 STAEUDTE, Andrea – 142 STEINERT, Steffen – 97 STEPHAN, Holger – 141 STEWART, A. Francis – 120, 150 STICHEL, Jan – 53 STOLZING, Alexandra – 53 STORCH, Alexander – 59 STRAETER, Norbert – 84, 98, 100, 101 STRAUBINGER, Reinhard Konrad – 45, 66 STROTMANN, Rainer – 44, 72 STRUHALLA, Marc – 94 STUMPP, Sascha Nico – 52 STUMVOLL, Michael – 67 SURENDRANATH, Vineeth – 120

T TARNOK, Attila – 139 TAYLOR, Arthur – 65 TEUCHER, Madeleine – 120 TEYRA, Joan – 106, 107 THEN BERGH, Florian – 118 TOMCZAK, Aurelie – 112 TRESSET, Guillaume – 97 TRETTNER, Susanne – 132 TRUTNAU, Mirko – 91 TUERKER, Can – 110 TUUKKANEN, Anne – 111

U UCKERMANN, Ortrud – 93 UHLMANN, Susann – 93

V VAN DIECK, Jan – 64 VATER, Corina – 128 VOGT, Carsten – 86 VOLLMER, Günter - 48 VON BERGEN, Martin – 86 VON EINEM, Sabrina – 135 VONAU, Winfried – 89

W WALCH-SOLIMENA, Christiane – 41        WALTHER, Anja – 128 WANDEL, Elke – 61 WASHEIM, Susanne – 84   WEBER, Jost – 151, 155, 15 WEGENER, Silke – 100   WEGMANN, Susanne – 150 WEGNER, Annett – 57 WEGNER, Florian – 153 WEGNER, Sebastian – 167 WEHRMEISTER, Ulf – 133 WEIDINGER, Gilbert - 40, 42 WEINZIERL, Konstanze – 134 WEIZENMANN, Nicole – 84 WETZEL, Richard – 97 WIEDEMANN, Peter – 93 WILHELM, Christian – 95 WINTER, Karsten - 133, 134 WIRTH, Manfred – 65 WOBUS, Manja – 61 WOLF, Gert – 89 WOLFRAM, Grit – 50 WOLTER, Anja – 65 WORCH, Hartmut – 63 WURM, Antje – 93

Z ZBORWOWSKI, Maciej – 164 ZEBISCH, Matthias – 101 ZERIAL, Marino – 73 ZHANG, Youming – 120 ZIELONKA, Anja – 54 ZIERAU, Oliver – 48 ZIMMERMANN, Wolfgang – 84, 113 ZLOBINSKY, Anja – 140 ZUR NIEDEN, Nicole – 132

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